Spring Conference 2011 - Society for General Microbiology

↑Contents 

Society for General Microbiology 

Spring 

Conference 

2011 

Harrogate 

International 

Centre 

11–14 April 

Abstracts 

Microbiology 

s o c i e t y f o r g e n e r a l 

www.sgmharrogate2011.org.uk

Sessions 

Spring Meeting 11–14 April 2011 

Harrogate – www.sgmharrogate2011.org.uk 

CONTENTS 

HA01 Seeing the cell through the ‘eyes’ of the virus 3 

HA02 Maths & microbes 10 

HA03 Social evolution in micro-organisms 13 

HA04 Vaccines 16 

HA05 Meningitis 27 

HA06 Guarding microbial diversity: the importance of fundamental infrastructure in underpinning the microbial sciences 34 

HA07 Mechanisms of DNA repair 37 

HA08 Microbial PAMPS 40 

HA09 Food biosecurity 44 

HA10 Insect symbiosis 47 

HA11 Working with the media 50 

HA12 Virology workshop: pathogenesis 52 

HA13 Intracellular life 59 

HA14 Virology workshop: replication & gene expression 65 

HA15 Osmotic & oxidative stress responses 73 

HA16 Virology workshop: virus structure & assembly 76 

HA17 Virology workshop: cell-to-cell transmission 81 

HA18 Virology workshop: vaccines & antivirals 84 

HA19 Life at zero growth rate 88 

Posters 

HA01 Seeing the cell through the ‘eyes’ of the virus 90 

HA02 Maths & microbes 91 

HA03 Social evolution in micro-organisms 91 

HA04 Vaccines 95 

HA05 Meningitis 97 

HA06 Guarding microbial diversity 101 

HA08 Microbial PAMPS 102 

HA09 Food biosecurity 102 

HA10 Insect symbiosis 106 

HA12 Virus pathogenesis 107 

HA13 Intracellular life 114 

HA14 Virus replication & gene expression 117 

HA15 Osmotic & oxidative stress responses 123 

HA16 Virus structure & assembly 127 

HA17 Cell-to-cell transmission of viruses 132 

HA18 Virus vaccines & antivirals 133 

HA19 Life at zero growth rate 135 

CMM Clinical & medical microbiology 136 

ENV Environment 146 

FB Fermentation & bioprocessing 149 

GM General microbiology 150 

SC Systems & cells 162 

Authors 164 

Additional abstracts 172 

Please note: Abstracts are published as received from the authors and are not subject to editing. 


Microbiology


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SESSION AbSTrACTS 

HA01 Seeing the cell through the ‘eyes’ of the virus 

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HA01 

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How animal viruses exploit endocytosis for cell entry 

Ari HElENiuS, Pierre-Yves lozach, Florian Schmidt, roger Meier, Jason Mercer 

Inst. Biochemistry, ETH Zurich, Switzerland 

using cellular, molecular, and systems approaches in combination with live cell imaging and electron microscopy, we are investigating how 

various animal viruses bind to cells, how they are internalized by endocytosis, and how they are delivered to organelles such as endosomes 

and the endoplasmic reticulum. We also analyze how the viruses and viral capsids escape into the cytosol or the nucleus, and how the 

genome is uncoated for replication. A variety of perturbation techniques and high and medium throughput sirNA screens have allowed 

identification of viral infectomes, i.e. the collection of cellular proteins and pathways involved in assisting entry of different viruses. Currently, 

the group is analyzing the entry of uukuniemi virus, a bunyavirus, via the DC-SiGN. We also focus on vaccinia virus (a large DNA virus of 

the poxvirus family), that triggers cell surface blebbing, macropinocytic internalization, capsid uncoating, and DNA replication. To induce 

uptake, mature particles (MVs) of vaccinia mimic apoptotic bodies. Once the core has been delivered to the cytosol and early transcription 

has taken place, proteosomes are needed to uncoat the viral DNA, and ubiquitination and again the proteasome to start replication of the 

viral DNA. 

The stepwise nature of adenovirus uncoating 

urs Greber 

Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland 

Viruses depend on host factors for propagation, and unfold unchartered complexity upon entry into cells. Entry is a coordinated and 

stepwise process. it typically involves attachment, movements on the plasma membrane, cell signalling, endocytic uptake, membrane 

penetration, cytoplasmic trafficking and genome release. Here i discuss how functional modules of the host coordinate the stepwise 

disassembly programme of human adenoviruses. Adenoviruses are small DNA-tumor viruses replicating in the nucleus. They infect the 

upper and lower respiratory, urinary and digestive organs, and give rise to epidemic conjunctivitis, and kill immunocompromised individuals. 

i will present data showing how adenoviruses take advantage of their receptors for actin and myosin-dependent movements on the plasma 

membrane, and thereby initiate their uncoating programme. This is key to activate membrane-lytic proteins, which are located within 

the intact virion. Partly uncoated cytosolic particles travel on microtubules to the nucleus, and release their genome at the nuclear pore 

complex. DNA-uncoating surprisingly requires conventional kinesin, a viral kinesin receptor, a kinesin-activating nucleoporin, and a virusbinding 

nucleoporin. This leads to disruption of the pore complex and enhances infection. 

Proteomics reveals new host cell proteins involved in viral entry and immune evasion 

Klaus Früh 

Oregon Health & Science University, Vaccine and Gene Therapy Institute, USA 

Many proteins of the cell membrane are dramatically altered during viral infection as a consequence of viral targeting of selected host 

pathways during entry, replication and egress. To obtain a global view of the effects of viral infection or selected viral immunomodulators on 

the membrane proteome we are using stable isotope labeling of amino acids in cell culture (SilAC) to quantitate changes in the abundance 

of membrane proteins. This approach revealed that Kaposi’s sarcoma associated herpesvirus and Human immunodeficiency virus eliminate 

the interferon-induced antiviral host protein BST2/Tetherin from the cell surface by employing viral or host ubiquitin-ligases. in contrast, 

we observed that BST2/Tetherin is used by Human Cytomegalovirus to gain entry into monocytes. Furthermore, SilAC revealed a central 

role for tetraspanin-enriched microdomains during HCMV entry. Quantitative membrane proteomics thus revealed novel functions of viral 

proteins and implicated novel host cell proteins and pathways in controlling or facilitating viral infection. 

Single-particle fluorescence studies of viral fusion 

Antoine Van Oijen 

Zernike Institute for Advanced Material, University of Groningen, The Netherlands 

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to 

observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. 

We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single influenza virus particles fusing with a target bilayer 

on an essentially fluid support. Analysis of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, 




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long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series 

of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be 

activated to initiate the fusion process. 

Capturing the bullet: the cell entry pathway of a rhabdovirus 

Sean Whelan 

Microbiology & Molecular Genetics, Harvard Medical School, USA 

using vesicular stomatitis virus (VSV), a prototype of the rhabdoviridae family, we have probed the cellular biology of virus entry and gene 

expression. VSV enters cells through clathrin dependent endocytosis. The dimensions of the virus particle are, however, larger than those 

of typical clathrin coated vesicles. We found that VSV is internalized through vesicles that are incompletely coated with clathrin, and that 

depend upon actin polymerization. Truncation of VSV particles through manipulation of the genome length, results in the uptake of virus 

through fully clathrin coated vesicles that no longer depend on actin polymerization. Panels of recombinant VSV that contain alternate 

glycoproteins which also mediate clathrin dependent entry, lend further support that the shape of the virus particle is a key determinant of 

the altered mode of clathrin mediated endocytosis. This work provides new insights into the interactions of virus with their host cell during 

their uptake. 

Offered paper A viral ubiquitin ligase has both SUMO-targeted and SUMO-independent specificities that counteract intrinsic 

antiviral defence 

CHriS BOuTEll, Delphine Cuchet, Emilia Vanni, Anne Orr, roger Everett 

MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow 

intrinsic antiviral resistance represents the first line of cellular defence to virus infection. During HSV-1 infection this response is inhibited by 

the viral ubiquitin ligase iCP0. Here we report that iCP0 has SuMO-Targeted ubiquitin ligase (STubl) activity that induces the widespread 

proteasome-dependent degradation of SuMO-conjugated proteins during infection. using PMl as an example, we demonstrate that iCP0 

preferentially degrades SuMO-modified PMl isoforms ii-Vi, but can additionally target PMl.i for degradation in a SuMO-modification 

independent manner. Mutation of SuMO interaction motifs within iCP0 not only affects its ability to degrade PMl, but also its capacity to 

complement and reactivate mutant HSV-1 viruses. in the absence of iCP0 we show that the SuMO pathway plays an important role in 

mediating intrinsic antiviral resistance, leading to the repression of viral replication. We conclude that iCP0 exhibits both SuMO-targeted 

and SuMO-independent specificities in order to counteract intrinsic antiviral resistance during HSV-1 infection. 

Offered paper Analysis of incorporation of tegument proteins VP1/2 and VP16 into virions during HSV-1 infection 

STANiSlAVA SVOBODOVA, Colin Crump 

Division of Virology, Dept of Pathology, University of Cambridge, Cambridge CB2 1QP 

Herpesviruses are large and complex viruses composed of three main structural components: the capsid, a genome containing icosahedral 

core; the tegument, a complex layer of virus proteins; and the envelope, a host derived lipid bilayer containing several viral membrane 

proteins. Compositionally, the tegument is the most complex part of herpesviruses containing up to 25 different proteins in herpes simplex 

virus type-1 (HSV-1). The incorporation of the tegument into the forming virus particles during assembly is poorly understood. There 

is increasing evidence for an ordered addition of the tegument during this process with discrete inner and outer tegument layers being 

composed of different subsets of viral proteins. 

The aim of the current study is to analyse tegument assembly in HSV-1, focussing on two essential tegument proteins VP1/2 and VP16, 

through the use of recombinant viruses. We have used these viruses to observe the dynamics and localization of the tegument proteins 

in both live and fixed cells during HSV-1 infection. Our data has shed new light on the order and localization of herpesvirus tegument 

assembly and suggest that at least some VP1/2 associate with capsids in the nucleus and that the majority of VP16 is incorporated into 

virions in the cytoplasm. 

Offered paper Cytoskeleton hijacking: microtubule dynamics upon HIV-1 infection 

ANA GuErrErO AlONSO, Matthias T. Dittmar 

Blizard Institute of Cell and Molecular Science, Queen Mary, University of London, London 

Despite huge efforts to try and understand its complex and unique life cycle there are still many aspects regarding HiV-1 cell entry to be 

deciphered. One of these aspects is the mechanisms by which HiV-1 hijacks the cytoskeleton, a process that is required for virus entry, 




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but also transport of the reverse transcription complex to the nucleus and exit of the cell. Key regulators of microtubule dynamics such 

as histone deacetylase 6 have been described to be modulated upon HiV-1 infection. recently, we have identified a motif located in the 

cytoplasmic tail of HiV-1 transmembrane protein (gp41) that modulates microtubule stability (tubulin acetylation) in tail- expressing cells, 

this effect being a unique feature of the lentiviral transmembrane protein (Malinowsky, K. et al., Virology 376: 69–78, 2008). Moreover, this 

modulation of microtubule stability reduced virus-cell fusion in Hela-CD4 cells and PM1 T-cells. it is therefore important to gain a more 

detailed knowledge of the upstream and downstream effectors that regulate microtubule dynamics in early HiV-1 infection. Currently, we 

are investigating the effects on microtubule stability (tubulin acetylation) upon expression of various gp41 cytoplasmic tail constructs in 

macrophages and T-lymphocytes using quantitative confocal microscopy. results will be discussed. 

Offered paper Dissecting the different mechanisms of calicivirus cell entry 

NiCOlE DOYlE1 , Terry Jackson2 , Andreas Gerondopoulos3 , Elisabetta Groppelli4 , Paul Monaghan5 , lisa roberts1 1 2 3 University of Surrey, Guildford, Surrey; Institute of Animal Health, Pirbright, Surrey; University of Liverpool, Cancer Research Centre, 

Liverpool; 4University College London, London; 5Australian Animal Health Laboratory, Geelong, Australia 

Caliciviruses are responsible for many diseases of man and animals, for example noroviruses cause outbreaks of human gastroenteritis. 

Although studies on norovirus replication have previously been hampered by the lack of an efficient culture system, the recently identified 

murine norovirus (MNV-1) has provided an excellent model for studying norovirus replication. it was known from previous work in our 

lab that MNV-1 infection of rAW264.7 macrophages does not use the clathrin, caveolin, or flotillin-dependent pathways. However, 

MNV-1 infection does require cholesterol and the presence of active dynamin. in contrast, previous work on the entry of feline calicivirus 

showed that it enters cells via a clathrin-dependent pathway, but is independent of caveolin and cholesterol; clearly these related viruses are 

using very different cell entry mechanisms. We are now using pharmacological inhibitors of signaling pathways (genistein, okadaic acid and 

wortmannin) and dominant negative inhibitors of proteins involved in endocytic and signaling pathways (rab proteins, Arf 6), as well as colocalization 

studies with specific endocytic markers to further elucidate the specific entry pathways used by these viruses. 

Offered paper A two-step mechanism for trafficking of influenza A virus vrNAs to the apical plasma membrane that involves 

the microtubule network 

MAriA-JOAO AMOriM, Eliot read, Emily Bruce, Amanda Stuart, Paul Digard 

University of Cambridge, Cambridge 

The segmented vrNA genome of influenza A virus (iAV) is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins 

(rNPs) and trafficked to the plasma membrane (PM). Cytoplasmic trafficking of rNPs is not well defined, either for route or when the 8 

segments meet to assemble a full genome. using fluorescent in situ hybridization to detect vrNA and live-cell imaging of rNPs, we show 

the initial stages of rNP trafficking involves accumulation of all segments at the pericentriolar recycling endosome (rE), in multiple cell types 

and viral strains. Here, rNPs surrounded gamma-tubulin and colocalized with rab11. As infection progressed, rNPs dispersed throughout 

the cytoplasm in structures of ~250–600 nm that again, contained multiple segments. GFP-tagged rNPs in living cells demonstrated rapid 

(average 1.3 micron/sec), bi-directional and saltatory movement, and characteristic of microtubule-based transport, inhibited by MT-drugs, 

and also co-trafficked with fluorescent rab11. We conclude that iAV rNPs concentrate at the pericentriolar rE, are loaded onto vesicles 

and traffic to the PM. This work shows an important understanding on how the 8 vrNA segments are funneled together to the PM, 

facilitating viral assembly by placing them at the same location where viral budding occurs. 

Offered paper Disruption of microtubule dynamics activates latent KSHV and HIV-1 

EDWArD TSAO1 , Paul Dobson2 , Katarzyna Wisniewska1 , Claire Pardieu1 , Sam Wilson1 , richard Jenner1 , Paul Kellam1,3 1 2 3 University College London, London; University of Manchester, Manchester; Wellcome Trust Sanger Institute, Cambridge 

latency establishment is a key strategy used by herpesviruses and retroviruses for viral persistence and immune evasion and the resulting 

viral reservoirs prevent the eradication of infection. latency is characterized by the ability of latent viral genomes to reactivate into the 

replicative cycle for producing virus progeny. This phase in the virus life cycle is particularly amenable to immune control and therapeutic 

intervention. understanding the molecular mechanisms through which latency is maintained allows it to be targeted therapeutically to clear 

the host of latent infection. Here, we report the use of a chemical genetic screen to both investigate pathways required for viral latency 

and to identify small molecules as potential therapeutics. We screened the NCi DTP Diversity set and identified 25 small molecules that 

reactivate Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency. We show one compound, uClB-15026, disrupts microtubule 

dynamics and triggers, through the extracellular signal-regulated kinase (ErK) pathway, KSHV lytic replication. We demonstrate enhanced 

killing of KSHV-associated tumor cells by ganciclovir when pre-treated with uClB-15026. Furthermore, we show uClB-15026, through 




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the ErK pathway, can induce transcriptional activity from the human immunodeficiency virus type-1 (HiV-1) long-terminal repeat (lTr) in a 

latency model, revealing commonalities between KSHV and HiV-1 reactivation from latency. 

Offered paper The hunt for a host protein that is crucial for arterivirus replication 

ADriAAN H. DE WilDE, Ali Tas, Eric J. Snijder, Martijn J. van Hemert 

Leiden University Medical Center, Leiden, Netherlands 

The replication complexes of positive-stranded rNA viruses are associated with cytoplasmic membrane structures and many steps 

of their replicative cycle heavily depend on cellular processes and host factors. Our research focuses on the replication/transcription 

complexes (rTCs) and host factors involved in nidovirus replication. We previously discovered that the in vitro rNA-synthesizing activity 

of semi-purified Equine arteritis virus (EAV) rTCs depends on a host factor that does not co-purify with rTCs. This host factor has now 

been characterized in more detail. Differential centrifugation of EAV-infected cell lysates yielded a pellet that contained heavy membrane 

structures and all of the viral rNA polymerase. The pelleted rTCs were inactive in an in vitro rNA synthesis assay unless supplemented 

with a host factor preparation. Basic biochemical analyses revealed that this crucial host factor is a cytosolic protein that is conserved 

among higher eukaryotes. We set out to identify this factor by purifying it from Hela cell lysates using size exclusion, ion-exchange- and 

hydrophobic interaction chromatography. Protein fractions were tested for their ability to reconstitute rTC activity. Both ‘serial’ and ‘parallel’ 

purification strategies were applied and the host factor is currently being analysed by conventional mass spectrometry and comparative 

quantitative mass spectrometry-based proteomics. 

Offered paper Using quantitative proteomics to understand how respiratory viruses interact with the host cell 

Brian Dove3 , rebecca Surtees1,3 , Thomas Bean3 , Diane Munday1 , Helen Wise2 , Paul Digard2 , John Barr1 , JuliAN HiSCOx1 1 2 3 University of Leeds, Leeds; University of Cambridge, Cambridge; Health Protection Agency, Porton Down 

The advent of stable isotope labeling with amino acids in cell culture (SilAC) coupled with lC-MS/MS allows different virus-host cell 

proteome interactions to be identified and quantified on a large scale, to the depth of several thousand proteins. Coupled to bioinformatic 

analysis using ingenuity Pathway Analysis provides the resolution of detailed signaling and canonical pathways that are activated/suppressed 

in virus-infected cells. using these approaches we have investigated how the avian coronavirus and human respiratory syncytial virus interact 

with the host cell proteome, mapping the balance between pro-viral and anti-viral activity, specifically focusing on sub-cellular organelles. 

More recently we have used the SilAC approach to simultaneously compare clinical isolates of influenza A virus focusing on 2009-swine 

origin H1N1 with A/Solomon islands/03/06, a seasonal H1N1 strain, in A549 cells, a model respiratory epithelial cell line. These data were 

integrated with transcriptomic and rNAi analysis to provide a detailed global overview of host cell changes in the context of virus-infection. 

A number of common and virus-specific affects were found including the sequestration of rNA hyper mutating enzymes to areas of virus 

replication and the recruitment of immune complex proteins to mitochondria. The functional implications of these results will be discussed. 

Offered paper repulsion of superinfecting virions: a mechanism for rapid virus spread 

VirGiNiE DOCEul1 , Michael Hollinshead1 , lonneke van der linden2 , Geoffrey Smith1 1 2 Imperial College London, Section of Virology, London; Radboud University Nijmegen, Nijmegen, Netherlands 

Viruses were thought to spread across susceptible cells by an iterative process of infection, replication and release, so the rate of spread is 

limited by replication kinetics. recently, we described a novel mechanism that enables vaccinia virus (VACV) to spread across one cell every 

1.2 hours, fourfold faster than predicted from its replication kinetics. using live video microscopy we showed that actin tails are formed 

early during infection on cells not producing any virions, indicating that infected cells are able to form actin tails upon contact with incoming 

virions. Early expression of VACV proteins A33 and A36 is critical for optimal virus spread and both proteins are necessary and sufficient 

for the formation of actin tails upon contact with extracellular enveloped virus (EEV) particles. These data reveal a novel mechanism 

for rapid virus spread by which infected cells repel superinfecting virus particles on actin tails toward non-infected cells. Current work is 

focussing on determining which protein(s) on the incoming EEV is required for induction of actin tail formation and the effect of A33 and 

A36 expression at the cell surface on the spread, membrane breakage and entry of VACV. 

Offered paper The solution structure of the hepatitis C virus (HCV) p7 protein identifies an external, membrane-exposed 

rimantadine binding site 

TOSHANA FOSTEr1,2 , Arnout Kalverda2 , Gary Thompson2 , richard Foster3 , Arwen Pearson2 , Steve Homans2 , Mark Harris2 , 

Stephen Griffin1,2 s o c i e t y f o r g e n e r a l 



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1 2 Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds; Institute of Molecular and Cellular Biology, Faculty of 

Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds; 3School of Chemistry, University of 

Leeds, Leeds 

The hepatitis C virus (HCV) p7 protein is essential for the production and release of infectious HCV particles. p7 is a viroporin which forms 

cation-conductive channels in vitro and acts as a proton channel in mammalian cells. Both channel activity and virion production can be 

targeted by small molecule inhibitors, rendering p7 an attractive drug target. We have determined the solution structure of monomeric p7 

in methanol to better understand channel function and define the mode of action for known inhibitor compounds. using a combination of 

chemical shifts, nuclear Overhauser effects, and paramagnetic relaxation enhancement as restraints, the structure of monomeric p7 confirms 

an α-helical hair-pin fold with the two trans-membrane helices separated by a flexible loop. Binding of rimantadine to both monomeric and 

oligomeric p7 resulted in perturbations to specific residues consistent with a binding site located within the C-terminal, membrane-exposed 

region of the channel, whereas this did not occur for a rimantadine-resistant mutant. Drug binding to this peripheral site is predicted to 

stabilise the closed channel conformation. in conclusion, the structure of monomeric p7 provides insights into the likely conformation of p7 

within an oligomeric assembly enabling future rational design of novel anti-viral compounds. 

Offered paper The role of hepatitis C virus in the pathogenesis of hepatocellular carcinoma 

GArriCK WilSON1 , Claire Brimacombe1 , Gary reynolds1 , Ke Hu1 , Peter Balfe1 , Stefan Hubscher2 , Jane McKeating1 1 2 Institute of Biomedical Sciences, University of Birmingham, Edgbaston, Birmingham; Dept of Cellular Pathology, Queen Elizabeth 

Hospital, Mindelsohn Way, Birmingham 

Hepatitis C virus (HCV) is associated with the development of hepatocellular carcinoma (HCC); however, the role of virus infection in the 

carcinogenic process is unknown. The limited progress in this area reflects the lack of model systems to study the viral life cycle. However, 

recent advances in the identification of HCV strains that assemble infectious particles in vitro enable studies to address the role of HCV on 

cellular oncogenic pathways. recent observations demonstrate that HCV stabilizes hypoxia-inducible factor-1α (HiF-1α), a regulator of the 

transcriptional response to oxygen deprivation and genes involved in angiogenesis, cell invasion and metastasis. HCV infected hepatocytes 

express elevated levels of vascular endothelial growth factor (VEGF), one of the major target genes for HiF-1α, and demonstrate increased 

proliferative and migratory capacity. importantly, inhibition of hepatocellular HiF-1α activity abrogates VEGF expression, restored the 

proliferative and migratory capacity of HCV infected cells and inhibited viral replication. These data highlight an essential role for HiF-1α and 

VEGF in the HCV life cycle and provide novel approaches for the treatment of HCV-associated HCC. 

Viral interior design: rewiring the host to generate organelle platforms for replication 

Nihal Altan-Bonnet 

Dept of Biological Sciences, Rutgers University, Newark, NJ, USA 

We have found that many rNA viruses manipulate multiple host components of the secretory pathway to generate organelles that are 

specialized for replication and are distinct in protein and lipid composition from host cell. We show that specific viral proteins promote 

recruitment of host phosphatidylinositol-4-kinase iiiβ (Pi4Kiiiβ) to membranes, while suppressing recruitment of membrane coat proteins. 

This results in the biogenesis of uncoated membrane platforms that are highly enriched in phosphatidylinositol-4-phosphate (Pi4P) lipids. 

We find that this Pi4P-rich lipid microenvironment is essential for viral rNA replication; and this unique lipid microenvironment may 

regulate the recruitment and assembly of viral replication machinery including viral rNA polymerases which we show specifically and 

preferentially bind Pi4P lipids. in addition, Pi4P lipid enriched membrane platforms along with host membrane curvature and cytoskeletonmodulating 

proteins may induce a membrane organization that can both concentrate replication machinery for efficient viral rNA synthesis 

reactions as well as shield them from host innate immune responses. Our findings reveal how rNA viruses can selectively exploit specific 

elements of the host to form specialized organelles, and identify Pi4Kiiiβ and cellular phosphoinositide lipids as key regulators of viral rNA 

replication and hence potential targets for the development of novel therapeutics. 

Hiding between the sheets – HCV replication at the Er membrane 

John Mclauchlan 

MRC-University of Glasgow Centre for Virus Research 

Chronic infection with hepatitis C virus (HCV) is a leading cause of liver disease across the globe. The virus replication cycle occurs in 

the cytoplasm and is intimately linked with membranes at the Er and the surface of lipid droplets, which are storage organelles. Viral 

rNA synthesis takes place at sites on the Er that are modified by NS4B, one of the viral non-structural proteins. These sites are likely to 




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enable not only replication of viral rNA but also protect newly-synthesized genomes from recognition by host defence mechanisms. The 

mechanisms used to create replication sites by NS4B are poorly understood but could be similar to the processes involved in the formation 

of Er tubules. in addition, the HCV core protein, which forms the capsid within virions, is targeted specifically to the membranous surface 

of lipid droplets. Disrupting the ability of core to target lipid droplets prevents release of infectious virus. using biophysical methods in 

live cells, the similarities and differences between HCV core and NS4B proteins and cellular components that target membranes will be 

described. These studies illustrate the importance of host membranes and the membrane-binding properties of viral proteins to productive 

HCV infection. 

Involvement of the cell nucleus in plant virus systemic infections 

M. TAliANSKY, S.H. Kim, E. ryabov, N. Kalinina, J. Shaw, S. MacFarlane, J.W.S. Brown 

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA 

The nucleolus and Cajal bodies (CBs) are prominent interacting subnuclear domains involved in crucial aspects of cell function such as rNA 

metabolism, the cell cycle and aging. Certain viruses interact with these compartments but the functions of such interactions are largely 

uncharacterized. in this work, we show that the ability of the Groundnut rosette virus OrF3 protein to move viral rNA long-distances 

through the phloem and mediate systemic infection strictly depends on its interaction with CBs, the nucleolus and the major nucleolar 

protein, fibrillarin. The OrF3 protein targets and re-organizes CBs into multiple CB-like structures and then enters the nucleolus by causing 

fusion of these structures with the nucleolus. This process is mediated by the interaction between OrF3 protein and fibrillarin leading to 

the formation of ring-like complexes. These rings formed by both OrF3 and fibrillarin proteins interact with viral rNA encapsidating it and 

re-organizing it into helical structures, and thereby play a key role in the assembly of umbraviral rNP complexes capable of long-distance 

movement and systemic infection. These results may have functional implications for other viruses, which will be discussed. 

Identification of a kinesin-I light chain binding signature in the human genome 

Mark P. Dodding, richard Mitter, Ashley Humphries, MiCHAEl WAY 

Cell Motility Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX 

(email michael.way@cancer.org.uk) 

During vaccinia virus infection, newly assembled virus particles, which fuse with the plasma membrane and remain attached to the outside 

of the cell recruit a signalling complex of Grb2, Nck, WiP and N-WASP to stimulate Arp2/3 complex-dependent actin polymerization to 

enhance their spread into neighbouring cells. Before vaccinia can induce actin polymerization it has to travel from its peri-nuclear site of 

assembly to the plasma membrane. it achieves this by recruiting kinesin-1 and undergoing microtubule-based motility. The viral protein A36 

interacts directly to the kinesin-1 light chain (KlC). We now show that this interaction is mediated by a bi-partite tryptophan based motif, 

which is also found in a number of cellular kinesin-1 binding proteins. Bioinformatic analysis reveals that a related bipartite motif is present 

in over 450 human proteins. using vaccinia as a surrogate cargo we show that the bi-partite motifs from a variety of cellular proteins can 

recruit KlC to promote virus transport in the absence of A36. Furthermore, these proteins can interact with the KlC outside the context 

of infection. Our observations suggest KlC binding depends on a common set of features found in a wide range of proteins with multiple 

cellular functions and disease associations. 

retrovirus cell-to-cell transmission 

Walther Mothes 

Section of Microbial Pathogenesis, Yale University School of Medicine, 295 Congress Ave, New Haven, CT 06536, USA 

The higher efficiency of HiV transmission via direct cell-cell contact over diffusion through the extra-cellular milieu is believed to be due 

to the ability of HiV to efficiently coordinate several steps of the retroviral life cycle at contact sites. using the murine leukemia virus 

(MlV) as a model retrovirus, we have shown that polarized assembly in infected cells contributes to the efficiency of viral spreading. in 

addition, studying HiV cell-to-cell transmission, we observe a potent entry block for cell-free HiV in certain T cells that can be overcome 

by the formation of an Env/CD4 dependent synapse. The ability of a virological synapse to overcome this block correlates directly to the 

expression levels of CD4, but not co-receptor. High CD4 expression led to tighter synapses and transmission became more resistant to 

neutralizing antibodies. As such, our mechanistic studies reveal details of how several retroviruses efficiently coordinate virus assembly and 

entry at sites of cell-cell contact. Current work in the laboratory is devoted to identifying the cellular factors required for virus cell-to-cell 

transmission and the isolation of small molecule inhibitors specifically interfering with virus transmission. 




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Offered paper Studying viral replication complexes in viral factories with high resolution imaging of proteins in cells 

Cristina risco 

Centro Nacional de Biotecnologia, CSIC, Madrid, Spain 

Viral factories are complex structures induced early in infection by recruitment of cellular and viral components. it is known that in these 

large scaffolds viruses insert their macromolecular complexes needed for genome replication and morphogenesis. Signalling events involved 

in their formation are mainly unknown. We are studying the structure and activities of factories built by several rNA viruses that are 

important pathogens for humans. One of our main goals is implementing methods for specific detection of viral macromolecular complexes 

in 3D maps of cells and to this purpose we have recently validated the first clonable tag for electron microscopy that works in live cells. 

With this new methodology we are studying how viral replicase molecules assemble active macromolecular complexes in intracellular 

membranes. These methods can provide us with a completely new vision of the structure-function relationships in complex biological 

systems such as those operating in viral factories. 

Offered paper Autophosphorylation of Kaposi’s sarcoma-associated herpesvirus thymidine kinase induces focal adhesion 

disassembly, cell contraction and blebbing via a FAK and rhoA-dependent pathway 

MiCHAEl Gill1 , Michael Way2 , rachel Turner1 , Mark Dodding2 , Philip Stevenson1 1 2 University of Cambridge, Cambridge; Cancer Research UK, London 

Paradoxically, the Thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside 

kinase, when compared to TKs from related herpes viruses. We now show that KSHV-TK, in contrast HSV-TK, associates with the actin 

cytoskeleton, induces extensive cell contraction and membrane blebbing. These dramatic changes in cell morphology are dependent the 

ability of the KSHV-TK kinase domain to auto-phosphorylate Tyrosine 65 and 85 in its N-terminus. Auto-phosphorylated KSHV-TK, which 

associates with FAK disrupts the integrity of focal adhesions leading to activation of rhoA-rOCK signalling to Myosin ii. KSHV-TK has no 

effect on cell morphology in the absence of FAK. Our observations suggest that KSHV-TK functions primarily as a tyrosine kinase that 

modulates cell morphology and signalling during infection by destabilizing focal adhesions. 

Offered paper Hepatitis C virus egress and release occurs through distinct Golgi-endosome compartments 

JAMEl MANKOuri1 , Stephen Griffin2 , Mark Harris1 1 2 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds; Leeds Institute of Molecular Medicine, 

St James’s University Hospital, Leeds 

The mechanism by which infectious hepatitis C virus (HCV) particles are released from infected cells remains poorly characterized. Whilst 

is it widely believed that components of the very low density lipoprotein (VlDl) assembly and secretion machinery are involved, recent 

studies have demonstrated that virus production requires an endosome-based secretory pathway. Here, we aimed to characterize the 

precise nature of the endosomal compartments required for HCV egress. 

using a panel of dominant negative rab GTPases, proteins representing a large family of ras-like enzymes that regulate many steps of 

membrane traffic, we demonstrate that the targeting of vesicles during endosome recycling to the Golgi, as well as intra-Golgi trafficking 

is required for the secretion of infectious HCV virions. We further demonstrate that HCV core localizes with lipid droplets and Golgiassociated 

clathrin adaptor complexes, confirming the importance of these compartments for HCV egress. importantly, when HCV release 

was impaired, we observed no inhibition of VlDl, apolipoprotein B and apoplipoprotein E secretion. 

These findings support the notion that HCV particles are secreted by a complex mechanism involving an exclusive Golgi-endosome 

trafficking pathway, which is distinct to a simple association with secreted VlDl. 

Offered paper Agents that modulate lipid droplets impair production of infectious hepatitis C virus 

JOlANDA liEFHEBBEr1 , Charlotte Hague1 , Graham Hope1 , Qifeng Zhang2 , Michael Wakelam2 , John Mclauchlan1 1 2 MRC-University of Glasgow Centre for Virus Research, Glasgow; Babraham Institute, Babraham Research Campus, Cambridge 

lipid metabolism plays an essential role in the life cycle of HCV, a positive single-stranded rNA virus. HCV core protein, which forms the 

viral capsid, localizes to the surface of lipid droplets, which are neutral lipid storage organelles. Disrupting the ability of core to target lipid 

droplets impairs virus production, suggesting that this interaction is important for assembly. Our aim is to investigate in greater depth the 

role of lipid droplets and lipid metabolism in HCV virion production. 

Here we present the effect of drugs that alter lipid metabolism. Compound 1 inhibits triacylglyceride synthesis leading to depletion of lipid 

droplets from cells. This results in a slight reduction in replication but formation of infectious virus is lowered to a greater extent, possibly 




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HA01 Cont. & HA02 

due to instability of core in the absence of lipid droplets. Compound 2 inhibits cholesterol ester synthesis resulting in large lipid droplets. 

Although this treatment does not apparently influence HCV rNA replication, infectivity is reduced. Mass spectrometry is in progress to 

assess the impact of these compounds on the lipid composition of cells. Overall, our data provide direct evidence for the importance of 

lipid droplets to virus assembly and suggest lipid modulators could offer a therapeutic route to treat infection. 

Offered paper Epstein–barr virus infection of polarized epithelial cell layers by b cell-mediated transfer infection: components 

of the virological synapses 

ClAirE SHANNON-lOWE, Martin rowe 

Cancer Research UK Centre for Cancer Studies, School of Cancer Studies, College of Medical & Dental Sciences, The University of 

Birmingham, Vincent Drive, Birmingham B15 2TT 

Epstein–Barr virus (EBV) exhibits tropism for both B- and epithelial cells, demonstrated by the strong association with B cell (Burkitt’s/ 

Hodgkin’s lymphoma) and epithelial cell malignancies (nasopharyngeal/gastric carcinoma). Direct infection of B cells is initiated by the 

interaction of the viral glycoprotein gp350 with B cell surface CD21. Fusion is triggered by gp42 interaction with HlA class ii, and is 

thereafter mediated by the core fusion complex, gHglgp42. in contrast, direct infection of CD21-negative epithelial cells is very inefficient. 

However, EBV can efficiently access the epithelium by first binding to the resting B cell and using this surface as a transfer vehicle. 

EBV binding to CD21 on resting B cells induces activation of B cell adhesion molecules, These co-cap with virus on the cell surface and 

induce firm binding to their cognate receptors on the epithelial cell. infection of polarised primary tonsillar epithelial cells by EBV is mediated 

by transfer from memory B cells at the basolateral surface. B cell adhesion is achieved by CD11b and CD48 interaction with the heparan 

sulphate moieties of CD44v3, with the involvement of lEEP-CAM. Thereafter, infection is achieved by EBV interaction with integrins 

specific for epithelial cells at the basolateral surface. 

Offered paper The cellular protein Hur relocalizes to the cytoplasm and stabilizes viral transcripts during sindbis virus 

infection of human cells 

AlExA DiCKSON, Kevin Sokoloski, Jeffrey Wilusz 

Microbiology, Immunology and Pathology Colorado State University, Fort Collins, Colorado, USA 

Background: Sindbis virus (SinV) stabilizes its mrNAs by utilizing the cellular Hur protein. SinV induces the dramatic relocalization of Hur 

from the nucleus to the cytoplasm during infection of human cells. We hypothesize that Hur relocalization is either a result of either a 

cellular stress response to viral infection or is actively induced by a virus-specific mechanism 

Results: SinV replication is required to induce Hur relocalization; adsorption of SinV to the cell is not sufficient. Nuclear-associated SinV 

nsp2 and nsp3 proteins were also not sufficient for Hur relocalization. Hur movement is species-specific, as it does not occur in SinV 

infections of mosquito cells. infections with measles virus do not cause Hur movement, suggesting that relocalization is not a generalized 

stress response of the cell to a viral infection. Changes in the phosphorylation state of Hur during SinV infection may present vital clues to 

the underlying mechanism of its relocalization. 

Conclusion: Hur protein movement out of the nucleus is selective for alphavirus infections and requires active viral gene expression. Based 

on the need for alphaviruses to use Hur to stabilize mrNAs and promote a productive infection, interfering with the induction of Hur 

relocalization could represent a novel avenue for antiviral therapeutics. 

HA02 Maths & microbes 

↑Contents 

Microbes make mathematics meaningful, beautiful, and useful 

John r. Jungck 

Dept of Biology, Beloit College, 700 College Street, Beloit, WI 53511, USA (Email: jungck@beloit.edu; Web: http://bioquest.org) 

Microbiology has historically been a leader in the use of mathematics in biology. Significant achievements were recognized by Nobel Prizes 

awarded in biology and medicine. Many of these contributions had fundamental mathematical models inherent in their work. Examples 

of mathematics applied to microbiology that are accessible to students in undergraduate beginning courses, associated with interactive 

computer resources, and illustrate the potential to engage students in mathematical exploration of microbiology because of their beauty, 

utility, power, understanding, and historically important conceptual developments include: (1) John Snow’s Ghost Map and computational 

geometry – cholera in london and the beginnings of epidemiology; (2) Fractals – bacteria in an ecosystem context – artistry of micro- 




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HA02 Cont. 

organisms; (3) Phylogenetics: (a) Fox and Woese’s three superkingdoms, (b) Margulis’ endosymbiotic theory, (c) Pace and relner’s 

microbiome; (4) Boolean models – Operons – Jacob and Monod; (5) Fluctuation test – luria-Delbrück – Poisson distribution; (6) logistic 

growth– environmental carrying capacity; (7) interval graphs – Benzer – fine structure of the gene; (8) Eulerian topology – Caspar and Klug 

– viral capsids – self-assembly; (9) Bioinformatics – gene order and content – pathogenicity hypothesis; (10) Fluid mechanics – ‘life at low 

reynolds Numbers.’ See our Microbes Count project: (http://bioquest.org/microbescount/) and MathBioEd issue: (http://www.lifescied.org/ 

content/vol9/issue3/cover.dtl). 

Using maths to understand biofilms 

Paul Stoodley 

School of Engineering Sciences, University of Southampton, University Road, Southampton SO17 1BJ 

The development of bacterial biofilms is influenced by both genetic (internal) and environmental (external) factors. Fluid flow is an 

important external factor since it controls two competing influences; 1) the mechanical stresses acting on the biofilm which tend to reduce 

biofilm through erosion and sloughing, and 2) the delivery of nutrients which tend to increase biofilm through growth. Fluid flow can also 

remove metabolites such as waste products and diffusible signal molecules from the biofilm which in turn will influence the concentration 

gradients which build up within the biofilms. Various important parameters will be described and examples will be used to illustrate how the 

hydrodynamic conditions (reynolds number, wall shear stress) of laboratory systems can be calculated from a few simple parameters such 

as the reactor geometry and flow rate. A simple model developed by Phil Stewart at the Center for Biofilm Engineering at Montana State 

university will be used to illustrate how various chemicals can diffuse through biofilms as a function of biofilm thickness and the diffusion 

coefficient. 

Massively-parallel microbial search: a new platform for synthetic biology 

Martyn Amos 

Novel Computation Group, School of Computing, Mathematics & Digital Technology, Manchester Metropolitan University 

Synthetic biology is a new discipline that is emerging at the intersections of biology, computing science, engineering, chemistry and maths. 

Practitioners in the field are particularly interested in applying rational engineering principles to biological systems, with the aim of persuading 

them to perform ‘useful’, human-defined tasks. in this talk, i will give a brief overview of the state-of-the-art, before describing our own 

work (as coordinator of the Eu BACTOCOM project) on a population-based, massively-parallel microbial search algorithm. This work 

will, we hope, lay the foundations for rapid and reliable synthetic biology, with potential applications in areas as diverse as drug production, 

environmental sensing, tissue engineering and energy. 

Microbial evolution in theory and practice 

ivana Gudelj 

Biosciences, University of Exeter 

Microbes are ubiquitous in nature and occupy virtually every environmental niche on earth. Contributing to this evolutionary success are 

diverse metabolic strategies as well as the ability to adapt to changing environments. Experimental evolution has provided an ideal setting 

for studying microbial diversification in action –experiments are conducted in controlled environments using culturable strains that are easily 

manipulated due to their known genetic structure. However this simplified approach to evolution poses the following questions: How do 

we know whether a given experimental outcome is particular to the laboratory system? What can we learn from laboratory experiments 

about micro-organisms in the wild? in this talk i argue that through the use of mathematical models we can begin to bridge the gap 

between laboratory and nature. i will present a mathematical model used to study microbial cooperation and demonstrate that this model 

can make good quantitative predictions of a given laboratory experimental setup. Subsequently i will discuss how these predictions can be 

generalized to other systems. 

HEA perspective and comments on maths teaching and graduate skills 

David J. Adams 

UK Centre for Bioscience, Higher Education Academy 

The maths and stats components of modern biology degree programmes are becoming increasingly demanding. There is widespread 

concern that students enter uK universities ill-equipped for the challenges associated with, for example, bioinformatics or a newly emerging 

area associated with so-called systems biology. This problem is exacerbated by a general lack of maths content in undergraduate biology 




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HA02 Cont. 

programmes; industrialists and research degree supervisors alike have complained of graduates lacking basic mathematical skills in research 

labs or other workplace settings. During 2010, the uK Centre for Bioscience, with BBSrC, sought to address these issues by holding a one 

day event on Mathematical Challenges for Biologists. Some of the major issues discussed at this meeting, along with key outcomes, will be 

presented. in particular the Centre has commissioned a survey that will assess the maths and stats content of uK undergraduate bioscience 

degree programmes and preliminary results from this survey will be discussed. in addition, examples of how the Higher Education Academy 

has supported, and continues to support, maths and stats teaching in uK universities will be described. Emphasis will be placed on how the 

Academy can best support microbiologists and other bioscientists within the new structure it will adopt from 2011/12. 

Offered paper Developing numeracy skills in biomedical sciences- a case study 

Pauline Fitzgerald 

Leeds Metropolitan University 

Since 1997, and the Dearing report recommendations that higher education courses should focus on key skills such as numeracy, 

universities have applied themselves to looking at the different ways in which these can be embedded into courses. Numeracy is one of 

the skills employers value highly, and yet students find maths difficult. So how can we develop these skills and improve employability in 

our graduates?The approach we have adopted at leeds Metropolitan university is to have an employability/ transferable skills strand at 

each level of the course. At level 1, the students undertake a ‘skills audit’, which involves some maths, so they can assess their knowledge 

and develop their numeracy skills. At level 2 they undertake a professional practice module which starts to apply their maths skills to the 

working environment. At level 3 they develop this further by undertaking a project management module. Their remit is to come up with 

a new biotech product, then show how they would develop the product, from the science behind it through to marketing and finance. As 

the course progresses, the students begin to appreciate the different ways that their maths skills can be promoted to potential employers. 

biomathtutor: what is it and could it help? 

Vicki Tariq 

University of Central Lancashire, Preston, Lancashire PR1 2HE 

Biomathtutor represents a prototype multimedia e-learning resource, which aims to support mathematics learning in the biosciences. A 

contextual, scenario-based problem-solving learning model has been adopted, in which a case study scenario, which covers aspects of 

haematology and microbiology, is presented via a stimulating high quality professionally produced narrated film. linked to the content of 

the film are thirty-three interactive questions for students to attempt on screen. Twenty-four additional practice questions, which cover the 

same range of basic mathematical concepts, presented in similar biological contexts, are also available for completion. in addition, students 

can access five relatively short face-to-face video mathematics tutorials in which a tutor explains some of the mathematical concepts 

students encounter in the interactive questions. 

During 2006/07, a project funded by the Higher Education Academy assessed the quality of the prototype learning materials developed 

and investigated their potential for integration into bioscience curricula. The methodology adopted involved the analysis of both quantitative 

and qualitative data from undergraduates and their tutors, using questionnaires, focus groups (students) and interviews (tutors). Overall, the 

reactions of students and their tutors toward biomathtutor were very positive, with both groups commenting favourably on aspects of its 

design and its potential to support mathematics learning. 

Mathematical predictions of antibiotic therapy efficacy 

robert Beardmore 

Dept of Biosciences, University of Exeter, Geoffrey Pope Building, Streatham Campus, Exeter EX4 4QD 

One of the main goals of applied mathematical research is to identify boundaries between different behavioral regimes in physical systems. 

For example, the phase transitions of physics are intimately related to the mathematical descriptions of nonlinear systems provided by 

bifurcation theory. Evolving biological systems are far more complex still and we should not be surprised if one model system displays 

a multitude of different behaviors. However, mathematics is one possible tool to decipher similar systems likely to behave differently 

and disparate systems likely to behave in the same way.Two examples of this philosophy will be given: a study of an evolving bacteriaphage 

microcosm and a theoretical epidemiological drug deployment problem from the literature. in the former, experiments designed 

to measure genetic diversity are sensitive to molecular details, in the latter, broad-scale conclusions on optimal drug deployment policies 

appear insensitive to many biological details. Finally, we discuss what can go wrong if inappropriate theoretical techniques are applied to 

mathematical formulations of such biological problems. 




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Improving engagement and achievement in science numeracy 

A. JANET HOrrOCKS, louise Milne 

University of Abertay, Bell Street, Dundee DD1 1HG 

Students starting our programmes typically have very little confidence with handling numerical problems and calculations (such as calculating 

molar concentrations). This lack of confidence even extends to confusion about the correct use of scientific notation and significant 

figures. We began to included basic numeracy within a first year module focused on laboratory skills however students were able to pass 

the module without engaging with the numeracy component and assessment so typically students who were intimidated by calculations 

avoided the numeracy part of the module. The numeracy section of the module was initially delivered via tutorials. For the session 2006/7 

the assessment was put online and supported with online tutorials and quizzes in addition to timetabled classes. The quizzes were designed 

so that students had to pass each section before progressing to the following section however engagement with online material was poor 

and student performance appeared to deteriorate. The following year students were required to pass a preliminary formative test to gain 

access to the summative test. The use of a preliminary test improved students performance however further gains in performance were 

made in later years by setting students clear short term goals and targets (supported by regular emails) and providing focused tutorial 

support sessions. 

HA03 Social evolution in micro-organisms 

↑Contents 

Spatio-genetic structure and prudent cooperation in microbes 

Kevin Foster 

University of Oxford 

Since Darwin, evolutionary biologists have been troubled by cooperative behaviours. in microbes, these behaviours are seen in the use of 

secretions and slow growth, which can both help neighbouring cells to divide and survive. What prevents cooperative cells being exploited 

by cheaters, which receive cooperative benefits without themselves contributing? i will discuss two solutions to the problem of cooperation 

in microbes. First, i will describe individual-based simulations and experiments that show genetic segregation in growing biofilms and 

colonies. This segregation is sufficient to ensure that bacteria are surrounded by their own genotype, which can favour the evolution of 

cooperation for the same reason that cells in your body cooperate with one another. Second, i will discuss a hypothesis that comes out of 

experiments with rhamnolipid secretion in the bacterium Pseudomonas aeruginosa. We find that cells only secrete rhamnolipids when it is 

cheap to do so, something we call metabolic prudence. This makes secretion effectively cost free and removes any advantage to cheater 

genotypes. in summary, spatio-genetic structure and prudent use of resources can both stabilise cooperation in microbial groups. 

A simple mechanism for complex social behaviour in Dictyostelium discoideum 

Chris Thompson 

Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT 

Despite the appearance of co-operation in nature, selection should often favour exploitative individuals that perform less of a costly 

cooperative act. This conflict of interests can lead to the evolution of complex, partner specific, social strategies. The social amoeba 

Dictyostelium discoideum provides a compelling model for studying conflict and cooperation. upon starvation, free-living amoebae aggregate 

and form a fruiting body composed of dead stalk cells and hardy spores. Different genotypes will aggregate to produce chimeric fruiting 

bodies, resulting in potential social conflict over who will contribute to the sporehead and who will ‘sacrifice’ themselves to produce the 

stalk. The outcomes of competitive interactions in chimera appear complex, with social success being strongly partner specific. Here we 

propose a simple mechanism, based on the production of and response to social signals governing developmental differentiation, to explain 

social strategies in D. discoideum. indeed, measurements of signal production and response can predict social behaviour of different strains, 

thus demonstrating a novel and elegantly simple underlying mechanistic basis for natural variation in complex facultative social strategies. 

This suggests that simple social rules can be sufficient to generate a diverse array of outcomes that appear complex and unpredictable 

when those rules are unknown. 




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A small rNA controls Myxococcus development and mediates a major social adaptation 

Yuen-Tsu Nicco Yu, xi Yuan, GrEGOrY J. VEliCEr 

Dept of Biology, Indiana University, Bloomington, IN 47405 USA 

See Additional Abstracts (pp. 172–173) 

↑Contents 

HA03 Cont. 

Cooperation and cheating in Pseudomonas quorum sensing 

Martin Schuster 

Dept of Microbiology, Oregon State University, USA 

Many bacteria demonstrate the hallmarks of a complex social life typically associated with higher organisms. Pseudomonas aeruginosa cells 

communicate with each other in a process termed quorum sensing (QS) to coordinate the transcription of hundreds of genes. We have 

shown by ChiP-chip (chromatin immunoprecipitation and microarray analysis) that secreted common goods and secretion machinery are 

the most overrepresented functions directly controlled by QS. In vitro evolution and co-culturing experiments under conditions that favor 

QS revealed that common goods production can be exploited by signal-blind regulator mutants, cheaters that gain the benefit of cooperation 

without contributing themselves. Mutants lacking either lasr, rhlr, or Pqsr, three key regulators in the P. aeruginosa QS network, 

varied greatly in their ability to invade wild type populations. These behaviors may explain the prevalence of certain QS mutants in natural 

environments. 

Social evolution in parasites 

Sarah reece 

Institutes of Evolution, Immunology & Infection Research, University of Edinburgh, School of Biological Sciences, Ashworth Laboratories, 

Edinburgh EH9 3JT 

Malaria (Plasmodium) and related parasites cause some of the most serious infectious diseases of humans, livestock, companion animals, 

and wildlife. research in my group uses an evolutionary framework to explain the social strategies that parasites have evolved to maximise 

in-host survival and between-host transmission. Malaria parasites replicate asexually in their vertebrate hosts, but must reproduce sexually 

to infect mosquito vectors and be transmitted to new hosts. As different stages are required for these functions, parasites must divide their 

resources between in-host replication and between-host transmission, and between the production of male and female sexual stages. Our 

experiments show that social interactions within the host shape the solutions to these resource allocation trade-offs. Moreover, parasites 

fine-tune their strategies in response to variation in relatedness and density within infections. Explaining within-infection processes is 

traditionally the domain of reductionist approaches, whereas evolutionary biology tends to focus on between-host processes, so our results 

represent a rare demonstration of how an evolutionary ecology framework can bridge these scales. More broadly, understanding how 

natural selection has solved the complex problems faced by parasites is central to biomedicine, yet the success of ‘evolutionary medicine’ 

hinges on a significantly better understanding of the evolutionary biology of parasites. 

Theory of social adaptation 

Andy Gardner1,2 1 2 Dept of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS; Balliol College, University of Oxford, Broad Street, 

Oxford OX1 3BJ (Email: andy.gardner@zoo.ox.ac.uk) 

Natural selection explains the appearance of design in the living world, but at what level is this design expected to manifest – gene, 

individual, society – and what is its function? Social evolution provides a window on this problem, by pitting the interests of genes, individuals 

and societies against each other. i review the foundations of Darwinian adaptation through the action of direct and indirect (kin selected) 

fitness effects, and how this leads organisms to appear designed as if to maximize their inclusive fitness. i also consider the possibilities for 

adaptation at the gene and group levels. 

The evolution of microbial cooperation and virulence 

Sam Brown 

Dept of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford OX1 3PS 

Microbes engage in a remarkable array of social behaviors, secreting shared molecules that are essential for foraging, shelter, microbial 

warfare, and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by nonproducing cheaters 

arising by gene loss or migration. in such conditions, how can cooperation persist? 




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↑Contents 

HA03 Cont. 

i review a series of models and data highlighting the importance of population structure in determining microbial social behaviours and 

consequent virulence, and highlight the peculiar role and importance of microbial mobile elements (plasmids, phages) in generating this 

structure. Finally, i discuss the possible therapeutic implications of understanding the social dynamics of microbial pathogens. 

Public goods, private shares 

JAN-ulriCH KrEFT1 , Stefan Schuster2 , Anja Schroeter2 1 2 Centre for Systems Biology, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT; Dept of 

Bioinformatics, School of Biology & Pharmaceutics, Ernst-Abbe-Platz 2, Friedrich Schiller University of Jena, D-07743 Jena, Germany 

(Email: j.kreft@bham.ac.uk; stefan.schu@uni-jena.de; an.schroeter@uni-jena.de) 

Extracellular effectors such as exoenzymes, siderophores, antibiotics, and autoinducers are public goods since they benefit not only 

producing but also non-producing cells, which are called cheaters as they do not pay the production costs. 

Cheaters outcompete the producers if the benefits are shared equally. This can be avoided in a spatially structured environment where 

neighbours are next of kin and receive more of the benefit. 

A simpler mechanism consists in partially ‘privatizing’ the ‘public’ good. For example, invertase produced by yeast remains bound to the cell 

wall, thereby generating glucose and fructose closer to the cell’s own transporters than any neighbours’. To some extent, the public good is 

made less public, and the behaviour less cooperative, but the point is that privileged access can act as an evolutionary refuge from cheaters, 

allowing the evolution of a mechanism for public good production from which cooperation can readily and repeatedly evolve. Similar 

arguments hold for autoinducer sensing. 

We show that partially privatized public good production is evolutionarily stable over a wide range of conditions in a model with minimal 

assumptions, essentially Monod kinetics. While this model is generic, it can explain the experimental observations of Greig & Travisano 

(2004). 

Ecological drivers of the evolution of public-goods cooperation in bacteria 

MiCHAEl BrOCKHurST1 , Michelle Habets1 , Angus Buckling2 , Andy Gardner2 1 2 Institute of Integrative Biology, University of Liverpool, Liverpool; Dept of Zoology, University of Oxford, Oxford 

The role of ecological processes in the evolution of social traits is increasingly recognized. We have explored, using general theoretical 

models and experiments with bacteria, the effects of common ecological variables (e.g., disturbance frequency and resource supply) on 

the evolution of cooperative biofilm formation. Our results demonstrate that cooperation tends to peak at intermediate frequencies of 

disturbance but that the peak shifts toward progressively higher frequencies of disturbance as resource supply increases. This arises due to 

increased growth rates at higher levels of resource supply, which allows cooperators to more rapidly exceed the density threshold above 

which cooperation is beneficial following catastrophic disturbance. These findings demonstrate the importance of ecological processes 

in the evolution of public-goods cooperation and suggest that cooperation can be favored by selection across a wide range of ecological 

conditions. 

Using bacterial lobster traps and scanning electrochemical microscopy to probe polymicrobial interactions 

Marvin Whiteley 

The University of Texas at Austin, Section of Molecular Genetics & Microbiology 

Prokaryotes are social organisms capable of coordinated group behaviors (referred to as quorum sensing, QS); however, how these 

behaviors proceed in the small populations commonly observed in nature (e.g., thousands of cells) are unknown. Here, i will discuss the use 

of two novel approaches for probing bacterial social interactions in small populations. The first approach utilizes multi-photon lithography to 

create picoliter-sized porous cavities capable of capturing a single bacterium and tracking growth and behavior of the resultant microcolony 

in real time. These bacterial ‘lobster traps’ allow for examination of behaviors in small, defined bacterial populations similar in size to 

those observed in nature. The second approach involves the use of scanning electrochemical microscopy (SECM) to monitor group 

behaviors in a bacterial biofilm through quantification of QS-controlled products. SECM has the unique ability to set the exact distance 

from an ultramicroelctrode sensing tip to a biological substrate through a feedback approach curve, and thus is able to measure the local 

concentration over a defined region (μm scale) of a biofilm. using these technologies we provide insight not only into the number of 

bacteria, but also the concentration of small molecules required to elicit social responses. 




16 




HA04 Vaccines 

↑Contents 

Exploiting host recognition of fungi for vaccine development 

Stuart M. levitz 

University of Massachusetts Medical School, Worcester, USA 

Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so they are primarily β1,3-d-glucans and free of mannans and 

proteins. We have loaded GPs with the model antigen ovalbumin (OVA) and tested GP-OVA as a receptor-targeted vaccine delivery 

system. immune responses in C57Bl/6 mice following immunization with GP-OVA were compared with OVA absorbed onto the 

adjuvant alum (Alum/OVA). While both GP-OVA and Alum-OVA formulations induced high igG titres, only GP-OVA stimulated antigenspecific 

CD4+ T cell lymphoproliferative and EliSPOT responses. Moreover, the T-cell responses induced by GP-OVA were Th1-biased 

(determined by iFN-γ EliSPOT) and Th17-biased (determined by il-17a EliSPOT). responses were long-lasting and robust even if with 

just two immunizations and with low doses of antigen. Encapsulation of antigen in the GPs was essential for optimal immunogenicity. 

Although GPs are phagocytosed by dectin-1 in vitro, in vivo immune responses were preserved in dectin-1 knockout mice, likely because of 

complement opsonization. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and 

Th1- and Th17-biased CD4 + T-cell responses. Studies examining protection against pathogens are ongoing. 

Fungal cell wall vaccines 

John Edwards 

Div of Infectious Diseases, Harbor/UCLA Medical Center, 1124 W Carson St, RB2, 2nd Fl, Torrance, CA 90502, USA 

During efforts to identify adhesins for Candida to human cells, we discovered that both Als1 and Als3 encoded cell wall proteins which 

were strong adhesins for human umbilical cord endothelial cells. To determine whether these adhesins could function as vaccines for 

prevention of hematogenously disseminated candidiasis (HDC) we produced recombinant proteins from the N-terminus of Als1p 

(r-Als1pN) and Als3p (r-Als3pN) in Saccharomyces and tested them in the mouse model for HDC. Considerable protection occurred 

in a lethal challenge for multiple strains and species of Candida. Focusing on r-Als3pN, both an adhesin and invasin for Candida, we found 

protection for oral/pharyngeal and vulvovaginal candidiasis also in the murine model. Structural, three dimensional homology studies showed 

Als3pN to have high levels of homology to staphylococcal surface proteins. The r-Als3p was also protective in the murine staphylococcal 

challenge model. The mechanism of action of the r-Asl3pN for both organisms is proposed to be through the Th1/Th17 pathway. This 

vaccine is currently in Phase 1 clinical development. 

Another Candida vaccine candidate in Phase 1 clinical development is based on the SAP2 antigen presented by a virosome delivery 

technology. A third Candida vaccine candidate based on diphtheria toxoid conjugated to cell surface beta-glucan is highly effective in murine 

models and is in late-stage preclinical development. Extensive investigation is in preclinical development stages for coccidioidomycosis, 

histoplasmosis, and blastomycosis also. 

Protecting against plague 

Diane Williamson 

Dstl Porton Down, Salisbury SP4 0JD 

Plague, caused by the bacterium Yersinia pestis, is an ancient disease, which still exists in the world today and which emerges with lethal 

effect from time-to-time. in endemic areas, the bacterium is maintained in enzootic reservoirs and is vectored by fleas. Man is an accidental 

host who may become infected by a flea that has fed on an infected animal. Existing killed whole cell vaccine fomulations afford protection 

against the flea-vectored bubonic form of the disease. Since pioneering work in the 1950s and 1960s into the pathogenicity of this 

bacterium, significant advances have been made in protecting people more comprehensively against this disease and particularly the most 

feared, pneumonic plague. This presentation will review the most recent advances in developing new and efficacious vaccines and describe 

how our understanding of this serious pathogen has advanced with modern technology to achieve a recombinant sub-unit vaccine currently 

in clinical development. 

HA04 

↑Contents 




17 




↑Contents 

HA04 Cont. 

Approaches to improving antigenicity of carbohydrate-based vaccines 

Brendan Wren 

Dept of Pathogen Molecular Biology, The London School of Hygiene & Tropical Medicine 

The most successful bacterial vaccines often consist of a glycan attached a protein carrier. Examples of such glycoconjugate vaccines 

currently licensed for humans include those used to combat Haemophilus influenzae, Neiserria meningitidis and Streptococcus pneumoniae. 

These possess a ‘best of both worlds’ vaccine formulation evoking both T cell-dependent and T cell-independent immune responses 

resulting in a more protective and longer lasting effect than either component alone. using traditional technology it is difficult to produce 

glycoconjugate vaccines that require the purification of the glycan from the native pathogen and chemical coupling to a protein carrier. This 

multi-step process is expensive and prone to variability and a recombinant approach that would eliminate the requirement for chemical 

coupling is highly desirably. in 2000, during the characterization of a novel N-linked general glycosylation system from the enteropathogen 

Campylobacter jejuni, a fortuitous discovery was made whereby it was discovered that a protein, a glycan and an oligsaccharyltransferase 

enzyme could be independently cloned and expressed in E. coli to produce for the first time recombinant glycoprotein (Wacker et al. 

Science 2002). Subsequent discoveries revealed that different glycans could be coupled to different proteins including de-activated bacterial 

toxins and the process has been termed Protein Glycan Coupling Technology (PGCT). This lecture will chart the progress of PGCT from 

the chance discovery, through multiple technological developments and the first clinical trials of recombinant glycoconjugate vaccines. 

Offered paper A new method to conjugate Burkholderia pseudomallei lipopolysaccharide to tetanus Hc fragment for use as a 

vaccine candidate for protection against melioidosis 

Sarah Ngugi1 , David Corser2 , Tim Atkins1 , rick Titball3 , JOANN PriOr1 1 2 3 Dstl Porton Down,, Salisbury; Fleet Bioprocessing Ltd, Fleet, United; University of Exeter 

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals. There is no existing licensed 

vaccine for melioidosis, and antibiotic therapy of the condition is not fully effective. Therefore an efficacious vaccine is urgently required. 

When lipopolysaccharide (lPS) extracted from B. pseudomallei strain K96243 was used as a vaccine candidate, it was shown to provide 

protection against an intra peritoneal challenge of 55 median lethal doses of B. pseudomallei. The mean time to death for vaccinated mice 

increased from 1.2 days to 30.5 days; with 66% survival 35 days post challenge. Vaccination with lPS alone produces a T cell independent 

immune response; however it has been reported that conjugation of polysaccharide to protein produces a T cell dependent response. 

Our aim is to use a novel conjugation methodology to conjugate lPS from B. pseudomallei to tetanus Hc fragment via a thiol-ether bond, 

creating a conjugate vaccine which we hope will stimulate a cell mediated immune response, which would lead to increased protection 

against a B. pseudomallei challenge. This new conjugate will now be evaluated in our murine model of melioidosis. 

© Crown Copyright. Dstl, 2011 

Offered paper Transcription of the gene encoding fHbp, a meningococcal vaccine antigen, is differentially regulated by iron 

availability 

HOllY SANDErS1,2 , Carina Brehony2 , rory Care1 , Martin Maiden2 , ian Feavers1 1 2 National Institute for Biological Standards and Control, Potters Bar; University of Oxford, Oxford 

Meningococcal Factor H Binding protein, fHbp, is an important component of several vaccines targeting serogroup B disease. Although 

fHbp is thought to be important for meningococcal survival in vivo, expression levels are variable, with current evidence suggesting oxygendependent 

regulation. in this study, quantitative rT-PCr was used to compare transcription levels of fHbp in total rNA extracted from 

98 meningococcal strains grown under iron-replete and iron-restricted conditions. Transcription of fHbp in the majority of strains tested 

was found to be iron-activated, while transcription in all ST32 strains investigated was iron-repressed. Differences in regulation may result 

from the exclusive ability of ST32 strains to produce a previously-described bicistronic rNA transcript containing both nmb1869 and 

fHbp reading frames. results of this study, along with previous evidence, support a model where regulation of fHbp is both oxygen- and 

iron-dependant, with transcription in ST32 strains also affected by regulation of nmb1869. This highlights the importance of using panels of 

strains from a variety of clonal complexes when investigating vaccine antigens, and suggests that results obtained from serum bactericidal 

assays, considered the correlate of protection for meningococcal disease, may not easily be extrapolated to protection from fHbp-specific 

antibodies in vivo. 




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Malaria in the mosquito: exquisite, but vulnerable biology 

r.E. Sinden 

Dept of Life Sciences, Imperial College London & Jenner Institute, University of Oxford 

The biological purpose of malaria infection in man is to produce the terminally, and sexually differentiated, haploid gametocytes which are 

responsible for the infection of the mosquito vector. These cells circulate in the peripheral bloodstream for extended periods until the host 

erythrocyte dies, or in a minority of cases until ingested by a female anopheline mosquito. The presentation will outline key developmental 

steps as the parasite infects the mosquito vector. in the mosquito bloodmeal the gametocytes emerge from the rBC (as gametes) in ~15 

mins. The female in the form of one large round cell, and the male – following 3 rounds of genome replication/mitosis, together with the 

structurally linked intracellular assembly of 8, 15um long axomenes, as 8 highly motile ‘sperm’. The surface organization of the gametes has 

been examined and aspects of the molecular basis for both gamete-recognition and gamete fusion have been described. Key ‘players’ in 

recognition are three members (P48; P47 and P230) of a large 6cys protein family, and HAP2 has a central role in gamete fusion. Following 

fertilization the zygote differentiates, over the subsequent ~24 hours, into a motile and invasive ookinete. Structural, fractionation and 

proteomic studies of the ookinete have identified key secretory organelles (micronemes), and their constituent proteins (e.g. CTrP, MAOP, 

WArP, PPlP5, CelTOS), some of which, together with the proteins resident on the ookinete surface (e.g. P25, P28) have been shown to 

be essential to the recognition and invasion of the mosquito midgut epithelial cell. Exposure of these parasite proteins on the cell surface 

is essential to their biological function in the mosquito bloodmeal, but simultaneously offers extended periods for their attack by immune 

mechanisms co-ingested in the bloodmeal. The talk will summarise some recent attempts to induce effective antibody responses to some 

of these vulnerable target molecules using viral vectored immunogens, and outline the key role ‘transmission-blocking’ vaccines may have in 

future control strategies. 

New strategies for development of a vaccine against San Joaquin Valley fever 

GArrY T. COlE, Brady Hurtgen, Chiung-yu Hung 

Dept of Biology & South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, Texas 

78249, USA 

San Joaquin Valley fever (coccidioidomycosis) is a human respiratory disease caused by inhalation of the spores of two members of a semiarid 

soil-borne mould, Coccidioides immitis and C. posadasii. The mycosis is endemic to the Americas. Pulmonary infection resolves without 

complications in the majority of individuals. Alternatively, the disease may persist as either a chronic or disseminated form in approximately 

5% of patients with symptomatic primary coccidioidomycosis. Persons who recover from symptomatic infection typically possess life-long 

immunity, suggesting that development of a human vaccine against this disease is feasible. Our approach to this task has been to generate 

a T-cell epitope-based chimeric protein vaccine together with an adjuvant that stimulate a robust and durable cellular immune response 

to Coccidioides infection. Epitopes of the chimeric vaccine were initially identified as components of immunoreactive, parasitic cell wall 

antigens by bioinformatic methods, and subsequently evaluated in cellular immunoasssays of synthesized peptide equivalents of the selected 

18- to 24-mer epitopes. Experimental adjuvants have been tested that enhance T-helper 1 (Th1) and Th17 activation, which appear to be 

essential for a protective response to this pathogen. Current efforts are focused on optimization of vaccine epitope content, dose, route of 

immunization and the delivery vehicle employed. 

Vaccination against Staphylococcus aureus: Isdb induces protection via both humoral and cellular immunity 

Amita Joshi, Tim Ebert, Greg Pancari, Sharon Smith, Hongxia Fan, Desmond Clark, robin Haimbach, leslie Cope, 

TESSiE MCNEElY 

Vaccine Basic Research, Merck Research Labs, West Point, PA 19486, USA 

Staphylococcus aureus is a well known cause of morbidity and mortality. iron regulated surface determinant B (isdB), a highly conserved, 

surface expressed antigen which functions in iron import, is being clinically investigated as a vaccine for S. aureus. Our study was initiated 

to investigate the mechanism of isdB mediated protection. An isdB specific monoclonal Ab (CS-D7) was used to investigate Ab mediated 

protection. CS-D7 had in vitro opsonophagocytic killing activity, and mediated protection in vivo. To investigate cellular immunity, isdB 

specific lymphocyte subsets were isolated. immune CD4+ and CD8+ T cells, B cells (CD19+) and plasmacytes (CD138highB220 intCD19lo ) 

were adoptively transferred, and only CD4+ T cells were protective. Supportive of this data, isdB immunized Jh mice (B cell deficient) 

were protected against lethal challenge, while nude (T cell deficient) mice were not. P19 KO mice were not protected, pointing to T 17 h 

lymphocytes as being critical. isdB immune splenocytes produced il-17A, but not iFN-γ nor il-2. Blocking il-17A in vivo with an il-17A 

mAb significantly increased lethality in isdB immune mice, while blocking iFN-γ did not. These findings suggest that il-17A production 

by T 17 cells plays an important role in isdB mediated defence against invasive S. aureus infection. Both efficacious antibodies and cellular 

h 

immunity can contribute to isdB mediated protection. 




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Offered paper The Burkholderia pseudomallei Mip protein, a virulence factor and drug target 

isobel Norville1,2 , Suxin Zheng3 , Katherine O’Shea1,2 , Mitali Sarkar-Tyson2 , richard Titball1 , Gabriele Varani3, NiCHOlAS HArMEr1 1 2 3 University of Exeter, Exeter, Devon; Dstl, Porton Down, Wiltshire; University of Washington, Seattle, WA, United States 

Prolyl-peptide isomerases (PPiases) are ubiquitous enzymes required for efficient protein folding. Additionally, some members of the 

FK506-binding protein (FKBP) PPiases have important roles in infection and intra-host survival in a range of bacterial and protozoan 

parasites. These so-called macrophage infectivity potentiators (Mips) have been characterized in a number of important pathogens where 

their loss affects the ability of infectious agents to infect macrophages in vitro. 

Burkholderia pseudomallei contains one gene (BPSS1823) that is a close homologue of the Legionella pneumophila Mip. Knock-outs of 

BPSS1823 in B. pseudomallei are significantly attenuated in macrophage and mouse models of infection. We solved the structure of B. 

pseudomallei Mip (BpMip) by x-ray crystallography and NMr. The 0.91 Å crystal structure shows BpMip binding to a peptide in a strikingly 

similar conformation to human FKBP12 binding to TGFb receptors. NMr structures of apo-BpMip and BpMip binding to a novel inhibitor 

highlight structural flexibility in the protein. The compound binds in a similar conformation to the peptide bound in the crystal structure, and 

shows significant differences to known inhibitors. 

Our results confirm that the B. pseudomallei Mip is a virulence factor, and highlight BpMip as a target for novel therapeutics. 

Offered paper Absent bactericidal activity of mouse serum against invasive African non-typhoidal Salmonella results from 

impaired complement function but not a lack of antibody 

Matthew K Siggins1 , Adam F Cunningham1 , JENNiFEr l MArSHAll1 , Jayne l Chamberlain1 , ian r Henderson1 , 

Calman A. Maclennan1,2 1Medical Research Council Centre for Immune Regulation & Clinical Immunology Service, Institute of Biomedical Research, School of 

Immunity & Infection, College of Medicine & Dental Sciences, University of Birmingham, Birmingham, B15 2TT; 2Division of Medical 

Microbiology, School of Infection & Host Defence, University of Liverpool, Liverpool, L69 3BX 

Nontyphoidal strains of Salmonella (NTS) are a major cause of fatal bacteremia in Africa. Developing a vaccine requires an understanding 

of the mechanisms of protective immunity, frequently derived from the in vivo mouse model of Salmonella infection. Antibody is important 

for protection against NTS and we previously found an important role for Ab in cell-free complement-mediated bactericidal activity against 

Salmonella in Africans. Mice immunized with heat-killed Salmonella Typhimurium strains D23580 (African invasive strain), Sl1344 and liveattenuated 

strain Sl3261 produced a Salmonella-specific Ab response. Sera from these mice deposited reduced levels of C3 on Salmonella 

compared with human sera and were unable to kill both wild-type and galE(-) rough mutant of D23580, indicating absent cell-free 

killing via classical and alternative complement pathways. Supplementing immune mouse sera with human complement enabled killing of 

Salmonella, whereas addition of human anti-Salmonella Ab to immune mouse sera had no effect. These findings indicate that mouse serum 

cannot effect cell-free complement-dependent killing of Salmonella due to the reduced ability of mouse complement to kill these bacteria 

compared with human complement. This difference in Ab-dependent immunity to Salmonella must be considered when applying findings 

from the mouse model of Salmonella disease to man. 

Global progress in Tb vaccine development 

Helen McShane 

The Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ 

(Email: helen.mcshane@ndm.ox.ac.uk) 

Tuberculosis remains a significant cause of mortality and morbidity throughout the world. The emergence of multi-and extensively drug 

resistant strains of Mycobacterium tuberculosis (M.tb) and the geographical overlap between the TB and HiV epidemics have compounded 

the problem. The only available vaccine, BCG, confers highly variable protection against pulmonary disease, which is responsible for much 

of the global burden of disease. However BCG administered at birth does confer reliable protection against disseminated disease in 

childhood, and novel regimens aiming to improve upon BCG alone are currently being developed. M.tb is an intracellular pathogen and a 

strong cellular immune response is essential for protection. understanding reasons underlying the variable efficacy of BCG is important in 

the development of an improved vaccine. The leading candidate vaccines currently being evaluated in clinical trials will be discussed, and the 

development of one of the leading vaccines, MVA85A will be reviewed to illustrate the development pathway for new TB vaccines. The 

main challenges in the field are lack of immunological correlates, lack of validated preclinical models, and finite capacity for efficacy testing. 




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Clinical proof of concept of a live attenuated ETEC vaccine: protection against traveller’s diarrhoea and an important public 

health tool for the developing world 

M.J. DArSlEY1 , A.l. Bourgeois2 , r. Walker2 , C. Harro3 1 2 3 TD Vaccines; PATH; Johns Hopkins 

immunity against CFAs and lT toxin are important for protection against ETEC. The development of a live attenuated ETEC vaccine has 

culminated in the testing of ACE527, comprising strains: ACAM2025 (CFA/i, lTB); ACAM2022 (CS5, CS6, lTB); ACAM2027 (CS1, CS2, 

CS3, lTB). We discuss the development of the vaccine, including the results of Phase i/ii clinical studies. 

A randomized, placebo controlled, double blind Phase i study was conducted in 36 subjects, followed by a Phase ii study in which 56 

subjects were evaluated in a well defined human challenge model. Subjects were vaccinated twice, three weeks apart, with ACE527 (29 

subjects) or placebo (27 subjects). Four weeks later subjects were admitted as in-patients and challenged. 

ACE527 was safe, well tolerated and immunogenic. The magnitude of the immune responses induced to lTB and CFA/i were comparable 

to those induced by challenge with the wild-type ETEC strain H10407. 

The vaccine demonstrated a biologically significant impact on diarrhoeal disease with statistically significant impacts on: subjects diarrhoea 

free; shedding of H10407; volume of diarrhoea in 24hrs; number with reduced activity. The 30% reduction in the incidence of moderate/ 

severe diarrhoea, the primary endpoint, did not reach statistical significance but we predict greater efficacy in field studies. 

Malaria vaccine development 

r.W. Sauerwein 

Dept of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 

The most effective way to reduce death and disease from malaria will be administration of an effective vaccine to susceptible populations. 

induction of long-lived immunity to Plasmodium falciparum (Pf), however, is a major obstacle to malaria vaccine development 

immunity to malaria is considered hard to acquire and artificial induction of sterile protection in humans has until now only been 

achieved by inoculating radiation-attenuated sporozoites through >1000 infective mosquito bites. We recently developed a protocol that 

demonstrates that sterile protection can be induced markedly more efficiently by inoculation of intact sporozoites under cover of a bloodstage 

anti-malarial drug. We further showed that protection is mediated by T cells eliminating liver parasites and antibodies targeting blood 

stage parasites. Cellular responses against Pf parasites, in particular iFNγ production, play an important role in anti-malarial immunity. In vitro 

cytokine (iFNγ, il-2) responses were measured by flow-cytometry prior to, during and over a year post-infection. We show that cellular 

responses against both sporozoites and Pf infected red blood cells are readily induced and remain virtually undiminished at least 14 months 

after even a single malaria episode. These observations indicate that anti-malarial immunity can be induced more readily than previously 

thought and support the concept of whole-parasite-based malaria vaccines. 

Vaccine antigens and immune reponses for prevention of schistosomiasis 

rASHiKA El riDi, Hatem Tallima 

Zoology Dept. Faculty of Science, Cairo University 

Schistosomiasis is a debilitating disease endemic in 76 countries of the Developing World. Many roadblocks hinder the development of an 

effective vaccine, among which our inability to identify an appropriate vaccine antigen and the major mechanisms of immune resistance to 

the infection. Parasite cytosolic antigens are hidden from the effector arms of the immune system and, thus, may not be used as candidate 

vaccine antigens. The effector cells and antibodies may also not access the developing larvae and adult schistosomes surface membrane 

antigens, as they are protected by a sphingomyelin-based hydrogen bond barrier. Accordingly, the excretory-secretory (ES) molecules of 

the migrating worms are the only parasite antigens available as target of the host immune responses. immune cells and antibodies interact 

with the ES antigens and, thus, subject the developing larvae to a hunt, which is certainly most effective in the lung capillaries, the main site 

for innate and adaptive immunity-mediated schistosome attrition. Since our selected ES antigens, prepared in a recombinant or multiple 

antigen peptide form, predominantly induce Th1 and Th17 immune responses, our use of the correct adjuvant helped to promote the 

hunting capabilities of the immune response effectors towards elimination of most, if not all, challenge parasites. 

Vaccines for leishmaniasis 

Paul Kaye1 , Ash Maroof1 , Juliane Schroeder2 , Naj Brown1 , Deborah Smith1 , Toni Aebischer2 1 2 Centre for Immunology & Infection, Hull York Medical School & Dept. of Biology, University of York, York; Institute of Immunology 

and Infection Research, University of Edinburgh, Edinburgh & Robert Koch Institut, Berlin, Germany 




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HA04 Cont. 

Of all the parasitic diseases of man, leishmaniasis is often referred to as an ‘easy’ target for vaccination, with a single life cycle stage 

responsible for disease and excellent epidemiological and experimental evidence that infection in man leads to protective immunity against 

re-infection. Nevertheless, no vaccines have yet been licensed for use in human leishmaniasis. Here, we will outline leishmaniasis vaccine 

development over the past two decades, illustrating how a combination of over-dependence on animal models, poor understanding of 

natural transmission, limited data on human immune responses, and inadequate funding may have all contributed to the lack of progress 

to date. We will discuss the role of therapeutic vaccines as a way forward in vaccine development, and present the results of our recent 

studies towards the development of an adenoviral-based therapeutic vaccine for visceral leishmaniasis. We will also discuss recent attempts 

to develop rational approaches to vaccine antigen selection using in silico methods, and will present data from recent studies where these 

approaches have been used to select antigens for inclusion in a live recombinant Salmonella vaccine for experimental cutaneous and visceral 

leishmaniasis. 

Platform approaches to address emerging biothreats 

John Hardham 

MSC, USN Office of the Assistant Secretary of Defense for Nuclear, Chemical & Biological Defense Programs 

With the growing threat of emerging infectious disease and genetically engineered biological threat agents, the ability of government 

agencies to develop agent-specific medical countermeasures is becoming limited. To address this gap, the united States Department 

of Defense utilizes a number of platform technologies to facilitate the rapid development of medical countermeasures. These platform 

technologies include antigen expression, vaccine, and therapeutic platforms that may be integrated with diagnostic capabilities to promote 

end-to-end response capabilities. 

Safety and efficacy of MF59 

Derek O’Hagan 

Global Head of Vaccine Delivery Research, Novartis Vaccines & Diagnostics, Cambridge, MA, USA 

New generation vaccines will increasingly comprise highly purified recombinant proteins. unfortuately, these antigens are often poorly 

immunogenic. Therefore, adjuvants will be required to enable these proteins to become effective vaccines. Although several novel adjuvant 

formulations have recently emerged, including 2nd generation approaches comprising more than one adjuvant, the approval of vaccines 

containing novel adjuvants has been slow, particularly in the uS. However, despite significant ongoing concerns, the necessary safety data is 

now emerging to show that new generation adjuvants can be safely used in diverse human populations. in combination with data showing 

the positive contributions of the adjuvants to the immune response, this safety data should allow several vaccines containing novel adjuvants 

to obtain licensure within the next few years. However, with the newer generation of adjuvants, it is becoming increasingly important 

to understand with greater clarity how they exert their effects in vivo. Moreover, the physicochemical characteristics of the adjuvant 

formulations need to be well defined. MF59 is a novel vaccine adjuvant that has been used commercially in Europe since initial licensure 

in 1997 in Fluad, a product comprising an improved flu vaccine for use in the elderly. This adjuvanted vaccine recently showed enhanced 

efficacy in a clinical trial in a pediatric population. 

Mechanisms of Action of ISCOMATrIX ® adjuvant 

E. MArASKOVSKY1 , A. Baz Morelli1 , N. Wilson3 , S. Koernig1 , A. Silva1 , K. Krstevska1 , D. Becher1 , M. Schnurr4 , G. Beltz2 , 

D. Drane1 1 2 3 CSL Limited, Parkville, VIC 3052, Australia; Walter and Eliza Hall Institute for Medical Research, Parkville, VIC 3052; Genentech, 

South San Francisco, USA; 4Medizinische Klinik, Innenstadt, Klinikum der Universität München, Germany 

The iSCOMATrix ® adjuvant has antigen delivery and presentation properties as well as immunomodulatory capabilities which combine 

to provide enhanced and accelerated immune responses. The responses are broad, including a range of sub classes of antibodies as well 

as both CD4 + and CD8 + T cells. A range of iSCOMATrix ® vaccines (iSCOMATrix ® adjuvant combined with antigen) have now been 

tested in clinical trials and have been shown to be generally safe and well tolerated as well as immunogenic, generating both antibody and 

T cell responses. The mechanisms by which iSCOMATrix ® adjuvant facilitates its immune effects is the scope of significant study and 

indicates that iSCOMATrix ® adjuvant (i) rapidly traffics Ag into the cytosol of multiple dendritic cell subsets, (ii) induces the induction of 

an array of cytokines and chemokines and (iii) links the innate and adaptive immune responses in vivo in a Tlr-independent but MyD88dependent 

manner. These data highlight the clinical utility of iSCOMATrix ® adjuvant in the development of prophylactic and therapeutic 

cancer vaccines. 




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HA04 Cont. 

New insights into vaccination against pulmonary pathogens 

Dennis W. Metzger 

Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY 12208, USA 

Nearly all pathogens enter the body through mucosal surfaces, yet there are few vaccines approved for human use that can provide optimal 

protection at these sites. Our work focuses on the role of interleukin-12 (il-12) as an intranasal vaccine adjuvant for inducing immunity 

against respiratory viral and bacterial pathogens, including agents that could be potentially used for bioterrorism. A well-known effect of 

il-12 is to stimulate T cells and natural killer cells to produce large amounts of interferon-gamma and thereby induce cell-mediated immune 

responses. We have now shown that il-12 also acts as a powerful adjuvant for antibody responses, both locally and systemically, and have 

directly examined the potential of il-12 co-inoculated with vaccines to increase protection against mucosal pathogens, including influenza 

virus, pneumococci, and Francisella tularensis, a Category A Biothreat. These results have shown that antibodies induced by this vaccination 

procedure are effective in preventing infection and can even protect the pulmonary tract in a therapeutic manner after pathogen exposure. 

The mechanisms responsible for protection, including the role of igA in mucosal immunity, will be discussed. 

Offered paper The development of a vaccine against non-typhoidal Salmonella infection 

Adam Cunningham 

MRC Centre for Immune Regulation, University of Birmingham, Birmingham B15 2TT 

Antibody present can protect against Salmonella infection and this is most clearly demonstrated by the protection conferred in adults against 

typhoid by vaccination with the T-independent antigen, purified Vi capsular polysaccharide. Whilst Vi antigen is just one of three vaccines 

against typhoid, there is no vaccine against non-typhoidal Salmonella, a leading killer of infants in sub-Saharan Africa or those of all ages with 

HiV. 

We have characterized the protective capacity of selected purified surface antigens to confer antibody-mediated protection against 

infection with S. Typhimurium in a murine model of disseminated infection. immunization with highly pure lPS, flagellin or OmpA fails to 

confer antibody-mediated protection to systemic infection despite inducing extensive and rapid antibody responses after immunization. 

in contrast, the porin protein OmpD, absent from S. Typhi, induces significant T-independent and T-dependent protection, although igG 

enhances protection 20-fold. lastly, porins induce antibody responses in infant mice without exogenous adjuvant, identifying their potential 

as a vaccine in susceptible age groups. This identifies a variable capacity of Salmonella antigens to offer protective immunity that is not 

necessarily related to their immunodominance or proximity to the cell surface. 

Offered paper An oral, heat stable vaccine against Clostridium difficile disease in hamsters 

PATiMA PErMPOONPATTANA1 , Hong Huynh1 , Jutarop Phetcharaburanin 1 , Jen Min Huang1 , Jenny Cook1 , Neil Fairweather2 , 

Simon Cutting1 1 2 School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey; Dept of Life Sciences, Imperial College London, 

London 

Clostridium difficile infection is an important nosocomial infection in the developed world for which no vaccine currently exists. Two toxins, 

A and B produced by most strains of C. difficile are implicated in disease. We have used bacterial spores (Bacillus subtilis) as a delivery 

vehicle to evaluate the carboxy-terminal domains of toxins A and B as protective antigens. Our findings show that oral delivery of the cell 

binding of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. 

Moreover, animals vaccinated with recombinant spores were able to survive reinfection that is important for treatment of a disease where 

patients are prone to relapse. Neutralizing antibodies to the toxin A domain were shown to be cross reactive with the reciprocal domain 

of toxin B and being of high avidity provided protection to challenge with an A + B + strain. We show that in contrast to parenteral delivery, 

mucosal immunization is required to generate secretory igA and that neutralizing sigA correlates with protection. This work demonstrates 

that an effective vaccine to C. difficile can be designed around two attributes, mucosal delivery and the cell-binding domain of toxin A. 

Nanoparticle-based antigen-adjuvant delivery systems 

ruSSEll J. MuMPEr1 , John A. McNeill2 1 2 Director, Center for Nanotechnology in Drug Delivery, Division of Molecular Pharmaceutics; UNC Eshelman School of Pharmacy, 

University of North Carolina at Chapel Hill, USA 

The overall goal of our research program is to engineer a safe and effective nanoparticle-based vaccine delivery system to co-deliver 

multiple HiV proteins and adjuvant(s) as a prophylactic or therapeutic HiV vaccine. 




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HA04 Cont. 

We have developed solid lipid nanoparticles and oil-filled nanocapsules (NPs) and have demonstrated enhanced humoral and cellular 

immune responses to a number of different HiV antigens (Tat, Nef, Env, Gag p24, Gag p41) and model antigens (OVA, β-Gal), with codelivery 

of selected adjuvants (i.e., CpG) that activate specific innate response pathways. Among other attributes, the NPs are antigen dose 

sparing, enhance MHC i presentation, and enhance the production of neutralizing antibodies. 

This talk will highlight engineering aspects of the NPs and how the surface properties, size, zeta-potential, and composition can be 

controlled to advantageously affect the biological properties in terms of (lack of) cell and gross toxicity and inflammation, and enhance 

antigen-specific immune responses. Current approaches are focusing on the engineering of lipid-based NPs with a small amount of surface 

accessible Ni for high affinity binding to multiple his-tagged HiV proteins. 

Are mucosal adjuvants needed to stimulate protective anti-HIV responses? 

Martin Cranage 

Centre for Infection and Immunity, St George’s University of London, London SW17 0RE 

The development of an effective vaccine against HiV is hampered by a series of critical issues not least of which is how can protective 

responses be induced at the mucosal portals of entry. This may be crucial for HiV as the virus infects the immune system directly and is 

rapidly spread from the mucosal portals of entry. A further significant factor is avoidance of local pro-inflammatory responses that are often 

induced by adjuvants, since this may facilitate primary infection. We have used trimeric gp140 of HiV-1 clade C to explore its mucosal 

vaccination potential with particular respect to the generation of antibodies. Non-adjuvanted gp140 administered intravaginally to rabbits 

was shown to be highly immunogenic; however, the same preparation administered to macaques was considerably less effective. Prior or 

subsequent systemic immunization with AS01-adjuvanted gp140 considerably enhanced the efficacy of vaginal immunization. Alternative 

mucosal-adjuvanted prepartions have so far failed enhance the responses seen with systemic vaccination, suggesting that access to antigen 

after mucosal administration may be a rate-limiting factor. Conjugation of transferrin to trimeric gp140 was shown, in a murine model, to 

enhance bio-availability after mucosal administration and to stimulate production of anti-gp140 antibodies. 

Offered paper In vitro selection and characterization of recombinant human antibodies from HIV-1-positive donors 

JiE TANG1 , Anatoliy Markiv1 , Hanna Dreja2 , Áine McKnight2 , Mingyue He3 , Angray Kang1 1 2 3 University of Westminster, London, Queen Mary, University of London, London, The Babraham Institute, Cambridge 

Human single-chain antibodies reacting with recombinant HiV-1 surface glycoprotein gp120 have been selected by in vitro ribosome display 

from two combinatorial antibody libraries.The immunoglobulin libraries were prepared from two long-term survivors whose serum showed 

cross-clade neutralizing activity against HiV-1. Total rNA was extracted from isolated peripheral blood mononuclear cells (from as little 

as 20ml fresh blood). Members of 8 heavy chain families, 4 κ light chain families and 9 λ light chain families were amplified by rT-PCr. 

Single-chain antibody libraries of igG1234 isotypes in the format of Vl-link–VH-CH were constructed, with all possible combinatorial pairs 

generated and analysed by gel electrophoresis. Both libraries were subjected to a cell-free eukaryotic ribosome display selection. Analysis 

of selected sequences reveals enrichment during selection and the isotypes of these antibodies (mostly igG1, with some igG2 and 4). 

More than 300 recombinant antibodies have been isolated. Seven recombinant antibodies from the heterologous library and three from 

autologous library exhibited significant binding to recombinant gp120 in EliSA. interestingly, similar CDr3 sequences of light and heavy 

chins were observed from the two libraries, suggesting the selection worked by ribosome display. 

Offered paper Assessment of novel tuberculosis vaccine antigens using MVA 

rOBErT WATSON, Yper Hall, James McCowen, Karen Buttigieg, Ann Williams, Miles Carroll 

Health Protection Agency, Porton Down 

The proven clinical safety record, induction of cellular antigen-specific immune responses and ability to boost a BCG prime, make Modified 

Vaccinia virus Ankara (MVA) a promising viral vector for delivery of novel tuberculosis vaccine candidates. We have identified 5 novel TB 

vaccine antigens that are potentially effective against multiple stages of disease. These were shown to give protection as plasmid DNA and 

adjuvanted protein against Mycobacterium tuberculosis infection in a guinea-pig aerosol challenge model. recombinant MVAs expressing 

these antigen candidates have been generated using transfer plasmids supplied by B. Moss (NiH) and assessed for immunogenicity in vivo. 

Following vaccination, antigen-specific iFN-g responses were observed as measured by ex vivo splenocyte EliSpot assay. Peptide pools 

spanning each whole protein were used to re-stimulate cells for 18 hours prior to enumeration of cells secreting cytokine, expressed as 

spot-forming units. These data demonstrated successful induction of a Th-1 cellular immune response by each of the antigens assessed. 

Furthermore, construction and evaluation of novel recombinant MVA was investigated with regards to the vaccinia promoter driving 




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HA04 Cont. 

transgene expression and vaccination route. Protection studies will follow to determine efficacy against M. tuberculosis challenge and the 

most promising candidates will be taken forward for further develoment. 

Adjuvants to enhance effector T cell responses 

Kingston H.G. Mills 

Immunology Research Centre, School of Biochemistry & Immunology, Trinity College Dublin, Ireland 

The design of effective subunit vaccines is dependent on identifying effective adjuvants to generate appropriate protective immune 

responses against the pathogen. Most licensed vaccines work by inducing protective neutralizing antibody responses. Although effective 

for prophylactic vaccination against virus and toxin-mediated bacterial diseases, antibodies alone are not adequate for many intracellular 

pathogens and may be ineffective for therapeutic vaccination. Here cellular immune responses mediated by different population of effector 

T cells may be more important. The induction of effector T cells is directed by innate immune cells, activated by pathogen-associated 

molecular patterns (PAMPs) or adjuvants. iFN-γ-secreting Th1 cells function to activate macrophages and enhance complement fixing 

antibodies in immunity to intracellular pathogens. immunity to extracellular pathogens required Th2 cells, which promote antibody 

production, but also Th17 cells that recruit neutrophils. Finally, regulatory T cells (Treg cells), can promote pathogen persistence by 

suppressing host protective immune responses. Therefore, a major obstacle to therapeutic vaccination for chronic infections, such as HiV, 

hepatitis C virus and Mycobacterium tuberculosis is to overcome the immunosuppressive effects of Treg cells. Targeting innate immune cells 

with designer adjuvants that turn off immunosuppressive molecules and Treg cells but promote effector T cells is one approach to achieve 

this objective. 

bacterial toxins as mucosal adjuvants 

Gill Douce 

IBLS, Division of Infection & Immunity, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ 

induction of immunity at mucosal surfaces is thought to be an essential feature in protection of the host against the many pathogens that 

gain access through these surfaces. Of the many different antigens tested, the most effective immunogens at these surfaces appear to be 

bacterially derived components and in particular bacterial toxins. Of those proteins studied to date, the highly homologous enterotoxins, 

Cholera toxin (CT) from Vibrio cholerae and heat labile toxin from enterotoxigenic Escherichia coli (lT) stimulate strong local and 

systemic anti-toxin response following mucosal presentation. in addition, co-administration of normally non-immunogenic antigens results 

in the production of antibody to these proteins. Whilst the high toxicity has made their use impractical for human vaccine development, 

generation and testing of site directed mutants has shown toxicity and adjuvant activity are not linked. Subsequent testing of these mutants 

in human trials demonstrated limited success. This talk will focus on the potential for the use of toxins in vaccine design, including the 

advantages and pitfalls that have been identified to date. This will include discussion of how our knowledge has and continues to allow 

rational design of recombinant proteins that more effectively stimulate immune responses at these surfaces. 

Understanding the mechanism of action of alum 

MirJAM KOOl1,2,3 , Monique M. Willart1 , Kaat Fierens1 , Menno van Nimwegen3 , Hamida Hammad1 , Bart N. lambrecht1,3 1Laboratory of Immunoregulation & Mucosal Immunology, Dept of Pulmonary Medicine, University Hospital Gent, Belgium; 

2 3 Dept for Molecular Biomedical Research, Flemish Institute for Biotechnology (VIB), Gent, Belgium; Dept of Pulmonary Medicine, 

Erasmus Medical Center, Rotterdam, The Netherlands 

Aluminum containing adjuvants (alum) are the most widely used adjuvants in human vaccines, however only recently we started to 

unravel the mechanisms by which they potentiate the immune system. it is common knowledge that alum predominantly induces humoral 

immunity. Surprisingly, in vitro studies showed no stimulatory effects on nature’s adjuvant, the dendritic cell (DC). However, in vivo after 

injection of alum the antigen was taken up, processed and presented by inflammatory monocytes that migrated from the peritoneum, thus 

becoming inflammatory DCs that induced a persistent Th2 response that re-circulated to other lymphoid organs. The stimulating effects of 

alum on both cellular and humoral immunity were completely abolished when CD11c + monocytes and DCs were conditionally depleted 

during immunization in CD11c-DTr transgenic mice. Strikingly, alum adjuvant induced high levels of uric acid. When uric acid crystals were 

injected as an adjuvant, they induced the same strong adjuvant effect as seen with alum adjuvant. One of the targets of uric acid is the 

Nlrp3 inflammasome. When DCs are exposed to alum adjuvant or uric acid crystals they release il-1β, and this is abrogated in cells lacking 

various Nlrp3 inflammasome components. The Nlrp3 inflammasome is also partially required in vivo for the innate immune response to 

ovalbumin in alum. The early production of il-1β and the influx of inflammatory cells into the peritoneal cavity is reduced in Nlrp3 deficient 




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mice, as well as the activation of adaptive cellular immunity to antigen-alum. These findings suggest that alum adjuvant is immunogenic by 

exploiting ‘nature’s adjuvant’, the inflammatory dendritic cell through induction of the endogenous danger signal uric acid. 

Progress towards vaccines against human cytomegalovirus 

P.D. Griffiths 

Centre for Virology, UCL Medical School, Rowland Hill Street, London NW3 2PF 

There are significant challenges to producing HCMV vaccines. reinfections of naturally immune pregnant women and allograft recipients are 

the most common forms of infection. The HCMV genome contains multiple immune evasion genes. While acknowledging these challenges, 

several positive factors must also be considered.The natural seropositive state provides some protection against infection and disease in 

pregnant women and allograft recipients. HCMV is not readily transmissible, so a vaccine which prevented 60% of primary infections could 

eradicate HCMV via herd immunity. The fact that HCMV has evolved countermeasures argues that immune responses are effective. 

in 2001 the institute of Medicine classified HCMV as most worthy of development on economic grounds and the NiH supported three 

separate investigator-led, Phase ii clinical trials of HCMV glycoprotein B (gB) vaccine plus MF59 adjuvant; one published and another 

completed. i will summarize these results in women of childbearing age and in allograft recipients respectively. in conclusion, the results 

reject the view of the pessimists and encourage development of vaccines containing gB plus additional immunogens against this important, 

but neglected, pathogen. 

recoding viral genomes by chemical synthesis: application for generating vaccines 

Steffen Mueller1 , Molly Arabov1 , Chen Yang1 , Anjaruwee Nimnual1 , Bruce Futcher1 , Charles Ward2 , Steve Skiena2 , 

ECKArD WiMMEr1 1 2 Dept of Mol. Gen. & Microbiology, Dept of Computer Science, Stony Brook University, Stony Brook, NY, USA 

A protein of 881 amino acids can be encoded in 10442 different ways. This huge number is due to the degeneracy of the genetic code. 

Encoding, however, is restricted in several ways. One prominent factor is codon bias (the preferred use in mammals that is different from 

that in bacteria), another factor is codon pair bias. The latter is the little known and little understood phenomenon that some codon pairs 

in our genes are underrepresented regardless of codon frequency. Example: On the basis of codon frequencies, the amino acid pair Ala- 

Glu is expected to be encoded by GCC-GAA and GCA-GAG about equally often. in fact, the codon pair GCC-GAA is strongly underrepresented 

(even though it contains the most frequently used Ala codon). That is: some codon pairs like each other, other pairs dislike 

each other. We have recoded the 881 amino acids referred to above that represent the coat proteins of poliovirus, by changing codon pair 

bias through chemical synthesis of the viral genome, and we will present the phenotypic consequences of such recoding. Similarly, we will 

present evidence that by changing codon pair bias through chemical synthesis of gene segments of influenza virus excellent influenza vaccine 

candidates can be generated. 

The evolving story of varicella-zoster virus vaccination 

Judith Breuer 

Dept of Virology, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF 

Despite its proven safety and efficacy for the prevention of both chickenpox and shingles the live attenuated vOka vaccine has a number 

of drawbacks. Waning of vaccine induced immunity occurs even when recipients continue to be exposed to circulating wild type virus and 

this is associated with reinfection and transmission. Vaccine associated adverse event, most commonly rash formation occur infrequently in 

healthy individuals but are more common in immunosuppressed individuals. Whole genome sequencing of viruses recovered from vaccine 

rashes provides insight into the genetic determinants of vaccine virulence 

Waning immunity and virus strain-specific antigenic differences as major factors in the resurgence of mumps outbreaks in 

highly vaccinated populations 

STEVEN ruBiN1 , Christian Sauder1 , Paul Duprex2 1 2 FDA/Center for Biologics Evaluation & Research, Bethesda, Maryland, USA; Boston University School of Medicine & National 

Emerging Infectious Diseases Laboratories, Boston, Massachusetts, USA 

Mumps virus causes an acute, communicable disease with symptoms ranging from parotitis and orchitis to more serious complications 

including encephalitis and deafness. During the pre-vaccine era, greater than 90% of persons had evidence of infection by late childhood. 

Thirty years of widespread use of mumps vaccines has resulted in near elimination of the disease; however, a global resurgence of mumps 




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has been underway over the past 5+ years, including highly vaccinated populations. Strikingly, a similar mumps virus genotype (G) has been 

isolated from these outbreaks, raising concern over the possibility of immune escape. Also of interest, the overwhelming majority of cases 

of this once childhood disease now occur in young adults, most of whom were vaccinated 10 or more years earlier, raising the possibility 

of waning immunity. To investigate these concerns, the virus neutralizing capacity of serum obtained from individuals at various times postvaccination 

was tested against a wide array of phylogenetically diverse mumps virus strains, including the genotype G strain. While we 

found no evidence of immune escape, neutralizing antibody titres were found to have declined with time post-vaccination, in many cases to 

potentially non-protective levels. These data indicate the need for revaccination during adolescence for long-term prevention of mumps. 

rational development of foot-and-mouth disease virus vaccines 

Bryan Charleston 

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF 

Current FMD virus vaccines are highly effective at inducing protective immunity in cattle. A single low microgram dose in adjuvant can 

generate protection from disease (though not necessarily infection) within 4–5 days. Nevertheless present vaccines are unsatisfactory 

in three respects. First, protection is not well maintained so that frequent booster doses are required. Second, vaccinated animals may 

become infected and shed virus, so that they may be a risk to unvaccinated livestock, and third, protection against heterologous virus types 

is poor. 

We have performed proof-of-principle experiments for a vaccine produced from non-infectious cultures. The implementation of methods 

to produce non-infectious FMDV capsids as vaccines, outside of high containment facilities, would significantly lower costs, improve 

production capacity and eliminate the risks associated with infectious virus during vaccine production and use. 

in addition, our initial work has demonstrated that a non-infectious source of virus capsids allows sequence manipulation to address the 

issue of antigen stability. implementation of improvements in vaccine stability would reduce the quantity of antigen required per vaccine 

dose, mainly by reducing losses during production and improving the shelf life of the formulated product. 

Increasing poxvirus immunogenicity by neutralizing virus immune evasion strategies 

Geoffrey l. Smith 

Section of Virology, Faculty of Medicine, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG 

Vaccinia virus (VACV) is an orthopoxvirus and the live vaccine used to eradicate smallpox. in 1982 VACV was developed as an expression 

vector and recombinant VACVs expressing antigens from other pathogens were shown to have potential as live vaccines. To increase the 

safety and immunogenicity of VACV, genes encoding virulence factors that suppress the host innate immune response are being identified 

and deleted from the virus genome. removal of a virus protein that inhibits components of the innate immune system may decrease 

virus virulence, because the virus is less able to combat the host response, and simultaneously increase immunogenicity, because a better 

innate immune response induces more potent adaptive immunity. The lecture will describe a family of intracellular VACV proteins that 

are members of the Bcl-2 family and which function inside the cell to inhibit apoptosis and or innate immune signalling pathways leading to 

expression of interferons, cytokines and chemokines. The mechanisms of action of these proteins and the consequences of deletion of the 

genes encoding these proteins on virus virulence and immunogenicity will be described. 

Marek’s disease vaccines versus pathogens: continuing race for dominance 

Venugopal Nair 

Head of Avian Viral Diseases Programme, Institute for Animal Health, Compton, Berkshire RG20 7NN 

Vaccination stands as one of the most successful achievements of the last century in the fight against infectious diseases both in human 

and veterinary medicine. The modern highly intensive poultry production practices rely heavily on the widespread use of vaccines against a 

multitude of pathogens for its sustainability and highly efficiency food production. One of the best examples of a vaccination approach for 

the control of poultry disease is Marek’s disease, a rapid-onset T-cell lymphoma, caused by the highly contagious Marek’s disease herpesvirus 

(MDV). With the widespread use over the last 40 years, vaccination with attenuated vaccine viruses has largely been the cornerstone of the 

control programme. However despite widespread vaccination, the virulence of MDV strains has shown continuing increase necessitating the 

introduction of new generations of vaccines at regular intervals, resembling biological arms race between the vaccines and the pathogens. 

Based on our recent studies, we propose that the continuing increase in MDV virulence is driven by vaccines themselves, particularly 

because of their inability to induce a sterilizing immunity. unless new generations of vaccines that can induce a sterilizing immunity are 

developed, continuing increases in MDV virulence could affect the sustainability of the current vaccination-based control strategy. 




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Harnessing cytomegaloviral immune evasion mechanisms for vaccine development 

Klaus Früh 

Oregon Health & Science University, Vaccine and Gene Therapy Institute 

The ultimate goal of our research is to understand the balance between immune stimulation and immune evasion of cytomegalovirus 

(CMV) and to use this understanding for the development of new treatments and vaccines. One of the major challenges for CMV 

vaccine development is the fact that CMV establishes secondary persistent infections in CMV-immune individuals despite the presence of 

significant antibody and T cell responses. However, this unique ability also represents an opportunity for the design of CMV-based vaccine 

vectors that can be used repeatedly despite pre-existing immunity to the vector. An additional unique feature of CMV-vectored vaccines 

is that persistent infection with CMV continuously stimulates a large percentage of T cells resulting in effector memory type T cell (TEM) 

responses. TEM-inducing vaccine vectors hold great promise for the development of vaccines for AiDS and other chronic infectious 

diseases. We are currently in the process to improve vaccine safety while retaining or increasing vaccine efficacy by modulating viral immune 

evasion mechanisms. recent results and developments will be discussed. 

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Neisseria population structure: integrating whole genome data with multilocus approaches to epidemiology and population 

biology 

Martin C.J. Maiden 

Dept of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS 

Pathogenesis has emerged from commensalism in the genus Neisseria on a number of occasions, once to give rise to the gonococcus 

and several times within N. meningitidis populations, resulting in the meningococcal hyperinvasive lineages. This emergence of virulence 

form organisms with a fundamentally non-pathogenic lifestyle is yet to be fully understood as it is a complex polygenic trait with little or 

no apparent advantage to the micro-organism. Comparative whole genome studies of multiple bacterial isolates with defined phenotypes 

provides a powerful approach to this question, but such studies present challenges in integrating and analysing large, complex datasets. We 

have adopted a scalable isolate-centric approach to whole genome analysis and have developed and implemented internet-based software 

that realises this paradigm (Bacterial isolate Genome Sequence Database, BiGSdb). This generalizable platform enables the integration 

phenotypes, multilocus sequence typing (MlST), whole genome sequence and other data in the study Neisseria population structure and 

evolution. Currently these combined data are illuminating the following areas of study: (i) the emergence of distinct microbiological species 

in the Neisseria; (ii) the structuring of Neisseria meningitidis populations into clonal complexes; (iii) the emergence of virulence within clonal 

complexes. 

Effect of vaccines on the population biology of the pneumococcus 

W.P. Hanage 

Dept of Infectious Disease Epidemiology, Imperial College London, Norfolk Place London W2 1PG 

While highly effective at preventing disease due to vaccine serotypes, and with a large beneficial herd effect in unvaccinated groups, 

pneumococcal conjugate vaccines target a small fraction of total serotype diversity. in vaccinated communities non-vaccine types have 

increased in prevalence, a process called ‘serotype replacement’ resulting in no overall change in the prevalence of pneumococcal carriage. 

The consequences for invasive disease have been mixed, apparently due to variation among the replacing serotypes in their propensity 

to cause invasive disease. in the uS for instance, there has been a sustained net reduction in invasive disease due to all serotypes, while 

in the uK the benefit of vaccination has been far smaller, apparently at least in part because of the invasive properties of the replacing 

serotypes. The pneumococcus, like other bacteria, has a remarkable capacity to respond to medical interventions. We will review and 

discuss replacement in different settings. using lessons learned from carriage data collected in 2001, 2004, 2007 and 2009 from children in 

Massachusetts, following vaccination starting in 2000, we will try to draw conclusions as to which non-vaccine serotypes are likely to enjoy 

the greatest benefit following the implementation of a 13-valent vaccine. 




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The influence of host and bacterial genotype on the development of tuberculosis disease 

Maxine Caws 

Oxford University Clinical Research Unit Vietnam, Oucru Hospital for Tropical Diseases, 190 Ben Ham To, Ho Chi Minh City, 

Vietnam 

Susceptibility to tuberculosis is still poorly understood. The vast majority of individuals infected with Mycobacterium tuberculosis will never 

develop active disease and among those who do there is a wide spectrum of clinical presentation. Approximately 5% will develop primary 

pulmonary tuberculosis with a further 5% initially containing the infection as latent tuberculosis but developing reactivation disease later in 

life. The most severe form of tuberculosis, tuberculous meningitis, represents only approximately 1% of clinical cases but has high morbidity 

and mortality, with only around one-third of treated cases having a good outcome. Outcomes in HiV co-infected individuals are extremely 

poor. 

Susceptibility to tuberculosis is influenced by host genetic, environmental and pathogen virulence factors all of which interplay to determine 

the outcome of exposure. Studies in Vietnam have begun to examine the interaction between innate immune host susceptibility factors 

with the major lineages of Mycobacterium tuberculosis and the impact of these factors on clinical diseases presentation and pathogenesis. 

Evidence suggests that the long co-evolution of Mycobacterium tuberculosis with human host populations may have led to complex 

geographical variations in susceptibility and virulence which we are only beginning to unravel. 

Streptococcus suis: a new emerging or an old neglected zoonotic pathogen? 

Marcelo Gottschalk 

Faculty of Veterinary Medicine, University of Montreal, Quebec, Canada 

infections caused by Streptococcus suis are considered as one of the most important economical problems in the swine industry worldwide. 

Moreover, S. suis is an agent of zoonosis that afflicts people in contact with pigs or pork-derived products. Described during the last 40 

years in Occident as a cause of ‘sporadic cases of S. suis meningitis’, S. suis is now one of the most common causes of meningitis in adult 

people in Asian countries. S. suis was in the spotlight last years due to a large human outbreak in China, where the severity of the infection 

(shorter incubation time, rapid disease progression and higher rate of mortality) attracted the attention from the scientific community 

and the general press. in fact, the number of publications on human S. suis infections significantly increased during the recent years. The 

pathogenesis of the infection caused by this pathogen is complex and far from being completely elucidated. However, studies in vivo (with 

a new developed animal model) and in vitro (with microglia cells, astrocytes, and brain microvascular endothelial cells) clearly showed that 

central nervous system inflammation induced by S. suis virulence factors is a key player in the development of clinical signs of meningitis. 

Virulence review of Streptococcus pneumoniae 

Peter Andrew 

University of Leicester 

Abstract not received 

Influenza A virus facilitates Streptococcus pneumoniae transmission and disease 

DiMiTri A. DiAVATOPOulOS 1† , Kirsty r. Short 1† , John T. Price 2 , Jonathan J. Wilksch 1 , lorena E. Brown 1 , David E. Briles 3 , 

richard A. Strugnell 1,4 , Odilia l. Wijburg 1,4 

1 Dept of Microbiology & Immunology, The University of Melbourne, Melbourne, Victoria, Australia; 2 Dept of Biochemistry & Molecular 

Biology, Monash University, Clayton, Victoria, Australia; 3 Dept of Microbiology, University of Alabama at Birmingham, Birmingham, 

Alabama, USA; 4 Australian Bacterial Pathogenesis Program, The University of Melbourne, Parkville, Victoria, Australia 

† These authors contributed equally to this study. 

Streptococcus pneumoniae (the pneumococcus) kills approximately 1.6 million people annually. Pneumococcal infections predominantly 

manifest as pneumonia, sepsis, meningitis and otitis media. S. pneumoniae is also a member of the normal nasopharyngeal flora, colonizing 

up to 80% of children. infection with influenza A virus (iAV) has been associated with both pneumococcal disease and transmission. 

However, to date no animal model has been available to investigate the role of iAV in the spread of S. pneumoniae. Here, we investigate 

pneumococcal-influenza synergism with a particular focus on the role of iAV on pneumococcal transmission. 

infant mice were colonized with S. pneumoniae and subsequently infected with iAV three days later. using this novel model we show 

increased pneumococcal colonization and disease in the presence of iAV. importantly, in vivo imaging showed that iAV was essential for the 

transmission of S. pneumoniae from colonized (‘index’) mice to their naïve co-housed littermates (‘contacts’). Transmission only occurred 




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when all mice were infected with iAV, and was prevented when an iAV-neutralizing antibody was used to inhibit iAV-replication in either 

index mice or contact mice. Together, these data provide novel insights into pneumococcal-influenza synergism and may indicate a 

previously unappreciated role of iAV in the spread of S. pneumoniae. 

Offered paper Suilysin contributes to invasion of Streptococcus suis in epithelial cells 

MArEN SEiTZ1 , Marcus Fulde1 , ralf Gerhard2 , Manfred rohde3 , Christoph G. Baums1 , ralph Goethe1 , Peter Valentin-Weigand 1 

1 2 3 University of Veterinary Medicine Hannover, Hannover, Germany; Hannover Medical School, Hannover, Germany; Helmholtz Centre 

for Infection Research, Braunschweig, Germany 

Suilysin (SlY) is a cholesterol-dependent pore forming cytolysin secreted by Streptococcus suis (S. suis), an important swine and zoonotic 

pathogen. The role of SlY in S. suis host-cell interaction is still unclear. invasion assays showed a higher invasion-rate of a sly-positive strain 

in comparison with a sly-negative mutant. Electron microscopy analysis of the invasion process revealed formation of membrane ruffles. 

Therefore, we hypothesized that activation of the actin cytoskeleton and rho-GTPases are involved in SlY-mediated invasion process. 

To resolve the molecular mechanism of SlY host-cell interaction a point mutation was introduced in the tryptophane-rich motif of the 

conserved undecapeptide of SlY, which might be responsible for the pore-forming function. The mutated SlY had a reduced haemolytic 

and cytotoxic activity. The role of rho-GTPases was investigated by pull-down analysis of activated GTPases and colocalization-studies. 

Pull-down assays demonstrated a time-dependent activation of rac1 via both wild-type and mutated SlY. in accordance, colocalization 

experiments showed associated of SlY with F-actin and rac1. These results suggest that SlY is able to activate GTPases, independent of 

pore-formation, which is associated with the formation of membrane ruffles and internalization of S. suis. 

Offered paper CcpA-dependent capsule expression affects virulence of Streptococcus suis 

JOErG WillENBOrG1 , Marcus Fulde2 , Astrid de Greeff3 , Manfred rohde2 , Hilde Smith3 , Peter Valentin-Weigand 1 , 

ralph Goethe1 1 2 University of Veterinary Medicine, Hannover, Germany; Helmholtz Center for Infection Research, Braunschweig, Germany; 

3Wageningen UR, Animal Sciences Group, Lelystad, Netherlands 

Streptococcus suis is one of the most important pathogens in pigs and an emerging zoonotic agent. Since the host environment seems to 

be an important regulatory component for virulence, we related expression of virulence determinants of Streptococcus suis to glucose 

availability during growth and to the catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, 

representing arcABC-operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao, and epf differed significantly 

between early exponential (high glucose) to early stationary (low glucose) growth of a highly virulent serotype 2 strain. Deletion of ccpA 

altered expression of the surface located virulence-associated factors arcB, sao, eno as well as the two presently proven virulence factors in 

pigs, ofs and cps2A, at early exponential growth. A cDNA expression array revealed 259 differentially expressed genes in strain 10ΔccpA. 

Among the lower expressed genes 18 could be related to capsule biosynthesis. Correspondingly, electron microscopical characterization of 

strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with a reduced resistance against killing by 

porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on capsule synthesis and virulence properties 

of Streptococcus suis. 

Group b streptococcal pili hijack integrin machinery to promote the development of meningitis 

Kelly S. Doran 

Dept of Biology, San Diego State University, Center for Microbial Sciences, San Diego State University, San Diego, CA, USA 

Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages known as Pili have 

been recently described in Gram-positive streptococcal pathogens, including GBS. The pilin adhesin, PilA, located at the tip of the pilus, 

contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the identity of the host receptor and the contribution 

of PilA in disease pathogenesis are largely unknown. using microarray analysis, defined bacterial mutants, recombinant PilA protein as well 

as in vivo neutrophil chemotaxis assays we demonstrated that PilA is both necessary and sufficient to activate host chemokine expression 

and drive neutrophil recruitment during infection. We further demonstrate that PilA binds collagen, which promotes GBS interaction with 

the α β integrin expressed on BBB endothelium. Both bacterial internalization and proinflammatory cytokine release were dependent upon 

2 1 

functional focal adhesin kinase. These findings were further substantiated in a murine model of hematogenous meningitis; mice infected 

with the PilA-deficient mutant exhibited delayed mortality, decreased neutrophil infiltration and bacterial CNS dissemination. Our results 

suggest that the bacterial pilus, specifically the PilA adhesin, plays a dual role in immune activation and bacterial entry into the CNS, and may 

represent an attractive target for therapeutic intervention. 




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Capsule biosynthesis in the pneumococcus 

Birgitta Henriques Norrmark 

Karolinska Institutet 

Pneumococci are major contributors to morbidity and mortality worldwide. They are the major cause of milder respiratory tract 

infections such as otitis and sinusitis, but also major contributors to severe diseases such as pneumonia, septicemia and meningitis. 

Several pneumococcal virulence attributes have been identified affecting disease development. Here important pneumococcal virulence 

components including pathogenicity islets will be discussed as well as interactions with the host. 

Offered paper Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples 

collected over a 5-year period in Edinburgh 

HEli HArVAlA1,2 , Nigel Mcleish2 , Jasmina Kondracka2 , Chloe l. Mcintyre2 , E. Carol McWilliam leitch22 , Jeroen Witteveldt2 , 

Kate Templeton1 , Peter Simmonds1 1 2 Clinical Virology Centre, Edinburgh; University of Edinburgh, Edinburgh 

Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of meningitis 

and neonatal sepsis-like disease. We determined the relative frequencies of EV and HPeV types over a 5-year period in Edinburgh. Highly 

sensitive EV and HPeV PCr screening of 4168 uncultured cerebrospinal fluid (CSF) specimens from hospitalised individuals collected 

between 2005–2010 identified 201 EV and 31 HPeV positive samples. Most of those available could be directly typed by sequencing of 

VP1 (97%, 176/182). Highest frequencies of EV infections occurred in young adults (n=43; 8.6%) although a remarkably high proportion 

of positive samples (n=98; 46%) were obtained from young infants (


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dependent on PAFr. Cell wall traffics to the fetal brain and drives strong neuroregeneration. in contrast, although PAFr supports entry of 

cell wall into neurons of the adult brain, neurons do not regenerate. Thus, phosphorylcholine-containing bacterial components such as cell 

wall induce host cellular response ranging from quiescence to severe pathophysiology. 

Offered paper Comparative study of two bacterial agents of meningitis: the capsular polysaccharide differently modulates 

interactions with dendritic cells 

MAriElA SEGurA 

University of Montreal, St-Hyacinthe, Quebec, Canada 

infections with encapsulated bacteria cause serious clinical problems. Besides being poorly immunogenic, the bacterial capsular 

polysaccharide (CPS) cloaks antigenic proteins thus allowing bacterial evasion of host immune system. Group B Streptococcus (GBS) 

and Streptococcus suis share the special characteristic of being the sole Gram+ bacteria containing sialic acid in their CPS. GBS type iii is 

a leading cause of neonatal infections and meningitis. S. suis type 2 is among the most common agents of human meningitis in Asia. We 

recently characterized the S. suis type 2 CPS. it shares common structural elements with GBS but sialic acid is α2,6- rather than 2,3-linked 

to Gal. Sialic acid expression by pathogens might results in modulation of immune cell activation. using CPS –/– mutants, we compared the 

interactions of these two meningitis pathogens with dendritic cells (DCs). We showed that S. suis CPS destabilizes lipid microdomains 

and prevents lacCer accumulation at the phagocytic cup during infection, allowing bacterial evasion of phagocytosis. Alternatively, GBS 

CPS engages lipid raft domains as a mechanism of entry and intracellular survival. The outcome of both interactions seems to differently 

alter cytokine production patterns, which might affect DC capacity to activate T cells and consequent orchestration of adaptive immune 

responses. 

Offered paper Interactions between galectin-3 and Neisseria meningitidis 

PAOlA QuATTrONi1 , Yanwen li1 , Davide lucchesi1 , Derek Hood2 , Martin Hermann3 , Hans-Joachim Gabius4 , Chris Tang1 , 

rachel Exley1 1 2 3 4 Imperial College London, London; University of Oxford, Oxford; University of Erlangen-Nuremberg, Erlangen, Germany; Ludwig- 

Maximilians-University, Munich, Germany 

Galectin-3 (gal-3) is a multifunctional protein which plays important roles in inflammation and interacts with both human cells and 

bacteria through recognition of beta-galactosides. Here we describe the interaction between gal-3 and Neisseria meningitidis, a commensal 

of the human respiratory tract and an important Gram negative human pathogen, which is able to cause septicaemia and meningitis. 

immunohistochemical staining showed that gal-3 is expressed at high levels in tissues from mice infected with N. meningitidis and co-localizes 

with bacterial colonies in human tissues from patients with meningococcal disease. Gal-3 binding to N. meningitidis was dependent on 

expression of lipopolysaccharide (lPS) and inhibition assays with lactose showed that the carbohydrate recognition domain (CrD) of gal-3 

is responsible for the binding, but the full length protein is necessary for interaction. in order to study the consequences of gal-3 binding we 

analysed the adherence of N. meningitidis to phagocytic and non phagocytic cells and the uptake of bacteria by macrophages. Our results 

show that while the addition of exogenous gal-3 to N. meningitidis did not affect the adhesion of bacteria to epithelial cells, there was a 

significant increase in the association of N. meningitidis to phagocytic cells, but with reduced bacterial uptake. 

Transversal of the blood brain barrier by protozoa 

Naveed Ahmed Khan1,2 1 2 School of Veterinary Medicine & Science, University of Nottingham, Sutton Bonington LE12 5RD; Dept of Biological & Biomedical 

Sciences, Aga Khan University, Karachi, Pakistan (Email: naveed.khan@nottingham.ac.uk; naveedahmed.khan@aku.edu) 

Several protozoal infections (such as malaria, toxoplasmosis, babesiosis, trypanosomiasis, amoebic encephalitis) are highly prevalent and are 

a serious health risk. The pathophysiology of these infections is better understood, while events leading to the constitution of brain infection 

are largely unknown. Traversal of the blood-brain barrier is a key step in their invasion of the central nervous system and accomplished 

by paracellular/ transcellular route or facilitated by Trojan horse mechanisms. The ability of protozoa to cross the blood-brain barrier 

continues to inspire researchers to understand the specific strategies and molecular mechanisms that allow them to enter the brain. Here, 

we summarize the current understanding of protozoa penetration across the blood-brain barrier, focusing on Acanthamoeba as a model 

organism. using primary human brain microvascular endothelial cells that constitute the blood-brain barrier, we describe parasite factors and 

immune-mediated mechanisms involved in the blood-brain barrier dysfunction, leading to neuropathogenesis. Advances in understanding 

the mechanisms of protozoal penetration across the blood-brain barrier offer unprecedented opportunities for the development of novel 

therapeutics. 




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↑Contents 

HA05 Cont. 

Cryptococcus: how it resists the host and what to do about it 

John r. Perfect 

Duke University, DUMC 3353, Durham, NC 27710, USA 

Cryptococcus neoformans and Cryptococcus gattii are presently in outbreak mode as a continued cause of substantial morbidity and 

mortality despite present treatments. in understanding this encapsulated yeast and its pathogenesis there has been substantial progress at 

the molecular level. in this presentation, there will be a focus on its central nervous system(CNS) life-style. We will examine the genetic 

controls of CNS entry with the identification of specific genes necessary for efficient movement of yeasts across the blood brain barrier, 

yeast survival within the CNS by the screen of a mutant library in cerebrospinal fluid, and the specific mechanisms that allow it to survive 

in this harsh environment. Specficially, with the use of yeast expression profiling at the CNS site of infection and site- directed mutants, we 

will discuss the impact of carbohydrate and nitrogen metabolism on yeast survival and describe a central regulatory pathway (Trehalose) 

which is unique for fungi compared to mammalian systems and thus a potential drug target. Furthermore, we will identify several host 

factors which appear to impact on cryptococcal survival and in summary, discuss specific areas of clinical practice and quidelines to meet the 

challenges of management for this life- threatening CNS pathogen. 

Pathogenesis of Escherichia coli meningitis: current concept 

Kwang Sik Kim 

Johns Hopkins University School of Medicine, 600 North Wolfe St, Park 256, Baltimore, MD 21287 USA 

Neonatal E. coli meningitis continues to be an important cause of mortality and morbidity, and. the incomplete knowledge of its 

pathogenesis contributes to the high prevalence of this disease. Most cases of E. coli meningitis develop as the result of hematogenous 

penetration of the blood-brain barrier. The current information on E. coli translocation of the blood-brain barrier is derived from studies 

using both in vitro model with human brain microvascular endothelial cells (HBMEC) and the animal model of experimental hematogenous 

meningitis. These studies demonstrate that E. coli penetration into the brain requires a high-degree of bacteremia, HBMEC binding 

and invasion, and traversal of the blood-brain barrier as live bacteria. E. coli binding to and invasion of HBMEC requires specific E. coli 

determinants and their interaction with host receptors, involving host signaling molecules. recent advances in microbial genome sequencing 

provided the unique opportunity to investigate the pathogenesis of bacterial meningitis, including E. coli meningitis, using functional genomic 

approaches (e.g., comparative genomics and microarrays). Genome-wide screen of microbial and host genomes is likely to elucidate 

microbial and host factors involved in microbial traversal of the blood-brain barrier and provide a novel approach to investigating the 

pathogenesis, prevention and therapy of bacterial meningitis. 

Factor H binding protein, meningococcal disease and vaccines 

Christoph Tang 

Centre for Molecular Microbiology & Infection, Imperial College London, Flowers Building, Armstrong Road, London SW7 2AZ 

Neisseria meningitidis is an important cause of bacterial meningitis and sepsis that has evolved to occupy a niche in the human nasopharynx. 

As a result of selection during surivival in its host, the bacterium possesses several distinct mechanisms to avoid killing by the human 

immune system, particularly the complement cascade.As well as utlizing other approaches, N. meningitidis recruits human regulators of the 

complement system to its surface to subvert immune responses. For example, factor H (fH) which inhibits the alternative complement 

pathway binds to the bacterium via a surface receptor fH binding protein (fHbp); fHbp is an immunogenic protein and also a key antigen in 

vaccines undergoing clinical trials. fH interacts with fHbp at higher affinities than any of its known human ligands, and this has implications for 

both microbial virulence and the prevention of disease by prophylactic immunization. 

Group b streptococcus – vaccine prospects 

Paul Heath 

Reader in Paediatric Infectious Diseases, Vaccine Institute & Division of Child Health, St George’s, University of London 

Streptococcus agalactiae (Group B streptococcus) is an important cause of disease in infants, pregnant women, the elderly and 

immunosuppressed adults. The disease burden in infants has been well defined in a number of Developed countries and it is clear that 

active measures are needed to prevent GBS because of its incidence, morbidity and mortality. An effective vaccine is likely to prevent 

the majority of infant disease (both early and late onset), to avoid the current real and theoretical limitations of intrapartum antibiotic 

prophylaxis (iAP) and to be cost-effective. The optimal time to administer such a vaccine would be in the third trimester of pregnancy. 

The main limitations on the production of a GBS vaccine are not technical or scientific but regulatory and legal. A number of candidates 




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HA05 Cont. 

including capsular conjugate vaccines using traditional carrier proteins such as tetanus toxoid and CrM197 as well as newly identified 

GBS-specific proteins; one or more GBS surface proteins; and mucosal vaccines have the potential to be successful vaccines. The capsular 

conjugate vaccines are the most advanced candidates. Changing the regulatory environment to allow the development of a vaccine for use 

in pregnancy would accelerate the development of this important prevention tool. Alternative strategies such as adolescent vaccination 

need further exploration. 

Group b streptococcus hypervirulence and meningeal tropism in neonates 

Asmaa Tazi1,2,3 , Olivier Disson4,5 , Samuel Bellais1,2 , Abdelouhab Bouaboud1,2 , isabelle Tardieux1,2 , Patrick Trieu-Cuot6 , 

MArC lECuiT4,5,7,† , Claire Poyart1,2,3,6,† 1 2 3 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France; INSERM, U1016, Paris, France; Assistance Publique 

Hôpitaux de Paris, Service de Bactériologie, Centre National de Référence des Streptocoques, Hôpital Cochin, Paris, France; 4Institut Pasteur, Groupe Micro-organismes et Barrières de l’Hôte, Paris, France; 5INSERM Avenir U604, Paris, France; 6Institut Pasteur, 

Unité de Biologie des Bactéries Pathogènes à Gram Positif, URA CNRS 2172, Paris, France; 7Université Paris Descartes, Assistance 

Publique-Hôpitaux de Paris, Service des Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants Malades, Paris, France 

† These authors contributed equally to this work 

Streptococcus agalactiae (group B streptococcus, GBS) is a normal constituent of the intestinal microflora and the major cause of human 

neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-Onset Disease (lOD), 

which is characterized by meningitis in infants after the first week of life. The pathophysiology of lOD remains poorly understood, but our 

epidemiological and histopathological results point to an oral route of infection. Here, we identify a novel ST-17-specific surface-anchored 

protein that we call HvgA, and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered 

more efficiently to intestinal epithelial cells, choroid plexus epithelial cells and microvascular endothelial cells that constitute the bloodbrain 

barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in non-adhesive bacteria conferred the 

ability to adhere to intestinal barrier and BBB constituting cells. in orally inoculated mice, HvgA was required for intestinal colonization, and 

translocation across the intestinal barrier and the BBB, leading to meningitis. in conclusion, HvgA is a critical virulence trait of GBS in the 

neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies. 

Serogroup b meningococcal vaccines 

ray Borrow 

Vaccine Evaluation Unit, Health Protection Agency, Manchester 

Serogroup B Neisseria meningitidis (MenB) is the leading cause of bacterial meningitis and septicaemia in most industrialised countries, due to 

the lack of a licensed, broad coverage vaccine. Two investigational vaccines are now progressing through trials. The first, termed 4CMenB, 

a recombinant vaccine, contains three antigens; Neisserial adhesion A (NadA), factor H binding protein (fHBP) and Neisserial heparin 

binding antigen (NHBA). These antigens have been formulated with outer membrane vesicles from the New Zealand epidemic strain. The 

immunogenicity of 4CMenB has been studied in a 2, 4, 6 month schedule in infants, showed good immunogenic against strains expressing 

homologous or related NadA and fHBP. Anamnestic responses were seen following a fourth dose at 12 months of age. responses were 

generally lower against strains expressing heterologous fHBP variants. 4CMenB clearly has the potential to protect infants from MenB 

disease. The potential coverage of this vaccine by both genotypic and phenotypic methodologies has been investigated in invasive disease 

isolates from epidemiological year 2007/8. The second investigational vaccine is a bivalent fHBP vaccine. This vaccine has successfully 

been through clinical studies in adults and adolescents, eliciting SBA activity in a high proportion of adults and adolescents. Vaccine antigen 

expression was found to be of importance for the SBA response. in conclusion, investigational vaccines are now progressing through clinical 

trials with hope of at least one reaching licensure in the near future. 

Offered paper Potential coverage of an investigational meningococcal group b vaccine in England and Wales 

JAMiE FiNDlOW1 , Stefanie Gilchrist1 , Danielle Thompson1 , Maria Stella2 , Giacomo Frosi2 , ray Borrow1 1 2 Vaccine Evaluation Unirt, Health Protection Agency, Manchester; Novartis Vaccines, Siena, Italy 

An investigational four component meningococcal group B vaccine (4CMenB) has proven safe and immunogenic in Phase i-iii trials. The 

vaccine contains three recombinant proteins; factor H binding protein (fHBP), Neisserial Heparin Binding Antigen (NHBA) and Neisserial 

adhesion A (NadA) formulated with Outer Membrane Vesicles containing the immunodominant PorA protein. Protection by 4CMenB 

depends upon antigen expression and cross-reactivity of induced antibody to antigen variants. Consequently, a meningococcal antigen 




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typing system (MATS) was developed to predict whether 4CMenB covers individual isolates. As MenB currently accounts for ~90% of 

disease in England and Wales, we investigated the potential coverage of 4CMenB using MATS. 

All MenB case isolates received at the Health Protection Agency Meningococcal reference unit (n = 535) from the epidemiological year 

2007/2008 were characterized genetically for PorA and phenotypically for recombinant antigens. 

We found that 63, 1, and 55% of isolates had positive MATS phenotype for fHBP, NadA and NHBA, respectively. Additionally, 20% 

harboured the homologous PorA. Coverage (>1 antigen with positive phenotype/homologous PorA genotype) was estimated at 73%. 

Among covered strains, 69% were positive for more than one antigen. We conclude that 4CMenB has the potential to protect against a 

significant proportion of MenB disease in England and Wales. 

Offered paper Haemophilus influenzae: disease and population structure in England and Wales 

DAViD liTT, Shamez ladhani, Carina Crawford, Anna Vickers, Mary Slack 

Health Protection Agency, London 

Haemophilus influenzae (Hi) causes invasive infections including meningitis, bacteraemia and pneumonia. Between 2000–2009, 5,965 invasive 

Hi cases across all age groups were reported in England and Wales, including 552 (9.3%) presenting with meningitis. The highest incidence 

of meningitis was in infants, followed by 1–4yr olds. Of the meningitis isolates, 53% were serotype b (Hib), 37% non-typeable (ntHi) and 

10% other Hi serotypes (notably Hif and Hie). 

The contribution of Hib to meningitis and other invasive diseases has been declining since the introduction of routine Hib immunization, 

and it lost its position as the most common capsulated Hi causing invasive disease for the first time in 2009 (8% of invasive Hi cases were 

due to Hib, 4% Hie and 9% Hif, with 79% due to ntHi). 

Characterization of Hi isolates showed that ntHi possessed widely disparate MlST types. By contrast, capsulated isolates formed discrete 

clusters (Hib around ST6, Hie around ST18 and Hif around ST124). These clusters were distinct from each other, with the exception of 

type ST18, which had been seen previously amongst Hib isolates. Hence, some Hie strains may have arisen from Hib strains via capsule 

switching. However, there is no evidence of major serotype replacement in Hi disease. 

Introduction of a new Group A meningococcal conjugate vaccine in the African meningitis belt 

F. Marc laForce 

PATH for the Meningitis Vaccine Project & Partners, Washington, DC, USA 

Epidemic meningitis due to Group A Neisseria meningitidis continues to be an important public health problem in Sub-Saharan Africa. Over 

the last decade the Meningitis Vaccine Project, a partnership between PATH and WHO, has developed, tested and licensed a new and 

affordable ($uS < 0.50 per dose) Group A meningococcal conjugate vaccine manufactured by the Serum institute of india. The vaccine was 

prequalified by WHO in June 2010 and extensive pharmacovigilance studies in Burkina Faso, Mali and Niger did not reveal any unexpected 

safety problems. Country-wide vaccination of 1–29 year olds (target population 10.5 million) began on December 6th 2010 in Burkina 

Faso. Smaller campaigns in specified districts were also done in Mali (3 million doses) and Niger (3 million doses). This is the first vaccine 

to be designed specifically for Africa that has received WHO prequalification. The vaccine is affordably priced and is available in quantity. 

introduction at public health scale is expected to result in herd immunity and prevent over one million cases and 100,000 deaths over 10 

years. identifying the necessary financial resources to introduce the vaccine across the entire meningitis belt remains a challenge. 

HA06 Guarding microbial diversity: the importance of fundamental 

infrastructure in underpinning the microbial sciences 

↑Contents 

Enhanced access to microbial resources 

Erko Stackebrandt 

DSMZ, Braunschweig, Germany 

Deposition of the type strain of novel species in at least two or more public culture collections located in two or more countries is 

mandatory. The same high importance should be acknowledged with respect to public access to all key strains published in journals as 

these resources are required for subsequent verification of published data in accordance with scientific principles and for consideration 

as reference materials in subsequent research. However, the journals’ encouragement to authors to make microbial biological resources 




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HA06 Cont. 

(strains, mutants, viruses and so on) available is too weak to convince most authors to share their strains with colleagues, not to mention 

deposition in culture collections. To blame authors in general would be an injustice to those who actually deposit selected strains, there 

are also several other stakeholders involved in a complex network of interactions. At least five partners can be identified, i.e., authors, 

public collections, funding agencies for research and for collections, as well as editors; in addition, the emerging Global Biological resource 

Centre Network (GBrCN; http://www.gbrcn.org/), offering an electronic platform for communication between authors and collections, 

may be involved. The dimension of the task requires a concerted action, an intense dialog collecting a broad spectrum of opinions from all 

stakeholders involved. This strategy will be presented. 

Maintaining taxonomic and nomenclatural standards in the era of ‘omics’ – a future challenge or a lost battle? 

Peter Kämpfer 

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Henrich-Buff-Ring 26-32 (IFZ), D-35392 Giessen Germany 

Taxonomy has been circumscribed as ‘the art of biological classification’ but is consisting of more than just classification (the orderly 

arrangements of recognizable units). in addition to classification, nomenclature, i.e. labelling of these units defined by classification and also 

identification of these units, are of central importance for a stable, practical and predictable system in all disciplines of microbiology. An 

essential aspect in classification is the comparison of two or more elements, whether they are genes or genomes, proteins or proteomes, 

biochemical pathways or metabolomes or whole organisms and the most important part is the definition of the ‘taxonomic units’ to be 

classified. The development of high throughput sequencing techniques has led to an enormous amount of data (genomic and other ‘omic’ 

data) and have also revealed an extensive diversity behind these data. Based on these findings, there is a strong tendency to classify and 

name the taxonomic units preferably on the basis of sequence data which are sometimes contrary to other informations and the biological 

meaning behind. 

The criteria used for classifications may change in the future, when we have a full insight into the complexity of the genomes (and other 

‘omes’) of micro-organisms. However, the maintance of stable and predictable taxonomic and nomenclatural standards is even more 

important in these ‘high-throughput’ times. 

Describing new microbial taxa: Quis custodiet ipsos custodes? 

iain C. Sutcliffe 

School of Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST (Email: iain.sutcliffe@northumbria.ac.uk; 

Tel: +44 (0)191 227 4071) 

Microbial taxonomy and systematics are vital in providing a sound framework for microbiology, particularly now that an explosion of 

molecular sequence-based approaches has revolutionised our understanding of microbial diversity. Formal descriptions of novel taxa 

remain the bedrock of taxonomy, along with the re-evaluation of established taxonomies. New descriptions face the challenge of proving 

genuine novelty in the face of an ever expanding catalogue of previously described taxa. The vast majority of descriptions of novel taxa are 

published in the International Journal of Systematic and Evolutionary Microbiology (iJSEM) and, furthermore, the valid publication of any new 

name (or combination) must be published in this journal. However, a small but significant number of descriptions of novel taxa appear in 

journals such as Antonie van Leeuwenhoek, which i represent as Editor-in-Chief. it is thus important that such studies conform to appropriate 

benchmarks that are upheld by rigourous peer review. Thus the guidelines for authors of the iJSEM rightly remain the gold standard for 

all descriptions of novel taxa. However, the future of taxonomy and our ability to attract young scientists into the field will depend on 

flexibility and pragmatism in defining minimal standards that are clear, open and not unnecessarily proscriptive. Thus Editors responsible for 

handling descriptions of novel taxa must remain encouraging custodians rather than zealous guards. 

reconstructing prokaryotes from diverse data sets – the importance of plausibility, predictability, correlation and verification 

Brian J. Tindall 

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Inhoffenstrasse 7b, Braunschweig, 38124, Germany 

Biology is rapidly moving towards two major goals, the availability of vast arrays of data and the possibility of high throughput sequencing of 

a large number of genomes. Nowhere are these aspects more apparent than in prokaryotes research. However, amid the euphoria there 

are also serious problems to be solved. Among these is the administration of the data, the correct annotation of the individual components 

and the realization that one should be equally rapidly working towards understanding how organisms work at the cellular/whole organism 

level. At present the data associated with a particular organism is held in distributed formats, ranging from public databases to publications 

and lab note books. Only when one attempts to the put the data into context is it possible to see it makes sense. it is the last step that 




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HA06 Cont. 

is the most difficult and requires putting individual data sets into context and attempting to determine whether it makes sense. it is at this 

point that a good working knowledge of prokaryote biology comes into play, and the elements of plausibility, predictability, correlation and 

verification take on central significance. unfortunately it is also the point at which one realises the pitfalls, flaws and errors that abound. 

The future of taxonomy 

A.l. Jones 

1Dept of Biology, Food and Nutritional Sciences, School of Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST 

Microbial systematics is a misunderstood scientific discipline. it is thought systematists use antiquated techniques to examine the molecular, 

morphological, physiological, and biochemical properties of micro-organisms. it is also thought that circumscribing novel taxa is not required 

and because of this, systematics has become a dying art. in truth this discipline is essential to all sciences, without the use of current 

techniques, descriptions of known and novel taxa cannot be correctly identified. Since the first publication on the use of 16S as a molecular 

tool, phylogenetic analysis of 16S rrNA genes has become an essential technique in microbial systematics. However this technique has 

limitations and it is becoming more apparent that full genome comparison is required for the full circumscription of novel taxa. The next 

generation of sequencing technology, is enabling more information to be incorporated into the full systematic picture. However the start 

of this era has shown that genomic data, via high throughput sequencing will compliment, rather than throwing a different light on the 

polyphasic data obtained. High-throughput sequencing is a cost effective method of genomic sequencing; however it is still years away from 

becoming one of the minimal standards. 

Culture collection federalization 

Pippa Bracegirdle 

CAMR, Porton Down, Salisbury, Wiltshire SP4 0JG 

The establishment of the Health Protection Agency in 2003 brought four of the existing uK national culture collections – the European 

Collection of Cell Cultures (ECACC), the National Collection of Type Cultures (NCTC), the National Collection of Pathogenic Viruses 

(NCPV) and the National Collection of Pathogenic Fungi (NCPF) – together within a single organization. 

The Health Protection Agency Culture Collections (HPACC) was established as an integrated, not-for-profit strategic business unit within 

the Health Protection Agency in 2005, with a mission to support and underpin laboratory healthcare science in the uK and worldwide. The 

HPACC is funded mainly by income from commercial sales supplemented by grant funding from the MrC, Wellcome Trust and Eu. 

The fundamental requirements of a Biological resource Centre apply to all four sister collections; however the specific technologies and 

processes can vary considerably. The commonalities and major divergences, and how the HPACC reconciles these, will be briefly reviewed. 

This presentation will also describe the ongoing process of integration and co-ordination of the four culture collections as a single unit, and 

as part of a large government health organization. 

A way forward? 

David Smith 

Curator of Genetic Resources Collection, CABI Bioscience UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY 

Culture collections secure the microbial genetic pool for research and to support the continued search for properties and novel 

products. The diminishing resource of specialist taxonomists makes ensuring correct identity of strains difficult and discovery of new 

diversity slow and haphazard. Collections strive to find ways to survive, not unlike the organisms they hold and supply. Harnessing the 

power of networking provides some of the answers. The World Federation for Culture Collections has been fighting the cause for 

over 4 decades (http://www.wfcc.info). in Europe, the European Culture Collection’s Organisation (http://www.eccosite.org) has been 

providing an incubator for pan-European initiatives. However, a lot of work and investment is still needed, by collections, Governments 

and bioindustry, if the power of microbial diversity is to be harnessed effectively. A recent European project, the European Consortium 

of Microbial resource Centres (http://www.embarc.eu) is addressing collection sustainability. However, project funding is itself not 

enough. The Global Biological resources Centres Network (GBrCN) (http://www.gbrcn.org) and the Microbial resources research 

infrastructure are investigating new strategies. Bringing together collections with stakeholders (users, policy makers, funders and microbial 

research efforts) to improve access and deliver scientific and technological and managerial excellence for research, education and 

technology. 




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HA07 Mechanisms of DNA repair 

↑Contents 

DNA repair in pathogenesis of tubercle bacilli 

Jaroslaw Dziadek 

Institute for Medical Biology, Polish Academy of Sciences, Lodz, Poland 

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis is exposed to a range of environmental insults, including the host’s 

immune response, during the infectious process. From the moment MTB are exhaled by infected individuals, through an active and latent 

phase in the body of a new host, till the time they reach the reactivation stage, MTB are exposed to many types of DNA damaging 

agents. like all cellular organisms, MTB have efficient DNA repair systems, and these are believed to play essential roles in mycobacterial 

pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different 

repair systems would be essential for progression to specific phases of infection. DNA double-strand breaks (DSBs), are the most lethal 

type of lesion that the genome can experience, as even a single unrepaired break can result in cell death. The DSBs in mycobacteria have 

to be repaired by homologous recombination (Hr) or non-homologous end-joining (NHEJ) DNA repair pathways. The role of ATPdependent 

ligases in the DSBs repair process and the essentiality of NHEJ and Hr in the early step of infection were investigated. 

DNA damage response in the archaeon Haloferax volcanii: the role of Mre11-rad50 

Stéphane Delmas, THOrSTEN AllErS 

School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH 

in polyploid species, the presence of multiple genome copies has profound implications for strategies of DNA repair. We studied DNA 

repair in the halophilic archaeon Haloferax volcanii, which maintains up to 20 genome copies. We have focussed on the Mre11-rad50 

complex, it is present in all domains of life and it plays a central role in the eukaryotic DNA damage response. Our results indicate 

that Mre11-rad50 play several roles in the DNA damage response of H. volcanii. On the one hand, it delays the use of homologous 

recombination, instead favouring micro-homology mediated end joining as an initial means of DNA double-strand break repair. On the 

other hand, Mre11-rad50 reorganizes the structure on the nucleoid in response to DNA damage, compacting genomic DNA inside the 

cytoplasm. We propose that the nucleoid compaction is part of the DNA damage response, in order to accelerate the localization of 

target sequences by DNA repair proteins. We suggest that polyploidy organisms use a DNA damage response that minimises the use 

of homologous recombination, since this repair strategy has the potential to create branched DNA structures that can prove difficult to 

resolve. 

removal of covalently bound topoisomerases from DNA by MrN and Ctp1 

Edgar Hartsuiker 

NWCRF Institute, School of Biological Sciences, Bangor University 

Camptothecins (CPTs) and etoposides are important cancer drugs which trap topoisomerases on the DNA. For a (cancer) cell to survive 

treatment the topoisomerases need to be removed. We have recently shown that the Schizosaccharomyces pombe rad32Mre11 nuclease 

activity is involved in the removal of both rec12Spo11 and Top2 from 5’ DNA ends, as well as Top1 from 3’ ends in vivo. A ctp1CtiP deletion 

is defective for rec12Spo11 and Top2 removal but over-proficient for Top1 removal, suggesting that Ctp1CtiP plays distinct roles in removing 

topoisomerases from 5’ and 3’ DNA ends. Analysis of separation of function mutants suggests that MrN-dependent topoisomerase 

removal contributes significantly to resistance against topoisomerase-trapping drugs. We have recently obtained preliminary evidence that 

the rad32Mre11 nuclease activity is also involved in providing resistance to another clinically important group of cancer drugs, the nucleoside 

analogues. These studies have important implications for our understanding of the role of the MrN complex and CtiP in resistance of cells 

to clinically important cancer drugs. 

Yeast global genome nucleotide excision repair promotes chromatin remodelling required for efficient repair of UV-induced 

DNA damage 

SiMON rEED, Shirong Yu, Yumin Teng, Mark Bennett, raymond Waters 

Dept of Medical Genetics, Haematology & Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN 

Following uV radiation induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin 

structure and these alterations are associated with efficient global genome nucleotide excision repair (GG-NEr) in yeast. Both these 

changes occur in response to uV radiation in the absence of functional GG-NEr. recently we reported that although these changes 

HA07 

↑Contents 




38 




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HA07 Cont. 

occur independently of functional NEr, they do depend on the rad16 GG-NEr protein. remarkably, we showed that constitutive 

hyperacetylation of histone H3 can suppress the requirement for both the rad7 and rad16 GG-NEr proteins during DNA repair. These 

observations hinted at a possible mechanistic link between the rad7 and rad16 proteins and the process of chromatin remodelling 

required for efficient DNA repair. Here we reveal how uV induced histone H3 acetylation is regulated during GG-NEr, and how this 

promotes chromatin remodeling necessary for efficient repair of DNA damage. We show that yeast rad7 and rad16 proteins drive uV 

induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. This is achieved via the concerted action of the 

ATPase, and C3HC4 riNG domains of rad16, which in concert regulate the occupancy of histone acetyl transferases on chromatin in 

response to uV damage. 

Offered paper Archaeal DNA repair helicase Hel308: structure–function and interactions with proteins of DNA replication 

and recombination 

isabel Woodman, Kirsty Brammer, EDWArD BOlT 

University of Nottingham, Nottingham 

Hel308 is an archaeal super-family 2 helicase with homologues in higher eukaryotes involved in repair of DNA strand crosslinks. 

Contributions of Hel308 to DNA repair processes are far from clear, including how it co-operates with other proteins. using archaeal 

Hel308 we identified physical interaction with rPA that required a conserved amino acid motif at the Hel308 C-terminus1 . This and 

previous analyses of Hel308 led us to propose that in archaea rPA acts as a platform for loading of Hel308 onto aberrant single-stranded 

DNA (ssDNA) arising at blocked replication forks. in line with data from a human Hel308 homologue, the helicase activity of archaeal 

Hel308 was only modestly stimulated by rPA, much less so than for other known interactions between helicases and ssDNA binding 

proteins. This supports a model for rPA localizing Hel308 to DNA damage sites, rather than stimulating the mechanism of helicase 

unwinding. We also present details of a winged helix domain structure in Hel308 that is conserved in rNA remodelling proteins Brr2, Mtr4 

and Prp43p, and that may function to maintain structural integrity of the overall Hel308 ring structure, and engages with nucleic acid. 

References: (1). Woodman il, Brammer K and Bolt El (2010) DNA repair. doi:10.1016/j.dnarep.2010.12.001 

Extreme gene machines: maintaining genetic integrity in boiling acid 

Malcolm F. White 

University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST 

Archaea frequently inhabit extreme environments and thus face challenges in maintaining functional cellular structures and macromolecules, 

in particular DNA and proteins. This evolutionary pressure has resulted in many adaptations that overlay the fundamental relationship 

between the archaea and eukarya in information processing systems. This talk will focus on recent research in our laboratory on the 

Nucleotide Excision repair (NEr) pathway in the model crenarchaeon Sulfolobus solfataricus, which grows in volcanic pools at 80°C, pH3. 

NEr removes bulky lesions such as photoproducts from DNA. lesions are detected, DNA unwound locally and nicks introduced on either 

side of the damage to release an oligonucleotide ‘patch’ containing the damage. The gapped product is then repaired by DNA synthesis. 

The enzymatic components of eukaryal NEr are the xPB and xPD helicases (components of transcription factor TFiiH) and the xPF 

and xPG nucleases. Most archaea have clear homologues of xPB, xPD and xPF, and these have been presumed to function in an NEr 

pathway that resembles a simplified version of the eukaryal process. recent work exploring the structures and mechanisms of the archaeal 

proteins will be discussed. 

The ends are nigh: proteins involved in binding to DNA ends in streptomycetes 

richard P. Bowater 

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ 

To maintain genomic integrity, all cells contain a variety of pathways that specialise in the repair and removal of particular damage-induced 

DNA lesions. DNA double-strand breaks (DSBs) are especially toxic to cells and different pathways act to ensure these are repaired. Nonhomologous 

end joining (NHEJ) is one pathway used to repair DSBs and it has been well characterized in eukaryotes. Homologous genes 

are present in a range of prokaryotes, and a functional NHEJ pathway has been confirmed in mycobacteria, where it is coordinated by two 

gene products. The bacterium Streptomyces coelicolor is an actinomycete with a complex life cycle, and it has homologues to the genes 

for mycobacterial NHEJ, but the biochemical activities for NHEJ appear to be encoded by at least 5 different genes. We have cloned the 

relevant genes and purified recombinant versions of the proteins, which include several DNA ligases, multiple Ku (end-binding) proteins, 

a nuclease and several primases. Preliminary experiments confirm that these proteins possess the expected biochemical activities in terms 




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HA07 Cont. 

of binding and processing of DSBs. Our data imply that S. coelicolor has NHEJ repair that functions in an intermediate fashion compared to 

NHEJ in mycobacteria and eukaryotes. 

repair mechanisms that prevent trinucleotide repeat instability 

Catherine H. Freudenreich 

Dept of Biology, Tufts University, Medford, MA 02155, USA 

Trinucleotide repeats can form secondary structures that interfere with replication fork progression and DNA repair. One potential 

mechanism for replicating through DNA structures is the use of non-replicative helicases, and i will present data on the role of the yeast 

Srs2 helicase and other helicases in replication through structure-forming repeats and prevention of repeat instability. in addition, we found 

that the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-rFC complex (Ctf18-rFC) prevents CAG/CTG repeat instability and fragility 

independently from its role in sister-chromatid cohesion. Both Srs2 and Ctf18-rFC prevent instability by suppression of recombination 

within the repeat. i will discuss the links between replication fork progression and regulation of recombination at expanded repeats, and 

how these processes collaborate to prevent repeat instability. 

Timing and spacing of ubiquitin-dependent DNA damage bypass 

Yasukazu Daigaku, Adelina A. Davies, irene Saugar, HEllE D. ulriCH 

Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD 

DNA damage tolerance, also called postreplication repair (Prr), is a pathway that enables cells to overcome replication problems and 

complete the duplication of their genomes in the presence of DNA lesions. in eukaryotes, the pathway is controlled by ubiquitylation 

of the replication clamp protein PCNA. Monoubiquitylation of PCNA activates translesion synthesis by specialised damage-tolerant 

DNA polymerases, whereas polyubiquitylation is required for an alternative, error-free pathway. We have designed an inducible system 

of damage bypass in budding yeast that has allowed us to analyse the effects of Prr in the course of the cell cycle and to separate its 

activity from bulk replicative DNA synthesis. We show that Prr can act not only during S phase, but similarly contributes to survival when 

activated after replication of the genome is largely complete. We find that uV lesions are processed predominantly by translesion synthesis, 

with error-free damage avoidance acting as a back-up pathway. Prr tracts analysed by fibre spreading are clearly distinct from replicative 

DNA synthesis, occurring in small patches along the chromosome. Our approach has for the first time revealed the distribution of Prr 

tracts in a synchronised cell population. 

Superfamily I helicase motors as modular components of DNA repair machines 

Mark S. Dillingham 

DNA:Protein Interactions Unit, School of Biochemistry, University of Bristol 

Superfamily 1 (SF1) DNA helicases are molecular motor proteins that couple the hydrolysis of nucleoside triphosphates to processive 

translocation and unwinding of duplex DNA into its component single strands. They are extremely abundant enzymes that, despite sharing 

a similar core structure, function in a diverse range of pathways and play critical roles in virtually all aspects of DNA metabolism. SF1 

helicases are highly modular, and their functional specificity appears to be governed by variable accessory domains and/or the interactions 

they make with other proteins. We are interested in understanding how helicase activity can be targeted, activated and catalytically 

modified by modifications to the core motor. in this talk i will discuss our work on the Bacillus subtilis AddAB complex, a helicase-nuclease 

that processes broken DNA ends for repair by homologous recombination. 

Offered paper Identification and characterization of E1b-AP5 as a novel DNA repair protein through its interaction with 

adenovirus E1b-55K 

ANDrEW BlACKFOrD1,2 , Sophie Polo3 , ross Chapman3 , linda Baskcomb3 , Serge Gravel3 , Andre rusch4 , Philippa Smith3 , 

Thomas Dobner4 , Malcolm Taylor2 , Andrew Turnell2 , Grant Stewart2 , roger Grand2 , Stephen Jackson3 1 2 3 4 University of Oxford, Oxford; University of Birmingham, Birmingham; University of Cambridge, Cambridge; University of Hamburg, 

Hamburg, Germany 

The cellular DNA repair machinery is a barrier to adenovirus (Ad) replication. During infection, two viral proteins, E1B-55K and E4orf6, 

form a ubiquitin ligase to inactivate host DNA double-strand break (DSB) repair pathways by targeting a number of DNA repair complexes 

for proteasomal degradation. Previously identified targets of E1B-55K/E4orf6 include the MrE11-rAD50-NBS1 (MrN) sensor complex, 

DNA ligase iV, TOPBP1, and the Bloom’s helicase BlM. E1B-55K-associated protein 5 (E1B-AP5) is a cellular protein that we previously 




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identified in a screen for E1B-55K-interacting proteins. We have now characterized E1B-AP5, and found that it plays a key role in cellular 

responses to DSBs through its interaction with the MrN complex. E1B-AP5 localizes to DNA damage sites in a manner that requires 

MrN, and promotes DNA end resection, ATr signalling, and DNA repair by homologous recombination, thereby contributing to cell 

survival following genotoxic stress. Finally, we have established that E1B-AP5 functions downstream of MrN and CtiP to promote the 

recruitment of BlM to DNA breaks. These results provide new molecular insights into how mammalian cells respond to DSBs, and 

highlights the relevance of studying viruses and their interactions with the host cell in order to define novel pathways in the cellular DNA 

damage response. 

DNA repair pathways acting on interstrand cross-links 

Thomas A. Ward1 , Zuzana Dudasova2 , Dana Vigašová2 , Miroslav Chovanec2 , PETEr J. MCHuGH1 1 2 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford; Dept of Molecular Genetics, Cancer Research Institute, 

Bratislava, Slovak Republic 

DNA interstrand cross-links (iCls) are an extremely toxic form of DNA damage that inhibit fundamental cellular processes such as DNA 

replication and transcription. Which DNA repair acts on iCls is heavily dictated by cell cycle phase. in budding yeast, a specific exonuclease 

(Pso2) is critical for repair in the G1 and G2/M phases of the cell cycle, but is only partly required in S-phase, during a process termed 

‘replication-coupled repair’. Consequently, we attempted to identify additional factors involved in replication-coupled iCl repair. using a 

combination of genetic and biochemical approaches, we have purified and characterized a new DNA repair complex containing a mismatch 

repair factor MutSa (Msh2-Msh6), the Mph1 helicase/translocase and a DNA-binding protein poorly characterized to date, Mgm101. We 

have termed this complex the M-M-M complex. When Pso2 is deleted in combination with any of the members of the M-M-M complex all 

replication-coupled iCl processing is abolished, leading to the massive accumulation of broken chromosomes in mutant cells, and cell death. 

The major components of the M-M-M complex are conserved in higher eukaryotes, suggesting that this complex might play a critical role in 

mammalian iCl repair, a process important for suppressing cancer and other developmental disorders. 

HA08 Microbial PAMPS 

↑Contents 

Entamoeba histolytica lipopeptidophosphoglycan and host response 

Nestor González-roldán1 , Hannelore lotter3 , Egbert Tannich3 , OTTO HOlST2 1 2 3 Divisions of Immunobiology; Structural Biochemistry, Research Center Borstel, D-23845 Borstel Germany; Bernhard Nocht Institute 

for Tropical Medicine, D-20359 Hamburg, Germany 

Entamoeba histolytica represents a human intestinal protozoan parasite that causes significant morbidity and mortality worldwide. The main 

symptoms of invasive amebiasis are severe haemorrhagic colitis or the development of extraintestinal (mostly liver) abscesses. In vitro as 

well as in vivo studies for experimental amoebic liver abscesses (AlA) documented that iFN-γ is an important factor in the early control of 

E. histolytica infections. in the experimental AlA mouse model, the role of activated natural killer T (NKT) cells in the defence against AlA 

was shown by their specific activation by α-galactosylceramide and their depletion in CD1d-deficient mice. Furthermore, it was found that 

the phosphatidylinositol moiety of the surface lipopeptidophosphoglycan of E. histolytica trophozoites (EhlPPG) induces iFN-γ secretion 

in NKT cells. its main activating component was the diacylated EhPib, 1-O-[(28:0)-lyso-glycero-3-phosphatidyl-]2-O-(16:0)-inositol. NKT 

cell activation occurred in a CD1d-restricted manner, however, simultaneously it depended on Tlr receptor signaling as indicated by the 

abrogation of iFN-γ secretion when bone marrow derived dendritic cells from il12p40–/–, MyD88–/–, and Tlr–/– mice were used for 

antigen presentation. The recognition of antigenic proteins occurred equal with sera from AlA patients and asymptomatic E. histolytica 

carrier, but differed with EhlPPG, implicating the presence of distinct virulent E. histolytica strains. 

Innate immune recognition of fungal β-glucans 

Gordon Brown 

Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD 

The innate ability to detect pathogens is essential for multicellular existence, and has been achieved through the evolution of germ-line 

encoded receptors which can recognise non-self structures, the so-called ‘pattern recognition receptors’ (Prr). One such receptor is 

Dectin-1, a type ii transmembrane glycoprotein with a single extracellular non-classical C-type carbohydrate recognition domain (CrD) 

and a cytoplasmic tail possessing an immunoreceptor tyrosine-based activation-like (iTAM) motif. Dectin-1 is predominantly expressed 




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HA08 Cont. 

on myeloid cells and recognises β(1–3)-linked glucans. We and others have demonstrated that this receptor mediates a variety of cellular 

responses to β-glucans, including phagocytosis, endocytosis and the oxidative burst and can induce the production of arachidonic acid and 

numerous cytokines and chemokines. These responses are triggered through the cytoplasmic iTAM-like motif of this receptor, utilizing novel 

signalling pathways involving a unique interaction with Syk kinase and collaborative signalling with the Tlrs. Dectin-1 is the first example of 

a signalling non-Toll-like pattern recognition receptor being involved in the induction of protective immune responses, and through these 

activities Dectin-1 plays a fundamental role in anti-fungal immunity in mice and humans. Here, we will new insights into the role of Dectin-1 

as well as other Prrs during fungal infections. 

NOD1 sensing of Helicobacter pylori peptidoglycan: the link between innate and adaptive immunity 

M. Kaparakis-liaskos 1 , A. irving2 , M. Gantier2 , l. Turner1 , A. Desra1, S. Mannering3, r. l. FErrErO1 . 

Centre for Innate Immunity & Infectious Diseases1 ; Centre for Cancer Research2 ; Monash Institute of Medical Research, Monash 

University, and St Vincent’s Hospital3 ; Melbourne (Victoria) Australia 

Gram-negative pathogens can deliver peptidoglycan (PG) into non-phagocytic epithelial cells via a novel mechanism involving outer 

membrane vesicles (OMVs). We showed that upon entry into host cells, the PG within OMVs was detected by the cytosolic host pathogen 

recognition molecule, NOD1. Moreover, NOD1 was essential for the development of OMV-specific innate and humoral immune 

responses in vivo. The aim of the current work was to elucidate the mechanism(s) of OMV entry and processing in non-phagocytic epithelial 

cells to further our understanding of the development of OMV-specific immune responses. We showed that chemical inhibitors targeting 

specific components of the major endocytosis pathways reduced both OMV entry and NOD1-dependent pro-inflammatory responses 

in epithelial cells. using confocal microscopy, we demonstrated that OMVs co-localized with lC3, a component of the autophagosome. 

Consistent with this finding, sirNA knockdown of lC3 resulted in reduced il-8 responses in cells. Moreover, studies with sirNA NOD1 

knockdown or NOD1 knockout cells showed that NOD1 was essential for OMV-induced autophagy. Collectively, our findings suggest that 

Gram-negative pathogens secrete OMVs that enter non-phagocytic epithelial cells via endocytosis, resulting in NOD1-driven host immune 

responses. 

Pathogen products in susceptibility and resistance to malaria 

louis Schofield 

Howard Hughes International Research Scholar, The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, 

Parkville 3050, Victoria, Australia 


NOD receptors and bacterial PAMPs recognition 

Thirumala-Devi Kanneganti 

St Jude Children’s Research Hospital, Memphis, TN 38105, USA 

The innate immune system comprises several classes of pattern recognition receptors (Prrs), including Toll-like receptors (Tlrs), 

Nucleotide binding and oligomerization domain-like receptors (Nlrs), and riG-1-like receptors (rlrs). Tlrs recognize microbes on the 

cell surface and in endosomes, whereas Nlrs and rlrs detect microbial components in the cytosol. Genetic variations in several Nlr 

members are associated with the development of a number of autoinflammatory and autoimmune disorders including Crohn’s disease, 

vitiligo, Blau syndrome, cryopyrinopathies, FMF, etc. Two Nlrs, NOD1 and NOD2, sense the cytosolic presence of the peptidoglycan 

fragments meso-DAP and muramyl dipeptide, respectively, and drive the activation of mitogen-activated protein kinase (MAPK) and the 

transcription factor NF-kappaB. A different set of Nlrs Nlrp3, Nlrp1 and ipaf are critical for the activation of inflammasomes, molecular 

platforms that mediate the activation of caspase-1 and processing of pro-il-1beta/il-18 into mature il-1 beta and il-18 in response 

to bacterial components. The results available so far suggest that Nlrs are critical mediators of innate immune responses and play an 

important role in inflammatory and infectious diseases 

Gut microbiota and mucosal immune homeostasis 

Denise Kelly 

Rowett Institute of Nutrition & Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen AB21 9SB 

Microbial colonization of the mammalian gut drives development, expansion and function of the mucosal immune system. Once established, 

resident commensal microbes actively contribute to immune homeostasis and gut health. This homeostasis relies on immunological 

tolerance to resident gut microbes and is influenced by a plethora of factors including microbial composition and compartmentalization, 




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HA08 Cont. 

innate immune sampling and recognition, as well as the local cytokine/chemokine milieu. The function of host pattern recognition receptors 

in microbial recognition, immune response and homeostasis has received significant attention over the last decade. in particular, Tolllike 

receptors which recognise molecular signatures on different classes of micro-organisms play a key role in activating and directing the 

immune response at mucosal surfaces. Tlr5, the receptor specific for bacterial flagellin, is crucially important in host-microbe interactions 

in the gut. This receptor responds differentially to activation by commensal and pathogenic flagellins; these responses may be specifically 

tailored to driving innate immune signals associated with either heightened or reduced flagellin responsiveness. The importance of these 

early innate signals in driving appropriate immune responses to pathogenic and commensal bacteria will be presented. 

C-type lectins in Candida albicans infection 

r.B. ASHMAN, C.A. Wells 

University of Queensland, Australia 

C-type lectins are important detectors of environmental signals. in particular, Dectin-1, Dectin-2, and the macrophage inducible C-type 

lectin, Mincle, (Clec4e), are pattern recognition receptors for the yeast Candida albicans. in mice, Mincle was found to be expressed across 

a wide range of tissues, whereas Dectin-1 and Dectin-2 were expressed constitutively in a tissue specific manner. Dectin-1 was generally 

expressed at low levels, but was abundant in the lung, whereas Dectin-2 was most readily detected in the spleen. in the lung, Mincle was 

present on a subset of F4/80+ monocytes, and neutrophils, as well as on a subset of E-cadherin+ pneumocytes. Within the spleen, it was 

induced at levels similar to Dectin-2, on the same sub-populations of cells. Following challenge with C. albicans, Mincle was the most highly 

induced of the three lectins, suggestive of a primary role in early responses to acute infection. unexpectedly, Mincle knock-out mice were 

susceptible to both oral and systemic infections. As oral infection is resolved via T-helper cell recruitment, whereas systemic infection is 

dependent on phagocytes for resolution, Mincle provides a crucial link between innate and adaptive immune responses to fungal pathogens. 

Offered paper Candida albicans mannans are important for immune recognition and phagocytosis 

rEBECCA A. HAll1 , Chirag C. Sheth1 , Hector Mora-Montes1 , Franscisco J. Alvarez1 , Jeanette Wagner1 , Mihai G. Netea2 , 

Alistair J.P. Brown1 , Frank C. Odds1 , Neil A.r. Gow1 1 2 School of Medical Sciences, University of Aberdeen, AB25 2ZD; Radboud University Nijmegen Medical Centre, 6525 GA, Nijmegen, 

The Netherlands 

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of C. albicans is comprised of a chitin-β-glucan inner skeleton 

surrounded by a dense outer layer of highly gylcosylated mannoproteins. This cell wall structure is shared by the majority of fungal species 

and is the first point of contact between the pathogen and the host’s immune system. As a result the mammalian immune system has 

evolved pathogen recognition receptors that recognise the main components of the fungal cell wall. The detection and removal of 

fungal pathogens is a complex process as the fungal cell wall is dynamic, continually changing in response to fungal morphology and host 

environmental cues. Here, we shown by systematically altering the glycan structure, that that the glycosylation status of C. albicans is 

important for activation of the host immune system, with stains displaying altered glycan structures failing to induce cytokine responses. in 

addition, we show that glycosylation plays an essential role in neutrophil phagocytosis, with strains defective in N-linked glycan accumulating 

on the phagocyte surface. The library of glycosylation mutants we are generating serve as an important tool in unravelling the precise 

carbohydrate structures which form the pathogen associated molecular patterns which the immune systems relies on to eradicate fungal 

infections. recent progress in defining the carbohydrate PAMPS required for innate immune recognition will be presented. 

Offered paper Identification of a natural MDA5 ligand during picornavirus infection 

Qian Feng, Stanleyson Hato, Jan Zoll, FrANK VAN KuPPEVElD 

Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands 

The riG-i-like receptors (rlrs) are newly identified Pathogen recognition receptors (Prrs) that recognize cytoplasmic viral rNAs 

and have crucial roles in host antiviral immunity by inducing type i interferons (iFN-α/β). MDA5, a member of the rlr family, has been 

suggested to recognize picornaviruses, a family of important animal and human pathogens such as polioviruses, coxsackieviruses, and 

encephalomyocarditisvirus (EMCV). 

using inhibitors to specific steps of the viral replication cycle, we studied the abilities of various viral rNA species (incoming ssrNA, dsrNA 

intermediate, nascent ssrNA genomic rNA) in stimulating host iFN-α/β response in a course of natural infection. Our results demonstrate 

that the dsrNA replication intermediate is the key iFN-α/β agonist in infected cells. Furthermore, experiments performed in MEFs derived 

from knockout mice confirmed that the observed iFN-α/β response resulted from specific MDA5 activation and downstream signaling 




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HA08 Cont. 

pathway. in addition, we studied the iFN stimulatory ability of viral rNA (vrNA), which carries a viral protein (VPg) covalently linked to its 

5’ end, versus VPg-lacking rNA transcribed in vitro (ivt rNA). interestingly, while vrNA could induce MDA5-mediated iFN-α/β response, ivt 

rNA failed to do so. This indicates an additional role of VPg in activating MDA5, possibly as a signal for ‘non-self’ rNA. 

TLr structural biology and the discrimination of Gram+ and Gram– bacteria 

Nick Gay 

Dept of Biochemistry, University of Cambridge 

Elucidation of the component parts and mechanism whereby Toll-like receptors (Tlrs) signal has involved over 10 years of research by 

many investigators. This has led to a reasonably detailed description of the main signaling pathways activated by Tlrs. Studying this system 

continues to provide insight into the initiation and control of the innate immune response, and the role dysregulation of these proteins and 

processes plays in infectious and inflammatory diseases. 

An important theme to emerge from molecular studies in the last two years is that Toll pathways are dependent on initial stimulus 

induced dimerization of the receptors by microbial pathogen associated molecules such as lipids and non-self nucleic acids. in particular the 

mechanisms by which Gram –ve lPS and Gram +ve di- and tri-acylated lipoproteins activate the the Tlr4 and Tlr2/(1,6) pathway have 

been elucidated. initial activation leads to the formation of a highly oligomeric, membrane associated complexes in the cytosol that act as a 

scaffold for numerous signaling and regulatory molecules that act in these pathways. Most remarkable has been analysis of the Myddosome, 

a complex formed by the death domains of the MyD88 and irAK signaling adaptors. 

C-type lectin signaling in infection and immunity 

Teunis B.H. Geijtenbeek 

Host Defense group, Center for Experimental & Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands 

(Email: T.B.Geijtenbeek@amc.uva.nl) 

Adaptive immune responses by dendritic cells (DCs) are controlled by pathogen recognition and. C-type lectins are crucial in tailoring 

immune responses to pathogens. upon pathogen binding, C-type lectins trigger distinct innate signaling pathways that induce specific 

cytokines to dictate T cell polarization. recent data demonstrate that C-type lectin signaling seems to control NF-κB activation, either by 

enhancing or inactivating transcriptional activity of specific NF-κB subunits. Dissecting the signaling pathways induced by C-type lectins and 

their effects on host immunity is essential to understand the molecular mechanisms leading to adaptive immune responses. 

Here i will discuss the molecular signaling pathways induced by the C-type lectin dectin-1 that control T cell polarization and il-1β 

processing to induce pathogen-tailored immunity. T helper type 17 (Th-17) responses are required to combat fungal infections and the 

cytokines il-1β, il-6 and il-23 secreted by human DCs induce Th-17 polarization. Fungi binding to Dectin-1 induce raf-1 that integrates 

with the Syk signaling at the level of NF-κB activation, which leads to specific cytokine responses. i will discuss the consequences of the Syk/ 

raf-1 signaling pathways and demonstrate that both are essential to induce T 1- and T -17-polarizing cytokines. Furthermore, i will discuss 

H H 

the importance of dectin-1 signaling in the processing of il-1β, which is crucial to Th-17 responses as well as innate immune responses. 

Induction and inhibition of type I IFN responses by rNA viruses 

Adolfo García-Sastre1,2,3 1 2 3 Dept of Microbiology, Dept of Medicine, Division of Infectious Diseases, Global Health & Emerging Pathogens Institute, Mount 

Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029, USA 

The cytoplasmic antiviral sensor riG-i plays a key role in rNA virus recognition and initiation of the innate immune response. Activation of 

riG-i by immunostimulatory rNA leads to its association with mitochondrially localized adaptor MAVS and downstream signaling resulting 

in production of type i iFN and other proinflamatory cytokines. immunoprecipitation of riG-i/rNA complexes from virus infected cells 

allowed us to analyze rNA molecules which specifically interact with riG-i during the course of viral infection. unbiased deep sequencing 

analysis along with biochemical characterization of isolated rNA revealed specific viral sequences associated with riG-i in Sendai and 

influenza virus infected cells. However, in influenza virus infected cells, induction of iFN is prevented due to expression of the viral NS1 

protein. This multi-functional protein interacts with and inhibits different components of the cell machinery, including TriM25 and CPSF30, 

preventing an optimal expression of iFN in virus-infected cells. Other rNA viruses, like flaviviruses, are potent inhibitors of iFN signaling. in 

the case of dengue virus, this is in part mediated by the viral protein NS5, that binds to STAT2 and targets it to degradation. The interplay 

between induction and evasion of iFN by rNA viruses plays a major role in viral pathogenesis. 




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recognition of paramyxoviruses by pattern recognition receptors 

Steve Goodbourn 

Biomedical Sciences Research Centre, St George’s, University of London, London SW17 0RE 

Paramyxoviruses are conventionally considered good inducers of type i interferons (iFN-α/β). recent studies have focused on the 

importance of the cytoplasmic pattern recognition receptor riG-i as a sensor of infection, apparently by recognition of uncapped 

5´-triphosphorylated virus-specific rNA molecules. Whilst riG-i is undoubtedly important in inducing iFN production, mda-5 also senses 

paramyxovirus infection and is correspondingly activated. The nature of the virus PAMPs that activate riG-i and mda-5 remain unclear, 

but our data demonstrate that virus molecules generated by aberrant transcription and/or replication events leading to the production of 

defective interfering viruses are the most effective inducers. 

Viruses in general mount a major challenge to the iFN system, and paramyxoviruses are no exception. indeed, most paramyxoviruses 

encode mechanisms to inhibit both the production of, and response to, type i iFN. The V proteins of many paramyxoviruses limit the 

production of iFN-β by direct interaction with mda-5, and the mechanism of this inhibition will be discussed. Since paramyxoviruses also 

activate riG-i it is probably necessary for these viruses to inhibit riG-i or the downstream consequences of riG-i activation. Our studies 

indicate that paramyxoviruses utilise a diverse range of strategies to limit the extent of riG-i activation. 

HA09 Food biosecurity 

↑Contents 

british meat teetering on a knife edge – what’s the value of traditional meat inspection? 

ANNETTE NiGSCH1 , Bojan Blagojevic1,2 , Nikolaos Dadios1 , Silvia Alonso1 1 2 Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield Hertfordshire AL9 7TA; Dept of Veterinary Medicine, Faculty of 

Agriculture, University of Novi Sad, Serbia 

background: Meat inspection (Mi), comprising ante- and post-mortem inspection, represents a fundamental control point for the various 

hazards associated with the meat production chain. Whereas at the time when Mi was established (19th c.) animal diseases presented with 

macroscopic lesions in specific organs of the animal and were easily identifiable during carcase inspection, today new threats have become 

more prevalent. While some of these hazards may cause clinical disease on the animal host, many exist sub-clinically. 

A debate has started at Eu level: policy makers and stakeholders suggest the traditional Mi system at the slaughterhouse is not fit anymore 

to protect the public against these diseases and a radical overhaul and a new way of thinking is necessary. 

Project description: To evaluate the risk of missing hazards for public health, animal health and animal welfare associated with current Mi 

practices a qualitative risk assessment was undertaken. in a second step changes in risk resulting from variation in post-mortem activities 

were assessed driven by following questions: a.) how does the risk change if specific carcase parts / organs are no longer inspected; b.) 

which alternative steps are suitable to achieve equal levels of protection if the traditional Mi is altered. 

Epidemiology of CTX-M ESbL Escherichia coli on cattle farms 

N.G. COlDHAM1 , M. Stokes1 , H. Wearing1 , l.C. Snow1 , M. Wootton2 , r.A. Howe2 , C.J. Teale1 1 2 Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB; Specialist Antimicrobial Chemotherapy Unit, 

Public Health Wales, Microbiology Cardiff, University Hospital Wales, Health Park, Cardiff CF14 4XW 

Extended spectrum beta lactamase (ESBl) enzymes are capable of inactivating 3rd and 4th generation cephalosporin antibiotics which are 

commonly used in hospitals as front line treatments. A year long passive surveillance study of CTx-M E. coli in a particular study area of 

Wales detected CTx-M sequence types 14 (group 9, n=3) and 15 (group 1, n=7) in faeces from cattle and sheep. Geographical analysis 

revealed that the CTx-M gene from veterinary sources was widespread. Next, network analysis was used to investigate the dissemination 

of CTx-M-14 E. coli from a study farm from which the dairy animals had been sold. The prevalence of CTx-M E. coli in the population of 

farms linked by animal movements to this farm was 59% (not significant) compared with 37% in the controls. However, farms which had 

used 3rd /4th generation cephalosporin antibiotics were 4 times more likely to have CTx-M E. coli. The veterinary isolates from these studies 

were compared to human CTx-M-14 ESBl isolates (n=19) from Wales using a multiplex PCr. This revealed that some CTx-M-14 isolates 

from human (n=3 of 19) and veterinary (n=6 of 14) sources shared a plasmid with similar backbone genes. 




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HA09 Cont. 

reducing Campylobacter in UK-produced chicken 

Kathryn Callaghan 

Food Standards Agency 

One of the Food Standard Agency’s (FSA) main priorities is to reduce food-borne disease in the uK. This is reflected in the FSA’s Strategic 

Plan 2010–2015, which states that food-borne disease will be reduced using a targeted approach, and that tackling Campylobacter in 

chicken is a priority. Campylobacter is the most common cause of food poisoning in the uK. it is found mainly in poultry but also in red 

meat, unpasteurised milk and untreated water. An FSA survey of Campylobacter in chicken on retail sale in the uK (2007/2008), reported 

that Campylobacter was present in 65% of the fresh chicken samples tested. An Eu baseline survey (2008) showed the uK estimated 

Campylobacter prevalence in broilers (caecal contents) was 75% and on broiler carcasses (skin samples) 86%. 

The findings from these surveys show that there are Campylobacter related challenges in our food-safety system. in order to achieve our 

strategic aim to reduce food-borne disease we have developed a Campylobacter risk Management Programme, encompassing a range of 

Government/industry partnership led projects at different points across the food chain. This talk describes the programme of work and the 

target to reduce Campylobacter in uK produced chicken. 

What have we learned from Campylobacter phage intervention trials? 

i.F. Connerton 

School of Biological Sciences, Division of Food Sciences, University of Nottingham 

Controlling campylobacters in poultry represents one of the greatest challenges to the agriculture and food industries if they are to achieve 

consumer and governmental demands to reduce human food-borne disease. research into the potential of bacterial viruses to treat 

bacterial infection (phage therapy) has increased greatly in recent times largely due to the quest to find alternative, sustainable methods to 

replace antibiotic treatments which no longer perform due to the dramatic rise in multi-drug resistant bacteria. Control of Campylobacter 

is an obvious target for phage therapy because a large proportion of poultry reared for meat harbour these organisms as a part of their 

intestinal flora with few practical alternatives for reduction. However, simply introducing bacteriophages to chickens is unlikely to result in 

the elimination the target bacteria, since like most predators phage seldom eliminate their prey in nature. Treatments that do not eliminate 

but reduce the numbers of campylobacters below critical thresholds will benefit public health. Combining phage therapy with other 

strategies such as physical and hygiene-control measures could reduce the numbers of human campylobacteriosis cases. Our studies to 

investigate the sustainable use of bacteriophages have revealed some important aspects of Campylobacter ecology in response to phage 

predation. 

Threats and hazards: emerging risks 

Alan Funnell 

Food Standards Agency 

Early identification of threats to food safety is fundamental to the effective management and implementation of timely, proportionate and 

decisive preventative actions. By developing emerging risk methodology we intend to identify the weaknesses and potential threats with 

respect to food safety in the global food chain. 

This programme aims to provide a co-ordinated approach to the collation and analysis of intelligence of food safety threats. it will provide 

a clearer picture of the drivers for such threats (e.g. economically or politically motivated issues) and will be used to develop strategies and 

capabilities to deal with these threats. 

This presentation will outline the Food Standards Agency (FSA) approach to ‘emerging risks’ and introduce food defence as a concept. 

Deterring, detecting and defeating malicious bio-attack on food 

Steve Barras 

Centre for the Protection of National Infrastructure (CPNI) 

We are born with some risks (like Salmonella food poisoning) and surprised by some risks (like fall-out from the Chernobyl disaster) 

while other risks emerge before us (like melamine in milk from China). Most food risks are adventitious and arise from environmental or 

ecological factors, or simply from lack of care. However the uK suffers from a low level of malicious contamination of food by the ‘Bad’, the 

‘Mad’ and the ‘Sad’, and now has to consider the possibility of food supply being disrupted by politically motivated groups. 

This paper will introduce food defence as a concept and the role of Publicly Available Specification (PAS) 96 which introduces ‘Threat 

Assessment Critical Control Points’ (TACCP) in mitigating the risk. 




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HA09 Cont. 

Futures techniques for food biosecurity 

Fiona lickorish 

Defra, Area 1A, Nobel House, 17 Smith Square, London SW1P 3JR 

The talk will focus on how futures research can support the identification of key challenges and opportunities for food bio-security and how 

overall, this can be achieved via long-term trend analysis supported by a continuous horizon scanning process. The presentation will start 

with a general introduction to futures research – what does it do, who is using it, what are the key methods. it will cover horizon scanning, 

making some rough suggestions on what a topic specific scanning system could look like, how it could be implemented and what types 

of benefits such a system would have. Finally, it will describe some of the key megatrends and what impact they might have on food biosecurity 

and will then focus on 3–4 interesting trends and will explain that these types of outputs are; what futures research produces and 

cover some key conclusions about impacts to show the value of the process to participants. 

New environments – new pathogens? 

Marion Wooldridge 

Centre for Epidemiology & Risk Analysis, Veterinary Laboratories Agency (Weybridge) 

Our environment is changing – and not just climate, but human demographics, society and culture, global politics and economics, and 

technology. All may affects pathogens, directly or indirectly. Pathogens may spread into new niches created by change; or may be 

transported internationally, rapidly, by new patterns of trade and transport; or may mutate at an increased rate, adapting to new conditions 

or hosts. For example, increasing urbanization associated with economic issues may lead to farm management changes, and possibly 

reduction of skilled and knowledgeable labour. The result may be new niches for pathogens, with any adverse effects going unrecognised 

by staff less familiar with normal patterns. Another example might involve politics – differences of opinion may sometimes lead to civil wars 

resulting in migration, untended crops and animals, reduced water supplies and hygiene, all of which can effect pathogen growth, spread, 

transport and mutation, and thus food (bio)-security. And environmental changes rarely occur in isolation. Their interactions and subsequent 

effects on pathogens, and thus potentially on food bio-security, is complex and difficult to predict. This presentation will look at a variety of 

these complex pathways, and illustrate the issues with some already observed changes in food-associated pathogens. 

Implications of climate change for pathogen defence in plants 

ADriAN C. NEWTON, ian K. Toth, Nicola Holden 

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA (Email: adrian.newton@scri.ac.uk) 

Climate change will have direct effects on both crop growth and yield and on plant pathogens as well as on the interactions between crops 

and pathogens. Many single factor climate change studies have demonstrated many and sometimes conflicting outcomes, but interactions 

are often complex with many trade-offs. Furthermore, outcomes such as effects on the fecundity and variability of pathogen populations 

are seldom considered. Much more needs to be done to understand the likely behaviour of such complex systems under climate change. 

However, in parallel an understanding of the key parameters of the biology of stability in host-pathogen is important for building-in resilience 

to the consequences of climate change. Most important amongst these is functional diversity. 

Plant pathologists focus on diseases and their causal agents. However, plants are hosts to complex communities of microbes including 

beneficial and benign fungi and bacteria as well as pathogens of other crops and animals including humans. As pathogens, microbes are 

often opportunists, normally occupying other ecological niches. Even specialised pathogens sometimes have extensive non-symptomatic 

phases. Either direct effects of climate change or indirect cropping pattern changes through policy or market drivers may also change the 

ecological relationships of microbes reducing or increasing pathogenic opportunity. understanding these relationships should inform our 

crop production and integrated crop protection strategies, aiming for greater stability rather than pathogen ‘zero tolerance’. Food security 

considerations bring in additional socio-economic, geographical and political factors. Enhancing resilience to the effects of climate change is 

important for all these systems. in general, increasing functional diversity is one of the most effective targets for improved sustainability. 

A new bacterial threat to potato production: Dickeya solani. Controlling its introduction and spread through monitoring and 

a targeted response 

GErrY SADDlEr, Greig Cahill, Karen Kelly, Malgorzata Kowalewska, David Kenyon 

Head of Diagnostics & Analytical Services Division, Scottish Agricultural Science Agency, 1 Roddinglaw Road, Edinburgh EH12 9FJ 

in Northern Europe and israel during the last five years potato production losses attributable to a previously unknown member of the 

bacterial genus Dickeya have risen alarmingly. This new species, provisionally named ‘D. solani’, causes wilt, blackleg-like symptoms and tuber 




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rots and is responsible for losses of α30 Million annually in the Netherlands alone. Since 2006, a monitoring programme has been running to 

ensure that Scottish potato production remains free of this pathogen. This programme targets imported potatoes for immediate processing, 

potato seed, growing crops and water samples from sources of irrigation. results from these surveys confirm that the majority of imports, 

crops and watercourses used to irrigate potatoes are free of the pathogen, but that a small number of imports, crops and one irrigation 

source are infected/infested with ‘D. solani’. results from recent MlST studies into strain diversity will be presented as will the growing 

body of data on disease epidemiology. How these latter data sets have shaped legislation to halt the further introduction and spread of this 

pathogen will also be discussed. 

Offered paper Environmental transmission of Escherichia coli in a cereal trial 

NiCOlA HOlDEN, Jacub Firowicz-Krosko, Frank Wright, Susan Mitchell, Tim Daniell, ron Wheatly 

Scottish Crop Research Institute, Dundee 

Escherichia coli comprise a large, diverse species commonly found in the intestinal tracts of mammals. However, a number of E. coli are able 

to adapt to alternative environments; some can colonize non non-mammalian host species, while others have become ‘naturalised’, that 

is they are adapted to persist in environmental habitats such as water and soil. recent work has also shown that food-borne pathogenic 

E. coli can colonize plant hosts and present a health risk in fresh fruit and vegetables. The aim of this study was to determine whether E. 

coli present in soil amendments applied to a barley trial were transmitted into growing crops. MlST of the E. coli isolates showed discrete 

populations grouped largely by their source: slurry, compost, soil, plant-associated. isolates with closely related STs from slurry or from 

compost were present in bulk soil sampled several months after the amendments were applied. Barley grain and barley rhizosphere isolates 

formed a distinct and separate group. Phylogenetic analysis showed the present of recombination in the population. Two of the bulk soil 

isolates and two of the barley grain isolates share ST with pathogenic isolates, suggesting that pathogenic bacteria persist in the soil and are 

present on harvested crops. 

Offered paper Dynamics of herd infection: multiple enteric pathogens in pigs 

HElEN DAViES, laura Hancox, Christine Dodd, Sabine Tötemeyer, Julian Wiseman, Kenneth Mellits 

University of Nottingham, Loughborough, Leicestershire 

incidence of enteric disease is highest in young pigs, particularly during the post-weaning period due to environmental and nutritional 

stressors. infections with various enteric pathogens can occur simultaneously; the presence of a secondary pathogen can cause extensive gut 

damage. Although checks have been established to control zoonotic pathogens such as Salmonella and Enteroxigenic E.coli (ETEC) on an 

individual basis, co-infections may increase susceptibility therefore control of one pathogen may aid control of another. 

Excretion of Salmonella, ETEC and rotavirus were tracked in pre and post weaning pigs from a commercial herd. Both semi-quantitative 

classical enrichment methods and a new rapid, sensitive PCr were employed to detect the bacterial pathogens. rotavirus was detected 

using an immunoCard STAT rotavirus kit. 

rotavirus, Salmonella and ETEC were all detected in sequential excretions in the pig herd. rotavirus was most predominant at weaning, 

preceding bacterial infection, which then occurred post-weaning. The predominant bacterial pathogen was Salmonella Typhurimum u288; 

however, additional Salmonella and ETEC strains were present. PCr detection enabled a rapid but accurate approach to testing for 

Salmonella and ETEC in pigs.Knowledge of co-infection dynamics will enable enhanced understanding and control of pathogens in pig herds, 

and potential transmission to the human food chain. 

HA10 Insect symbiosis 

↑Contents 

Genome reduction of symbiotic micro-organisms: how small can they get? 

John McCutcheon 

University of Montana – Missoula, USA 

Bacterial genomes vary in size over two orders of magnitude. The 580 kilobase Mycoplasma genitalium genome has historically defined 

the extreme small end of this spectrum, and has therefore heavily informed theoretical and experimental work aimed at determining the 

minimal gene content necessary to support cellular life. recent genomic data from insect symbionts have revealed bacterial genomes that 

are incredibly small—two to four times smaller than M. genitalium—and these tiny genomes have raised questions about the limits of 

genome reduction and have blurred the once-clear distinction between autonomous cellular life and highly integrated organelle. New data 




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HA10 Cont. 

from various systems with symbiotic bacterial or archaeal partners have begun to shed light on how these bacteria may function with such 

small gene sets, but major mechanistic questions remain. Comparative genomic data for the five smallest cellular genomes will be presented, 

along with a discussion of the minimal gene content necessary to survive as a symbiotic bacterium. 

Novel biological functions conferred by insect endosymbionts 

Takema Fukatsu 

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan (Email: t-fukatsu@aist.go.jp; 

website http://staff.aist.go.jp/t-fukatsu/English%20Top.html) 

in nature, organisms are living in close association with surrounding physical environment as well as other organisms. Thus, each of the 

organisms is regarded as a component of the ecosystem. Considering the diverse microbial community found inside the organisms in 

general, each of the organisms can also be regarded as constituting an ecosystem. Many animals and other organisms constantly harbor 

micro-organisms inside their body, which has been referred to as ‘endosymbiosis’. Due to the close spatial proximity, extremely intimate 

biological interactions and inter-dependency are commonly found between the partners called host and symbiont. Novel biological 

properties are often generated through such associations. in many cases, host and symbiont are integrated into an almost inseparable entity. 

Here we present a series of our works on the evolutionary origins and biological functions of insect endosymbionts. 

recombination and gene transfer in Wolbachia infecting Drosophila 

Siv Andersson 

University of Uppsala, Norbyvägen 18 C S-752 36 Uppsala, Sweden 


Metabolic coevolution between symbiotic micro-organisms and their hosts 

Angela E. Douglas 

Sarkaria Institute of Insect Physiology & Toxicology, Dept of Entomology, Cornell University, Ithaca, NY, USA 

intracellular symbiotic bacteria with a long evolutionary history of obligate vertical transmission tend to have much reduced genomes, as 

a consequence of relaxed selection and genomic deterioration. Genes lost commonly include those coding for metabolic pathways also 

present in the host. The sequenced genomes of the intracellular symbiont Buchnera and its aphid host reveals additionally that Buchnera 

has incomplete metabolic pathways for the synthesis of key nutrients transferred to the host, and that metabolism genes of the host can 

potentially compensate for these deficiencies, resulting in coupled metabolic pathways between the partners. This condition is evident for 

five of the ten essential amino acids that Buchnera provides to the host, supplementing the low essential amino acid content of the aphid 

diet of plant phloem sap. Coupled metabolic pathways between host and symbiont, as inferred from analysis of the annotated gene content 

of the partners, is supported by evidence from metabolic modeling, quantitative analysis of the proteomes of Buchnera and the aphid host 

cells, and studies on metabolite flux between the partners. 

Understanding whole system immunity: the coupling of host defences and symbiont-conferred protection in the pea aphid 

immune response 

Nicole Gerado 

O. Wayne Rollins Research Center, Emory University, 1510 Clifton Road NE, Room 2006, Mail Stop #1940-001-1AC, Atlanta, GA 

30322, USA 

Disease in animals is mitigated both through immune-system based defences and through non-immunological mechanisms of resistance and 

tolerance. These non-immunological mechanisms include behaviors to avoid infection, increasing reproduction prior to death, and reliance 

on symbiotic microbes that can provide protection and disease threats. The reliance of pea aphids on bacterial symbionts that provide 

protection, and the availability of new genomic tools to study the immune system of these insects, provides an opportunity to allow us 

to understand how immune-system based and non-immunological defences may be paired to define the total immune capacity of these 

host insects. Here, i will briefly review our current understanding of immunity in these hosts, focusing on responses to fungal and bacterial 

pathogens. i will also explore the possibility that symbiont-conferred protection may have shaped the evolution of the total aphid immune 

system. 




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HA10 Cont. 

Coevolution between microbes and insect behaviour – defensive symbionts in beewolf antennae 

Martin Kaltenpoth 

Max Planck Institute for Chemical Ecology, Research Group Insect Symbiosis, Hans-Knoell-Str. 8, 07745 Jena, Germany 

Microbial symbionts represent a major source for evolutionary innovation in insects. Although nutritional symbioses are the most common 

types of interactions, mutualistic microbes can fulfil a variety of other functions, notably the protection against pathogenic micro-organisms. 

Beewolves of the genera Philanthus, Trachypus, and Philanthinus (Hymenoptera, Crabronidae) cultivate defensive Streptomyces bacteria in 

specialized antennal glands. The bacteria are transmitted vertically by an unusual route that involves specialized adaptations in the beewolf’s 

behaviour: After secretion from the mother’s antennae into the brood cell, the larva detects and acquires the bacteria from the brood 

cell environment and applies them to the cocoon silk, from where they are later taken up by the adult beewolf into the antennae shortly 

before emergence. On the cocoon, the symbionts provide protection against a wide range of potentially detrimental fungi and bacteria by 

producing a cocktail of at least nine different antibiotic substances. in addition to this protective function, the antennal secretion containing 

the symbionts confers directional information to the larva that is crucial for successful cocoon-spinning and adult emergence. Thus, 

beewolves constitute a fascinating model system in which the dual function of the microbial symbionts is tightly intertwined with the host’s 

ecology and behaviour. 

Fungal partners in the diversification of gall midges 

Patrick Abbot 

Dept of Biological Sciences, Vanderbilt University, 7280A MRB III, 465 21st Ave South, USA 

Symbiosis with prokaryotic and eukaryotic micro-organisms is a common feature of life for insects, and recent studies have emphasized the 

degree to which microbial symbionts can act as sources of adaptive phenotypic complexity for their hosts. However, despite the ubiquity 

of symbiosis, only a handful of organisms have been studied in depth, meaning that our understanding of how co-evolutionary interactions 

between host and symbiont contribute to phenotypic complexity remains incomplete. i describe co-evolutionary patterns between the 

ectosymbiotic fungal symbiont and the goldenrod galling midge, Asteromyia carbonifera. Phylogenetic reconstruction placed the fungal 

symbiont within the same clade as Botryosphaeria dothidea, a typically free-living, generalist plant pathogen and endophyte. Symbiont isolates 

from four divergent midge lineages and several geographic locations demonstrated a striking phylogenetic discord between the two, despite 

evidence indicating that the interaction is an obligate one for the midge. However, new genomic data is revealing the extraordinary degree 

to which a history of interaction with fungi has shaped the midge genome, ultimately acting as a source of evolutionary innovation and 

ecological opportunity. 

Offered paper Leafcutter ant symbionts and their antifungal compounds 

rYAN SEiPKE1 , Jörg Barke1 , Sabine Grüschow1 , rebecca Goss1 , Douglas Yu1,2 , Matthew Hutchings1 1 2 University of East Anglia, Norwich; Kunming Institute of Zoology, Kunming, China 

leafcutter ants live in a tripartite symbiosis with the fungus Leucoagaricus gongylophorus, which they farm for food, and antifungal-producing 

actinobacteria in genus Pseudonocardia that have been proposed to be the sole, co-evolved, mutualists of leafcutter ants. Next generation 

sequencing phylogenetic studies revealed that many other genera of actinobacteria are present on the ant cuticle, raising the possibility 

that Pseudonocardia may not be the only antifungal-producing mutualist. in order to address this possibility we isolated actinomycetes from 

the leafcutter ant species Acromyrmex octospinosus and obtained two Pseudonocardia spp. (P1-P2) and nine Streptomyces spp. (S1-S9). 

Antifungal bioassays revealed that isolates P1, S3, S4, S5 and S9 inhibited the growth of the garden parasite, Escovopsis weberi. We further 

characterized P1 and S4 using a genomics-guided chemistry approach. This approach demonstrated that P1 produces a novel polyene 

antifungal related to nystatin, and that S4 produces the antifungal candicidin and also identified a novel antifungal biosynthetic cluster whose 

product remains unknown. These data support an environmental acquisition model in which A. octospinosus recruits useful actinomycetes 

from the soil to protect the fungal garden, and furthermore suggest that the environmental acquisition and co-evolution models are not 

necessarily mutually exclusive and may be occurring simultaneously. 

Offered paper Modelling metabolite flux between the pea aphid and its endosymbiont Buchnera aphidicola APS 

SANDY MACDONALD1 , Gavin Thomas1 , Angela Douglas1,2 1 2 Dept of Biology, University of York, York, North Yorkshire; Dept of Entomology, Comstock Hall, Cornell University, Ithaca, NY, USA 

The pea aphid and its bacterial endosymbiont Buchnera aphidicola APS form an obligate symbiosis, with the bacterium synthesizing essential 

amino acids (EAAs) lacking in the phloem sap diet of the aphid. A recent genome scale metabolic model of Buchnera reinforced the role 




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of Buchnera in synthesizing EAAs for the aphid, and suggested the possibility that the profile of EAAs synthesised by the Buchnera could be 

altered by differing supply of carbon and nitrogen sources to the Buchnera from the aphid. Here, we used this metabolic model, in tandem 

with 454 pyrosequencing, to investigate the basis for observed variation in the dietary requirement for EAAs among four pea aphid clones. 

First, we sequenced the Buchnera from the four pea aphid clones, and showed that the differing dietary EAA phenotypes of the aphids 

could not be attributed to sequence variation in the Buchnera. Second, using flux balance analysis of the Buchnera metabolic model, we 

showed that the four profiles of EAAs could be reproduced by supplying distinctly different profiles of carbon and nitrogen sources to the 

Buchnera, suggesting that the differing nutritional phenotypes seen are governed by aphid metabolism and not Buchnera genotype. 

The microbiota of Drosophila as a model system for insect interactions with symbiotic micro-organisms 

Nichole A. Broderick 

Global Health institute, Ecole Polytechnique Federale de lausanne (EPFl), Switzerland 

Eukaryotes and microbes have co-existed and co-evolved for billons of years. The importance of this association is particularly evident 

when considering the contributions of gut microbes to animal health and physiology. While the importance of gut microbes in vertebrate 

health is widely appreciated, the complex diversity of these communities complicates their decipherment. in contrast, the relative simplicity 

of invertebrate gut microbial communities, especially in insects, provides a simpler model. Here i will describe how the genetically tractable 

model Drosophila melanogaster provides a useful system to elucidate mechanisms underlying the complex relationships between a host and 

its microbiota. The gut of Drosophila parallels that of other animals and the microbiota of lab stocks is simple in composition and diversity. 

However, this simple consortium has important contributions to the host, as flies lacking their microbiota are altered in their basal immune 

status and pathways controlling gut homeostasis. This basal response is in turn an important regulator of the abundance, diversity, and 

spatial localization of microbiota. Furthermore, gut microbiota of Drosophila can influence susceptibility to infection through interactions with 

host immune and stress responses. Thus, Drosophila provides a useful model to decipher mechanisms by which gut microbiota impact gut 

physiology and homeostasis. 

benefit and dependence in insect–microbial symbioses 

Fabrice Vavre 

Laboratory of Biometry & Evolutionary Biology, University Lyon I, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, 

France (Email: Fabrice.vavre@univ-lyon1.fr) 

Numerous insects interact with vertically transmitted symbionts that exhibit very diverse effects. Many of these symbionts are mutualists 

and bring important advantages to their hosts, notably through the complementation of their diet. Others manipulate the reproduction 

of their host to their own advantage. Such manipulations include feminization of genetic males, male-killing, induction of parthenogenesis 

or cytoplasmic incompatibility. These two types of symbiosis have classically been studied separately, but recent advances show that this 

dichotomy is not as pertinent as previously thought. First, it is becoming clear that reproductive manipulators can also provide advantages to 

their hosts. Second, some examples show that the dependence of the host upon its symbiont may rapidly evolve even when the symbiont 

does not bring benefit to its host. These recent developments in the field of insect symbiosis provide interesting new scenarios for the 

evolution of obligatory host-symbiont relationships. 

HA11 Working with the media 

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Science in the media 

Julia Wilson 

Sense About Science 

Sense About Science is a small charity that equips people to make sense of science and evidence. This session will discuss science related 

controversies in media reporting and what scientists can do to respond to misconceptions in public discussions. Do you think it is important 

for good science and evidence to be communicated to a wider audience? What happens when research announcements go wrong; 

statistics are manipulated; risk factors are distorted; or discussions become polarised? Not yet the leaders in the field what can early career 

researchers do to encourage good science and evidence in the public domain? using examples of myth-busting and evidence hunting 

projects of the Voice of Young Science network of early career researchers, Julia will discuss the impact and importance of standing up for 

science. 




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HA11 Cont. 

Getting science into the media: the role of press officers 

laura udakis 

Society for General Microbiology, Marlborough House, Basingstoke Road, Spencers Wood, Reading RG7 1AG 

Press officers are usually the people who initiate science stories that appear in the media. Spotting newsworthy research that might interest 

the general public is often not straightforward and demands a high level of integrity. As well as pitching science to journalists through media 

releases, press officers act as the facilitator between scientists and journalists, helping each group understand the needs, wants and concerns 

of the other. This session will explain the responsibilities of press officers, how they can support scientists working with media when a story 

takes off and the advantages to scientists in maintaining a good relationship with their press office. 

Getting science into the media: the role of journalists 

richard Gray 

Science Correspondent, The Sunday Telegraph 

Arguably there has never been a better time for science in the media – newspapers, magazines, TV news and radio are packed with daily 

reports on the latest findings from some truly fascinating research. Even radio 4’s Today programme manages regular blusterings on the latest 

scientific endeavours. Coverage of scientific research by the media, however, can often seem baffling, and even frustrating, for scientists. Why 

can’t science journalists get all the facts right? Why do they need to speak to me right now? Why do they have to seek alternative views? 

Why is the headline so misleading? And why do they only cover the irreverent? Hopefully this talk will give some insight into the murky 

mindset of the science journalist and impenetrable world of their editors. it will reveal what makes a good science news story, what journalists 

are looking for when they speak to scientists and provide some advice on how to get your own research into the media. 

Working with journalists 

Gerard Fleming 

National University of Ireland, Galway 

This presentation will give a personal perspective on dealing with the media as a research-active microbiologist. This is based on the author’s 

experience of scientific findings being selected for public dissimilation by the publisher’s press office. it will deal with the process of liaising 

with the press officer in formulating the press release. it will relate the author’s experience in working with print and broadcast journalists post 

release. The talk will explore concerns, pitfalls and opportunities and offer practical advice with regard to engaging with the media. 

The role of the Science Media Centre 

Jonathan Webb 

Science Media Centre 

The Science Media Centre is first and foremost a press office for science when science hits the headlines. We provide journalists with 

what they need in the form and time-frame they need it when science is in the news – whether this be accurate information, a scientist 

to interview or a feature article. in between these big stories, we are busy building up our database of contacts on the areas of science 

most likely to feature in the news. This allows us to be pro-active and puts us in a position to facilitate more scientists to engage with the 

media when their subjects hit the headlines. We also run a series of longer term activities to improve the interaction between science and 

media, such as advice guides for scientists talking to the media, background briefings for journalists and ‘Science in a Nutshell’ cheat sheets 

for newsdesks. Our aim is to ensure that when a major science story breaks, we can quickly offer news desks a list of scientists available to 

comment, a summary of the main scientific points involved and details of which press officers or web sites to go to for further information. 

Feedback from journalists has been very positive. 

The impact of ‘new media’ 

Toby Shannon 

British Science Association 

The session will explore the use of ‘new media’ techniques by researchers, such as blogging and social networking, to engage the public 

with science. Over the course of 15–20 minutes, the session will cover case studies of successful and engaging use of social media to 

communicate science and address some practical issues surrounding the use of social media. 

The session is targeted at researchers who may have had no experience of social media as a public engagement tool but also to researchers 

with some experience that wish to discuss other issues, possibly by asking questions via Twitter during the session. 




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HA12 Virology workshop: pathogenesis 

↑Contents 

Offered paper Encephalitis due to virus invasion of alien ticks? 

Tamara Gritsun 

University of Reading, Reading 

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers 

to fatal encephalitis but the factors that determine disease severity are not fully defined. Here we propose that the emergence of atypical 

antigenically-deficient variants of TBEV in Europe is linked to their adaptation to ticks previously ‘alien’ to their habitat. This process appears 

to be driven by the selection of variants with single mutations that render the virion surface more hydrophobic and positively charged thus 

enhancing the efficiency of TBEV entry into alien tick species. These mutations also appear to enhance the ability of TBEV to cross the human 

blood-brain barrier, thus offering an explanation for the increased incidence of fatal encephalitis. Future research will reveal if these emerging 

TBEV strains continue to invade Europe by adapting to the local tick species and if they are associated with severe forms of this disease. 

Offered paper The host response to a neuroinvasive West Nile virus infection. 

BEN HAxTON1,2 , Karen Mansfield1 , Alejandro Nunez1 , Daniel Hicks1 , Nicholas Johnson1 , Anthony r. Fooks1 1 2 Veterinary Laboratories Agency – Weybridge, Woodham Lane, Addlestone, Surrey KT15 3NB; King’s College London, Division of 

Immunology, Infection & Inflammatory Diseases, Dept of Immunobiology, 2nd Floor Borough Wing Guy’s Hospital, London SE1 9RT 

The mosquito-borne Flavivirus, West Nile virus is a positive sense single-stranded rNA virus, where human infection has occasionally been 

associated with neurological disease. The aim of this study was to develop a murine model of neuroinvasive West Nile Virus. infection 

by the intranasal (iN) route was highly effective in inducing immunopathology within the central nervous system. increased transcriptional 

activity of the cytokines iFN-γ and TNF-α; and the chemokines CCl5 and CxCl10 were measured by quantitative PCr. 

immunohistochemistry demonstrated chemokine expression in the brain parenchyma. Based on cell morphology, it appears that neurons 

predominantly expressed CxCl10, whilst astromicroglial cells expressed CCl5. in addition, the integrity of the blood brain barrier (BBB) 

was assessed. The adhesion molecule, iCAM-1, was increased suggesting modulation of the BBB, whilst detection of murine immunoglobulin 

G in the brain indicated that the BBB integrity was reduced in response to WNV infection. Cellular infiltrates associated with perivascular 

cuffs were observed and CD8 specific transcripts were found to be significantly increased in brain tissues following iN infection. Further 

cellular phenotying is required to identify the cells responding to infection and cellular damage. 

Offered paper Analysis of entry by a Gammaherpesvirus in vivo 

riCArDO MilHO, Philip Stevenson 

Division of Virology, Dept of Pathology, University of Cambridge, Cambridge 

Gammaherpesviruses infect >95% of humans and can cause a variety of cancers. Two human gammaherpesviruses are known – Epstein- 

Barr virus (EBV) and the Kaposi’s Sarcoma-associated Herpesvirus (KSHV). Once a gammaherpesvirus establishes infection, it persists for 

life. An efficient control of infection requires a deep understanding of host entry, but this remains an ill-defined aspect of Gammaherpesvirus 

biology. Here we use Murid Herpesvirus-4 (MuHV-4), a gammaherpesvirus of mice closely related to KSHV, to investigate how a 

gammaherpesvirus first enters its host. We have previously identified the nose as a likely entry point, based on luciferase imaging of MuHV- 

4-exposed mice. We first aimed to identify the primary cell target in the nose. A histological analysis revealed that MuHV-4 specifically 

infects sustentacular cells in the olfactory neuroepithelium. We then addressed whether an interaction with heparan sulfate (HS) is 

important for nose infection, since MuHV-4 heavily relies on HS for binding in vitro. interestingly, we found that both sustentacular cells and 

olfactory neurons express Syndecans (HS-bearing proteoglycans). Furthermore, scanning electron microscopy showed how MuHV-4 must 

penetrate through a dense layer of neuronal processes before reaching sustentacular cells. Our work sheds a new light at the obstacles 

faced by gammaherpesviruses during entry in vivo. 

Offered paper The influence of the herpes simplex virus type 1 (HSV-1) latency-associated transcripts on latency 

establishment in the rOSA26r reporter mouse model 

MiCHAEl NiCOll, João Proença, Viv Connor, Stacey Efstathiou 


During HSV-1 latency, virus transcription is restricted to the latency-associated transcripts (lATs). These transcripts possess anti-apoptotic 

functions and encode mirNAs that down-regulate virus immediate early gene expression. Despite intensive study, the precise in vivo role 

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HA12 Cont. 

of lATs remains controversial. Studies of lAT-deficient mutants in animal models have shown that they are not essential for any aspect of 

latency. However, whilst there is general agreement that such mutants reactivate with reduced efficiency, it is unclear whether the deficit is 

at the level of latency establishment or reactivation. 

Our research has focused upon investigating the efficiency of latency establishment of lAT-positive and lAT-negative viruses encoding Cre 

recombinase, using rOSA26r reporter mice. This model system facilitates the permanent marking of latently infected neurones permitting 

accurate assessments of latent viral loads. 

using this system we have observed that, in comparison to lAT-positive virus, infection of mice with lAT-negative mutants results in 

increased numbers of latently infected neurones within mouse trigeminal ganglia. Furthermore, we observe that this balance can be shifted 

to favour enhanced latency establishment of lAT-positive virus when the inoculum dose is greatly increased. The implication of these 

findings to proposed functions of lATs in the regulation of HSV-1 latency will be discussed. 

Offered paper Human cytomegalovirus modulates the cellular immune response by altering the microenvironment of latency 

GAViN MASON, Emma Poole, John Sinclair, Mark Wills 

University of Cambridge, Dept of Medicine, Cambridge, Cambridgeshire 

Primary human cytomegalovirus (HCMV) infection, even in the presence of a functional immune response, is not cleared. The virus 

establishes latency in cells of the myeloid lineage, lasting for the life of the host. latent infection of CD34+ myeloid progenitor cells, 

an established model of HCMV latency, results in an altered profile of secreted cellular proteins (the secretome). Our data show that 

this latency induced secretome attracts a subset of CD4+ T cells bearing the chemokine receptors CCr3 and CCr5. We have also 

determined that this recruitment is dependent on the cytokine CCl8, a known ligand for CCr3 and CCr5. Furthermore, the latency 

induced secretome specifically inhibits CD4+ T cell mediated cytokine production and cytotoxicity, effector functions associated with 

Th1 cells. using neutralization assays we show that this reduced Th1 cytokine production occurs due to the increased levels of il-10 and 

TGF-β present in the latency associated secretome. Taken together our data suggest a possible strategy, whereby cellular chemokines and 

cytokines induced by latent virus mediate avoidance of immune recognition and/or clearance of the virus during latency. 

Offered paper Epstein–barr virus (EbV)-associated inflammation is contributory to its oncogenic pathology: a multistage in 

vivo model of carcinogenesis 

ASiF QurESHi, Joanna Wilson 

University of Glasgow, Glasgow 

it has long been postulated that chronic inflammation is tumourigenic but the mechanisms involved are poorly understood. EBV associated 

cancers, including Nasopharyngeal carcinoma, are heavily infiltrated with leukocytes, which could contribute to tumourigenesis. To 

explore the role of the lMP1 oncogene of EBV in inflammation, we used a transgenic mouse model of multistage carcinogenesis where 

lMP1CAO is expressed in the epidermis. The skin of these mice undergoes progressive inflammatory pathology prior to neoplasia and is 

infiltrated with CD4 + -T-cells, CD8 + /GranzymeB + -T-cells, CD4 + /CD25 + /FoxP3 + -T -cells, mast cells and neutrophils. In vivo blockade of 

reg 

leukocyte recruitment resulted in delayed progression of the pathology. Furthermore, the critical role of mature B and/or T-lymphocytes 

in the advancing pathology was evidenced by their genetic elimination (lMP1/rAG-null mice), which limited the pathology to an early 

benign stage. STAT3, a key mediator of inflammation, is activated in the transgenic tissue along with up-regulation of several inflammatory 

proteins including TGF-ß1, CD30, CD30l, CD40, l-Selectin, il-3, il-1β, and s100A9. lMP1/D6-null mice show accelerated inflammation 

and carcinogenesis, suggesting a role of chemokines in lMP1 induced cancer predisposition. The preneoplastic skin also shows dermal 

deposition of immunoglobulin and increased levels of C-3. Studying this multistage model of carcinogenesis may provide new insights for 

cancer therapies. 

Offered paper Genome-scale ordered rNA structure and rNAi evasion in plant viruses 

CrYSTAl HOrNSEY1 , Eugene ryabov1 , Peter Simmonds2 , David Evans1 1 2 University of Warwick, West Midlands; University of Edingburgh, Edinburgh 

recently, a new type of sequence order-dependant rNA structure termed Genome-scale Ordered rNA Structure (GOrS) has 

been described. in animal viruses the presence of GOrS correlates with the ability to establish a persistent infection in the natural, 

immunocompetent host and so may contribute to the evasion of innate immune responses. GOrS is also readily detected in many plant 

viruses. Since plants use rNAi to combat viral pathogens and it is known that rNA structure affects the efficiency of the sirNA responses, 

we speculate that the extensive structure found within GOrS-containing viruses may contribute to viral pathogenesis. We set out to test 




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this using the highly structured, Potato leaf roll virus as a model system. Variant genomes have been generated where an extensive region 

has been engineered to have different levels of bioinformatically determined structure. Virus recovery of these variant genomes indicates 

that GOrS is not obligatory for replication. However, a luciferase-based reporter gene system demonstrates that GOrS containing 

sequences are less effective at inducing rNAi-mediated responses. Current studies involve mapping differences in the structure of the 

variant genomes and determining whether GOrS sequences are also poor targets for rNAi responses. 

Offered paper Progression of liver pathology in acute GbV-b infection 

Jessica Dale1 , Simon Hood1 , James Greenhow1 , Helen Bright2 , Neil Berry1 , Peter Karayiannis3 , NiCOlA rOSE1 1 2 3 HPA-NIBSC, Potters Bar, Hertfordshire; Pfizer Research and Development, Sandwich, Kent; Imperial College London 

The infection of tamarins with the hepatotropic GB virus, B (GBV-B) is an accepted surrogate animal model of HCV infection in humans. 

This model can be exploited in assessing immunotherapeutic strategies against HCV, particularly if they can be seen to reduce viral load and 

minimise or reverse damage to affected organs. A better understanding of virus-induced liver pathology will allow us to effectively evaluate 

the impact of anti-viral intervention. 

We have previously observed liver damage even when virus can no longer be detected in the periphery, however, little is known about 

pathology earlier in infection. To this end we infected tamarins with GBV-B and using histology and immunohistochemical analyses we 

examined liver sections obtained from animals at various time-points post infection Minimal changes are observed 2 weeks post-infection 

(wpi) compared to tissue from uninfected animals. By 7 wpi, however, hepatocyte organization is disrupted, there is an influx of B and 

T(CD4+) cells and there is evidence of increased cell activation and iFNγ production. We are currently studying samples taken at 24 wpi at 

which point virus is no longer detectable in the periphery. These data add to the scant knowledge of GBV-B-associated changes in the liver. 

Offered paper Comparative tissue pathology following either a short or long induction of the doxycycline-dependent 

SIVrtTA. 

DEBBiE FErGuSON1 , Simon Hood1 , Neil Berry1 , Mark Page1 , richard Stebbings1 , Martin Cranage2 , Atze Datz3 , Ben Berkhout3 , 

Neil Almond1 1 2 3 HPA-NIBSC, Potters Bar, Herts; St George’s Centre for Infection & Immunity, University of London, London; AMC University of 

Amsterdam, Amsterdam, The Netherlands 

The mechanisms associated with the potent protection conferred by live Attenuated SiV against wild-type SiV remain elusive. We used the 

doxycycline-dependent conditional live attenuated SiVrtTA derived from SiVmac239Δnef to investigate the role of vaccine persistence and 

local tissue responses in this protection. Groups of cynomolgus macaques were vaccinated with SiVrtTA in the presence of doxycycline for 

either 20 or 3 weeks prior to heterologous challenge with SiVsmE660. A further group was vaccinated in the presence of doxycycline for 3 

weeks followed by a 17 week doxycycline free period prior to challenge. 

immunohistochemical analyses of tissues collected at termination 20 weeks following SiVsmE660 challenge are currently underway. Within 

the spleen consistent group results for S100 and CD20 staining were observed following the 20 week window between vaccination and 

challenge irrespective of doxycycline removal. CD68, Fas and MHCii staining was variable within the long vaccination groups with consistent 

group results being found following 3 weeks vaccination. CD40 staining was consistent where the continued presence of doxycycline 

maintained SiVrtTA replication. Fasl and plasma cell staining was consistent across all groups. 

Analyses of GAlT are underway including location of virus infected cells, innate responses, cellular trafficking and apoptosis. 

Offered paper Dissecting the expression mechanism of Pb1, Pb1-F2, and Pb1-N40 and their contributions to influenza A 

virus pathogenicity 

BrETT JAGGEr1,2 , Helen Wise1 , John Kash2 , Jeffery Taubenberger2 , Paul Digard1 1 2 University of Cambridge, Dept of Pathology, Division of Virology, Cambridge; Viral Pathogenesis and Evolution Section, Laboratory of 

Infectious Diseases, NIAID, NIH, Bethesda, MD, USA 

Genome segment 2 of the influenza A virus (iAV) usually encodes three polypeptides from a single, unspliced mrNA: the PB1 viral 

polymerase, the pro-apoptotic PB1-F2, and PB1-N40, whose function is unknown. While these proteins have accepted and emerging 

functions in viral replication and pathogenesis, the mechanisms of their translation are incompletely understood; however, leaky ribosomal 

scanning is likely to play a key role. using diverse virus backgrounds, we have generated mutant viruses with altered expression levels 

of PB1-F2 and/or PB1-N40 and are characterizing their replication and pathogenicity in cell culture and mice. importantly, the strategy 

of mutating AuG4 to eliminate PB1-F2 expression utilized in previous studies resulted in a marked overexpression of PB1-N40 in all 




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virus backgrounds tested. Furthermore, while mutations shown to individually cause the loss of either PB1-F2 or PB1-N40 on the Pr8 

background had no or minimal effects respectively on virus replication and pathogenicity, loss of PB1-F2 in conjunction with PB1-N40 

overexpression decreased pathogenicity and replication in mice. The simplest interpretation of these results is that overexpression of 

PB1-N40 is deleterious to virulence. Our findings have important implications for the design of mutants aimed at understanding the 

contributions of segment 2 gene products to iAV pathogenicity. 

HA12 Cont. 

Offered paper Vaccinia virus protein C6 modulates innate immunity and contributes to virulence 

rEBECCA SuMNEr1 , Daniel Mansur1 , leonie unterholzner2 , Andrew Bowie2 , Geoffrey Smith1 1 2 Imperial College London; Trinity College Dublin, Dublin, Ireland 

The innate immune system provides the first line of defence against viruses and its activation results in the production of interferons (iFNs), 

proinflammatory cytokines and chemokines that combat the infection and activate adaptive immunity. During their evolution viruses have 

evolved mechanisms to counteract these defences, and as such can be useful tools to study and better understand the immune system. 

Vaccinia virus (VACV), a large DNA virus used as the vaccine to eradicate smallpox, expresses many proteins that modulate the host 

response to its benefit. Protein C6 is one such example. C6 is predicted to be a member of the family of nine VACV Bcl-2 proteins, and 

was shown to be a small intracellular protein expressed early during infection that inhibits iFN-β production by targeting the irF3, but not 

NF-κB, innate immune signalling pathway. The level at which C6 blocks this pathway was determined and TANK, an important adaptor 

molecule for iFN-β activation, was identified as a binding partner. Deletion of C6 from VACV had no effect on replication and spread 

in vitro, but significantly attenuated the virus in two murine models of infection. These results demonstrate C6 is a virulence factor that 

contributes to virulence by modulating innate immunity. 

Offered paper Isolation and direct whole-genome sequencing of varicella-zoster virus from a wide range of clinical samples 

DANiEl DEPlEDGE1 , Eleanor Gray1 , ravinder Kanda1,2 , Simon Watson3 , Emily leProust4 , Judith Breuer1 1 2 3 4 University College London, London; Imperial College London, London; Wellcome Trust Sanger Insitute, Cambridge; Agilent 

Technologies, California, USA 

Whole genome sequencing of herpesvirus genomes from clinical material has hitherto depended on the isolation of viral DNA from 

tissue culture or long PCr and gel purification for direct amplification material. Neither method is adequate for low volume samples 

containing low copy number DNA, particularly in the presence of contaminating host material. To overcome this we have adapted Agilent’s 

SureSelect Target Enrichment system to isolate viral DNA from a range of clinical samples (vesicle fluid, cerebrospinal fluid, red blood cells, 

peripheral blood mononuclear cells and saliva), prior to whole genome sequencing. We present here an overview of the methodology and 

demonstrate our success in generating high coverage, high quality, full genome sequences from these samples. 

Offered paper XMrV Infection in human disease 

MArK rOBiNSON1 , Otto Erlwein1 , Steve Kaye1 , Philip Tuke2 , Kate Tettmar2 , richard Tedder2,3 , Myra McClure1 1 2 Imperial College, Infectious Diseases, Jefferiss Trust Laboratories, London; Transfusion Microbiology R&D, National Transfusion 

Microbiology Laboratories, NHS Blood and Transplant, London; 3Blood Borne Virus Unit, Viral Reference Dept, Centre for Infections, 

Health Protection Agency, London 

The novel gammaretrovirus, xenotropic murine leukemia virus-related virus (xMrV), was identified in human prostate cancer tissue in 

2006, confirmed in 2009 and later linked to a second human condition, chronic fatigue syndrome (CFS). These investigations were all 

carried out in the uSA. The results have not been reproduced in Europe or in China. We found no evidence for xMrV infection in CFS. 

Moreover, we failed to find evidence of xMrV infection in uK prostate cancer patients and in prostate cancer tissue taken from patients 

in india, Korea, Thailand and Japan, or in cancers other than that of the prostate. Our uK CFS patients were always xMrV-free. We did, 

however, generate false-positive results from prostate cancer patient tissue, despite the fact that the no-template controls in our PCr were 

consistently negative and the PCr for murine mitochondrial DNA was often also negative. Sources of this contamination will be discussed 

in our presentation. 

Offered paper Investigating the role of the Merkel cell polyomavirus small T antigen in Merkel cell carcinoma pathogenesis 

DAViD GriFFiTHS, Kathryn richards, Eric Blair, Andrew Macdonald, Adrian Whitehouse 

University of Leeds, Leeds 

Merkel Cell Carcinoma (MCC) is an increasingly common, aggressive tumour arising in epidermal, mechanoreceptor Merkel cells. in 2008, a 

novel human polyomavirus known as Merkel Cell Polyomavirus (MCV), was identified and is now strongly implicated in MCC pathogenesis. 




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The proposed mechanism involves monoclonal viral integration prior to tumourigenesis, followed by the expression of oncogenic viral 

genes. 

Studies are being undertaken to assess the functions of one MCV early protein: the small T antigen. This shares homology with the other 

polyomavirus small T oncogenes required for cellular transformation in vitro and in vivo. 

Due to the lack of MCV reagents, recent work has involved generating bacterial and eukaryotic small T antigen expression vectors, 

as well as production of a stable cell line which inducibly expresses MCV small T. To investigate the role of small T in MCV mediated 

tumourigenesis, quantitative rT-PCr arrays have been performed. Findings show that small T expression results in the disruption of 

a number of cellular signalling pathways associated with cancer development, such as those involved in NfkB activation and matrix 

metalloproteinase expression. Further work has investigated the mechanisms used by small T to affect these pathways and their implications 

with regard to cellular transformation. 

Offered paper Importance of HTLV-1 basic leucine zipper (HbZ) in the outcome of HTLV-1 infection 

SilVA HilBurN, Maria Antonietta Demontis, Charles Bangham, Graham Taylor 

Imperial College London 

Whilst expression of HTlV-1 Tax protein has been extensively associated with HTlV-1 pathogenesis much less is known about the 

HTlV-1 basic leucine zipper protein (HBZ), which is encoded on the antisense strand of the px region of HTlV-1. We have previously 

used the detection of anti-HBZ specific CD4 and CD8 T-lymphocytes to infer expression of HBZ in vivo, noting both a lower frequency of 

HBZ specific T-cells than Tax-specific T-cells and an association of HBZ-specific CD8 T-lymphocytes with low viral load and asymptomatic 

carriage. using quantitative real-time PCr we have demonstrated that Tax mrNA expression is usually detected only after 4 hours 

unstimulated culture whereas HBZ mrNA is detected, at low level, at baseline. After 4 hours Tax mrNA concentrations are at least 10 

fold higher than HBZ mrNA. Of 30 patients with HTlV-1 infection, Tax mrNA was detected at baseline in 3 (all of whom had HTlV-1associated 

myelopathy and high HTlV-1 viral load) whilst HBZ mrNA was detected in 18 again being associated with high HTlV-1 viral 

load. in summary we found HBZ mrNA to correlate positively and anti-HBZ CD8 T-lymphocytes inversely with viral load and conclude 

that HBZ plays a major role in the pathogenesis of HTlV-1 infection which is driven by high viral load. 

Offered paper Characterization of virus infection of murine embryonic stem cells. 

rACHAEl WASH1 , Sabrina Calabressi1 , Stephanie Franz1 , David Goulding1 , Bill Skarnes1 , Wendy Barclay3 , Juergen Haas4 , Stacey 

Efstathiou2 , Paul Kellam1,5 1 2 3 4 Wellcome Trust Sanger Institute, Cambridge; University of Cambridge, Cambridge; Imperial College London; University of Edinburgh, 

Edinburgh; 5University College London, London 

recent rNAi studies have identified host genes which modify the phenotype of virus infection. We have mapped these genes to knockout 

mouse and embryonic stem (ES) cell resources, so far identifying 146 mice and 1212 ES cells of interest. Comparison of mouse and human 

genomes has revealed that the majority of genes are found in both species, showing that murine systems are relevant as models for human 

infections. Murine ES cells may therefore be a useful resource for further investigation of the roles cellular genes play in host-pathogen 

interactions. ES cells can be genetically modified and there are resources available where the aim is to inactivate all known mouse genes. 

Furthermore, mutant ES cell lines have the potential to be differentiated into other cell types, as well as being used to generate knockout 

mice for in vivo studies. 

To investigate whether murine ES cells can be used to explore host-virus interactions, we assessed responses following exposure to herpes 

simplex virus-1 (HSV-1). infection was confirmed by flow cytometry and Western blotting. Growth kinetics showed that HSV-1 replicates 

in ES cells although they are clearly less permissive than other cell lines. replication is enhanced when ES cells are differentiated using 

3-methoxybenzamide. 

Offered paper Quantitative analysis of mitochondria in A549 cells infected with human respiratory syncytial virus reveals 

recruitment of immune complex proteins 

DiANE MuNDAY, John Barr, Julian Hiscox 


Human respiratory syncytial virus (HrSV) causes serious lower respiratory tract disease in infants, and infections recur throughout life. Viral 

rNA synthesis and virion assembly occur in the cytoplasm inducing a stress response in infected cells as replication alters specific host cell 




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gene expression. We previously used quantitative proteomic analysis of HrSV-infected cells which identified changes in mitochondrial 

activity. These were further defined using a focused transcriptomic analysis of the genes that encode mitochondrial respiration, biogenesis 

and function in conjunction with a SilAC-based quantitative proteomic analysis of purified mitochondria from HrSV-infected cells. The 

data indicated very specific changes in the mitochondrial proteome. interestingly, immune complex proteins including Mx1, STAT1 and 

iSG15 were identified as significantly increased in abundance in the mitochondrial fraction from HrSV-infected cells, potentially having been 

recruited there as part of the antiviral response. The mitochondrial import receptor Tom70 (also identified in our studies) was recently 

shown to play an important role in the innate immune response to Sendai virus infection by recruiting antiviral proteins to the mitochondrial 

signalling platform. Our study, focusing on the mitochondrial proteome and transcriptome of HrSV-infected cells, may serve to further 

characterize links between negative sense virus infection, mitochondria and antiviral defence. 

Offered paper The interactome of the nucleocapsid protein of Crimean Congo haemorrhagic fever virus in 293T cells 

rEBECCA SurTEES1,2 , roger Hewson2 , Miles Carroll2 , Cheryl Walter1 , Julian Hiscox1 , John Barr1 1 2 University of Leeds, Leeds; Health Protection Agency, Porton Down 

Crimean Congo Haemorrhagic Fever Virus (CCHFV) is a potentially lethal human pathogen and one of the most widespread of the severe 

BSl4 haemorrhagic fever viruses. The nucleocapsid protein (NP) of CCHFV is essential for viral replication and encapsidation of the viral 

genome. upon infection, NP localizes to perinuclear inclusion bodies in infected cells in a temporal and actin-dependent manner. NP is 

known to directly bind this cytoskeletal protein, in addition to Mx1, an anti-viral, interferon stimulated protein. To further investigate the 

cellular binding partners of NP, a GFP nanotrap was used in combination with stable isotope labeling of amino acids in cell culture (SilAC) 

coupled to lC-MS/MS. This technique allows differentiation between GFP-NP specific, and non-specific interactions. NP was fused to the 

C-terminus of GFP, and GFP-NP or GFP only constructs were transfected into cells grown in ‘light’ (unlabelled) media or ‘medium’ (r6K4) 

media. 24 hours later, GFP and GFP-NP were immunoprecipitated and the specific binding partners of NP were identified by lC-MS/ 

MS. Significant pathways were highlighted using ingenuity Pathway Analysis (iPA) and specific interactions validated using a combination of 

western blotting, immunoflourescence and functional assays. Here, we present novel host interaction partners associated with the CCHFV 

NP. 

Offered paper Understanding the mechanisms of HCV-mediated induction of autophagy and its role in virus pathogenesis 

BJOrN-PATriCK MOHl, Mark Harris 


Autophagy is up-regulated in response to Er stress, pathogen infection, starvation or growth factor deprivation and its role in disease 

pathogenesis following viral infection is beginning to be elucidated. Autophagy is a catabolic process of self-degradation of cellular 

components whereby double-membrane autophagosomes sequester portions of the cytoplasm or organelles and fuse with lysosomes 

or vacuoles. The aim of this study is to elucidate the mechanisms by which HCV induces autophagy and how this induction contributes 

to virus pathogenesis. We demonstrate that HCV mediated autophagy induction is independent of, and precedes the virus induction of 

Er stress. Although the virus proteins do not colocalize with autophagosomes, we observed that a recognized autophagy marker, lC3-ii, 

closely associates with lipid droplets (lD) in virus infected cells. Whilst lD are known to play a critical role in multiple aspects of the virus 

lifecycle; these data suggest that the virus mediated induction of autophagy may contribute to the accumulation of lD during infection. 

Offered paper Aberrations in lipid metabolic pathways during hepatitis C infection 

MArK rOBiNSON, Arvind Patel, John Mclauchlan 

MRC-University of Glasgow Centre for Virus Research, Glasgow 

The replication of hepatitis C virus (HCV) is intricately linked with hepatocyte lipid metabolism and lipid droplet formation. There 

is mounting evidence that HCV directly modulates essential cellular pathways involved in lipid metabolism, lipoprotein signalling and 

cholesterol metabolism. Furthermore chronic HCV infection is associated with liver steatosis, which is observed frequently in patients with 

genotype 3 HCV infections prevalent in the uK. The current study aimed to better understand the impact of HCV infection on cellular 

pathways involved in lipid homeostasis. Targeted quantitative PCr profiling of cellular pathways was used to investigate the effects of HCV 

in in vitro sub-genomic replicon systems and the infectious HCV-Jc1 cell culture system. The use of targeted mrNA profiling of specific 

cellular pathways has provided novel information on the abnormalities of lipid metabolism during HCV infection. 




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Offered paper bovine viral diarrhoea virus – mechanisms of virulence 

STEPH J.F. rEED, Joe Brownlie, Carole Thomas 

RVC, Hertfordshire 

in cattle, infection with Bovine Viral Diarrhoea Virus manifests frequently as a subclinical syndrome, resulting in reductions in reproductive 

and commercial productivity, and increased susceptibility to secondary infections. However, virus strains can emerge that cause severe 

outbreaks of disease, associated with thrombocytopenia, haemorrhages and even death. Virulent infections can be experimentally 

reproduced, indicating the inherent role of virus related factors. 

Virulent strains of BVDV appear to replicate faster with demonstrably higher levels of antigen in infected tissues. Our research is 

investigating the underlying mechanism. 

Virus growth curves showed that the high virulence strains replicate more rapidly and to a higher titre than a subclinical strain. rT qPCr 

results suggest that higher virulence viruses are, in fact, better adapted to rNA replication and thus more efficient virus production. 

To investigate the initiation of protein translation of these strains, the irES region (which initiates binding of the translation complex) was 

compared. Following cloning of virus irESes into a dicistronic reporter vector, their relative functionality was evaluated by luciferase assay. 

The results and implications of this work will be discussed in this short presentation. 

Offered paper Investigation of the effects of a non-cytopathic dengue virus type 2 mutant on host cell processes 

ANDrEW DAViDSON, rebecca Ward, Helga Kroschewski, rosmani ismail, Holger Hannemann 

School of Cellular & Molecular Medicine, University of Bristol, Bristol 

Flaviviruses are known to cause persistent infections, although this has been rarely reported for dengue virus (DENV). We isolated a 

DENV-2 mutant that can establish a persistent, non-cytopathic (ncp) infection in cultured cells. Sequence analysis of the DENV-2 genome 

isolated from a number of persistently infected cell lines revealed two nucleotide changes in the NS4B gene, encoding an amino acid 

substitution. NS4B is a small integral membrane protein required for virus replication and implicated in perturbing the type i interferon (iFN) 

response. The NS4B mutations were confirmed to cause the ncp viral phenotype by reverse genetic analysis. However transient expression 

of the wild type (wt) and mutant NS4B proteins showed no differences in protein localization or their effects on iFN signalling. The growth 

kinetics of the wt and ncp viruses in cells with competent or defective iFN signalling pathways were not significantly different. DENV is 

known to induce the unfolded protein response (uPr) in infected cells. Examination of one of the signalling arms of the uPr, that of xbp1 

mrNA splicing, showed that xbp1 splicing occurred in cells infected with the wt but not the ncp virus. Current studies aim to understand 

how this effect is mediated. 

Offered paper Nairobi sheep disease virus (NSDV) blocks the host innate immune response 

BArBArA HOlZEr1 , Anne Bridgen2 , Michael D. Baron1 1 2 Institute for Animal Health, Pirbright, Surrey; Newtonmore, Scotland 

Nairobi Sheep Disease Virus (NSDV) is highly pathogenic in sheep and goats, causing acute hemorrhagic gastroenteritis and abortion. 

NSDV and the Asian variant Ganjam virus (GV) are members of the genus Nairovirus within the family Bunyaviridae and are closely related 

to Crimean-Congo hemorrhagic fever virus that causes a similar severe disease in humans. Many viruses have evolved systems to overcome 

innate immune responses. in this study we have tried to understand how NSDV is interfering with the innate immunity. We analysed 

interferon (iFN) induction in NSDV infected cells and examined the ability of NSDV to interfere with iFN action. Vero cells infected with 

NSDV or GV show a delayed iFN induction compared to cells infected with the Newcastle disease virus (NDV) and an ongoing GV or 

NSDV infection makes cells less responsive to NDV-induced up-regulation of iFN expression. Our results show that NSDV and GV are 

able to avoid an early iFN induction. Furthermore we could demonstrate that NSDV and GV are able to inhibit type i and type ii iFNdependent 

gene expression in VErO cells. We were also able to identify the viral protein which is responsible for inhibiting iFN induction 

as well as iFN action. 

Offered paper role of cellular caspases, nuclear factor-kappa b and interferon regulatory factors in bluetongue virus infection 

and cell fate 

MErEDiTH STEWArT, Polly roy 

Dept of Pathogen Molecular Biology London School of Hygiene and Tropical Medicine Keppel Street, London 

Bluetongue virus (BTV) infection causes haemorrhagic disease in ruminants and induces cell death. We investigated BTV-induced apoptosis 

in cell culture using chemical inhibitors and knock-out cell lines and showed that both extrinsic and intrinsic pathways are activated 




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independently. in addition to caspase-8, -9 and executioner caspase-3 activation, we identified cleavage of caspase-7 and PArP. Cleavage of 

PArP indicated that BTV-induced cell death appeared to be due to apoptosis rather than necrosis, which was further substantiated by data 

that showed HMBG-1 was not translocated from the nucleus. Further, we examined if NF-κB response is related to BTV-induced apoptosis 

similar to reovirus. Our data suggested that NF-κB response was not linked to the induction of apoptosis. The response was rapid during 

the early stage of infection and was suppressed during the late stage of BTV replication. Furthermore, CPE was visible earlier and virus titres 

were higher in the presence of NF-κB inhibitor (SN50), indicating that NF-κB has a role in initiating an antiviral environment. in addition, we 

show that BTV infection induces interferon regulatory factors (irF-3 and irF-7). The induction of irF responses occurred at early stage of 

infection and mirrored the timing and response of NF-κB. 

Offered paper A novel immune evasion mechanism for human papillomavirus: the E7 oncoprotein increases expression of 

the anti-inflammatory protein IL-18 binding protein 

KATHrYN riCHArDS, Mohammed Haider, Eric Blair, Miriam Wittman, Andrew Macdonald 

University of Leeds, Institute of Molecular & Cellular Biology, Leeds 

Human Papillomavirus (HPV) oncoprotein E7 has been implicated in a number of immune evasion mechanisms, including disruption 

of interferon production and Toll-like receptor signalling. interleukin-18 (il-18) is a pro-inflammatory cytokine produced in response 

to infection, stimulating T-cell production and induces Th1 cell responses. il-18 Binding Protein (il-18BP) binds to il-18, counteracting 

its activity. Because previous data has shown E7’s ability to evade the innate immune response, we looked at how E7 impacted the 

inflammatory response. We measured il-18BP output by EliSA from human cell lines stably expressing E7s from low- and high-risk HPV 

types in response to stimulation with iFN-γ. We found that these cells produced increased amounts of il-18BP compared to control 

cells. il-18BP mrNA transcripts were also increased in cells expressing E7, suggesting that E7 modulates transcription of this critical antiinflammatory 

cytokine. Current studies are focused on whether E7 has an impact on the il-18BP promoter, levels of mature il-18BP, which 

regions of E7 may be responsible for this upregulation, and analysing these phenomena in primary keratinocytes, a more physiologically 

relevant cell type. results from these studies will shed light on how HPV E7 disrupts the inflammatory response in a much more global 

manner than was originally thought. 

Offered paper regulation of the E6 PDZ protein binding function during the HPV18 life cycle 

CrAiG DElurY, Sally roberts 

University of Birmingham, Birmingham 

The malignant potential of high-risk Human Papillomaviruses (HPV) is due to the transforming activities of E6 and E7. The E6 protein 

contributes to cell transformation by multiple mechanisms including proteosomal degradation of cellular proteins such as p53, and PDZ 

domain-containing proteins involved in regulation of cell polarity and proliferation. A PDZ binding motif (PBM) present in high-risk E6 

proteins facilitates binding with PDZ domain-containing proteins such as discs large. 

To understand the role of this E6 domain in viral pathogenesis, we have constructed mutations within the high-risk HPV18 genome that 

alter E6-PBM function. These mutations include deletion of the PBM and mutation of the protein kinase A (PKA) recognition sequence of 

E6, thereby inhibiting PKA negative regulation of E6 PDZ-protein binding. These mutants retain all other functions of E6, but display striking 

differences in their cellular phenotypes and link E6-PBM function to maintenance replication of viral episomes and growth regulation of 

episome-containing cells. loss of negative regulation of this E6 domain was associated with hyperproliferation and invasion of the episomecontaining 

cells; properties that could also be induced in the wild-type genome-containing cells by inhibition of PKA signalling. Thus, changes 

in PKA-signalling during HPV infection may contribute to development of HPV-malignancies. 

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Mechanisms of mycobacteria interference with cell-intrinsic immunity processes 

Natascha Sattler, xuezhi Zhang, Sonia Arafah, Aurélie Guého, THiErrY SOlDATi 

Dept of Biochemistry, Sciences II, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva-4, Switzerland 

Pathogenic mycobacteria such as M. tuberculosis and M. marinum utilise common strategies to invade innate immune phagocytes, 

manipulate their bactericidal phagocytic apparatus and increase the success of cell-to-cell transmission. M. marinum is the closest relative to 

mycobacteria of the tuberculosis complex and provides a powerful model to study the pathogenesis of tuberculosis. using the soil amoeba 




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HA13 Cont. 

Dictyostelium discoideum as a host, we identify and characterize mycobacterial and host factors that modulate resistance to infection and cellto-cell 

spreading. We study how Dictyostelium senses bacteria and analyse mutants with altered growth on Gram+ and Gram- pathogenic 

and non-pathogenic bacteria. We investigate the role of NADPH-oxidases in production of reactive Oxygen Species and bacteria killing. 

We also dissect the involvement of paralogs of the CD36 family of scavenger receptor type B in the specific uptake of M. marinum and 

the establishment of a bactericidal phagolysosomal environment. After phagosome maturation arrest, M. marinum escapes its vacuole to 

the cytosol. We study the complex importance of autophagy in the genesis, maintenance and breakage of that replication compartment. 

This illustrates the high degree of conservation of virulence strategies and defence mechanisms, highlighting Dictyostelium as a powerful and 

simple host model system to study mycobacteria infection. 

Evolved to multitask: Chlamydia trachomatis regulates secreted effector proteins and host proteins with a single protease 

raphael H. Valdivia 

Dept of Molecular Genetics & Microbiology & Center for Microbial Pathogenesis, Duke University Medical Center, Durham NC 

27710, USA 

Chlamydia trachomatis, the causative agent of blinding trachoma and many sexually transmitted diseases, injects a large number of effector 

proteins into the cytoplasm of epithelial cells to manipulate host functions important for bacterial survival. Here we describe proteomic 

approaches to identify Type iii secretion effector proteins translocated by Chlamydia during invasion. We also report that an effector 

translocated later in infection, the protease CPAF, degrades a subset of these early effectors, especially those required for chlamydial entry 

and for the establishment of the pathogen-containing vacuole (‘inclusion’). We probed the significance of the proteolysis of host and 

bacterial proteins by generating cell-permeable CPAF-specific inhibitory peptides. These peptides blocked the cleavage of bacterial effectors, 

severely compromised inclusion integrity, and ultimately led to a Caspase1-dependent death of infected epithelial cells. We propose that 

CPAF acts as ‘master’ effector that regulates the function of multiple effectors, remodels the bacterial protein content of the inclusion 

membrane and manipulates cytoskeletal elements to ultimately maintain the integrity of the inclusion. Overall, these findings establish CPAF 

as an essential virulence factor and an attractive candidate for the development of protease inhibitors with potent anti-chlamydial activity. 

Subversion of host cells by Salmonella 

David Holden 

Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN 

Following entry of Salmonella into host cells, this pathogen remains in a membrane-bound compartment called the Salmonella-containing 

vacuole. Bacteria sense the vacuolar environment and activate the expression of the SPi-2 type iii secretion system (T3SS), to form the 

envelope-spanning secretion system and associated translocon pore in the vacuolar membrane. Bacteria then sense the near-neutral pH 

of the host cell cytoplasm; this results in dissociation and degradation of a bacterial membrane-bound regulatory complex, which activates 

effector translocation. 

We are currently studying the functions of various effectors of the SPi-2 T3SS, which have been implicated in several physiological activities, 

including avoidance of killing by macrophages, bacterial replication in a variety of host cell types, interference with immune signalling and the 

induction of cytotoxicity. 

Offered paper The Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) is a key mediator for bacterial 

escape from infected cells and for the spread of the infection in vivo 

Andrew Grant, Fiona Morgan, Duncan Maskell, PiETrO MASTrOENi 


The pattern and outcome of an infection depends on the precise dynamics of growth, spread and death of the pathogen in vivo at the 

single cell level. Salmonella enterica exemplifies a pathogen that lives within spleen and liver phagocytes and replicates inside a specialized, 

membrane-bound vacuole. 

Salmonella infections have a truly dispersive nature with intracellular infection densities remaining low due to escape of the bacteria from 

infected cells and emergence of new foci at distant sites. This leads to avoidance of the spatial and nutritional intravacuolar constraints that 

are predicted to reduce bacterial division rates. 

Both host and pathogen variables determine the dynamics of the infection. We have recently identified a novel key role for the Salmonella 

pathogenicity island 2 (SPi-2) type iii secretion system (T3SS) in the shaping of in vivo infection dynamics. We found that a major function of 




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SPi-2 T3SS in vivo is to enable bacterial escape from infected cells and facilitate spread in the organs. What is more, SPi-2 T3SS mutants are 

able to grow to high numbers within the intracellular environment, which is not what has been reported as happening in many studies of 

infected macrophages in vitro. 

Offered paper biochemical characterization of C. trachomatis Incb and IncC 

TOM HiNSON, Maud Dumoux, richard Hayward 

Institute of Structural & Molecular Biology, University College London & Birkbeck, University of London 

Chlamydia trachomatis is an obligate intracellular bacterium that invades eukaryotic host cells and survives and replicates within a modified 

intracellular compartment termed an ‘inclusion’. A family of ~40 bacterial effector proteins termed inclusion proteins (incs) integrate into 

the inclusion membrane from where they subvert host processes. The structure, function and targets of these key virulence proteins 

remain largely unknown. Here we describe the expression and purification of full-length incB and incC. These form large heat-stable homooligomers 

and integrate into liposomes in vitro, providing a model system to study their structure and topology. When expressed in cultured 

cells, incB localizes to calreticulin- positive membranes whereas incC traffics to the cell periphery, inducing membrane deformation. Studies 

using chimeras reveal that incC membrane deformation requires the hydrophobic domain whereas targeting requires additional information 

within hydrophilic regions 

role of type IV secretion systems for the intracellular lifestyle of Bartonella 

Christoph Dehio 

Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland 


The intracellular life style and habitat of Leishmania mexicana parasites: insights generated from a proteomic parts list 

Toni Aebischer 

FG16 Mycology/Parasitology Robert Koch Institute, Berlin, Germany 

The molecular understanding of habitats of intracellular pathogens and their adaptations to it is thought to lead the way to new approaches 

to control these infections. We have recently adopted a fluorescence activated particle sorting strategy to isolate the habitat of Leishmania 

parasites –protozoan parasites that cause leishmaniasis threatening more than 350 million people worldwide – to enable proteome analysis 

of both, the respective phagosome compartment and the intracellular pathogen. Bioinformatic analysis of more than 1700 parasite proteins 

allowed inferring metabolic adaptations to intracellular life and in combination with reverse genetics to assess the relevance of these 

adaptations, in particular the role of fatty acid catabolism. Further development of this methodology enabled the identification of >600 

proteins that contribute to the host cell-derived phagosome containing the parasites. This phagosomal proteome data set was compared 

to data sets from phagosomes containing inert latex beads that were publicly accessible. Thereby specific properties of the parasite’s habitat 

can be predicted. 

Analysis of vesicular traffic in Toxoplasma gondii 

Markus Meissner 

Institute of Infection, Immunity & Inflammation College of Medical, Veterinary & Life Sciences, University of Glasgow 

in order to invade and survive within host cell apicomplexan parasites evolved specialised secretory organelles (micronemes and rhoptries) 

that contain important virulence factors. Although trafficking motifs in proteins transported to these organelles have been identified, 

the machinery involved in protein sorting is only poorly understood. rab-GTPases are small G-proteins that play a central role in the 

compartment specific transport of vesicles from a donor to an acceptor membrane. interestingly the genome of apicomplexan parasites 

contains a rather reduced set of rab-GTPases that does not reflect the cellular complexity of these organisms. using Toxoplasma gondii as 

a model system, we generated transgenic parasites with tuneable copies of rab-GTPases for localization and overexpression studies. We 

found that most conserved rab-GTPases localize to the same compartment as their orthologues in other eukaryotes, indicating that the 

secretory pathway in apicomplexans is conserved. Conditional overexpression of 5 rab-GTPases results in tight and specific phenotypes 

and we identified the rab-GTPases, rab5A and rab5C, as important trafficking factors for the transport to the secretory organelles. 

Surprisingly, only a subset of micronemal proteins requires functional rab5A and C for their transport to the apical complex. 




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Offered paper Host cell exploitation by Microsporidia 

EVA HEiNZ1 , Alison Gregory1 , John Mifsud2 , Paul Dean1 , Peter G. Foster3 , Edmund r. Kunji2 , T. Martin Embley1 1 2 3 Newcastle University, Newcastle upon Tyne; The Medical Research Council, Cambridge; Natural History Museum London, London 

Microsporidia are a group of obligate intracellular reduced fungi with a broad host range. They have highly streamlined genomes and a 

highly reduced metabolic potential. To compensate for this loss, a common strategy of intracellular parasites is the use of transporters to 

steal these substrates from their host cell. Microsporidia express a unique group of nucleotide transport proteins (NTTs) otherwise only 

described in obligate intracellular bacteria, where they are used to import nutrients from the host cell to the parasite, who lost the ability for 

their de novo generation. 

We are investigating the evolution of these transporters and the patterns of NTT gene duplications, and thus lineage specific and general 

adaptations to the intracellular lifestyle among Microsporidia. Preliminary results indicate that Microsporidia re-invented mitosomal targeting 

of the NTT proteins at least twice during the evolution of the extant species. To study this, we are investigating the location of the NTT 

proteins of Trachipleistophora hominis, a Microsporidian that has been reported to infect patients with HiV/AiDS. We are also forming 

hypotheses of their possible transport substrates based on comprehensive analysis of microsporidian genomes, and will complement this by 

functionally characterizing the T. hominis NTTs. 

Offered paper Deciphering the role of autophagy during the infection of Dictyostelium with Mycobacterium marinum 

SONiA ArAFAH1 , Monica Hagedorn2 , Thierry Soldati1 1 2 University of Geneva, Geneva, Switzerland; Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany 

The soil amoeba Dictyostelium discoideum functions like innate immune phagocytes and is used as a host to study the mechanisms of 

infection by Mycobacterium marinum, a close cousin of M. tuberculosis. After uptake by Dictyostelium, M. marinum survives and replicates in 

a phagosome which does not acidify nor fuse to lysosomes, resulting in a friendly niche, before it escapes to the cytosol after about 24h. 

recent results indicate a contribution of M. marinum factors, such as its type Vii secretion system, as well as the autophagy pathway in niche 

biogenesis, maintenance and rupture. indeed, at sites of niche breakage, some mycobacterial and host proteins are ubiquitinated and, via 

the adaptor protein p62/Sqstsm1, appear to recruit GFP-Atg8. We investigate the localization of major Atg proteins like Atg9 or Atg18 to 

the bacterial vacuole. Strikingly, in atg1-null cells the M. marinum vacuole ruptures after only a few hours, resulting in massive ubiquitination 

of cell-wall material. We are studying the contribution of ESAT6, a potential secreted pore forming toxin from M. marinum in this process. 

Alltogether our data suggest that autophagy might play a complex and surprising role in biogenesis and maintenance of the mycobacterial 

vacuole and in bacterial survival inside its host. 

The virulence machinery of malaria parasites 

TANiA DE KONiNG-WArD1 , Silvia Haase1 , Kathryn Mifsud1 , Paul Gilson2 , Brendan Crabb2 1 2 Deakin University, Waurn Ponds, 3217, Australia; Burnet Institute, Melbourne, 3004, Australia 

The success of the protozoan parasite that causes malaria resides in its ability to extensively remodel its host red blood cell (rBC). This 

process involves the export of hundreds of parasite proteins into rBC cytosol and in some cases to the rBC surface, where these proteins 

play key roles in parasite virulence and survival. The identification of a targeting motif in most exported proteins has suggested that a 

common trafficking pathway is utilised to export proteins beyond the confines of the parasite. recently we have revealed the identification 

of a novel molecular machinery comprising five proteins through which exported proteins pass. As a common portal for many essential 

proteins it is hence not surprising that we found the core components of this translocon are essential for parasite survival. Hence, the 

translocon has now become a strongly validated drug target. The fifth component, a thioredoxin protein termed Trx2, could be genetically 

disrupted in rodent malaria parasites although parasites were significantly impaired in their ability to cause cerebral malaria. This indicates 

that Trx2, whilst not essential, plays an important regulatory role in protein export, the mechanisms of which we are currently investigating. 

The intracellular fate of Histoplasma capsulatum 

J.D. Nosanchuk 

Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Ullmann Building, Room 107, Bronx, 

NY 10461, USA 

Histoplasma capsulatum is the most prevalent thermally dimorphic endemic mycoses in the Americas. The fungus efficiently avoids 

host effector responses by residing within phagosomes in macrophages. We have shown that antibodies to cell surface antigens of H. 

capsulatum, including heat shock protein 60 (Hsp60), histone 2B, and the M antigen, can modify the pathogenesis of histoplasmosis. 




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Exploration of the mechanisms involved in the altered host-pathogen interactions in the presence of antibody have illuminated interesting 

aspects of pathogenesis, including new findings regarding phagocytic processes, intracellular survival and T cell stimulation. interestingly, 

these studies can also inform us about basic antibody function, such as the differential impact of antibody isotype on disease outcome. 

Furthermore, we have recently utilized H. capsulatum-binding antibodies to study physical interactions, such as fungal cell aggregation and its 

influence on pathogenesis. The antibodies have also facilitated the study of the recently described secreted fungal vesicles and have opened 

avenues for exploring protein-protein interactions that have thus far allowed us to gain new insights into the regulation and trafficking of key 

cellular proteins. Hence, monoclonal antibodies are an extraordinary component of our toolbox for exploring the interplay between 

H. capsulatum and host cells. 

The assorted interactions of Cryptococcus with host immune cells 

SiMON A. JOHNSTON, robin C. May 

School of Biosciences, College of Life & Environmental Sciences, University of Birmingham, Birmingham B15 2TT 

Cryptococcus is a fatal fungal pathogen of humans causing death through meningitis. Cryptococcus is a major pathogen of the 

immunocompromised, especially AiDS patients, and causes an estimated six hundred thousand deaths per year. The interaction between 

Cryptococcus and the host immune system is highly complex as demonstrated by the huge variation in outcome, from asymptomatic to fatal 

systemic disease, even in the immunocompetent. 

Much of this complexity arises from the ability of Cryptococcus to efficiently parasite macrophages. in this talk i will discuss part of our work 

on two major aspects of macrophage parasitism by Cryptococcus; intracellular proliferation and non-lytic escape via vomocytosis. i will 

present data showing how we study these processes at three levels: 

i. sub-cellular, where the host actin cytoskeleton is a negative regulator of Cryptococcus escape by vomocytosis. 

ii. cellular, using chicken macrophages as a model for avian vectors of Cryptococcus. 

iii. organismal, using zebrafish to study these cell biological processes in the whole organism to discover their role in disease outcome. 

Divide et impera: strategies used by the transforming parasite Theileria 

Dirk Dobbelaere 

Division of Molecular Pathology, University of Bern, Laenggassstrasse 122, P.O. Box 8466, CH-3001 Bern, Switzerland 

Theileria parasites possess the unique ability to transform the cells they infect, inducing uncontrolled proliferation and protection against 

apoptosis, resulting in a clonal expansion of parasitised host cells. To achieve this, Theileria usurps host cell signaling pathways that regulate 

cellular proliferation and survival. This is facilitated by the fact that the transforming schizont, a multinuclear syncytium, resides freely in the 

host cell cytoplasm where it can interact with pivotal regulatory components of signaling pathways and also host cell cytoskeletal structures. 

A striking example is the recruitment and activation at the parasite surface of the kinase iKK, a central regulator of the NF-αB pathway. 

Schizonts are strictly intracellular and, as only cells that harbour the schizont maintain the transformed phenotype, we investigated how 

the parasite ensures that both daughter cells remain infected at each host cell division. At the onset of mitosis, the schizont associates with 

newly formed astral microtubules ensuring its localization together with the chromosomes at the equatorial region during metaphase. As 

chromosomes separate, the parasite becomes incorporated into the central spindle and is divided equally over the two daughter cells 

during cytokinesis. interestingly, this process requires catalytically active host cell PlK1, which accumulates at the parasite surface. 

Pathogen exploitation of the actin cytoskeleton 

Matthew Welch 

Dept of Molecular & Cell Biology, University of California, LSA Rm. 305, Berkeley, CA 94720-320, USA 

Rickettsia species in the spotted fever group mobilize the actin cytoskeleton of host cells to enable cell invasion and to drive intracellular 

motility and cell-to-cell spread. Because the molecular mechanisms used by Rickettsia to exploit actin during invasion and motility are distinct 

from other pathogens and poorly understood, we sought to identify the bacterial and host proteins important for these processes. using 

an rNAi-based screen in Drosophila S2r+ cells, we defined a set of core proteins involved in invasion that includes the Arp2/3 and WAVE 

complexes, as well as the rho GTPases Cdc42 and rac. in mammalian cells, the Arp2/3 complex was also crucial for invasion, while the 

WAVE complex and individual GTPases played an important but less prominent role, suggesting differences in the degree of functional 

redundancy for different cell types. A second rNAi screen delineated a set of core proteins — profilin, fimbrin/plastin, capping protein 

and cofilin — as crucial for actin-based motility. Moreover, we discovered that a bacterial surface protein, Sca2, is the first known bacterial 

mimic of host formin proteins and functions to nucleate and elongate actin to enable motility. Our results highlight the unique evolutionary 

strategies and molecular mechanisms employed by Rickettsia to mobilize host actin. 




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Offered paper Dynamic disulfide bonding in structural components of the Type-III secretion system apparatus of Chlamydia 

trachomatis 

Helen Betts-Hampikian, KEN FiElDS 

University of Miami Miller School of Medicine, Miami, FL, USA 

The Chlamydial elementary body exhibits disulfide bonding among several cysteine-rich outer-membrane proteins that confer a highly 

cross-linked outer envelope. Conversely, these disulfide bonds become reduced in the vegetative reticulate body form, linking redox status 

of cysteine rich envelope proteins with progression of the developmental cycle. it is well established that the activity of the chlamydial 

type-iii secretion system (T3SS) is also intimately linked to development. Several structural components of the T3S apparatus contain an 

unusually high number of cysteine residues in comparison to homologues from other pathogens. These include the needle protein CdsF, 

the putative tip protein CT584, and the OM secretin CdsC. We have found that disulfide bonding occurs within at least EB-localized CdsC 

and CdsF. Additionally, we have determined that disulfide bonding within these proteins responds to development and behave similarly 

to non-T3SS, cysteine-rich envelope proteins. Disulfide bonding of CdsF was not required for CdsF polymerization, raising the possibility 

that this Chlamydia-specific feature could contribute to stabilization of CdsF needles in infectious particles. in aggregate we surmise that the 

redox status of these T3SS apparatus proteins is intimately linked to the developmental cycle and may be important in host-cell sensing and 

signaling rB to EB conversion. 

Offered paper Investigating how Group b streptococci survive within phagocytes 

NiCOlA CuMlEY, robin May 


The opportunistic human pathogen Streptococcus agalactiae, or group B strep (GBS), is a leading cause of neonatal septicaemia and 

meningitis. The organism is a commensal of the gastrointestinal and genitourinary tract in approximately 30–50% of the population and the 

vast majority of individuals exhibit no symptoms of infection. Although GBS has a predominantly extracellular lifestyle, we and others have 

previously shown that it survive for extended periods within phagocytic cells. The ability of the organism to exploit this intracellular niche 

may be an important step in the organism’s progression from commensal to systemic infection. 

using the mouse derived macrophage like cell line J774, we now shown that the GBS containing phagosome matures normally and indeed 

that phagosome acidification is required for the organism to survive intracellularly, since pharmacological inhibition of maturation leads to 

GBS death. in addition, we demonstrate that most previously characterized GBS virulence factors are dispensable for intracellular survival. 

However, the global regulator Covr/S, known to control the response of the organism to changes in environmental pH, is essential for 

persistence within the phagosome, suggesting that the ability of the organism to sense and adapt to environmental changes is critical for the 

resistance to phagocytic killing. 

New insights into Shigella–gut epithelium interaction 

Chihiro Sasakawa 

Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 Japan 

Bacteria-gut epithelium interplay and the host innate defence response are the most critical issue in determining the fate of bacterial 

infection and severity of the diseases. Shigella spp, the causative agent of bacillary dysentery, are highly human-adapted pathogens capable of 

invading and colonizing the intestinal epithelium, which results in strong inflammatory colitis. Shigella secrete many number (more than 50) 

of effectors via the type iii secretion system during infection, some of which are delivered into the surrounding space and some others into 

the host cell cytoplasm and nucleus. The delivered effectors mimic and usurp the host cellular function, and modulate host cellular signal 

pathways and immune response, thus playing key roles in promoting bacterial infection and evading host defence systems. in this workshop, 

i would like to highlight our current topics related to Shigella infectious stratagems executed by the effectors, those include bacterial evasion 

from autophagy, modulation of inflammatory response, and repairing epithelial damage. 

Getting in and out: how Listeria monocytogenes breaches host cell barriers 

Trinad Chakraborty 

Centre for Medical Microbiology & Virology, Faculty of Medicine, Justus-Liebig University Giessen, Frankfurterstrasse 107, 35392 

Giessen Germany 

Autophagy acts as intrinsic defence system against intracellular bacterial survival. recently, multiple cellular pathways that target intracellular 

bacterial pathogens to autophagy have been described. These include, the Atg5-lC3 pathway that targets Shigella, the ubiquitin (ub)- 




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NDP52-lC3 pathway that targets Group A Streptococcus (GAS) and Salmonella typhimurium, the ub-p62-lC3 pathway that targets 

Mycobacterium tuberculosis, Listeria monocytogenes, and S. typhimurium, and the diacylglycerol-dependent pathway that targets S. typhimurium. 

in addition, the bacterial invasive process is targeted by theNOD1-Atg16l-lC3 pathway. Bacterial pathogens with an intracytosolic lifestyle 

i.e., those capable of inducing actin polymerization and cell-to-cell spread, also employ diverse tactics to evade autophagic recognition. 

Thus autophagy-based restriction of bacterial growth of Shigella, L. monocytogenes, and Burkholderia pseudomallei is overcome by deploying 

highly evolved systems to evade autophagic recognition. i review the current knowledge related to host recognition of L. monocytogenes 

by the innate immune system, and highlight how mechanisms of autophagic recognition by the host is overcome by effective bacterial 

countermeasures. 

HA14 Virology workshop: replication & gene expression 

Offered paper Characterizing the role of interferon-responsive elements within the promoter of murine herpesvirus-68 

ORF50 gene in virus lytic infection 

BruNO MANSO 1 , James Stewart 2 , Bahram Ebrahimi 1 

1 Institute of Integrative Biology, University of Liverpool, Biosciences Building, Liverpool; 2 Institute of Infection and Global Health, 

University of Liverpool, Liverpool 

in γ-herpesviruses, the balance between lytic infection and reactivation from latency depends entirely on expression of an immediateearly 

viral gene (ORF50) encoding the replication and Transcriptional Activator (rTA) protein. Paradoxically, the promoter for this vital 

protein contains a complex array of cellular interferon-related transcription binding sites such as interferon-regulatory factors. Moreover, the 

presence of these binding sites in the context of viral lytic infection and reactivation from latency remain unclear. We have used the murine 

model MHV-68 to decipher the potential role irF-binding elements, present within the viral ORF50 promoter, may play in regulating virus 

lytic infection. We show that the transcription activity of a region of 700 bp upstream of ORF50 OrF is down regulated when cells are pretreated 

with type i interferons. Detailed analysis of this domain revealed a series of binding sites associated with iFN response elements, 

including interferon regulatory factors 3 and 7 (irF-3 and irF-7). Site-directed mutagenesis of these viral promoter binding sites resulted in 

the up-regulation of ORF50 promoter activity in transient transfection assays. We provide a model of how these cellular factors modulate 

the expression of rTA with implications for virus lytic infection and reactivation from latency. 

Offered paper Investigating the role of the cellular rNA helicase DDX3 in HCV replication 

SEAMuS STACK, Allan Angus, Arvind Patel 

Centre for Virus Research, University of Glasgow, Glasgow 

↑Contents 

The cellular DEAD-box rNA helicase DDx3 is essential for Hepatitis C Virus (HCV) replication. However, the exact stage of the viral 

lifecycle at which DDx3 functions is unknown. To investigate this, we analysed various parts of the HCV lifecycle using a number of model 

systems, including HCV psuedoparticles (HCVpp), subgenomic replicons (SGr) and the HCV cell culture system (HCVcc). The effect of 

DDx3 knockdown on these systems was determined by transducing target cells with lentivirus encoding short hairpin rNA against the 

C-terminus of DDx3. The HCVpp system is used to study virus entry. DDx3-knockdown cells did not alter HCVpp infectivity, indicating 

that DDx3 does not affect virus entry. SGr’s are autonomously replicating mini-genomes used to study viral rNA replication. Knockdown 

of DDx3 from SGr-containing cells caused no disruption to rNA replication, suggesting DDx3 is not involved in rNA synthesis. HCVcc 

is based on a full-length cloned viral genome that replicates and produces infectious virus particles, thus allowing the full viral lifecycle to be 

studied. DDx3 knockdown from target cells severely impaired HCVcc infectivity. Collectively, these results indicate that DDx3 functions at 

an early step in the viral lifecycle, acting after entry but before release of the genome into the cytoplasm. 

Offered paper Intrinsic resistance to HSV-1 infection involves SUMO pathway-dependent recruitment of cellular repressors 

to viral genomes 

Delphine Cuchet-lourenco, Chris Boutell, Vera lukashchuk, Kyle Grant, Amanda Sykes, Jill Murray, Anne Orr, 

rOGEr EVErETT 


Components of PMl nuclear bodies (ND10) are recruited to sites associated with parental HSV-1 genomes. This cellular response is 

linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein iCP0. We report that the SuMO interaction motifs of 




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PMl, Sp100 and hDaxx are required for recruitment of these proteins to HSV-1 induced foci, which also contain SuMO conjugates and 

a SuMO E3 ligase. SuMO modification of PMl and elements of its TriM are also required for recruitment in cells lacking endogenous 

PMl. Mutants of PMl isoform i and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of iCP0 null 

mutant HSV-1 infection mediated by their normal counterparts. We conclude that recruitment of ND10 components to sites associated 

with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SuMO modification pathway. 

Offered paper A paramyxovirus lacking its interferon (IFN) antagonist does not induce IFN in the absence of defective 

interfering viruses 

MAriAN KilliP1 , Dan Young1 , Kay Childs2 , Stephen Goodbourn2 , richard randall1 1 2 Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife; Division of Biomedical Sciences, St George’s, University 

of London, London 

it is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (iFN) induction are 

produced during virus replication and, to limit the activation of the iFN response by these PAMPs, viruses encode iFN antagonists. We have 

studied iFN induction by parainfluenza virus type 5 (PiV5) at the single-cell level, using a cell-line expressing GFP under the control of the 

iFN-α promoter. We demonstrate that a recombinant PiV5 (termed PiV5-VΔC) that lacks a functional V protein (the viral iFN antagonist) 

does not activate the iFN-β promoter in the majority of infected cells; rather, as we observe for wild-type PiV5, it is the presence of nonviable 

defective interfering (Di) viruses within PiV5-VΔC virus stocks that activate the iFN response. Furthermore, we show that these 

Di viruses can activate the iFN-β promoter in the absence of virus protein synthesis (and hence virus replication) and that a co-infecting 

non-defective virus is not required. We conclude that viral PAMPs capable of activating riG-i/MDA-5 are not produced/exposed during 

the replication of non-defective PiV5, and that the V protein functions to block activation of the iFN response by PAMPs produced by Di 

viruses that are regularly generated during the replication of non-defective virus. 

Offered paper Cell gene expression correlating with EbEr expression in Epstein–barr virus-infected lymphoblastoid cell lines 

rACHEl BOSSHArD, Goran Gregorovic, robert E. White, Claudio Elgueta Karstegl, Paul J. Farrell 

Section of Virology, Imperial College Faculty of Medicine, London W2 1PG 

Novel Epstein-Barr Virus (EBV) strains with deletion of either EBEr1 or EBEr2 and corresponding revertant viruses were constructed and 

used to infect B lymphocytes to make lymphoblastoid cell lines (lCls). The lCls were used in microarray expression profiling to identify 

genes whose expression correlates with the presence of EBEr1 or EBEr2. Functions of regulated genes identified in the microarray analysis 

include membrane signalling, regulation of apoptosis and the interferon/antiviral response. Although most emphasis has previously been 

given to EBEr1 because it is more abundant than EBEr2, the differences in cell gene expression were greater with EBEr2 deletion. in this 

system, deletion of EBEr1 or EBEr2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting 

lCls. using the recombinant viruses and novel EBEr expression vectors, the nuclear redistribution of rpl22 protein by EBEr1 in 293 

cells was confirmed but in lCls almost all the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not 

detectably changed by EBEr1. The changes in lCl gene expression identified here will provide a basis for identifying the mechanisms of 

action of EBEr rNAs. 

Offered paper Functional profiling of the murine norovirus genome 

luCY THOrNE, Dalan Bailey, ian Goodfellow 


Human norovirus is a major cause of viral gastroenteritis; understanding the function of the viral proteins and their interactions with hostcell 

factors is central to the development of anti-viral targets and rational approaches to attenuation. Functional profiling by insertionalmutagenesis 

can reveal protein domains and rNA elements important for replication. Here, transposon-mediated insertional-mutagenesis 

was used to create 5 libraries of mutagenised murine norovirus infectious clones, each containing a 15-nt sequence randomly inserted 

within a defined region of the genome. Transfection of in vitro transcribed capped rNA allowed recovery of infectious virus from each 

library, which was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCr 

profiling. Genome-wide profiling revealed functional protein domains and structural mapping of the insertion sites in the viral capsid protein 

indicated constraints in the receptor binding domain. Analysis of the insertions in a predicted rNA stem-loop structure corroborated 

previous results on the importance of a host protein binding site for replication. As proof-of-principle, several insertion sites were 

introduced into the wild-type clone and allowed the recovery of infectious virus. Further characterization of these viruses could also 

facilitate engineering the first reporter-tagged murine norovirus for replication and attenuation studies. 




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Offered paper Next-generation sequencing of mrNA in adenovirus-infected cells 

Vanessa Evans, Gary Barker, DAViD MATTHEWS 

University of Bristol, Bristol 

We have used next generation sequencing (also known as deep sequencing) of mrNA isolated from the cytoplasm of adenovirus infected 

Hela cells at 8 and 24 hours post infection. Analysis of the sequence data using Bowtie/TopHat/Cufflinks suite of software on the Galaxy 

server has enabled a simultaneous analysis of changes in cellular and viral transcription. This analysis shows that at 8 hours post infection 

adenovirus derived sequences make up approximately 0.3% of mapped sequences which rises to almost 82% of mapped sequences by 24 

hours, illustrating the dominance of adenovirus transcripts at this time point. Analysis of splicing patterns in the adenovirus mrNA reveals a 

small number of previously unknown splice events as well as correctly identifying previously characterized splice events. Moreover, for the 

first time we have a preliminary dataset from which to infer the relative abundance of adenovirus transcripts which confirms, for example, 

that hexon is the most abundantly expressed transcript. Analysis of cellular genes shows expected widespread changes transcription and 

highlights changes in exon usage as a result of adenovirus infection. Thus, the data reveals the power of next generation sequencing to 

simultaneously monitor viral and cellular transcription. 

Offered paper The cellular rab11 pathway is involved in late stages of influenza assembly and budding 

EMilY BruCE, Maria Amorim, Eliot read, Agnes Foeglein, robert Mahen, Amanda Stuart, Paul Digard 


influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of spheres or 

elongated filaments. We previously reported that cellular rab11 (a small GTP-binding protein involved in endocytic recycling) is involved 

in both filament formation and the final budding step of spherical particles. Significantly, A549 cells depleted of rab11 showed a marked 

defect in ribonucleoprotein trafficking, as observed by immunofluorescence and fluorescent in situ hybridization of nucleoprotein and 

vrNA respectively. We also detected a relocalization of endogenous rab11, and a high degree of colocalization between rab11 and NP. 

Co-precipitation experiments revealed specific interactions between a tagged form of rab11 and rNP components (NP, PB1, PB2, PA and 

vrNA) but not M1 or M2. Furthermore, we were able to show a direct interaction between rab11 and PB2, most likely responsible for 

mediating rNP transport. Finally, we observed a 40% decrease in levels of surface M2 after rab11 depletion, though the significance of this 

is still under investigation. Based on these findings, it is clear that the cellular rab11 pathway plays an important role in rNP transport and 

possibly budding site organization which may be responsible for the observed budding and filament formation defects. 

Offered paper Novel genes and splicing revealed by deep sequencing of the human cytomegalovirus transcriptome 

Derek Gatherer1 , Charles Cunningham1 , Katarina Baluchova1 , ralph Hector1 , Derrick Dargan1 , Julie Galbraith2 , Pawel Herzyk2 , 

Gavin Wilkinson3 , ANDrEW DAViSON1 1 2 MRC–University of Glasgow Centre for Virus Research, Glasgow; Sir Henry Wellcome Functional Genomics Facility, University of 

Glasgow, Glasgow; 3Dept of Medical Microbiology, School of Medicine, Cardiff University, Cardiff 

Transcription of the 236 kbp genome of human cytomegalovirus (HCMV) has been studied for several decades by many research groups. 

Deep sequencing now promises to add an unprecedented level of detail on the transcription of every nucleotide. in the present study, 

illumina sequence data were derived from polyadenylated rNAs expressed by HCMV strain Merlin at a stage during infection of human 

fibroblasts when infectious virus was being produced. Analysis of these data focused on a systematic exploration of splicing patterns and 

identification of novel transcripts. The most prominent findings were then investigated by additional rNA mapping experiments. Extensive 

splicing was revealed in the HCMV genome, some of which related to expression of protein-coding regions and some to expression of 

antiparallel, apparently non-coding transcripts. in regions of the genome that had not been recognized as encoding proteins, six novel genes 

were characterized, four of which were predicted to encode proteins and two of which appeared to encode non-coding transcripts. 

Offered paper Understanding the role of ribosome translocation on bunyavirus rdrp-mediated transcription of viral rNA 

templates 

CHErYl WAlTEr, John Barr 

University of Leeds, Leeds, West Yorkshire 

The Bunyaviridae family of segmented negative stranded rNA viruses contains many serious human pathogens. it has previously been 

shown that transcription of the bunyavirus rNA template by the viral rNA-dependant rNA polymerase (rdrp) is obligatorily coupled to 

translation of the newly transcribed mrNA. Pausing ribosome translocation leads to premature rdrp-directed transcription termination 




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at specific sites on the rNA template that possess transcription termination activity, and this phenomenon represents the only known 

example of coupled transcription/translation in a eukaryotic system. Our aim is to understand the molecular basis of how the translocating 

ribosome is able to so profoundly influence rdrp activity. 

We use an assay that allows the generation and visualization of transcription products from cDNA-derived bunyavirus rNA templates. 

By introducing an active transcription termination signal into a bunyavirus template, and performing a comprehensive mutagenic analysis 

of both its conserved sequence and structural elements, we have identified rNA template features that dictate the requirement for 

the obligatory coupling phenomenon, and inform on the possible mechanism that is responsible. We will discuss our findings and 

complementary approaches used towards understanding this unique feature of bunyavirus rNA synthesis. 

Offered paper Understanding KSHV vIrF-2 cell interactions 

MOHAMED MuTOCHEluH1 , Cristina Aresté1 , Wenbin Wei1 , John Arrand1 , Carmel McConvile1 , Kym lowry2 , David J. Evans2 , 

David J. Blackbourn1 1 2 School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT; School of Life Sciences, University of 

Warwick, Coventry CV4 7AL 

Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes the lytic cycle vIRF-2 gene that shares homology with cellular 

interferon (iFN) regulatory factors. The antiviral mechanisms mediated by type i iFNs are important pathways of innate host cell defence, 

limiting viral replication and spread. Our aim is to understand how virF-2 protein contributes to KSHV biology. 

Methods: 293-derived cell clones were engineered to express doxycycline-inducible vIRF-2. iFN responses were induced with recombinant 

(r) iFN-aB2 and measured by iFN stimulated response Element (iSrE)-luc reporter gene assay. The effects of virF-2 on the transcriptome 

in response to riFN-aB2 were determined by DNA microarray analysis and confirmed by western blot. Encephalomyocarditis virus (EMCV) 

was titred by plaque assay on l929 cells. 

Results & conclusion: virF-2 protein inhibited activation of iSrE-luc by over 50% and significantly (p


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To examine whether these longer lived precursors might have a functional role, we sought to determine the effect of enhancing the 

rate of cleavage at the NS4B5A boundary on viral replication. introduction of a double valine substitution at the P3 and P4 positions of 

the boundary led to a dramatic increase in the rate of NS4B5A cleavage and blocked replication. using a replicon library with a targeted 

degenerate coding region, second site compensatory mutations were found that restored replication. importantly some of these second 

site compensatory mutations fell exclusively on the NS5A side of the boundary and all resulted in a reduction in the rate of boundary 

processing. Our data suggest that NS4B5A containing precursors have a function to fulfil in viral replication which requires they exist for a 

minimum period of time after translation for this to be achieved. 

Offered paper Effect of the Pb2 627K mutation in Eurasian lineage H5N1 highly pathogenic avian influenza viruses for avian 

hosts 

JASON lONG1,2 , Wendy Barclay1 , Jill Banks2 1 2 Imperial College London, London; Veterinary Laboratories Agency, Weybridge, Surrey 

residue 627 of the PB2 subunit of influenza virus polymerase shows genetic variation in a host specific manner. However, since 2005 the 

Eurasian-lineage H5N1 highly pathogenic avian influenza viruses bear the mammalian signature 627K. The significance of this for the avian 

host is not understood. 

The polymerase genes of a Eurasian lineage and pre-Eurasian lineage H5N1 virus were expressed, both bearing either 627K or 627E (avian 

signature) on the PB2 gene. An in vitro polymerase activity assay was conducted in chicken cells (DF-1) at varying temperatures. At 39°C 

presence of PB2 627K decreased polymerase activity relative to the 627E variant. However, at the lower temperature of 37°C, PB2 627K 

resulted in increased activity. We hypothesise that current Eurasian lineage viruses bearing PB2 627K may preferentially replicate in the 

cooler respiratory tract of the bird, compared to the warmer temperature of the gut. This is supported by reports that Eurasian lineage 

viruses often show buccal rather than cloacal shedding. Some Eurasian-lineage viruses have changes in the HA gene, that may lead to a 

change in tissue tropism. Whether PB2 627K may have driven HA changes or vice versa is unclear. Further investigation is underway to 

understand the implication of these findings. 

Offered paper bluetongue virus VP1 polymerase activity in vitro: template, dinucleotide priming and cap dependency 

EiKO MATSuO, Polly roy 

London School of Hygiene and Tropical Medicine, London 

Despite its structural similarity, bluetongue virus (BTV) protein, VP1, is known to possess a function as polymerase on its own, unlike 

rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with polymerases of other members of 

family Reoviridae, BTV VP1 has not been characterized well. Here, we demonstrate that BTV VP1 possesses sequence-independent in vitro 

catalytic activity but requires some cis-acting element shared by the members of family Reoviridae allowing synthesis of rotavirus genome. 

BTV VP1 could synthesize the ten dsrNAs simultaneously from BTV core-derived ssrNA templates in a single in vitro reaction as well as 

genomic dsrNA segments from rotavirus core-derived ssrNA templates that possess no sequence similarity with BTV. in contrast, dsrNAs 

were not synthesized from non-viral ssrNA templates by VP1, unless those were fused with some specific BTV sequences. Further, we 

showed that the polymerase activity was stimulated by two different mechanisms: an allosteric modulation by cap structure and a priming 

of dinucleotide. Synthesis of dsrNAs from capped ssrNA templates was significantly higher than that from uncapped ssrNA template and 

addition of dinucleotide enhanced the activity as long as the last nucleotide of ssrNA template was complemented by a dinucleotide. 

Offered paper The KSHV rTA protein is a SUMOylation targeted ubiquitin ligase (STUbl) 

JENNiFEr WOOD, David Hughes, Adrian Whitehouse 

Institute of Molecular & Cellular Biology, University of Leeds, Leeds 

Kaposi’s sarcoma associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), a cancer of immunosuppressed individuals. 

like all herpesviruses, KSHV has a biphasic life cycle, with a dormant latent phase and a productive lytic phase. This lytic phase is initiated by 

expression of the virally-encoded rTA protein. rTA functions by binding KSHV lytic promoters and recruiting cellular transcription factors, 

leading to lytic gene expression. in addition, it functions as an E3 ubiquitin ligase, actively degrading repressor proteins thought to maintain 

the virus in the latent state. The aim of this study was to investigate the mechanism by which rTA recognises these cellular targets, which 

it marks for degradation. We have identified that rTA functions as a SuMOylation targeted ubiquitin ligase (STubl), which recognises its 

poly-SuMOylated targets via SuMO interacting motifs (SiMs). We show that the transcriptional repressor protein Hey1 (a known rTA 

target) is in fact SuMOylated in vivo, and by mutating rTA’s SiM domains (either singly or in combination) rTA-mediated degradation of 




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Hey1 is attenuated. We are currently confirming these data using cell-free assays. This work has identified yet another novel mechanism by 

which viruses interact with the ubiquitin-proteosome system. 

Offered paper Extensive investigation of the HCV IrES variability suggested new functions to some nucleotides and revealed 

possible importance of domain II for the therapy 

ANAS KHAWAJA1 , Vaclav Vopalensky1 , ludek roznovsky2 , Jakub Mrazek3 , Martin Pospisek1 1 2 Charles University in Prague, Faculty of Science, Prague, Czech Republic; University Hospital Ostrava, Infectious Diseases and AIDS 

Clinic, Ostrava, Czech Republic; 3Institute of Public Health Ostrava, Dept of Molecular Genetics, Ostrava, Czech Republic 

Synthesis of the hepatitis C virus polyprotein is fully controlled by an internal ribosome entry site (irES) located within 5’uTr of the viral 

rNA. We developed a flow-cytometry-based approach for monitoring patient to patient differences in irES activities of their whole viral 

populations. Simultaneously, we employed conventional methods comprising cloning of PCr fragments, DGGE, TGGE, sequencing and 

bicistronic reporter assay for finding HCV irES mutations and measuring their activities. Sequence data from patients’ samples along with 

analyses of migration patterns by DGGE and/or TGGE allowed us to compare and estimate the variation that may have persisted over time 

within one or group of patients individually and collectively. As a part of our effort aimed to get more inside the relation between particular 

irES mutations and corresponding changes in irES structure and function, we compiled from the literature a comprehensive database 

comprising over 1200 irES mutations further categorized by frequency, structural and functional behavior, experimental parameters, clinical 

data etc. Surprisingly, database mining revealed that patients non-responsive to iFN-ribavirin therapy are less prone to accumulate mutations 

within the HCV irES domain ii. Possible explanation of this phenomenon and information about novel mutations as regards their influence 

on irES function will be discussed. 

Offered paper Identification of a 13th influenza A virus protein; a novel splice variant form of the M2 ion channel 

HElEN WiSE1 , Edward Hutchinson1,2 , Zi Kang1 , Nicole robb2 , John Kash3 , Brett Jagger1,3 , Ervin Fodor2 , Andrew Firth1 , Julia Gog4 , 

Jeffrey Taubenberger3 , Paul Digard1 1 2 3 Division of Virology, University of Cambridge, Cambridge; Sir William Dunn School of Pathology, University of Oxford, Oxford; Viral 

Pathogenesis and Evolution Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National 

Institutes of Health, Bethesda, USA; 4Centre for Mathematical Sciences, University of Cambridge, Cambridge 

Four mrNAs are produced from segment 7 of influenza A virus. unspliced transcripts encode M1, while spliced mrNA2 encodes the 

M2 ion channel, which functions in virus entry and assembly and is a target for antiviral and vaccine therapies. No protein products have 

been identified from spliced mrNAs3 and 4. Serial passage of virus engineered to lack M2 through loss of the mrNA 2 splice donor site 

identified a single nucleotide pseudoreverting mutation. This mutation was necessary and sufficient to restore wild-type growth in cell 

culture and virulence in mice. The reversion mechanism did not restore mrNA2 production; instead mrNA4 was strongly upregulated. 

We show that mrNA4 encodes a variant 99 amino-acid M2 protein (designated M42) with an alternative ectodomain. Furthermore, we 

demonstrate that production of M2 and M42 is in balance and that single nucleotide changes in the background of otherwise WT Pr8 

or udorn viruses upregulated mrNA4/M42 expression at the expense of mrNA2/M2 production. importantly, this includes a change 

previously identified as a 14C2 antibody escape mutation. We conclude that segment 7 can express at least three polypeptides and that 

this suggests a ready route by which influenza A virus could escape from an M2e vaccine. 

Offered paper UL144 is expressed during latency but in a strain-specific manner 

EMMA POOlE, Kathy raven, Matthew reeves, John Sinclair 

Cambridge University, Cambridge 

it is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34+ bonemarrow 

progenitor cells. The precise mechanisms that control the establishment and maintenance of latency remain poorly understood. 

using a model of experimental latency we have analysed changes in cellular protein secretion during HCMV latency (the latency-associated 

secretome). We find that whilst two different clinical strains of virus we have tested have very similar secretome profiles, there were a few 

notable differences. in contrast to latent infection with the Merlin strain of HCMV, which robustly upregulated secretion of the chemokine 

CCl22, latent infection with the TB40E strain of virus showed no such increase. 

We have previously shown that the viral gene product responsible for induction of CCl22 during lytic infection is ul144. Consequently, 

we analysed whether induction of CCl22 during latency was also due to ul144 expression.intriguingly, we observed expression of ul144 

during latent infection with Merlin but not TB40E. This fits well with the observed ability of Merlin but not TB40E to induce CCl22 

secretion during latency. 




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These data show that ul144 may be a novel latency-associated viral gene but that latent expression of ul144 may be restricted to certain 

viral strains. 

Offered paper Splicing factor SrSF2 (SC35) controls HPV16 E6 gene expression during cervical tumour progression 

MElANiE MCFArlANE, Sheila V. Graham 


Human papillomavirus type 16 E6 oncoprotein rNA is alternatively spliced, with at least four rNA isoforms expressed during tumour 

progression. using a model system for cervical cancer progression we show that E6 splicing is altered in transformed cells. Cellular 

constitutive and alternative splicing is positively regulated by the essential serine-arginine-rich splicing factors (SrSFs). in the cervical cancer 

model a subset of SrSFs, SrSF1–3, are specifically upregulated upon tumour progression. in order to determine whether any of the 

upregulated SrSFs are responsible for altered E6 splicing during transformation, sirNA knockdown of SrSF1–3 was carried out. Surprisingly, 

none of the knockdowns resulted in a different splicing pattern; however SrSF2 knockdown caused a significant decrease in total E6 rNA 

expression. This was accompanied by a reduction in the rate of cell division and increased cellular senescence. We hypothesise that SrSF2 

is involved in regulating E6 transcription or E6 rNA stability in cervical cancer cells and is therefore a possible therapeutic target for cervical 

cancers. 

Offered paper Genomic gymnastics of rift Valley fever virus 

BENJAMiN BrENNAN, richard M. Elliott 

University of St Andrews, St Andrews 

rift Valley fever virus is a mosquito-borne pathogen of both livestock and humans. The virus genome contains 3 segments of negative (or 

ambisense) rNA; termed large (l), Medium (M) and Small (S). using reverse-genetics we have generated recombinant viruses lacking 

both the non-structural proteins NSs and NSm. This was achieved by replacing the coding sequence of the NSs protein on the S segment 

with the coding sequence for the glycoprotein precursor, generating a two-segmented rVFV (r2MP12). As the newly generated virus 

only contained the l and S viral genomic segments, we investigated whether the virus has the ability to acquire a third rNA segment. 

Here we demonstrate with reverse-genetic experimentation that it is possible to rescue and passage the r2MP12 virus with additional 

genomic segments. We show that the virus can acquire an extra S- or M-like segment, with or without the acquisition conferring a selective 

advantage to the virus. We also present preliminary evidence of a selective excision event, in which the coding sequence for the viral 

glycoproteins in the chimeric S segment is deleted when r2MP12 is rescued and passaged with the authentic MP12 M segment. 

Offered paper Engineering the bunyavirus genome to prevent reassortment 

BérYl MAZEl-SANCHEZ, richard M. Elliott 

University of St Andrews, St Andrews, Fife 

Viruses within the Bunyaviridae family have a negative-sense, tripartite rNA genome. Each segment contains an open reading frame (OrF) 

flanked by untranslated regions (uTrs) that promote transcription, replication and encapsidation of the viral genome. 

using reverse genetics, we have produced recombinant viruses that contain deletions within either their 3’ and/or 5’ uTrs in one, two or all 

three segments of Bunyamwera virus (BuNV). Some of these viruses are grossly attenuated in tissue culture, producing smaller plaques or 

even no plaques, and reduced host-cell protein shut-off compared to wild-type virus. 

After blind serial passage in tissue culture, all viruses partially recovered fitness, generating higher titres and showing larger plaque size. 

We determined the complete nucleotide sequence for each virus. The deleted uTrs sequences were maintained but a few amino acid 

mutations were observed in the polymerase protein. We can now use these viruses to investigate whether they are capable of reassorting 

with wild-type virus to test the hypothesis that evolved viruses are genetically inert. Such viruses could be used to design live-attenuated 

vaccines against serious pathogens within the family. 

Offered paper Protein methylation regulates the immediate early KSHV OrF57 protein 

MArKO NOErENBErG, Adrian Whitehouse 


lytic reactivation of Kaposi’s Sarcoma associated Herpesvirus (KSHV) has been associated with tumourogenesis in immunocompromised 

patients. Therefore, essential, immediate early proteins like KSHV OrF57 are ideal targets for drug intervention. OrF57 is a multifunctional 

protein which interacts with a variety of cellular proteins to facilitate the processing and export of viral intronless rNA into the cytoplasm 




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and enhance efficient translation of viral target mrNAs. However, it remains largely unknown how these different aspects are orchestrated. 

Here we show that protein methylation regulates OrF57 function. We demonstrate that OrF57 is posttranslationally methylated and 

inhibition of methylation has a dramatic effect on the ability of OrF57 to export intronless viral rNA out of the nucleus. Experiments 

were undertaken to examine the role of methylation of OrF57 on its ability to bind proteins and rNA, nucleolar trafficking, nucleolarcytoplasmic 

shuttling as well as its possible role in protecting viral rNAs from degradation. initial results suggest methylation may have an 

effect on the rNA binding properties of the OrF57 protein. in addition, we have identified a cellular protein methyltransferase which 

interacts with OrF57, providing a novel candidate for drug development. 

Offered paper bluetongue virus encodes a novel non-structural protein 

MAxiME rATiNiEr, Andrew Shaw, Giulia Franzoni, Salvatore Gulletta, Marco Caporale, Massimo Palmarini 

MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, 

Veterinary and Life Sciences, University of Glasgow, Glasgow 

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been accepted 

that the 10 segments of the dsrNA genome of BTV encodes for 7 structural and 3 non-structural proteins. The non-structural proteins 

(NS1, NS2, NS3) play different roles in the viral replication cycle. recently, bioinformatics analysis has revealed a conserved open reading 

frame in segment 9 of the BTV genome overlapping the open reading frame encoding VP6. This open reading frame is predicted to 

encode a protein of 77/79 amino acid residues. By western blotting and immunofluorescence, we revealed that BTV expresses this protein 

both in mammalian and insect cells and in experimentally infected mice. We also show that this novel protein, that we termed NS4, is 

a non structural protein. By confocal microscopy, we showed that NS4 is expressed early after infection and localizes in the nucleoli of 

BTV infected cells. By reverse genetics, we show that NS4 deletion mutants replicate in mammalian and insect cells as efficiently as wild 

type BTV. However, NS4 confers a replication advantage to BTV in cells treated with interferon. These data suggest that NS4 may play a 

fundamental role in virus-host interaction. 

Offered paper rNAseq analysis of the KSHV latent and lytic cycle in PEL 

DOMENEC FArrE1,2 , Anne Palser1 , Paul Kellam1,2 1 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge; University College London, London 

in order to study gene expression dynamics of Kaposi’s sarcoma herpesvirus (KSHV) and its host, we performed Transcriptomic Sequencing 

(rNA-Seq) using the illumina/SOlExA sequencing pipeline and developed methods to analyse the transcriptome of primary effusion 

lymphoma (PEl) cell lines at different times of the viral cycle, from latency to lytic state. 

Enrichment for polyadenylated rNA was used to reduce the quantity of ribosomal rNA present in the samples and paired-end sequencing 

was applied. Paired-end short read sequences were mapped, using MAQ and Bowtie, to the virus and human genomic sequences using 

TopHat and Cufflinks to directly obtain gene expression values. To correctly calculate the level of expression of the KSHV genes and 

transcripts, a new annotation of the complete transcripts of KSHV was manually generated after exhaustively searching the bibliography. 

We applied these methods to study different KSHV infected PEl cell lines (JSC-1, BCBl-1, BC3, HBl-6, CroAP-5, CroAP-6) in latency 

and in different time points after inducing lytic viral replication cycle (0h, 24h, 48h, 72h). We observe significant gene expression variability 

between the different PEl cell lines in latency, and dynamic changes in virus gene expression during lytic replication. 

Offered paper Identification of residues within domain II of the hepatitis C virus protein NS5A that are essential for the virus 

life cycle 

DOuGlAS rOSS-THriEPlAND, Mair Hughes, Mark Harris 


The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) is known to be essential for genome replication, assembly and release 

of infectious HCV virions, as well as perturbing numerous host pathways in favour of HCV persistence. Of the three identified domains 

of NS5A, domains i and iii have been shown to be involved in rNA replication and virion assembly respectively, whilst the function(s) 

of domain ii remains undefined. To address this deficiency, we have performed alanine scanning mutagenesis of the C-terminal region 

of domain ii in order to identify residues critical for HCV replication. We demonstrate that a large number of residues within this region 

are absolutely required for rNA replication in a sub-genomic replicon system. We have further characterized mutations that presented 

a normal replication phenotype in the context of the assembly or release of infectious virions. These data imply that domain ii of NS5A 

is required at multiple stages within the virus lifecycle and forms the foundation of further work aiming to elucidate the biochemical 

mechanisms underpinning these independent requirements. 




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Offered paper Expression and function of mir-199/214 cluster in cytomeglaovirus infection 

NOuF lAQTOM, Diwakar Santhakumar, Amy Buck 

University of Edinburgh, Edinburgh 

mir-199/214 cluster in CMV infection and demonstrated its broadly potent antiviral effect on the replication capacity of different 

herpseviruses (Santhakumar et al., PNAS 2010). However, the molecular mechanisms involved in mir-199/214 regulation have not been 

clear. Our recent data suggests that Twist-1 is an important regulator of pri-mir-199/214 cluster expression in CMV infection. Also, gene 

expression profiling of messengers rNAs that respond to mir-199-3p over-expression versus inhibitation demonstartes that this mirNA 

regulates genes involved in Pi3K/AKT and oxidative stress pathways. Here we report this new data, which suggests that the mir-199a/214 

cluster is regulated by transcription factors associated with innate immune signaling and controls cellular genes required during multiple 

stages of the cytomegalovirus life cycle. 

Offered paper Influenza A virus NS1 protein is required for efficient segment 7 mrNA nuclear export 

CAriNA PErEirA, Eliot read, Helen Wise, Maria Amorim, Paul Digard 


The rNA genome of influenza A virus is transcribed in the cell nucleus, necessitating nuclear export of viral mrNA. For some segments, 

this involves the cellular NxF1-dependent mrNA export pathway but how the viral mrNAs are recruited is unknown. 

using transfection to recreate viral rNPs with single vrNA templates, the requirements for segment 4 and 7 mrNA export were studied. 

Fluorescence in situ hybridization (FiSH) showed that under these circumstances, segment 4 mrNA accumulated in the cytoplasm, as seen 

in virus infected cells. However, segment 7 mrNA was largely retained in the nucleus, suggesting an additional viral protein was needed for 

its export. Addition of further viral proteins identified this as NS1 and showed that intact effector and rNA-binding domains were required 

for the activity. Analyses of segment 7 protein synthesis and mrNA splicing as well as FiSH with an intron-specific probe showed that NS1 

promoted efficient export of the unspliced M1 transcript. Furthermore, nuclear export of segment 7 mrNA was similarly defective in cells 

infected with NS1 mutant viruses. 

We conclude that NS1 is required for efficient export of M1 mrNA, potentially playing a role as a adaptor protein between the viral rNA 

synthesis machinery and cellular export pathway. 

HA15 Osmotic & oxidative stress responses 

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Environmental stress response pathways in yeast and its relatives 

Christoph Schüller 

Max F. Perutz Laboratories, University of Vienna 

Cells exposed to starvation or harmful conditions adjust the balance between protection and proliferation. This so-called environmental 

stress response has been explored in some detail in S. cerevisiae and relates to the observation that a large set of genes are co-ordinately 

activated under different acute environmental insults such as osmo-stress or nutrient starvation. in yeast Msn2 function as central 

transcriptional activators of ESr genes while specific stress response systems such as the Hog pathway or the Yap1/Skn7 factors function 

in parallel. Furthermore, starvation conditions change activity of growth and nutrient signaling systems such as PKA leading to activation 

of stress response and autophagy. These factors and mechanisms are also present in the opportunistic human fungal pathogen Candida 

glabrata, which is closely related to yeast. Which traits help C. glabrata to adapt to the host environment? The main differences seem to 

include an extended repertoire of adhesin genes, high drug resistance, an enhanced ability to sustain prolonged starvation and adaptations 

of the transcriptional wiring of stress response genes. We suggest that in C. glabrata the key regulatory mechanisms and genes providing 

stress protection are similar to yeast, but have been adopted to meet stress and starvation conditions possibly encountered in a hostpathogen 

confrontation. 

Macromolecule diffusion and confinement in prokaryotic cells 

BErT POOlMAN, Jacek Mika 

Dept of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands (Email: b.poolman@rug.nl; 

website: www.rug.nl/gbb/enzymology) 

recent observations on the mobility of macromolecules and their spatial organization in live bacterial cells will be presented. We outline 

the major fluorescence microscopy-based methods to determine the mobility and thus the diffusion coefficients (D) of molecules, which 




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is not trivial in small cells. The extremely high macromolecule crowding of prokaryotes is used to rationalize the reported lower diffusion 

coefficients as compared to eukaryotes, and we speculate on the nature of the barriers for diffusion observed for proteins (and mrNAs) 

in vivo. Building on in vitro experiments and modeling studies, we evaluate the size dependence of diffusion coefficients for macromolecules 

in vivo, both in case of water-soluble and integral membrane proteins. We comment on the possibilities of anomalous diffusion and provide 

examples where the macromolecule mobility may be limiting biological processes. 

References: Mika, J.T. & Poolman, B. (2010) Molecule diffusion and confinement in prokaryotic cells. Curr Opin Biotech 22, 117–126. 

Mika, J.T., van den Bogaart, G., Veenhoff, l., Krasnikov, V.V. & Poolman, B. (2010) Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress. Mol 

Microbiol, 77, 200–207. 

ramadurai S, Duurkens r., Krasnikov VV, & Poolmam B.(2010) lateral diffusion of membrane proteins: consequences of hydrophobic mismatch and lipid composition. Biophys J 99, 1482–1489. 

ramadurai S, Holt A, Schäfer lV, Krasnikov VV, rijkers DTS, Marrink SJ, Killian AJ & Poolman B (2010) influence of Hydrophobic Mismatch and Amino Acid Composition on the lateral 

Diffusion of Transmembrane Peptides. Biophys J 99, 1447–1454. 

ramadurai, S., Holt, A., Krasnikov, V., van den Bogaart, G., Killian, J.A. & Poolman, B. (2009) lateral diffusion of membrane proteins. J Am Chem Sci 131, 12650 

van den Bogaart, G., Hermans, N., Krasnikov, V., & Poolman, B. (2007) Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress. Mol. Microbiol 64, 858–871. 

Activation and termination of oxidant-responsive Yap1 transcription factor function 

W. Scott Moye-rowley 

Dept of Molecular Physiology & Biophysics, University of Iowa, Iowa City, Iowa 52242, USA 

The Saccharomyces cerevisiae bZip-containing transcription factor Yap1 is induced upon exposure to oxidants. Yap1 normally cycles 

between the cytoplasm and nucleus but rapidly accumulates in the nucleus during oxidative stress. Nuclear localization is the result of 

oxidant-specific folding of Yap1 that acts to prevent Yap1 association with the exportin Crm1. H O induces formation of two different 

2 2 

disulfide bonds that cause nuclear accumulation of Yap1. Formation of these bonds requires the presence of the glutathione peroxidase 

homologue Gpx3 and the presumptive chaperone protein Ybp1. These auxiliary factors are not necessary for Yap1 nuclear localization 

upon challenge with oxidants like diamide. We have found that Ybp1 appears to be a key determinant of the level of Yap1-dependent 

H O tolerance. Ybp1 serves to designate a pool of Yap1 that is competent to respond to H O stress while the remaining Ybp1-free 

2 2 2 2 

Yap1 mediates resistance to diamide. More recent experiments indicate that termination of Yap1 activity involves proteasome-mediated 

degradation the nucleus. Mutant forms of Yap1 that are constitutively localized to the nucleus are also subject to constitutive proteolysis. 

Our data indicate that oxidatively modified Yap1 is degraded after executing its role in positive transcriptional regulation. 

Dynamic regulation of hyperosmotic stress response in the budding yeast 

HAruO SAiTO, Kazuo Tatebayashi, Katsuyoshi Yamamoto, Keiichiro Tanaka, Hui-Yu Yang 

Institute of Medical Sciences, University of Tokyo, Tokyo, Japan 

When challenged with high external osmolarity, the budding yeast Saccharomyces cerevisiae initiates an adaptive program that includes: 1) 

synthesis and accumulation of the compatible osmolyte glycerol; 2) transient cell cycle arrest; 3) transient inhibition of protein synthesis; and 

4) a global change in gene expression pattern. These responses are controlled by the Hog1 MAP kinase (MAPK), which is activated by high 

osmolarity stimulus through the HOG (High Osmolarity Glycerol) signal pathway. Previously, we have shown that the HOG pathway can 

be activated by two alternative osmosensing mechanisms, termed the SlN1 branch and the SHO1 branch. A signal emanating from either 

branch converges on the Pbs2 MAPK kinase (MAPKK) that activates Hog1. 

The SlN1 branch employs the Histidine kinase-based signaling mechanism that is homologous to the bacterial two-component systems. in 

contrast, the working principle of the SHO1 branch remains relatively obscure. in this presentation, we will discuss our current view that the 

activity of the SHO1 branch is regulated by dynamic interactions among four transmembrane proteins, namely Hkr1 and Msb2 (the putative 

osmosensors), Sho1 (the membrane anchor for the Pbs2 MAPKK), and Opy2 (the membrane anchor for the Ste11 MAPKKK), as well as 

between these membrane proteins and their cytoplasmic partners. 

Oxidative stress-specific regulation of the fission yeast MAP kinase Sty1 by a mitochondrial phosphatase 

Yujun Di, Amna Butt, Keren Dawson, Nic Jones, CArOliNE r.M. WilKiNSON 

Paterson Institute for Cancer Research, Cell Regulation Group, University of Manchester, Wilmslow Road, Manchester M20 4BX 

(Email: njones@picr.man.ac.uk; cwilkinson@picr.man.ac.uk) 

Cells need to respond in an appropriate and timely manner to a broad spectrum of stress conditions. A key feature of stress responses is 

the mobilization of appropriate defence and repair mechanisms. in fission yeast, many of these responses are orchestrated through the Sty1 

MAP kinase. like all MAPK pathways, Sty1 signalling is highly regulated to control the magnitude and duration of signalling. This is achieved 

through co-ordination of positive and negative acting signals. Positive signals include stress conditions that result in the phosphorylation of 




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HA15 Cont. 

Sty1 and negative signals involve phosphatases that inactivate Sty1 through dephosphorylation. Ptc4 is a PP2C-family phosphatase which 

regulates both the magnitude and duration of Sty1 activation in response to H 2 O 2 but not other stresses. Surprisingly, Ptc4 localizes to 

the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS). A fraction of Sty1 also localizes to the 

mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of Ptc4’s MTS is greatly reduced 

specifically upon H 2 O 2 , resulting in the full length form of the phosphatase; this displays a stronger interaction with Sty1 thus suggesting a 

mechanism for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4. 

Osmoadaptation in Candida glabrata 

Ken Haynes 

University of Aberdeen, Imperial College London & University of Exeter 

The pathogenic yeast Candida glabrata is highly resistant to osmotic stress. This osmoadaptation, as in many fungi, is at least partially 

mediated by the stress-activated protein kinase Hog1. C. glabrata hog1 mutants exhibit reduced growth compared to wild-type cells in 

salt, but both accumulate internal glycerol to the same level. This demonstrates that internal glycerol accumulation alone is not sufficient 

for osmoadaptation. The question arises, how does Hog1 mediate osmoadaptation in C. glabrata? To answer this question a dynamic C. 

glabrata osmoadaption network was generated from time-series expression data. This along with conventional analysis of wild-type, hog1 

and ste11 expression data suggested that C. glabrata responds, at least in terms of regulated genes, to osmotic stress in a similar way 

to S. cerevisiae, but that the circuitry is re-wired (this was confirmed by 2-hybrid analysis). And additionally there is a C. glabrata specific 

component to the response. This was verified by phenotypic analysis of a C. glabrata transcription factor knock-out collection. These data 

show that two closely related species have solved the same biological problem in a different manner and adds further support to the 

growing view that transcriptional ‘wiring’ varies from specie to species. 

How does Escherichia coli insert Fe–S clusters into over 150 protein species? 

Frédéric Barras 

LCB-CNRS and Aix Marseille University, 31 Chemin Joseph Aiguier, Marseille 13009 France 

iron-sulfur (Fe-S) clusters are ubiquitous cofactors present in a myriad of proteins controlling processes as diverse as DNA replication, 

gene expression, photosynthesis, respiration and gene regulation. Their assembly and delivery into apo-proteins are catalysed by different 

multi-protein systems conserved throughout prokaryotes and eukaryotes. Because so many cellular processes are dependent upon Fe-S 

proteins, alteration of the Fe-S clusters, or of the systems that make them, has profound impact on cellular physiology. in particular, stresses 

such as oxidative and nitrous stresses, metal excess or iron limitation constitute adverse environmental conditions for Fe-S biosynthesis and/ 

or stability. remarkably, Escherichia coli possesses, iSC and SuF, the two best conserved Fe-S biogenesis systems. in E. coli, the iSC and SuF 

systems are thought to function under non-stress and stress conditions, respectively. in addition, ‘non-iSC, non-SuF’ components might 

intervene at different steps in the process. After a brief overview of the overall Fe-S biogenesis process, i will describe selected aspects of 

the delivery step and the role of a new actor, NfuA, a ‘non-iSC, non-SuF’ component. Altogether, these issues should permit a discussion of 

the mechanisms allowing E. coli to keep making and distributing Fe-S clusters under fluctuating conditions, including stressful ones. 

Offered paper The impact of combinations of osmotic and oxidative stresses upon the pathogenic yeast Candida albicans 

DESPOiNA KAlOriTi1 , Mette Jacobsen1 , Anna Tillmann1 , Miranda Patterson2 , Ken Haynes3 , Jan Quinn2 , Neil Gow1 , Al Brown1 1 2 School of Medical Sciences, University of Aberdeen, Aberdeen; Faculty of Medical Sciences, Newcastle University, Newcastle; 

3Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter 

Stress responses are important for Candida virulence. responses to individual stresses have been studied in considerable detail. However, 

following contact with its human host, Candida is often exposed simultaneously to combinations of stress. Our aim is to define the 

impact of such combinatorial stress responses upon Candida albicans. The initial focus is on osmotic and oxidative stress responses. Our 

hypothesis is that simultaneous exposure to these stresses might cause complex interactions between the osmotic and oxidative stress 

signalling pathways, leading to antagonistic or synergistic effects upon Candida viability. As predicted, flow cytometry-based assays revealed 

synergistic effects of osmotic and oxidative stresses upon C. albicans killing. Further investigation of the molecular mechanisms that underlie 

these combinatorial stress responses was performed using microarrays. This genome-wide expression profiling has suggested interactions 

between the osmotic and oxidative stress signalling pathways. This view has been reinforced by examining directly the activation of key 

stress regulators in response to combinatorial stresses. in conclusion, we show for the first time that combinations of medically relevant 

stresses have a major impact upon C. albicans, exerting synergistic effects upon the killing of this pathogen. 




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Offered paper The osmotic stress response of Listeria monocytogenes: role and regulation of the stress-inducible sigma factor 

Sigb 

CONOr O’BYrNE, Emily Starr, Marta utratna 

NUI Galway, Galway, Ireland 

Listeria monocytogenes is a highly adaptable pathogen of humans that can tolerate a wide range of physical and chemical stresses. its 

tolerance to osmotic stress is underpinned by an osmoregulatory system that includes three compatible solute transporters. The alternative 

stress-inducible sigma factor SigB (σB ) is central to the osmotic stress response of L. monocytogenes, partly because of its role in regulating 

the expression of these compatible solute uptake systems. in the present study we used a combination of proteomics and transcriptomics 

to determine the overlap between the osmotic stress stimulon and the σB regulon in L. monocytogenes. Although the osmotic stress 

stimulon is large (>200 genes) a surprisingly small number of genes (30) were induced by osmotic stress in a σB-dependent manner. 

Analysis of transcript levels in response to increasing levels of NaCl showed that σB-dependent gene expression is induced in proportion to 

the extent of the osmotic stress. The activation of σB in response to osmotic upshock was found to be rapid but transient. Together these 

data highlight the important role played by σB in protecting L. monocytogenes against osmotic stress and give new insights into the regulation 

of σB itself. 

Physiological proteomics and stress/starvation responses in Gram-positive bacteria 

Michael Hecker 

Universität Greifswald, Institut für Mikrobiologie, F.-L.-Jahn-Straße 15, D-17487 Greifswald, Germany 

Proteomics is an excellent tool to ‘bring the genome sequence to cell physiology’. Because of their low complexity bacteria are useful 

model systems to transfer the ‘virtual life of the genes to the real life of the proteins’ shown for our model bacteria Bacillus subtilis and 

Staphylococcus aureus. using a reasonable combination of gel-based and gel-free approaches almost 70 to 80% of the proteins expressed 

under specific circumstances can be detected and quantified. This entire proteome is ready for physiological applications. in the second part 

of the talk it will be shown that a combination of gel-based and gel-free proteomics is a powerful tool to address physiological issues. This 

will be shown for heat and oxidative stress in Bacillus subtilis and for oxygen starvation in Staphylococcus aureus. Proteomic can be used to 

define the structure of regulons, their integration into gene expression networks, to visualize protein damage and protection by oxidative 

stress and finally to elucidate signal transduction networks from the stress stimulus to gene expression. 

HA16 Virology workshop: virus structure & assembly 

↑Contents 

Offered paper How to build a coronavirus 

Benjamin W. Neuman1 , GABriEllA KiSS1 , Andreas H. Kunding3 , David Bhella6 , M. Fazil Baksh1 , Stephen Connelly2 , 

Ben Droese2 , Joseph P. Klaus9 , Shinji Makino5 , Stanley G. Sawicki7 , Stuart G. Siddell8 , Dimitrios Stamou3 , ian A. Wilson2 , 

Peter Kuhn2 , Michael J. Buchmeier4 1 2 3 University of Reading, Reading, Berkshire; The Scripps Research Institute, La Jolla, CA, USA; The University of Copenhagen, 

Coppenhaga, Denmark; 4The University of California, Irvine, CA, USA; 5The University of Texas, Medical Branch, Galveston, Texas, 

USA; 6Medical Research Council Virology Unit, Glasgow; 7University of Toledo, Ohio, USA; 8University of Bristol, Bristol; 9University of Vermont, Vermont, USA 

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host 

factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size 

using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations 

and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a 

relatively narrow range of membrane curvature. in contrast, compact M protein is associated with flexibility and low spike density. Analysis 

of several types of virus-like particles and virions revealed that S protein, N protein and genomic rNA each help to regulate virion size 

and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus 

assembly. 




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HA16 Cont. 

Offered paper Cryo-EM reconstructions of equine rhinitis A virus (ErAV) reveal a putative disassembly intermediate 

SASKiA BAKKEr, Arwen Pearson, Peter Stockley, David rowlands, Neil ranson 

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds 

Equine rhinitis A virus (ErAV), a member of the Aphtovirus genus of the Picornaviridae, is closely related to foot-and-mouth disease virus 

(FMDV). 

using cryo-EM and single-particle image processing with icosahedral averaging, we have made 3D reconstructions of native ErAV and 

an empty particle. Our model of the native virus corresponds well to the published crystal structure. Additional density is present in the 

interior of the capsid, which can be attributed to the rNA genome. 

The empty particle shows an ~20% expansion compared to the native particle and exhibits large pores in the capsid. Twelve pentamers 

of the low-pH crystal structure could be fitted into the electron density map as rigid bodies. The resulting model suggests that the interpentamer 

contacts are mediated by hydrophobic and electrostatic interactions at the vertices of the pentamers. 

Although the formation of empty (i.e. lacking rNA) particles from mature virions has been described for both ErAV and FMDV, the 

mechanism for the ejection of the rNA is not known. it is possible that the particle described here is a disassembly intermediate with a 

crucial role in the cell entry process. 

Offered paper Picornavirus–host cell interactions elucidated by cryo-electron microscopy and image reconstruction 

Jani Seitsonen1 , Petri Susi2 , Outi Heikkilä2 , Pasi laurinmäki1 , robert Sinkovits3 , Timo Hyppiä2 , SArAH BuTCHEr1 1 2 3 University of Helsinki, Helsinki, Finland; University of Turku, Helsinki, Finland; University of California, San Diego, USA 

Structural analysis combined with functional characterization has considerably improved our ability to study and understand the interactions 

between molecules that drive biological processes. The study of host cell to virus interactions, for instance, can be used to inform the 

development of antiviral drugs and to limit the damage caused by the host cell response. We have studied the structure and interactions 

of human parechovirus and coxsackievirus with cellular integrins by biochemical assays, and cryo-electron microscopy and image 

reconstruction to subnanometer resolution. Hence we can define the interaction sites and provide a probable basis for our observed 

differences in binding of the viruses to two different integrins. 

Offered paper Single molecule studies of rNA genome encapsidation 

AlExANDEr BOrODAVKA, roman Tuma, Tomas Wilkop, Peter Stockley 


Single stranded rNA (ssrNA) viruses are complex rNA processing systems that perform packaging, replication and transcription of their 

genomes. We have developed a single molecule method based on fluorescence correlation spectroscopy (smFCS) for monitoring virus 

assembly in real time in vitro. The method was tested on small ssrNA bacteriophage MS2 using fluorescently labeled coat protein. This 

allowed us to monitor kinetics of virus assembly and to delineate effect of the rNA length using subgenomic rNAs. By using labeled 

subgenomic and full genomic rNAs we can now follow folding and encapsidation of these long rNA molecules in real time. 

The kinetics of encapsidation of the three tested subgenomic rNAs differs substantially and refolding of the viral rNA appears to be a 

prerequisite for successful packaging. smFCS yields comparable results irrespective whether the coat protein or the small rNA that triggers 

capsid assembly is labelled. 

Thus smFCS constitutes a versatile technique for studying virus assembly and folding of large rNA molecules during encapsidation. 

This work was funded by the Wellcome Trust Fund and supported by the university of leeds. 

Offered paper A Hamiltonian path approach to rNA virus assembly 

NiCHOlAS GrAYSON, Eric Dykeman, reidun Twarock 

University of York, York 

A significant number of rNA viruses assemble their protein containers and genomic material simultaneously. Proposed here is an assembly 

model for rNA viruses where the interactions between the rNA and the capsid proteins play a pivotal role in the assembly process. We 

demonstrate our theory for bacteriophage MS2 which has a capsid formed from 90 protein dimers that have two types of interactions with 

the rNA. We show that different assembly pathways correspond, from a mathematical point of view, to Hamiltonian paths of different 

lengths on an rNA cage within the capsid. information on these Hamiltonian paths is then incorporated into tile assembly models. We use 

these models to make predictions on the relative concentrations of the assembly intermediates. 




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HA16 Cont. 

Offered paper High-throughput SHAPE analysis reveals a structural switch in the feline immunodeficiency virus packaging 

signal rNA 

JuliA KENYON1 , Sian Tanner1 , Michal legiewicz2 , Pretty Phillip3 , Tahir rizvi3 , Stuart le Grice2 , Andrew lever1 1 2 University of Cambridge Dept of Medicine, Level 5 Addenbrookes Hospital, Hills Rd, Cambridge CB2 0QQ; RT Biochemistry Section, 

HIV Drug Resistance Program, National Cancer Institute, Frederick, MD, USA; 3Depts of Microbiology and Immunology, Faculty of 

Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates 

Feline immunodeficiency virus (FiV) infects many species of cat and causes an AiDS-like illness. it is a potential non-primate model for HiV 

and is being developed as a gene therapy vector for use in humans, yet little is known of its molecular biology. 

unspliced retroviral rNA acts as the viral genome and also encodes viral proteins. The virus must distinguish between full-length viral 

rNA and spliced viral and cellular rNAs in order to package two copies of its genome into each virion, and it does this by recognition of 

packaging signals within the genomic rNA. in FiV these are known to be within two regions at the 5’end of the genome. 

Here, we have examined the rNA packaging signal structure by high-throughput SHAPE (selective 2’hydroxyl acylation analysed by primer 

extension) and mutational analysis and show that the monomeric packaging signal rNA is present in two conformations; one of which has a 

lower free energy and promotes dimerization of the rNA; the other has a higher free energy and a more accessible AuG. Based on these 

structures, we propose a model for viral dimerization and assembly. 

Offered paper Lethal combinations of packaging signal mutations suggest a genome assembly checkpoint during influenza A 

virus morphogenesis 

KATEriNE KuDrYAVTSEVA, Helen Wise, Edward Hutchinson, Julia Gog, Paul Digard 


influenza A virions incorporate one copy of each of the eight genomic segments, which is thought to be achieved through segment-specific 

packaging signals located in the terminal regions of the vrNAs. The mechanism by which these signals function is uncertain, but their 

mutation can lead to drops in virus titres of ~10–1,000-fold. 

We performed a systematic analysis of pairwise combinations of packaging mutations in different segments and, surprisingly, we found that 

most, but not all double mutants exhibited a synergistic reduction of over 10,000-fold in virus growth with certain combinations resulting in 

a lethal phenotype. The extent of the defect suggests that random packaging cannot compensate for this deficiency and that a checkpoint 

prevents assembly of virions with grossly defective genomes. 

To test these hypotheses, we are currently studying several highly attenuated packaging double mutants. initial analysis suggests that the 

block to efficient virus replication occurs post nuclear export of the genome but before virus budding. Moreover, it has been possible to 

isolate revertant viruses from serial passage that maintain the initial lesions. interestingly, none of the suppressor mutations were located in 

the previously identified packaging signals. 

Offered paper In vitro reconstitution of bTV rNA–protein complexes and core assembly pathway 

SOFiA lOurENCO, Polly roy 

Dept of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 

London 

x-ray crystallography and electron cryo-microscopy of Bluetongue virus (BTV) particles revealed several aspects regarding the organization 

and structure of the capsid. BTV is a non-enveloped virus possessing two capsids: composed of VP2-VP5 (outer capsid) and VP3-VP7 

(inner capsid or core), the later one enclosing the replication complex formed by VP1 (rNA polymerase), VP4 (capping enzyme) and VP6 

(helicase), protecting the 10 dsrNA genomic segments. However, the assembly and packaging mechanisms remain unclear. 

in this study, we established a novel tool to reconstitute BTV rNA-protein complex in a cell-free system and have used the system to 

investigate the requirements and order of core assembly pathway. The data obtained indicated that the rNA is the driving force for 

the assembly of the replicase complex and its absence affects strongly core assembly. Furthermore, we demonstrate that the in vitro 

reconstituted cores were infectious in insect cells and were capable to synthesise BTV proteins. The system can be used for further studies 

on mechanisms of rNA-protein interactions during BTV core assembly and in identification of rNA packaging signals. 




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HA16 Cont. 

Offered paper Entry of HSV-1 and the role of the major structural protein VP1-2 

FErNANDO ABAiTuA1 , Torhu Daikoku2 , Colin M. Crump3 , Marianne Bolstad1 , Peter O’Hare1 1 2 Section of Virology, Faculty of Medicine, Imperial College London, London; Dept of Virology, University of Toyama, Toyama, Japan; 

3Division of Virology, University of Cambridge, Cambridge 

Herpesvirus VP1-2 (ul36) is a structural protein assembled into the tegument and essential both for entry and assembly. Evidence for 

multiple critical roles of VP1-2 originated from analysis of the temperature-sensitive mutant tsB7. At non-permissive temperature, tsB7 

capsids accumulate at the nuclear pore with defective genome release and little virus gene expression. We identified the amino acid 

alterations in VP1-2 responsible for tsB7 defect and constructed a virus with a single residue change in VP1-2. This single change confers 

a temperature dependent 4–5-log reduction in plaquing efficiency and distinct entry and assembly defects. in further analysis of VP1-2 we 

have identified a highly conserved basic motif within the N-terminal region. This motif functions as a Nuclear localization Signal (NlS), and 

when deleted completely abrogates complementation of a ul36-ve virus. We have now constructed a virus lacking just this basic motif. 

in non-complementing lines there is a profound reduction in plaquing efficiency and essentially no virus production. Both the ts and NlS 

deletion viruses show a similar cell-type dependent effect in infection, producing abnormal virus-induced clusters in certain cells, which may 

reflect alternate modes of entry, differences in cell survival and division, or immune signalling triggered by the restricted infection. 

Offered paper The interactions between herpes simplex virus type 1 tegument and human ESCrT machinery 

YuDAN rEN1 , Susanne rauch2 , Susanne Bell1 , Juan Martin-Serrano2 , Colin Crump1 1 2 Division of Virology, Dept of Pathology, University of Cambridge, Cambridge; Dept of Infectious Diseases, King’s College London, 

London 

Mature herpes simplex virus type 1 (HSV-1) particles are formed by the budding of tegumented viral capsids into the lumen of cytoplasmic 

membrane compartments during secondary envelopment. The budding and scission of the viral envelope from the host membrane share 

topological similarities with the formation of intraluminal vesicle in multivesicular bodies, a process requiring the cellular Endosomal Sorting 

Complexes required for Transport (ESCrT) machinery. There are four multiprotein complexes and many associated proteins that function 

in ESCrT-mediated membrane budding. it has been previously found in our lab that VPS4 and the ESCrT-iii complex are required for 

HSV-1 envelopment. However, the direct interactions between viral and host cell proteins that mediate ESCrT recruitment are unknown. 

To identify protein-protein interactions, 25 HSV-1 tegument proteins were tested against a panel of ESCrT and ESCrT-associated proteins 

in yeast two-hydrid assays. GST-pulldown and protein co-localization assays were performed to confirm interactions. Defects in HSV-1 

production caused by the depletion of identified cellular proteins using sirNA were analysed. Our data suggest that HSV-1 has evolved 

multiple mechanisms to hijack this essential membrane scission machinery to facilitate its assembly. 

Offered paper The inner tegument protein, pUL36, attaches to the capsid verticies in HSV-1 virions 

WAN HO FAN1 , Ashley P. roberts3 , David Pasdeloup2 , David Bhella1 , Frazer J. rixon1 1 2 MRC-University of Glasgow Centre for Virus Research, Glasgow; Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, 

France; 3Centre for Biomolecular Sciences, University of Nottingham, Nottingham 

Structural analysis of the Herpes Simplex Virus type 1 (HSV-1) virion has revealed that the capsid is connected to the surrounding 

proteinaceous tegument layer through connections at the capsid vertices. The tegument proteins that form these connections are 

unidentified, although large protein pul36 (330kDa) has been proposed as a candidate based on its close association with the capsid 

during the initial stages of infection and its role in virion maturation and envelopment. 

pul36 interacts with a second tegument protein, pul37, and virus mutants lacking either of these inner tegument proteins are blocked 

for full virion assembly; accumulating as capsids in the cytoplasm of infected cells. We have determined the structure of DNA containing 

cytoplasmic capsids produced by these mutants using cryo-EM and 3D reconstrustions. The tegument density at the capsid verticies was 

present in pul37 minus capsids but missing in capsids from the pul36 mutant. Analysis of the mutant capsid compositions strongly support 

the identification of pul36 as the major constituent of the vertex associated tegument. This result identifies pul36 as a major component 

of the capsid-tegument interface and accounts for its essential role in tegument addition and virion maturation. 




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HA16 Cont. 

Offered paper Characterization of a herpesvirus multi-component glycoprotein–tegument complex involved in virion 

assembly 

KEViN MAriNGEr, Gill Elliott 

Imperial College London, London 

The herpes simplex virus (HSV) particle consists of the viral genome-containing capsid surrounded by a structure called the tegument, 

which contains up to 23 virus-encoded proteins, and the viral glycoprotein-containing envelope. While the molecular interactions 

involved in tegument assembly and envelopment remain poorly defined, it is proposed that at least some tegument proteins are recruited 

to the virion by binding the cytoplasmic tails of envelope glycoproteins. Previous work by our lab and others demonstrated that the 

major tegument protein VP22 interacts with glycoproteins E and M. Furthermore, pull-down of VP22 from infected cells specifically coprecipitates 

gE and gM together with two other tegument proteins, VP16 and iCP0. We have now investigated the role of this protein 

network in virion assembly and show that VP22 can be packaged into the virion by gE or gM. However, iCP0 requires all three proteins 

to be assembled. These four proteins form a discrete complex in infected cells, with VP22 required to bridge the two glycoproteins, an 

interaction that enables iCP0 recruitment. interestingly, VP16 plays no part in this complex despite being a major binding partner of VP22. 

Therefore, this work reveals the first distinct multi-component complex involved in HSV tegument-envelope assembly. 

Offered paper Identification and isolation of intermediates in the assembly of hepatitis b core particles and use of core 

particles composed of fused dimers for antigen presentation 

KriS HOlMES1 , Dale Shepherd1 , David rowlands1 , robert Gilbert2 , Nicola Stonehouse1 1 2 University of Leeds, Leeds; University of Oxford, Oxford 

The hepatitis B virus particle consists of a nucleocapsid comprising the DNA genome and the core antigen (HBcAg) surrounded by a 

lipoprotein envelope including surface antigen molecules (sAg). HBcAg assembles into virus like particles (VlPs) composed of 180 or 

240 dimers with T=3 or T=4 symmetry, respectively, however, the assembly pathway is not fully understood. We have been able to 

disassemble and reassemble HBcAg VlPs and have identified three assembly intermediates using mass spectrometry. Furthermore, the 

use of fused HBcAg dimers (tethered via a GGS repeat linker) results in the presence of more stable intermediates allowing isolation. This 

fused dimer system is being developed as an antigen presentation platform, in collaboration with iQurltd. introduction of the linker results 

in VlPs isomorphic to wild type in addition to larger assemblies. Variability in VlP size can be reduced by linker shortening and morphology 

improved after disassembly/reassembly. 

Fused dimers incorporating part of the sAg have been expressed to form VlPs that can be classified into three sizes by cryoEM. Two 

size classes possess icosahedral symmetry but the third has octahedral symmetry. Surface antigen alone can self-assemble into octahedral 

particles, raising the possibility of interplay between the assembly properties of the two viral proteins. 

Offered paper Understanding viral budding: the structure of human respiratory syncytial virus matrix protein 

ViCTOriA MONEY, Helen McPhee, Scott Watson, John Sanderson, Paul Yeo 

Durham University, Durham 

Human respiratory Syncytial Virus (hrSV) is a negative-sense, single-stranded rNA virus from the viral order Mononegavirales, which 

contains some of the most economically and socially important human pathogens including: measles, mumps and Ebola. The matrix protein 

(M) forms a layer between the viral holo-nucleocapid and the host-derived lipid bilayer envelope. M is responsible for a number of critical 

roles in the viral replication-cycle among which is the recruitment of host proteins to the viral envelope and the initiation of the budding 

and the final assembly process. 

We have solved the three-dimensional structure of hrSV M to a resolution of 1.6 Å by x-ray crystallography. This is the first structure of a 

full-length matrix protein from the Mononegavirales and provides a mechanism for driving association with the negatively charged lung cell 

membrane. in addition, the presence of a flexible linker region between the two domains of the protein demonstrates the possibility of that 

M may adopt different domain orientations according to the specific role it is required to play at any particular time. 

The results of tensiometry measurements which cast light on the nature of the interaction of the matrix protein with the cell membrane will 

be presented. 




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Offered paper Unravelling lactococcal phage baseplate assembly by mass spectrometry 

DAlE SHEPHErD1 , David Veesler2 , Julie lichière2 , Nicola Stonehouse1 , Alison Ashcroft1 , Christian Cambillau2 1 2 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds; Architecture et Fonction des Macromolécules Biologiques 

(AFMB), UMR 6098 CNRS and Universités Aix-Marseille I & II, Campus de Luminy, Marseille, France 

lactococcal Phages infect Lactococcus lactis, a bacterium used extensively in the dairy industry. They are composed of a capsid head, a 

long non-contractile tail and a large structure at the end of the tail termed a baseplate, which mediates adhesion to the bacterial host. 

High resolution techniques such as CryoEM and x-ray crystallography have been used previously to study baseplate structure. However, 

with these techniques it has not been possible to study dynamic processes in solution, for example assembly processes. We have used 

Electrospray ionization Mass Spectrometry (ESi-MS) to study the baseplate complexes of the p2 and TP901-1 lactococcal phages. Due to 

the ability of ESi-MS to analyse heterogeneous mixtures of proteins in a single experiment, analysis of the complete organelles, as well as 

some key subcomplexes, has been undertaken. The mass and stoichiometries of the intact complexes have been confirmed, and analysis 

of subcomplexes has allowed the low resolution mapping of protein interfaces within the baseplates. These data, together with information 

from electron microscopy and crystallography, has led to a proposed lactococcal phage baseplate assembly mechanism. 

Offered paper The abundant generation in plants of empty virus-like particles suitable for bionanotechnological applications 

KEiTH SAuNDErS, Pooja Saxena, Paolo lenzi, Eva Thuenemann, George lomonossoff 

John Innes Centre, Norwich 

Knowledge concerning the structure, stability, and assembly of Cowpea mosaic virus (CPMV) particles has been obtained due to the 

ready generation of these particles via infection of plants. Such particles, and those modified through genetic engineering or by chemical 

conjugation, are now being developed for use in many bionanotechnological applications. However the uses of such particles maybe limited 

by the fact that they contain viral nucleic acid and therefore retain their ability to infect plants and spread in the environment. Furthermore, 

presence of nucleic acid within the particles precludes them being loaded with heterologous material which would be desirable for certain 

applications. 

We are now able to generate rNA-free (empty) CPMV virus-like particles in plants. The procedure involves the co-expression of the coat 

protein precursor (VP60) with its cognate proteinase (24K), the cleavage of the former by the latter resulting in the abundant production 

of empty virus-like particles. Particles created are devoid of nucleic acid and hence there are minimal bio-safety concerns involved with their 

use. in addition the space within the particles is now available for the encapsulation of chemical moieties such as drugs, imaging agents and 

metal ions, thus extending the potential uses of CPMV in bionanotechnology. 

HA17 Virology workshop:cell-to-cell transmission 

↑Contents 

Offered paper The HTLV-1 virological synapse 

MOHAMED NEJMEDDiNE1 , isabelle Clerc1 , Yuetsu Tanaka2 , Graham P. Taylor1 , Charles r.M. Bangham1 1 2 Imperial College London, London; Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan 

Human lymphotropic virus type-1 (HTlV-1) is a complex retrovirus. Naturally infected lymphocytes produce virtually no cell-free HTlV-1 

particles in vivo, and cell contact is required for efficient transmission of HTlV-1 between cells and between individuals. HTlV-1 spreads 

efficiently between T-cells via a tight and highly organized cell-to-cell contact known as the virological synapse (VS) by analogy to the 

immunological synapse. The polarization of the microtubule-organizing center (MTOC), in the infected cell toward the target cell, is an 

important feature for the establishment of a functional VS. This polarization is induced by a synergistic combination of signals from HTlV-1 

Tax protein within the cell and from cross-linking of adhesion molecules at the cell surface. it is now clear that other retroviruses, including 

the human immunodeficiency virus type-1 (HiV-1) and murine leukemia virus (MlV), also spread efficiently by cell-to-cell contact via a 

virological synapse. However, the mechanisms by which the virions are delivered to the VS and the role played by virus proteins is still 

not completely understood. i shall review here the recent advances on HTlV-1 VS and present new data on the contribution of HTlV-1 

proteins to the formation of the VS and the role of the actin cytoskeleton. 




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Offered paper Measles virus transmission from infected dendritic cells to T cells in DC/T cell conjugates 

SuSANNE KOETHE, Sibylle Schneider-Schaulies 

Institute of Immunology & Virology, University of Wuerzburg, Wuerzburg, Germany 

Dendritic cells (DC) act as sentinels for sensing invading pathogens. Measles virus (MV) exploits dendritic cells as Trojan horses for accessing 

secondary lymphoid organs where transmission to T cells is accomplished. Within DC/T cell conjugates organized transmission sites are 

formed, referred to as virological synapses, where viral structural proteins and entry receptors accumulate. DCs support MV infection in 

vitro (MV-DCs) and in vivo and this promotes maturation accompanied by mobilization. Expectedly, the frequency of autologous MV-DC/T 

cell conjugates in vitro is low, yet can be increased in number on the amount of T cells conjugated per MV-DC upon pre-activation of T 

cells. MV proteins accumulate in MV-DCs and their subcellular redistribution towards the contact site is currently analysed. CD150, the MV 

receptor, is upregulated on activated T cells and on MV-DCs, yet massive redistribution towards the contact site does not occur. However, 

inclusion of CD150 specific antibodies efficiently reduced MV transmission to T cells. Though this may relate to direct block of MV entry 

into these cells, it may indicate that homotypic or heterotypic (with MV H protein) CD150 interaction may act to stabilize conjugate 

stability which, in the absence of antigen recognition, is low. This hypothesis is currently analysed. 

Offered paper Cell-to-cell fusion properties of the infectious bronchitis virus (IbV) spike glycoprotein 

EriCA BiCKErTON, Paul Britton 

Institute for Animal Health, Compton 

The spike (S) glycoprotein of iBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular 

tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in cell-to-cell fusion. The iBV Beau-r 

strain is able to replicate in both primary chick kidney cells and Vero cells but does not cause syncytia. Beau-r was further adapted to 

syncytia formation on Vero cells by serial passage and syncytia started to form after nine passages (P ). This new growth phenotype 

9 

was initially identified by brightfield microscopy and subsequently by confocal microscopy using indirect immunofluorescence. The P10 virus population was sequenced and deep amplicon sequencing was carried out using three Beau-r P isolates in order to compare the 

10 

adapted Beau-r S gene sequences to the non-adapted Beau-r S gene sequences with the aim of determining which amino acids of the 

S glycoprotein are involved in cell-cell fusion. Sequence analysis showed that there were many amino acid differences however, few were 

shared between isolates. This indicated that syncytia formation results from multiple amino acid changes and that no simple or common 

changes are responsible for this phenomenon. 

Offered paper Delineating routes of HCV infection: cellular and viral factors that define cell-to-cell transmission 

luKE MErEDiTH1 , Garrick Wilson1 , Helen Harris1 , Claire Brimacombe1 , Joe Grove2 , Peter Balfe1 , Jane McKeating1 1 2 HCV Research Group, Institute for Biomedical Research, University of Birmingham, Birmingham; MRC Laboratory for Molecular Cell 

Biology, University College London, London 

Hepatitis C virus (HCV) is an important human pathogen of the Flaviviridae family that induces a chronic infection in 3% of the world’s 

population. The major reservoir supporting HCV replication is hepatocytes and infected subjects develop progressive liver disease. HCV 

initiates infection by cell-free particle diffusion or via direct cell-to-cell transfer, where the latter confers resistance to neutralizing antibodies 

targeting the viral glycoproteins. Cell-to-cell infection potentiates the localized spread of genetically distinct viruses in the liver, providing 

a strategy for HCV to escape anti-viral therapies. recombinant viruses expressing glycoproteins cloned from chronically infected subjects 

show variable dependency on scavenger receptor Bi that limits cell-to-cell spread, allowing the delineation of glycoprotein residues that 

define routes of viral dissemination. Our recent observation that hepatocellular scavenger receptor Bi is low and heterogeneous in the 

human liver support a role for this viral receptor in limiting HCV spread within the liver and transmission between hosts. These studies 

increase our understanding of viral mechanisms driving HCV escape from host immune responses and anti-viral therapies. 

Offered paper Influence of phosphoinositides on bluetongue virus assembly and maturation 

BiSHNuPriYA BHATTACHArYA, Polly roy 


Bluetongue virus (BTV) is an insect-borne orbivirus within the family Reoviridae. BTV may cause high morbidity and mortality in ruminants. 

The virion is composed of seven discrete proteins organized into two concentric capsids and ten segmented double-stranded rNA 

genome. We have shown previously that one of the outer capsid proteins, VP5 and NS3, the only glycosylated non-structural protein 

encoded by BTV co-segregate with membrane raft domains. in this study we have examined the role of lipid raft, focusing particularly 




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on phosphatidylinositol (4,5) bisphosphate [Pi(4,5)P 2 ], a predominantly plasma membrane residing phosphoinositide that has also been 

implicated in vesicle formation and trafficking. The possibility of this lipid influencing BTV production was explored by overexpressing 

cellular enzymes that either depletes cellular Pi(4,5)P2, or induces the formation of Pi(4,5)P2-enriched endosomal structures. The data 

obtained from these studies indicated perturbation in virus production and showed altered localization of the viral proteins, in BTV 

infected cells. This is the first demonstration of the essential role of negatively charged membrane lipid molecules in the later stages of virus 

replication of non-enveloped viruses in infected cells. 

Offered paper Interaction of calpactin light chain (S100A10/p11) and NS3 protein is essential for intracellular trafficking of 

bluetongue virus 

CriSTiNA CElMA, Polly roy 


Bluetongue virus (BTV) particles, in addition to cell lysis, are also released from the plasma membrane of infected cells by budding. The 

NS3 viral protein has been implicated in this process. NS3 is expressed as a full-length protein and as a shorter NS3A that lacks the first 13 

residues. Previously we reported that NS3 interacts with the cellular trafficking protein S100A10/p1 via its N-terminal end. in this report by 

using reverse genetics system we investigated the role of NS3 and NS3A on virus release, both in mammalian and insect vector-derived 

cells. Our data showed that a mutant virus expressing only NS3 was still able to bud from mammalian cells; however, when expressing 

only NS3A, BTV particles were arrested in the cytoplasm. Further, three single substitution mutations in the N-terminal domain had similar 

drastic effects on virus release. in contrast, while at late stages of infection, the wild-type particles in insect cells were associated within 

cytoplasmic vesicles, the mutant viruses were scattered throughout the cytoplasm, but not confined within the vesicles. These results 

suggest that the release of BTV from mammalian and insect cells occurs through different mechanisms. Our data supports the role of 

S100A10/p11 in BTV trafficking 

Offered paper Adaptations required of avian influenza NAs to facilitate human cell-to-cell transmission 

DEENA BluMENKrANTZ, Neeltje van Doremalen, Holly Shelton, Kim roberts, Wendy Barclay 


Introduction: influenza virus’ mechanism of cell-to-cell transmission requires efficient apical release followed by penetration of the airway 

mucosal barrier. Neuraminidase (NA), a sialidase expressed on the surface of infected cells and incorporated into the viral envelope, must 

cleave sialic acid (SA) from glycoproteins both on the cell’s surface and in secreted mucosa to allow infection of naive cells. SA linkages 

differ between humans and the influenza virus’ natural host, birds. Therefore to cause a pandemic, the NA of a virus must adapt to cut 

through human barriers. 

Aim: To identify adaptations required of avian influenza NAs that facilitate human cell-to-cell transmission. 

Methods: reassortant viruses with 7 genes from a recent pandemic strain were combined with various NAs from different hosts. inhibition 

of viral infectivity by mucus was determined by plaque assay, NA activity at the cell surface was measured by FACS and viral spread across 

differentiated airway epithelial cells was imaged. 

Results: Viruses with avian-adapted NAs showed a significant reduction in activity compared to viruses with human-adapted NAs indicating 

the shorter NA stalk in poultry viruses was partly responsible. Conclusion: The next pandemic influenza virus will probably not contain a 

NA gene from a virus that circulated in gallinaceous poultry. 

Offered paper Structure, function and evolution of nidovirus haemagglutinin esterases; molecular basis for sialic acid subtype 

preference 

Martijn A. langereis, Stephin Vervoort, Qinghong Zeng, Gerrit J. Gerwig, Eric G. Huizinga, Johannis P. Kamerling, 

rAOul J. DE GrOOT 

Utrecht University, Utrecht, Netherlands 

Viruses that use sialic acids (Sias) as receptor determinant for host cell attachment come with a treasure trove of lectins and sugar-modifying 

enzymes. For example, toro- and coronaviruses (family Coronaviridae, order Nidovirales) possess envelope glycoproteins, hemagglutininesterases 

(HEs), that mediate reversible virion attachment to O-acetylated Sias. The HEs do so by acting both as lectins and as receptordestroying 

enzymes (sialate-O-acetylesterases), functions exerted by separate protein domains. Originating from (an) influenza C-like 

hemagglutinin-esterase fusion protein(s), the HEs were added to the corona- and torovirus proteomes relatively recently through separate 

horizontal gene transfer events. We demonstrate that HEs differ in their fine specificity towards distinct O-acetylated Sia species and that in 




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each case lectin ligand specificity and sialate-O-acetylesterase substrate preference are in perfect accord, indicative for co-evolution of the HE 

lectin and esterase domains. Structure-guided mutational analyses of esterase catalytic pockets and receptor-binding sites provide insight into 

the molecular basis for substrate binding, substrate recognition and receptor selection in this important class of virion proteins. Although at 

present the biological significance of HE fine specificity is not known, Sia subtype preference seemingly correlates with viral host tropism. 

Offered paper Contrasting effects of tetherin on the growth of CD134-dependent and CD134-independent strains of feline 

immunodeficiency virus 

isabelle Dietrich1 , Elizabeth McMonagle1 , Greg Towers2 , Margaret Hosie1 , BriAN WillETT1 1 2 MRC-University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow; MRC Centre for Medical Molecular Virology, 

University College London, London 

Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, 

we characterize the feline homologue of tetherin and assess its effects on the replication of feline immunodeficiency virus (FiV). Feline 

tetherin was expressed in many feline cell lines and was induced by interferons (α, ω or γ). like human tetherin, feline tetherin displayed 

potent inhibition of FiV and HiV-1 particle release, however, this activity resisted antagonism by either HiV-1 VPu, or the FiV Env and 

‘OrfA’ proteins. Further, as over-expression of a complete FiV genome in trans could not overcome feline tetherin, these data suggest that 

FiV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FiV, 

indeed, syncytia formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) 

strains of FiV (FiV Fca-F14 and FiV Pco-ColV). Thus, while tetherin may prevent the release of nascent viral particles, cell to cell spread 

remains efficient in the presence of abundant viral receptors and tetherin up-regulation may enhance syncytia formation. Accordingly, 

tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell to cell spread. 

Offered paper The β2-Integrin CD18 maintains the HIV-1 assembly compartment in macrophages 

SEBASTiAN GiESE, Annegret Pelchen-Matthews, Mark Marsh 

Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, University College London, London 

in primary macrophages, HiV undergoes assembly in internal compartments (iC) that are connected to the cell surface by closely apposed 

membrane sheets. These iCs are also found in uninfected macrophages. The aim of this study was to identify key structural proteins of the 

iCs in monocyte-derived macrophages (MDM) and to investigate the role of the iCs in viral transmission. 

Electron microscopy studies show that some of the membranes associated with the iCs bear ~30 nm thick coat structures on their 

cytoplasmic sides. These domains are enriched on the extracellular surface with the β2 integrin CD18, while the cytoplasmic coat appears 

to link CD18 to actin via talin, vinculin and paxillin. Depletion of CD18 by rNAi leads to a marked reduction in the number of coat 

structures in both uninfected and HiV-infected MDMs and perturbs the integrity of virus-containing iCs in infected cells. These observations 

suggest that CD18 is involved in maintaining the structure of the iCs of MDMs. 

Though our studies indicate that cell-free transmission of HiV is not impaired by down-regulation of CD18, the iCs may be involved in cellto-cell 

transfer of HiV across virological synapses between macrophages and T cells. 

HA18 Virology workshop: vaccines & antivirals 

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Offered paper An in depth analysis of original antigenic sin in dengue virus infection 

ClAirE MiDGlEY1 , Martha Bajwa-Joseph1 , Sirijitt Vasanawathana2 , Wannee limpitikul3 , Bridget Wills4 , Aleksandra Flanagan5 , 

Emily Waiyaiya1 , Hai Bac Tran1 , Alison Cowper1 , Pojchong Chotiyarnwon1,7 , Jonathan Grimes5 , Sutee Yoksan6 , Prida Malasit7,8 , 

Cameron Simmons4 , Juthathip Mongkolsapaya1,7 , Gavin Screaton1 1 2 Dept of Medicine, Hammersmith Hospital Campus, Imperial College London, London; Paediatric Dept, Khon Kaen Hospital, 

Ministry of Public Health, Khon Kaen, Thailand; 3Paediatric Dept, Songkhla Hospital, Ministry of Public Health, Songkhla, Thailand; 

4Oxford University Clinical Research Unit, Wellcome Trust Major Overseas Program, Hospital for Tropical Diseases, Ho Chi Minh 

City, Vietnam; 5Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, 

University of Oxford, Oxford; 6Center for Vaccine Development, Institute of Molecular Biosciences, Mahidol University, Bangkok, 

Thailand; 7Medical Molecular Biology Unit, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; 8Medical Biotechnology Unit, National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development 

Agency, Pathumthani, Thailand 




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The evolution of dengue viruses (DENV) has resulted in four antigenically similar yet distinct serotypes. infection with one serotype likely 

elicits life-long immunity to that serotype, but not against the other three. DENV infection, although frequently mild, can lead to dengue 

haemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary infections implying a role for the adaptive 

immune response in disease. There is currently much effort towards the design of dengue vaccines but these efforts are made difficult 

by the challenge of inducing durable immunity to all four viruses. Domain 3 of the DENV envelope protein (ED3) has been suggested 

as a candidate because it contains neutralizing epitopes and it was originally thought that few cross-reactive antibodies were directed to 

this domain. We performed a detailed analysis of the anti-ED3 response in a cohort of patients experiencing either primary or secondary 

DENV infections. The results showed dramatic original antigenic sin, both in terms of binding and enhancement activity. This has important 

implications for vaccine design and, in addition, we propose a simple in vitro EliSA assay to diagnose the original DENV infection in 

secondary dengue cases. 

Offered paper A medium-throughput inhibitor screen for hepatitis C virus entry 

VANESSA COWTON, Allison Marty, Ania Owsianka, Nicola Mamczur, Arvind Patel 

Medical Research Council (MRC)-University of Glasgow Centre for Virus Research, Glasgow 

The entry step of viral infection is vital in establishing infection therefore is a potential target for anti-viral therapy. Hepatitis C virus (HCV) 

is believed to enter cells via the clathrin-mediated endocytosis pathway. The aim of this study was to develop a medium-throughput screen 

to identify inhibitors of HCV entry. Our interest was twofold: 1) to identify novel potential targets for antiviral therapy and 2) to gain 

some insight into the signalling pathways involved in HCV entry. in order to satisfy both aims we chose to screen a kinase inhibitor library. 

The screen we developed has four stages; the inhibitors were screened initially in the JFH-1 cell culture (HCVcc) system using a reporter 

cell-line. Subsequently, compounds of interest were screened using the HCV pseudoparticle (HCVpp) system before titration curves 

were performed to determine the iC50 values of the compounds. Finally the compounds were tested in the Vesicular Stomatitis Virus 

pseudoparticle (VSVpp) system. VSV also uses the clathrin-mediated endocytosis pathway for cell entry enabling us to classify compounds 

as HCV-specific or more general inhibitors. This study identified 13 inhibitors of HCV entry, work is ongoing to determine the mechanisms 

involved. This process can be scaled up to screen more compounds. 

Offered paper Identifying inhibitors of the HIV-1 Nef protein 

MArK VErOW, Colin Fishwick, Mark Harris 


using the x-ray crystal structure of full length HiV-1 Nef- solved at the university of leeds- we have been identifying inhibitors to this 

protein using a variety of in-silico methods. Currently we have produced a range of third generation molecules. This has enabled us to 

investigate the structure-activity relationship of these Nef inhibitors. 

Offered paper Characterizing the role of anti-retroviral innate immune responses in the mechanism of protection conferred 

by live attenuated SIV 

GiADA MATTiuZZO1 , Claire Ham1 , Nicola rose1 , Neil Almond1 , Martin Cranage2 , Greg Towers3 , Neil Berry1 1 2 Division of Retrovirology, NIBSC-HPA, South Mimms, Hertfordshire; Centre for Infection and Immunity, Division of Clinical Sciences, 

St George’s University of London, London; 3MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University 

College London, London 

live attenuated simian immunodeficiency virus (SiV) vaccines can confer potent protection against wild-type SiV challenge. While correlates 

of protection amongst adaptive immune responses have been difficult to identify, persistence of the vaccine appears crucial. Hence, the 

role of specific antiretroviral innate immune responses in vaccine protection is currently being studied. We have recently shown that 6/8 

Mauritian-derived cynomolgus macaques (MCM) vaccinated with a nef-disrupted SiVmac251 clone were completely protected against 

detectable infection following, uncloned, heterologous virus, SiVsmE660 challenge. in unvaccinated MCMs SiVsmE660 replicates to high 

levels. Sequence polymorphisms of the restriction factor TriM5α have been associated with a differential outcome of SiV infection in rhesus 

macaques. To investigate whether TriM5α polymorphism contributes to vaccine protection, we characterized the TriM5α and SiV capsid 

sequences recovered from vaccine breakthrough MCM and compared with controls. We are also investigating in the differential expression 

of TriM5α and other interferon-inducible genes in vaccinates at central sites of virus infection in vivo. The results will further characterize the 

SiV/MCM model, and establish whether innate immunity contributes to the protection conferred by live attenuated SiV. 




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Offered paper Modelling the probability of outbreaks of equine influenza as a result of genetic changes in the virus 

JANET DAlY1 , Andrew Park1,2 1 2 University of Nottingham, Sutton Bonington Campus; University of Georgia, Athens, Georgia, USA 

Equine influenza is a respiratory infection of horses caused by influenza A virus. Despite extensive use of influenza vaccines in some equine 

populations, outbreaks continue to occur. Antigenic drift, the accumulation of amino acid changes in the surface glycoproteins, particularly 

haemagglutinin (HA), the main targets of virus-neutralizing antibodies, leads to variant viruses that are no longer recognised by antibodies 

raised to an earlier strain. As a result of antigenic drift, the strains included in human influenza vaccines are updated frequently. A strain 

surveillance programme is in place for equine influenza. As for human influenza, decisions whether vaccine strains should be updated 

are based mainly on antigenic data from haemagglutination inhibition assays. Although genetic data is also considered, linking amino acid 

substitutions in HA to the likelihood of vaccine breakdown has been problematic. We used data from experimental infections of ponies 

immunized with vaccines containing strains that matched or did not match the challenge strain to inform mathematical models to address 

this issue. Modelling suggests that in a population with mixed susceptibility, as few as two amino acid differences in sites exposed to 

antibodies can lead to outbreaks occurring. The challenge remains to link these results to epidemic dynamics. 

Offered paper Compound C75 inhibits rotavirus replication – further data on the relationship of viroplasms with lipid 

droplets 

Winsome Cheung, Andrew lever, ulriCH DESSElBErGEr 

University of Cambridge, Dept of Medicine, Cambridge 

rotaviruses (rVs) are triple-layered virus particles containing a genome of 11 segments of dsrNA. Early rV morphogenesis and rNA 

replication occur in cytoplasmic inclusion bodies termed viroplasms (Vpls). A close physical and functional interaction between Vpls and 

the cellular organelles lipid droplets (lDs) has been demonstrated (Cheung et al., J Virol 2010; 84: 6782–6798). Two classes of chemical 

compounds affecting lD homoeostasis (isoproterenol + iBMx, triacsin C) were shown to decrease Vpl formation, dsrNA formation and 

the yield of infectious rV progeny. Here we show that compound C75 (3-carboxy-4-octyl-2-methylenebutyrolactone), a fatty acid synthase 

inhibitor, also blocks rV replication. Thus, interference with lD formation and metabolism by three distinct modifiers operating on disparate 

processes (lipolysis following perilipin A phosphorylation, fatty acid-CoA synthetase inhibition and fatty acid synthase inhibition) all of which 

inhibit rV replication, confirms the functional interrelationship of these cellular organelles with rV-specific inclusion bodies. 

Offered paper Conditional live attenuated SIVrtTA protects against heterologous SIVsmE660 

NEil AlMOND1 , Neil Berry1 , Mark Page1 , richard Stebbings1 , Debbie Ferguson1 , Martin Cranage2 , Atze Das3 , Ben Berkhout3 1 2 3 NIBSC-HPA, Potters Bar, Hertfordshire; St George’s, University of London; Laboratory of Experimental Virology, AMC University of 

Amsterdam, Netherlands 

live attenuated SiV confers potent protection against wild-type SiV challenge. However the mechanism of protection and the role of 

adaptive immunity is unclear. 

We have utilised a novel doxycycline-dependent conditional live attenuated vaccine SiVrtTA derived from SiVmac239αnef to investigate the 

role of duration of vaccination and the persistence of the vaccine virus in protection against intravenous challenge with the heterologous 

wild-type virus SiVsmE660. 6 of 6 MHC-typed Mauritian derived Macaca fascicularis were protected against detectable infection when 

vaccinated for 20 weeks with SiVrtTA in the continued presence of doxycyline. When vaccinated for 3 weeks, only 1 of 6 macaques was 

protected. When vaccinated for 20 weeks with SiVrtTA, but doxycyline was removed after 3 weeks, 3 of 6 macaques were protected 

against detectable infection with SiVsmE660. By contrast vaccination of indian rhesus macaques with the same vaccine for 26 weeks 

protected only 2 of 8 vaccinates against HOMOlOGOuS challenge with wild type SiVmac239. 

We are investigating why superior potent protection against heterologous virus challenge is obtained in cynomolgus macaques and in 

particular whether a) the kinetics of vaccine virus replication, b) the relative persistence of vaccine virus or c) innate anti-retroviral responses 

contribute to the protection observed. 

Offered paper Analysis of the avian coronavirus infectious bronchitis virus as a potential vaccine vector 

KirSTEN BENTlEY, Maria Armesto, Paul Britton 

Institute for Animal Health, Compton 

The avian coronavirus infectious bronchitis virus is an important respiratory pathogen of poultry. Coronaviruses have been identified as 

potential vaccine vectors, in part due to large genome size. iBV encodes genes for accessory proteins, 3a and 3b and 5a and 5b, and a short 

intergenic untranslated region (iuTr), which have all been shown not to be essential for replication in vitro. These genome regions may 




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offer sites at which heterologous proteins can be introduced giving vaccines affording protection against both iBV and a second virus based 

on the heterologous protein. recombinant iBVs were generated replacing 5a/5b, the iuTr or 3a/3b with reporter genes eGFP or hrluc, to 

investigate the genetic stability of iBVs expressing foreign proteins. recombinants were passaged in chick kidney cells and embryonated eggs. 

loss of reporter genes was observed between passage 4 and passage 8. These unstable viruses were found to be defective-like populations 

and a repeat of passaging with highly diluted virus shows a stable virus population can be maintained. The appearance of defective-like 

populations is unpredictable and raises questions over the suitability of such recombinants for vaccines. Further work characterizing 

recombinants and defective-like populations is required to assess the potential use as a vaccine vector. 

Offered paper Whole-genome sequencing of VZV vaccine-rash strains reveals new mutations 

DANiEl DEPlEDGE1 , Eleanor Gray1 , Meleri Jones1 , ravinder Kanda1,2 , Shaun Tyler3 , Judith Breuer1 1 2 3 University College London, London; Imperial College London; Public Health Agency of Canada, Winnipeg, Canada 

immunization against the varicella-zoster virus (the causative agent of chickenpox and shingles) is achieved using a live vaccine vOka which 

is attenuated for replication in skin compared to the wild type progenitor strain pOka. vOka differs from pOka at 45 loci, however, all bar 

3 sites are polymorphic with the result that the vaccine contains a mixture of vOka strains. Approximately 5–10% of vaccinees develop 

chickenpox/shingles like blisters. We have previously shown that the vOka vaccine strains recovered from vaccine associated blisters are a 

subset of the original population of strains in the vaccine preparation and that selection for the wild type allele occurs at three polymorphic 

loci. To determine whether newly acquired viral mutations may have also have contributed to the rash forming phenotype, we used 454 

and illumina methodologies to generate whole viral genome sequences from 8 vaccine-rash viruses including 4 from a vOka vaccinepreparation 

that was too virulent to be taken forward for licensure. We report the identification of several new SNPs that differ from both 

parental and vaccine strains and speculate on their possible role in rash formation. 

Offered paper Developing novel inhibitors of adamantine-resistant influenza A virus M2 ion channels 

ranjitha Tatineni1 , Toshana Foster1 , Joseph Thompson2 , Wendy Barclay3 , richard Foster2 , Paul Targett-Adams4 , STEPHEN 

GriFFiN 1 

1 2 Leeds Institute of Molecular Medicine, St James’ University Hospital, University of Leeds, Leeds; School of Chemistry, University of 

Leeds, Leeds; 3Imperial College London; 4Pfizer UK, Sandwich 

The rapid emergence of pandemic influenza and associated severe disease requires immediate control using antiviral chemotherapy. 

Historically, monotherapy using adamantane based inhibitors of the M2 proton channel represented 1st line treatment/prophylaxis, yet 

resistance to these drugs arose rapidly and is now present in virtually all circulating strains. Modern neuraminidase (NA) inhibitor drugs are 

similarly becoming less effective due to widespread resistance resulting from their application as monotherapies, despite showing distinct 

modes of action. As combinations of M2 and NA inhibitors are known to suppress the generation of resistance in vitro, new M2-specific 

compounds capable of blocking adamantane-resistant channels could provide new therapeutic options. 

We have employed in vitro channel forming assays, in silico drug binding and cellular assays to identify novel inhibitors of M2 with 

chemotypes distinct from adamantanes and which appear to block channel activity via distinct modes of action. Their effects on 

adamantane-resistant (S31N) M2 channels will be described. Compounds based on such molecules will provide a means to revisit a 

clinically proven drug target to complement future antiviral strategies. 

Offered paper Genotype-associated differences in susceptibility and resistance development of hepatitis C virus towards the 

protease inhibitors VX-950 (Telaprevir) and ITMN-191 

ingrid imhof, PETEr SiMMONDS 

University of Edinburgh, Edinburgh 

Protease inhibitors (Pis) have proven to be effective adjuncts to interferon / ribavirin treatment of hepatitis C virus (HCV) infections. little 

clinical or in vitro data exists however, on their effectiveness for non-type 1 genotypes that predominate in Europe, the Middle East, Africa 

and most of Asia. 

To investigate susceptibility and resistance development of other genotypes, NS3 protease and NS4B genes from each were inserted into 

the JFH clone to produce replication competent intergenotype chimaeras. These showed marked differences in susceptibility to iTMN-191 

and Vx-950; gt1, gt4 and gt6 showed iC values of 2–3 nM, >100-fold lower than genotypes gt2/gt3/gt5. Vx-950 susceptibilities varied 

50 

five-fold, with gt1/gt2 most susceptible, gt4/gt5 most resistant. Culture of gts1–6 in Pis induced numerous mutations in the NS3 protease 

domain, variable between genotypes in position and amino acids substituted. Mutations each conferred a resistant phenotype, usually 

without affecting replicative fitness. 




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Major differences were found between genotypes in their susceptibility and resistance development to Pis. However, equal sensitivities of 

genotypes 1, 4 and 6 to Danoprevir and a broader efficacy range of Telaprevir between genotypes than initially conceptualised provide 

strong evidence that Pis might be effectively used beyond their genotype 1 target group. 

Offered paper Development of a small animal model for equine influenza virus vaccine cross-protection studies 

ADAM rASH, Alana Woodward, Neil Bryant, Deborah Flack, Beverley Walladge, lyndsey Dunn, Debra Elton 

Animal Health Trust, Newmarket, Suffolk 

Traditionally cross-protection studies for equine influenza virus vaccines have been carried out in the well-established Welsh mountain 

pony model. Although this is the most relevant model, these experiments are prohibitively expensive for use on a regular basis and from 

an ethical perspective it is preferable to avoid the use of companion animals where possible. We have developed a mouse challenge model 

to test the ability of antigenically distinct strains to cross protect against recently circulating field strains. Groups of mice were immunized 

with one of four vaccine strains, which included the current OiE recommended strains, prior to challenge with recently circulating Florida 

sublineage viruses. Detection of virus rNA in daily nasal washes was successful, avoiding the use of euthanasia at regular time points. Mice 

vaccinated with an older American lineage strain shed higher levels and for a longer period compared to the other vaccine groups. There 

was no difference in shedding between the groups vaccinated with either of the recommended strains. These results mirror those obtained 

from a recent pony cross-protection study using both of the recommended vaccine strains and the same Florida sublineage challenge strain, 

suggesting that the mouse may be a good small animal model for these studies. 

Offered paper Imino sugars as antivirals against human influenza A viruses 

SAirA HuSSAiN1 , Joanna Miller2 , Peter rosenthal3 , Nicole Zitzmann2 , John McCauley1 1 2 Division of Virology, National Institute for Medical Research, London NW7 1AA; Oxford Antiviral Drug Discovery Unit, Dept of 

Biochemistry, University of Oxford, Oxford OX1 3QU; 3Division of Physical Biochemistry, National Institute for Medical Research, 

London NW7 1AA 

Current therapies for human influenza A viruses targeting viral proteins have led to the emergence of drug resistant strains. in the case of a 

pandemic, multiple drug therapy might be advised. Compounds targeting host cell enzymes would further reduce viral loads and minimise 

occurrence of multiple drug resistant strains. imino sugars exhibit anti-viral activity as they inhibit endoplasmic reticulum α-glucosidases 

which are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. 

The anti-viral potential of an imino sugar, N-nonyl deoxynojirimycin (NN-DNJ), was tested on circulating human influenza A viruses. in a 

single cycle of replication, NN-DNJ caused a greater than 10-fold reduction in both haemagglutinin titre and infectious virus titre of two 

H3N2 strains, but there was a reduced inhibition of a pandemic H1N1 strain. 

We determined the effects of NN-DNJ treatment on the two virus glycoproteins, the Haemagglutinin (HA) and Neuraminidase (NA). 

An alteration in electrophoretic mobility of the HA protein was seen by radio-labelling virus proteins in NN-DNJ treated cells. Sialidase 

activity of all three viruses in NN-DNJ treated cells decreased to 50% when compared with untreated cells. Therefore, NN-DNJ has shown 

potential as therapy for influenza A viruses due to its anti-viral effect. 

HA19 Life at zero growth rate 

↑Contents 

resuscitation of dormant mycobacteria and their relatives: how does it work? 

Michael Young 

Institute of Biological Environmental and Rural Sciences, Cledwyn Building, Penglais Campus, Aberystwyth University, Ceredigion 

SY23 3DD 

The resuscitation-promoting factors are a family of secreted or membrane-anchored proteins found in the actinobacteria. The founder 

member, rpf, was isolated from Micrococcus luteus culture supernatant. it had the remarkable property of resuscitating dormant bacteria 

– as do the rpf-like proteins found in several other organisms, including Mycobacterium tuberculosis. Originally predicted to act as signalling 

molecules because of their remarkable potency, it now seems very likely that the resuscitation mediated by rpf proteins is a direct or 

indirect consequence of their demonstrated muralytic activity. All rpf proteins contain a transglycosylase-like domain (PFAM 06737) 

and although lytic transglycosylase activity has been widely inferred, it has not yet been confirmed experimentally. The precise nature of 

the trigger that breaks dormancy is still unknown. The most likely possibilities are either the cleavage of bonds in the bacterial cell wall 

peptidoglycan or, alternatively, the detection of the muropeptide products released by a receptor located in the cell envelope. 




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HA19 Cont. 

Yeast quiescent machines 

isabelle Sagot 

IBGC CNRS UMR 5095, 1, rue Camille Saint Saëns, 33077 Bordeaux cedex, France 

Quiescence is a widespread cellular state defined as a temporary arrest of proliferation. little is known about the molecular determinants that 

orchestrate quiescence establishment and exit. Furthermore, the connections between the control of cell cycle progression and quiescence 

are not fully understood. recently, we have described a new actin cytoskeleton structure, named Actin Bodies, which is specifically assembled 

in quiescent yeast cells. Further, we have discovered that, in quiescent cells, another cellular machine, the proteasome, is drastically relocalized 

from the nucleus into a cytoplasmic structure that we have called Proteasome Storage Granules. These results establish that yeast 

quiescent cells are not simply G1 resting cells but are ultra-structurally committed to the quiescent state. using these structures as specific 

quiescence markers, we found that yeast can enter quiescence not only from G1 but from all cell cycle phases. Moreover, we established that 

quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose. importantly, glucose needs to 

be catabolized all the way down glycolysis to allow quiescence exit, but strikingly ATP replenishment appears not to be the exit signal. These 

findings clearly establish that quiescence entry and exit primarily rely on the cellular metabolic status. 

Fungal spores as survival vehicles in space and time 

Jan Dijksterhuis 

Applied and Industrial Mycology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands 

Fungal spores are characterized by a dormant state and moderate stress-resistance. These are persistent structures that survive adverse 

conditions including the presence of antifungal compounds. upon contact with a moist substrate, germination of conidia occurs which 

includes a change from a dormant stabilized state towards a growing vegetative cell. This process takes a number of hours dependent on 

many environmental cues. Some fungal spores show extreme persistence and dormancy and somehow survive in time over long periods. 

They produce ascospores that are as resilient as the bacterial spores of Bacillus subtilis. They survive ultrahigh pressure, temperature and 

drought and are only activated to germination after a rigorous physical trigger as a short high temperature-treatment. Dormant ascospores 

contain high concentration of compatible solutes and have a high density and internal microviscosity, which drops strongly during 

germination. This contribution deals with aspects of dormancy and the breaking of dormancy in fungal spores. We follow the fate of spores 

during germination and will see that the change from dormancy to vegetative cell is impressive. 

Sporulation in mycobacteria 

lEiF A. KirSEBOM1 , Santanu Dasgupta1 , Bhuphender Singh1 , B. M. Fredrik Pettersson1 , Nurul M. islam1,2 1 2 Dept of Cell & Molecular Biology, Uppsala University, Uppsala, Sweden; Dept of Botany, Dhaka University, Dhaka, Bangladesh 

Mycobacteria inhabit various environmental reservoirs e.g. ground and tap water, soil, animals and humans. Many mycobacteria are also 

highly successful pathogens that cause disease in both humans and animals, e.g. M. tuberculosis and M. avium supsp paratuberculosis. They can 

also establish latent asymptomatic infections of which very little is known. We have provided evidence that mycobacteria can form spores. 

Our data opens up for the possibility that mycobacteria might use sporulation as a strategy for dormancy and persistent pathogenicity, as 

well as for survival under different environmental conditions. Our current focus is to describe and understand sporulation in mycobacteria 

and recent findings will be discussed. 

Transcription and splicing in Microsporidia spores 

Naomi M. Fast 

University of British Columbia, Canada 

Microsporidia are highly derived fungi that are intracellular parasites of animals and some protists. Outside of their hosts, microsporidia 

exist as hardy spores, assumed to be largely dormant. intracellular, replicative forms of the parasite are referred to as meronts. Among 

eukaryotes, microsporidia possess a number of unusual features such as extremely small genomes – many in the size range of those of 

bacteria. indeed, the effects of genome reduction are evident at every level of microsporidian biology, including key processes such as 

transcription and rNA processing. Examining rNAs from both dormant and replicative forms of the parasite illustrates stark differences 

in the two forms. All transcripts remain unspliced in the spore, and possess extremely long 5’ untranslated regions (uTrs), frequently 

overlapping with upstream genes. Conversely, meront transcripts look much more typical: 5’uTrs are shorter and transcripts lack introns. 

The presence of unusual transcripts in the spore raises questions about their role in the dormant stage of the parasite; the possibility exists 

that these transcripts have a structural rather than an informational function, or perhaps they serve no function, and are merely a byproduct 

of the parasite’s transition to dormancy. 




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POSTEr AbSTrACTS 

HA01 Seeing the cell through the ‘eyes’ of the virus 

↑Contents 

HA01/01 The impact of macrophage activation on African swine fever virus replication and its potential role in persistence 

PAMElA liTHGOW1 , David Chapman1 , Haru Takamatsu1 , Dirk Werling2 , linda Dixon1 1 2 Institute for Animal Health, Pirbright; Royal Veterinary College, North Mymms 

African swine fever virus (ASFV) replicates in macrophages and infection has been shown to correlate with expression of cell surface 

markers, including CD163, characteristic of intermediate and late stages of differentiation. To determine if macrophage activation state 

influences ASFV infection, porcine monocytes were stimulated to form classically or alternatively-activated macrophages using media 

supplemented with iFNγ or il4. Cell surface marker expression was assessed using FACS analysis to confirm the induction of the predicted 

macrophage phenotypes. The percentage of macrophages infected at 24 hours post-infection (hpi) with the Benin 97/1 or uganda isolate 

increased by about 15% when cells were treated with il4; whereas the percentage infected with OurT88/1 or lisbon57 decreased 

by about 20%. These results show that the macrophage activation state and the virus isolate determine replication in macrophages. At 

72hpi the percentage of infected cells was high (80–100%) in il4 treated macrophages compared to other treatments (0–60%). When 

treated with il4 cell numbers increased by 10–200% post-infection. Virus titration showed a lag in production of extracellular virions 

from il4 treated macrophages but production was similar to other treatments by 72 hpi. This data suggests that alternative activation of 

macrophages may favour virus persistence over longer periods. 

HA01/02 An investigation of the cellular functions of LEDGF/p75 

SiOBHAN HuGHES, Victoria Jenkins, Peter Cherepanov 


lens epithelium-derived growth factor (lEDGF) is a critical co-factor of lentiviral integration that accounts for the characteristic bias of 

the retroviral genus towards integration within active transcription units of the host genome. it’s well-characterized role in viral replication 

notwithstanding, cellular functions of lEDGF are poorly understood. 

in spite of its apparent ubiquitous association with active transcription units, gene expression profiling of lEDGF-null mouse embryonic 

fibroblasts (MEFs) revealed that less than 200 genes were significantly different between lEDGF-null and wild type MEFs. These data 

strongly suggest that lEDGF does not play an essential nor direct role in the regulation of gene expression. 

A tandem affinity purification strategy identified the heterodimeric S-phase kinase, Cdc7:ASK, as a novel interactor of lEDGF. Both kinase 

subunits co-immunoprecipitated with lEDGF from human cells; moreover, fractionation experiments suggest that lEDGF may affect 

chromatin binding of the kinase. Biochemical analyses showed that the integrase-binding domain of lEDGF interacts with the C-terminus 

of ASK. Furthermore, lEDGF potently stimulated the kinase activity of Cdc7:ASK in vitro, while removing the C-terminal region of ASK 

resulted in a hyper-active kinase. These results indicate that the interaction with lEDGF relieves autoinhibition of Cdc7:ASK kinase activity, 

imposed by the C-terminus of ASK. 

HA01/03 The mechanism of retroviral integration through X-ray structures of its key intermediates 

GOEDElE MAErTENS, Stephen Hare, Peter Cherepanov 

Division of Infectious Diseases, Imperial College London, St Mary’s campus, Norfolk Place, London W2 1PG 

To establish successful infection, a retrovirus must integrate a copy of its genome into a host cell chromosome. This reaction is catalysed 

by the viral enzyme integrase (iN). A tetramer of iN binds and synapses viral DNA ends, forming a highly stable complex, referred to as 

intasome. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the 

viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) superfamily of recombinases 

were reported, the mechanics of target DNA capture and strand transfer by these enzymes have not been established. Here, we report 

crystal structures of the intasome from prototype foamy virus in complex with target DNA, illuminating the pre-integration target DNA 

capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between iN dimers within the 

intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced iN active sites to access the scissile 

phosphodiester bonds. Our results elucidate the structural basis for retroviral DNA integration and moreover provide a framework for the 

design of iNs with altered target sequences. 

HA01 

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HA01 Cont., HA02 & HA03 

HA01/04 Targeting HPV16 E6 and E7 oncoproteins using rNA aptamers 

TAMArA BElYAEVA1 , Clare Nicol1 , Gilles Travé2 , Eric Blair1 , Nicola Stonehouse1 1 2 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT; IREBS, Ecole Supérieure 

de Biotechnologie, 67412-Illkirch, France 

Human papillomavirus 16 (HPV 16) is associated with the development of cervical cancer mediated by the E6 and E7 oncoproteins. E6 

promotes degradation of the tumour suppressor p53, whereas E7 acts to destabilise the cell cycle control protein prb. in addition to these 

well-characterized roles, E6 and E7 have been shown to interact with over 50 additional cellular proteins. in this study, E6 and E7 rNA 

aptamers have been selected with the aim of using these to modulate protein-protein interactions and therefore dissect the activities of the 

oncoproteins in cellular transformation. We found that several of the aptamers were able to induce apoptosis in HPV16-transformed cells, 

actively expressing both E6 and E7. Two of the E6 aptamers (termed F2 and F4) had a preference for binding to C-terminus of E6 (the site 

of interaction with PDZ-domain containing proteins). Both were able to interfere with this interaction, with F2 being the most effective. 

One of the E7 aptamers (A2) bound to the N-terminal region of E7 and treatment of cells with A2 resulted in a dramatic loss of the E7 

oncoprotein and a rise in cellular prb levels. We now aim to develop the possible therapeutic potential of these molecules. 

HA02 Maths & microbes 

HA02/01 Effect of infection history on influenza in a population: insights from mathematical models 

ADAM KuCHArSKi, Julia Gog 

Dept of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge 

in diseases where multiple infections are possible during an individual’s lifetime, such as influenza, a host’s history of infection and immunity 

will determine outcome of future exposures. in turn, the suite of varying individual infection histories in the population will shape the 

population dynamics of the disease. The immune profile of the population dictates the success or otherwise of new and re-emerging strains, 

but exploring the consequences of precisely how immunity is acquired has proven rather difficult as the possible number of combinations 

of infections that an individual could have seen becomes staggeringly large over time. 

using new developments in the mathematical modelling of diseases with multiple strains, we have a created an easily manageable 

framework to explore the effect of assumptions about immune acquisition on disease behaviour. in particular, we see that ‘original antigenic 

sin’ could lead to some non-intuitive patterns of immunity in some age groups, which could be responsible for some unusual phenomena 

seen in past influenza epidemics. 

HA03 Social evolution in micro-organisms 

↑Contents 

↑Contents 

HA03/01 rhomboid proteases in microbial signalling and development 

EliNOr P. THOMPSON1,2 , Beverley J. Glover1 1 2 University of Greenwich, Chatham Maritime, Kent, University of Cambridge, Cambridge 

Communication within and between cells is necessary for growth and development of multicellular organisms and of unicellular microbes 

in their changing environment. Prokaryotic and eukaryotic signalling mechanisms converge at the process of regulated intramembrane 

proteolysis, in which cleavage of a membrane-bound precursor allows signalling by the released peptide. The membrane proteases involved 

include those of the rhomboid family, almost-ubiquitous serine proteases described in Drosophila (EGFr signalling), the apicomplexa 

(adhesion, infection) through to yeast (mitochondrial regulation). Having investigated plant rhomboid mutants and localization, we became 

interested in microbial counterparts—little known in prokaryotes beyond Providencia stuartii’s AarA rhomboid. The latter cleaves an 

N-terminal extension of the TatA transport channel, thus mediating quorum sensing. 

We searched for further proteases and possible substrates within bacteria and also social micro-organisms: we are carrying out 

complementary work in prokaryotes and eukaryotes, including a Dictyostelium rhomboid mutant. Query sequences included our plant 

chloroplast rhomboid and P. stuartii AarA and TatA. Putative proteins’ active-site motifs and essential residues were identified. Since we 

hypothesise that rhomboids are ideal candidates for signalling networks, it is notable that rhomboids were not found in obligate intracellular 

microbes. We report on our investigations and discuss rhomboids in relation to microbes’ development, attachment and biofilms. 




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HA03 Cont. 

HA03/02 Population density and the fitness benefit of bacterial quorum sensing 

SOPHiE E. DArCH1 , Stuart A. West2 , Klaus Winzer1 , Stephen P. Diggle1 1 2 Centre for Biomolecular Sciences, University of Nottingham, Nottingham, Dept. of Zoology, University of Oxford, Oxford 

it has been argued that bacterial cells coordinate behaviours at the population level, by a process that has been termed quorum sensing 

(QS). The underlying assumption made is that extracellular behaviours coordinated by QS are more beneficial when carried out at higher 

cell densities. However, this fundamental assumption has never been experimentally tested. We demonstrate this using the opportunistic 

pathogen Pseudomonas aeruginosa, by independently manipulating bacterial density and the response to QS signal. A minimal growth 

medium – Quorum Sensing Media (QSM) was used to culture strains of P. aeruginosa in varying concentrations of CASamino acids (CAA) 

and bovine serum albumin (BSA) to investigate the benefits of QS at a high cell density. using a set of growth curve experiments we 

demonstrate that a signal negative QS mutant (lasi-) receives greater fitness benefits from QS induction when grown at a high cell density 

when compared to a low cell density culture. We found that the benefit of QS was relatively greater at higher cell densities, and was due 

to more efficient use of QS-dependent public goods. Overall, these results suggest that the function of QS is to coordinate the switching 

on of social behaviours when they provide the greatest benefit. 

HA03/03 Social dilemmas and the implications for virulence in Staphylococcus aureus populations 

EriC J.G. POlliTT1 , Stuart A. West2 , Shanika A. Crusz1 , Stephen P. Diggle1 1 2 University of Nottingham, Nottingham; Oxford University, Oxford 

Staphylococcus aureus coordinates the production of virulence factors at a group level using quorum sensing (QS), which involves the 

production and sensing of autoinducing peptide (AiP) signal molecules by the agr locus. QS is costly to perform for individual cells and social 

evolution theory predicts that it is vulnerable to exploitation by non-cooperating ‘cheats’. 

We experimentally tested this theory by introducing mixed populations of rN6390B wild type and an agr mutant (QS cheat) into a 

waxworm (Galleria mellonella) virulence model. results demonstrated that the agr mutants invade cooperating WT populations during 

infections and that cheat invasion also leads to a reduction in infection severity. We also investigated the effect of transmission rate on the 

viability of the QS cheats in a population. Our work shows that QS is a social trait in S. aureus and provides an explanation as to why agr 

mutants readily occur in clinical populations of S. aureus. it also suggests that asocial cheats could be developed to treat infections, which has 

major implications for the treatment of antibiotic resistant organisms such as S. aureus. 

HA03/04 Coercion, common interest and the reliability of signalling in bacteria 

rOMAN POPAT1 , Sam Brown2 , Ashleigh Griffin2 , Stuart West2 , Stephen Diggle1 1 2 University of Nottingham, Nottinghamshire; University of Oxford, Oxfordshire 

Honest signalling poses a Darwinian dilemma. if a signal can be faked by the sender, selection to ignore the signal acts on the receiver. 

Three mechanisms can maintain signal reliability; (1) to be costly (handicap); (2) to be a reliable index of quality that cannot be faked 

(e.g. deer roar), or (3) common interest in communication between emitter and receiver. The last is likely to be important in intercellular 

signalling in micro-organisms (e.g. due to high relatedness within populations). Pseudomonas aeruginosa uses Quorum Sensing (QS) to 

regulate a number of social behaviours. However, where QS regulates the production of public goods it is vulnerable to exploitation by 

cheating. using experimental evolution, common interest of signalling was varied to study the evolution of signal reliability. The results 

indicate that selection for reliable signalling is maintained by common interest in the cooperative trait regulated by QS. We also provide 

evidence that a deviance in the QS strategy can invade the resident WT population and that a suboptimal signal level removes selection for 

QS by altering the cost:benefit ratio. These results help to explain the diversity of QS phenotypes and genotypes present in infections and 

aid us in understanding the evolution of reliable signalling. 

HA03/05 Meticillin-resistant Staphylococcus aureus (MrSA) interaction with Acanthamoeba and macrophages 

Mihaela Cardas1 , Naveed Khan2 , SElWA AlSAM1 1 2 University of Essex, Colchester; University of Nottingham, Leicestershire 

Staphylococcus aureus is a leading cause of nosocomial infections, and can produce serious and life-threatening diseases. With the emergence 

of methicillin-resistance, S. aureus has gained particular attention and has become a major problem in the successful treatment. Despite the 

application of eradication strategies, infections due to MrSA are rising. Notably, Acanthamoeba, a ubiquitous protozoan pathogen is known 

to harbour microbial pathogens. The fact that Acanthamoeba resembles human macrophages in their phagocytic activity and that they 

exhibit parallel mechanisms in their interactions with MrSA, suggest that Acanthamoeba may provide an alternative model to study MrSA 




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HA03 Cont. 

pathogenesis. The overall aim of the present study was to determine Acanthamoeba and macrophages interactions with S. aureus, MrSA 

and S. epidermidis. using association, invasion and intracellular assays, it was observed that all bacterial isolates exhibited interactions with 

Acanthamoeba and macrophages. Next, we determined whether bacteria survive inside Acanthamoeba during the encystment process and 

that viable bacteria can be isolated from mature cysts. using encystment assays, the findings revealed that MrSA and S. aureus but not S. 

epidermidis exhibited survival inside Acanthamoeba cysts. Given that Acanthamoeba cysts are air-borne, these findings suggest cysts may act 

as Trojan horse to help spread S. aureus and MrSA to the susceptible hosts. 

HA03/06 Competence-stimulating peptide allelic variants expressed in nasopharyngeal colonization events with multiple 

pneumococcal strains 

MArCuS lEuNG1 , Mark Schiebler1 , Stephen Gillespie2 , Bambos Charalambous1 1 2 University College Medical School, Royal Free Campus, University College London, London; University of St Andrews, North Haugh, St 

Andrews, Fife 

Background: Transformation and biofilm development in pneumococci is regulated by the quorum sensing network initiated by the 

Competence-Stimulating Peptide (CSP) encoded by comC. There are genetic variants or pherotypes of CSP, and we have explored those 

that occur in multiple serotypes colonizing the nasopharynx simultaneously. 

Methods: Nasopharyngeal swabs were obtained from Tanzanian children under 6 years of age. Pneumococcal serotyping was performed by 

conventional serology and confirmed by DNA based methods. Genomic DNA was obtained from heat lysed cells and the comC gene was 

amplified by PCr. restriction fragment length polymorphism of the comC was used to identify pherotypes CSP1, 2 and 4. 

Results: Forty four strains from six multiple colonization events, as defined by serotype, were pherotyped. Five out of the six mixed 

colonization events expressed heterogeneous pherotypes. Pherotypes CSP1, 2 and 4 were found together in two events, CSP1 and 2 in 

two events and CSP2 and 4 in one event. One colonization event had serotypes 35A and 6B with pherotype 4. 

Conclusion: This study revealed that the majority of colonization events with heterogeneous serotypes had mixed pherotypes. Further work 

is underway to establish whether there is cross-talk between these CSP variants. 

HA03/07 The evolutionary ecology of the peptide-based quorum sensing system in Bacillus thuringiensis 

liQiN ZHOu1 , Didier lereclus2 , Christina Nielsen-leroux2 , Ben raymond1 1 2 Royal Holloway, University of London, Egham, Surrey; Institut Pasteur, Paris, France 

Micro-organisms can exhibit diverse cooperative behaviours associated with virulence. However, the evolutionary forces that maintain 

these cooperative behaviours have rarely been investigated in naturally co-evolved host-pathogen systems. We investigated the 

evolutionary biology of the peptide-based quorum sensing (QS) system in Bacillus thuringiensis. The gene plcR controls the production of 

various extracellular proteins, often involved in virulence, in response to an autoinduced heptapeptide signal produced by the gene papR. 

Approximately 1% of naturally occurring strains are null QS mutants (or cheats) in this system. We tested the hypothesis that both signal 

production and the virulence genes activated by QS peptides are social traits, and measured the invasion of isogenic cheats at varying 

pathogen doses and cheat frequencies. Wild-type strains with functioning papR and plcR genes had higher group level fitness than cheats 

(measured as spores produced per host). However, cheats could not outcompete wild-type bacteria when in vivo infections were allowed 

to fully sporulate. in contrast, experiments with homogenized insects indicate that cheats can outcompete wild-types, corroborating 

previous in vitro work with Pseudomonas. Although investigation of this system is at an early stage, we hypothesize that spatial structure 

within insect cadavers limits the fitness of null QS mutants. 

HA03/08 Parasitic growth of Pseudomonas aeruginosa in co-culture with the chitinolytic bacterium Aeromonas hydrophila 

Nina Jagmann, BODO PHiliPP 

University of Konstanz, Dept of Biology, Microbial Ecology, Konstanz, Germany 

Polymer-degrading bacteria face exploitation by opportunistic bacteria that grow with the degradation products without investing energy 

into production of extracellular hydrolytic enzymes. This scenario was investigated with a co-culture of the chitinolytic bacterium Aeromonas 

hydrophila strain AH-1N and Pseudomonas aeruginosa strain PAO1 as opportunist with chitin as sole source of carbon, nitrogen, and energy. 

Co-cultures of both strains had a biphasic course. in the first phase, strain PAO1 grew along with strain AH-1N without affecting it. The 

second phase was initiated by a rapid inactivation of and a massive acetate release by strain AH-1N. Both processes coincided and were 

dependent on quorum sensing-regulated production of secondary metabolites by strain PAO1. Among these the redox-active phenazine 

compound pyocyanin caused the release of acetate by strain AH-1N by blocking the citric acid cycle through inhibition of aconitase. Thus, 




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HA03 Cont. 

strain AH-1N was forced into incomplete oxidation of chitin with acetate as end product, which supported substantial growth of strain 

PAO1 in the second phase of the co-culture. For identifying molecular mechanisms underlying this parasitic growth strategy of strain 

PAO1 transposon mutagenesis was carried out. Six mutants showed a strongly altered phenotype in co-culture with strain AH-1N and are 

currently being analysed. 

HA03/09 Effect of antibiotics on Pseudomonas aeruginosa populations in artificial sputum media 

Elli WriGHT, Joanne Fothergill, Andrew Greenhalgh, Craig Winstanley 

University of Liverpool, Liverpool 

P. aeruginosa lung infections are the major cause of morbidity in cystic fibrosis (CF). The liverpool Epidemic Strain (lES) represents a 

successful clone of P. aeruginosa associated with transmissibility and increased morbidity. Although these chronic lung infections are never 

cleared despite intensive antibiotic usage, the P. aeruginosa populations diversify phenotypically. The aims of this study were to model this 

diversification during growth in artificial sputum medium (ASM) and to determine whether ASM can be used to predict lES population 

responses to antimicrobial challenges. P. aeruginosa lESB58 cultured in ASM (with and without antibiotics) for 7 days was investigated for 

diversification with respect to phenotypic and genotypic characteristics. Diversification of lESB58 populations was observed with respect to 

colony morphology, hypermutability, pyocyanin production and antibiotic susceptibilities, with increased diversity in the presence of some 

commonly used antibiotics. These data suggest that P. aeruginosa undergoes diversification in ASM that resembles the situation in the CF 

lung, and that ASM is a suitable alternative to animal models for studying bacterial population divergence and responses to antibiotics. 

Acknowledgements: Funded by the Dr Hadwen Trust for Humane research, the uK’s leading medical research charity funding exclusively 

non-animal research techniques to replace animal experiments. 

HA03/10 Socioecology and evolution of Bacillus subtilis 

Štefanic Polonca1 , Francesca Decorosi2 , Carlo Viti2 , Janine Petito3 , Frederick M. Cohan3 , iNES MANDiC-MulEC1 1 2 Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia; Dept of Agricultural Biotechnology, University of Florence, 

50144 Florence, Italy; 3Dept of Biology, Wesleyan University, Middletown, CT 06459, USA 

The ComQxPA quorum sensing locus shows dramatic diversity within the species Bacillus subtilis, resulting in different communication 

groups (or pherotypes), even among strains isolated from microscale sampling of soil. Members of one pherotype can exchange signals and 

consequently induce co-members of the population to express adaptive traits, while members of different pherotypes cannot communicate 

efficiently. The ecological significance of pherotype diversification within Bacillus species is not yet understood and we tested the hypothesis 

that pherotypes represent ecologically distinct groups with similar genetic and eco-physiological traits. using the Ecotype Simulation model 

we showed that, although microscale strains separate into three putative ecotypes, pherotypes do not correspond strictly to putative 

ecotypes, carbon utilization patterns or colony morphotypes, rejecting the proposed hypothesis. However, dominance of one pherotype 

within the putative ecotype clusters and the patterns of pherotype distribution across ecotypes provide a framework for an alternative 

model for evolution of social interactions. This model explains preservation of diversity of pherotypes among closely related individuals and 

consequent communication between non-kin. 

HA03/11 rhamnolipid biosynthesis gene expression in Pseudomonas aeruginosa 

MiCHEllE ruDDEN, roger Marchant, ibrahim Banat 

University of Ulster, Coleraine 

Synthesis of rhamnolipid biosurfactants is governed by the complex hierarchical quorum sensing (QS) regulatory system present in 

P. aeruginosa which also controls several other virulence associated traits. Our primary research interest is the biosynthetic control of 

rhamnolipid synthesis; we believe that targeting the genetics of this system will provide insights for novel production strategies. The final 

steps involved in rhamnolipid synthesis are catalysed by rhamnosyltransferase enzymes rhlB and rhlC. The expression of these two 

genes was studied in P. aeruginosa PAO1 under standard laboratory conditions used for rhamnolipid production. Quantitative-real time 

PCr analysis revealed a fluctuating gene expression pattern for both genes. Elevated levels of gene expression were detected during late 

exponential and stationary phases of growth with expression declining in late stationary. Most significant was the observed difference in 

timing and expression levels for both genes with increased levels of rhlB expression preceding maximum rhlC expression. HPlC-MS analysis 

shows the effect of such stringent regulation rhamnolipid yields and ratios of mono- and di-rhamnolipids produced. These results indicate 

the biosynthesis of rhamnolipids is specifically transcriptionally regulated, further experiments will investigate expression of rhamnolipid 

genes in different a growth environment for enhanced transcriptional expression and sustained rhamnolipid production. 




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HA03/12 Sharing of quorum sensing signals and social interactions in a bacterial community causing a bacterial plant disease 

Taha Hosni12 , Chiaraluce Moretti1 , Giulia Devescovi2 , Zulma rocio Suarez-Moreno2 , M’ Barek Fatmi3 , Corrado Guarnaccia2 , 

Sandor Pongor2 , roberto Buonaurio1 , ViTTOriO VENTuri2 1Dipartimento di Scienze Agrarie e Ambientali, sezione di Arboricoltura e Protezione delle Piante, Università degli Studi di Perugia, Via 

Borgo XX Giugno, 74-06121 Perugia (Italy); 2International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 

Trieste, Italy; 3Institut Agronomique et Vétérinaire Hassan II, Complexe Horticole d’Agadir, BP. 18/S, Agadir Morocco 

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. in the knot disease 

of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, 

namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been 

very often found to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species 

isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of quorum sensing signals. The luxI/R family 

genes responsible for production and response to these signals in all three bacterial species have been identified and characterized. 

Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on quorum sensing, however 

the lack of signal production can be complemented by wild type E. toletana or P. agglomerans. it is also apparent that the disease caused by 

Psv is aggravated by the presence of the two other bacterial species. The potential role of quorum sensing in establishing a stable consortia 

leading to a polybacterial disease is discussed. 

HA04 Vaccines 

↑Contents 

HA04/01 Development of glycoconjugate vaccine for Streptococcus suis 

VANESSA SOFiA A. TErrA, Jon Cuccui, rebecca H. langdon, Brendan W. Wren 


Streptococcus suis is a Gram-positive bacterium and causative agent of pneumonia, meningitis, endocarditis and septicaemia. recently, 

S. suis crossed the species barrier infecting humans and causing deaths. Therefore, the development of an efficient and cheap vaccine 

is paramount. Glycoconjugate based vaccines have been shown to be effective as proven by available vaccines for S. pneumoniae, H. 

Influenza and N. meningitidis. These vaccines consist of a polysaccharide produced and purified from the native organism, chemically 

coupled to a carrier protein. The chemical conjugation is difficult and expensive, creating a limitation to the process. The development 

of protein glycan coupling technology may solve these issues. using previously identified and characterized CjPglB we were able to show 

that in vitro glycosylation is possible, however CjPglB does not transfer non-acetylated glycans. Our group is in possession of a collection 

of CjPglB orthologues from ε and δ proteobacteria. These will be tested using the in vitro glycosylation system in order to understand if 

their specificity is relaxed allowing transfer of non-acetylated glycans. Furthermore, in search of a suitable carrier protein a rapid Virulence 

Annotation assay will be done by using the in vivo model Galleria mellonella. 

HA04/02 Development of a novel Francisella tularensis subunit vaccine using protein–glycan coupling technology 

Jon Cuccui1 , Madeleine Moule1 , rebecca Thomas2 , DOMiNiC MillS1 , Michael Kowarik3 , Michael Wacker3 , Joann Prior2 , 

Timothy Atkins2 , Brendan Wren1 1 2 3 London School of Hygiene and Tropical Medicine, London; Dstl, Porton Down; GlycoVaxyn, Schlieren, Switzerland 

Francisella tularensis is the causative agent of tularaemia, commonly known as rabbit fever, a potentially fatal human disease. The organism is 

an ACDP category 3 pathogen, and to date only an unlicensed vaccine, providing incomplete immunity whilst demonstrating considerable 

side effects, is available. The O-antigen glycan of F. tularensis has been shown to be immunogenic. To date, glycoconjugates have proven 

successful as vaccines against other bacterial pathogens such as Streptococcus pneumoniae. However, these vaccines have been synthesised 

by chemical coupling of the desired glycan to a protein adjuvant, a process which is both expensive and inefficient. The Campylobacter jejuni 

oligosaccharyl transferase PglB has been shown to catalyse the transfer of glycans from a lipid carrier to a protein containing the acceptor 

sequon D/E-x-N-Y-S/T if present in an exposed secondary structure. This project demonstrates that recombinant expression of PglB in 

Escherichia coli together with an acceptor protein and the locus encoding the F. tularensis O-antigen biosynthesis results in the generation 

of protein glycosylated with the F.tularensis O-antigen glycan. This glycoprotein is currently being assessed for its protective capability in a 

BAlB/c mouse model of tularaemia. 




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HA04/03 The development of novel glycoengineering tools 

SHEriF ABOuElHADiD1,2 , Jon Cuccui2 , Mohamed rahman2 , Brendan Wren2 1Dept of Biotechnology, Institute of Graduate Studies and Research, Alexandria University 163 Horreya Avenue, Chatby, Alexandria, 

Egypt, Alexandria, Egypt; 2Dept of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene 

and Tropical Medicine Keppel Street, London WC1E 7HT 

Polysaccharide subunit vaccines are effective against a wide spectrum of pathogen but have the drawback that they elicit T-cell 

independent immune responses, leading to short lasting protection. Coupling the polysaccharide to a protein carrier overcomes this 

problem by producing a T-cell dependent immune responses and also increases the efficacy of the vaccine. Chemical conjugation suffers 

the drawback of being a complex, time consuming procedure and often resulting in low yields. Biological conjugation can be 

achieved in E.coli carrying three plasmids; one coding for polysaccharide, another for an acceptor protein and the last coding for the 

oligosaccharyltransferase PglB. However, the use of three plasmids may lead to complications of selection of both compatible origins of 

replication and antibiotic marker. A complex part of the process involves isolating and expressing the polysaccharide coding region. To 

overcome this problem the glycoengineering machinery can be transformed into a bacterium that naturally generates the polysaccharide, 

reducing the number of plasmids required to two. However, some hosts are also unable to maintain even these. We present a novel tool 

designed to overcome these problems by enabling the insertion of the oligosaccharyltransferase coding gene into the chromosomal DNA 

of the host. 

HA04/04 Development of a novel Streptococcus pneumoniae vaccine using protein–glycan coupling technology 

lAurA YATES, Jon Cuccui, Vanessa Terra, rebecca langdon, Brendan Wren 

LSHTM, London 

Streptococcus pneumoniae, a leading cause of pneumonia and meningitis, is responsible for over one million deaths annually. The current 

vaccine has several limitations; it is expensive to produce and protects against only 13 of the 90 known serotypes. Serotype prevalence 

varies around the world, and the vaccine is optimised for use in the developed world; it is of limited use in the developing world where 

it could be of greatest benefit. The Campylobacter jejuni oligosaccharyltransferase CjPglB transfers glycans onto protein carriers. it was 

hypothesised that CjPglB could be used to transfer S. pneumoniae capsular polysaccharides (CPS) onto an immunogenic protein carrier, 

providing a cost effective means of producing glycoconjugate vaccines, tailor-made for specific populations. Preliminary work was 

conducted using serotype 4 S. pneumoniae. The CPS locus was cloned in E. coli, however no recombinant serotype 4 CPS production was 

detected. rT-PCr and Coomassie staining suggest that the genes are transcribed and translated. An in vitro peptide-based glycosylation 

assay indicated that CjPglB is capable of transferring serotype 4 CPS onto an acceptor protein. Further studies are ongoing to achieve 

recombinant expression of S. pneumoniae CPS in E. coli and to test alternative serotypes as substrates. 

HA04/05 Development of a capsular polysaccharide conjugate vaccine to protect against Burkholderia pseudomallei 

infection 

A.J. George1 , M. Sarkar-Tyson1 , S. Ngugi1 , A. Scott1 , i.S. roberts2 , J.l.Prior1 1 2 Defence Science & Technology Laboratory, Porton Down, Salisbury, SP4 0JQ; Faculty of Life Sciences, University of Manchester, 

Manchester M13 9PT 

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of high mortality in endemic areas. B. pseudomallei is known to 

express several surface polysaccharides, including a capsule (CPS), lipopolysaccharide (lPS) and an exopolysaccharide (EPS). The CPS is 

well characterized and a known virulence factor, having shown immunogenicity and protection in murine models. Generally, polysaccharides 

do not confer T cell-dependent immunity and therefore immunity is short-lived. By covalently linking polysaccharide to protein, the 

polysaccharide moiety of the conjugate becomes T cell-dependent and immune memory is induced. The aim of this project is to conjugate 

CPS to tetanus Hc fragment protein and evaluate this as a vaccine candidate to protect against B. pseudomallei infection. CPS was extracted 

from B. pseudomallei K96243 and purified using affinity chromatography. This purified material was conjugated to the tetanus Hc fragment 

protein using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and an adipic acid dihydrazide (ADH) linker. results suggest 

the presence of a conjugate but yields were low. Future work will investigate optimization of the conjugation chemistry and the resulting 

conjugates will be evaluated in a murine model of infection. 




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HA05 Meningitis 

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HA05/01 Transcription of the gene encoding fHbp, a meningococcal vaccine antigen, is differentially regulated by iron 

availability 

HOllY SANDErS1,2 , Carina Brehony2 , rory Care1 , Martin Maiden2 , ian Feavers1 1 2 National Institute for Biological Standards and Control, Potters Bar; University of Oxford, Oxford 

Meningococcal Factor H Binding protein, fHbp, is an important component of several vaccines targeting serogroup B disease. Although 

fHbp is thought to be important for meningococcal survival in vivo, expression levels are variable, with current evidence suggesting oxygendependent 

regulation. in this study, quantitative rT-PCr was used to compare transcription levels of fHbp in total rNA extracted from 

98 meningococcal strains grown under iron-replete and iron-restricted conditions. Transcription of fHbp in the majority of strains tested 

was found to be iron-activated, while transcription in all ST32 strains investigated was iron-repressed. Differences in regulation may result 

from the exclusive ability of ST32 strains to produce a previously-described bicistronic rNA transcript containing both nmb1869 and 

fHbp reading frames. results of this study, along with previous evidence, support a model where regulation of fHbp is both oxygen- and 

iron-dependant, with transcription in ST32 strains also affected by regulation of nmb1869. This highlights the importance of using panels of 

strains from a variety of clonal complexes when investigating vaccine antigens, and suggests that results obtained from serum bactericidal 

assays, considered the correlate of protection for meningococcal disease, may not easily be extrapolated to protection from fHbp-specific 

antibodies in vivo. 

HA05/02 The meningococcal transcriptome during prolonged association with human respiratory tract epithelial cells – 

modelling nasopharyngeal colonization 

AriANN HEY, Ming-Shi li, Simon Kroll 


To study the meningococcal transcriptome during prolonged epithelial association (modelling colonization: preparatory to comparison with 

in vitro biofilm- and blood-grown organisms) microarray analysis of gene expression was performed on rNA extracted from meningococci 

(strain MC58) co-cultivated for up to 21d with the bronchial epithelial cell line 16HBE14. results have initially been compared to the 

transcriptome of bacteria grown for 3h in tissue-culture medium. Consistent with the well-established reduction in capsulation during 

colonization, polysaccharide biosynthesis genes synx, siaB and siaC, and export genes ctrA-C, were found to be down-regulated at 24h and 

96h. By contrast, genes encoding pilus components were up-regulated to varying extents, including in particular the major subunit PilE, PilQ 

and PilT. Transcription of genes encoding putative non-pilus adhesins was also increased, including the outer membrane protein NMB2095 

and the autotransporter adhesin NadA. Of the five components of the investigational MenB vaccine 5CVMB – NadA, NMB2091, FHBP, 

NMB1030 and NMB2132 – the first two were up-regulated and the last two down. However, the additional vaccine candidate PorA was 

transcribed at a high level. in this model, early stages of ‘epithelial colonization’ are accompanied by significant changes in the meningococcal 

transcriptome, although by 24h a transcriptional plateau appears to be established. 

HA05/03 Host responses to Neisseria meningitidis and Neisseria lactamica indicate different colonization strategies at the 

respiratory tract 

HAZEl EN EN WONG1,2 , Ming-Shi li2 , J. Simon Kroll2 , Martin Hibberd1 , Paul langford2 1 2 Infectious Diseases, Genome Institute of Singapore; Dept of Medicine, Imperial College London, St Mary’s Campus, London 

Neisseria meningitidis is a bacterium that colonizes the nasopharyngeal mucosal surface. Occasionally, it can invade to cause potentially 

life-threatening meningitis and septicaemia. in contrast, closely related bacteria such as the commensal Neisseria lactamica, also colonize 

the nasopharynx but do not cause disease. Early interactions of these two different species of Neisseria within the nasopharynx during 

colonization could affect host cell responses, and these may be capable of altering the outcome of the infection. Characterizing the host 

responses to these two different neisserial species may uncover how pathogenic and commensal bacteria modulate the host in different 

ways. Human bronchial epithelial cell responses to N. meningitidis and N. lactamica were determined by genome wide microarrays, with 

validation using qrT-PCr and EliSAs. Genes involved in host energy production processes were associated with N. meningitidis and N. 

lactamica, suggesting that they both utilise host resources for energy. in contrast, the data indicated that while N. meningitidis down-regulates 

host defence genes, N. lactamica initiates a proinflammatory response, suggesting specific colonization processes that may lead to different 

clinical outcomes. Neisserial secreted proteins were involved in some of these differential responses, suggesting novel mechanisms for 

modulation of the host response. 

HA05 

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HA05/04 Minor pilins as novel vaccine candidates against Neisseria meningitidis 

ANA CEHOViN, Vladimir Pelicic 

Imperial College London, Centre for Molecular Microbiology and Infection, London 

There is an urgent need for development of vaccines against meningococcal infections. N. meningitidis expresses type iV pili (Tfp) which 

are key virulence factors. Tfp are composed of subunits of the major pilin PilE and minor pilins Pilx, PilV and ComP with important roles in 

Tfp biology. Tfp have been attractive candidates for vaccine development for a long time, however development of a pilus-based vaccine 

was halted due to extensive antigenic variation of PilE. We therefore tested whether minor pilins might display vaccine potential. We first 

investigated the prevalence and sequence variation of genes encoding minor pilins in a large collection of clinical isolates. These genes were 

present in all clinical isolates and, unlike pilE, were found to be highly conserved. Phylogenetic analysis revealed that their sequences cluster 

within clonal complexes. We then tested whether antibodies directed against these proteins interfere with Tfp-linked functions and/or 

mediate bactericidal activity. Although sera directed against minor pilins did not have high bactericidal titres, some of them interfered with 

Tfp functions. Our results suggest that it is worth pursuing the studies on minor pilins as they may have the potential for inclusion in a broad 

vaccine against N. meningitidis. 

HA05/05 Studying the assembly machinery of a universal bacterial virulence factor: the type IV pilus 

MiCHAEllA GEOrGiADOu, Vladimir Pelicic 


Type iV pili (Tfp) are one of the most widespread bacterial virulence factors. They mediate a wide spectrum of functions which contribute 

to the pathogenesis in many bacterial species, including the important human pathogen Neisseria meningitidis. Since the molecular 

mechanisms of Tfp biogenesis are poorly understood, our group started a systematic analysis using N. meningitidis as a model. it was 

determined that 15 Pil proteins, all of which are highly conserved in other piliated species, are necessary for Tfp biogenesis. A genetic 

approach suggested that seven of these are involved in pilus assembly per se: the pilin PilE, the prepilin peptidase PilD, the ATPase PilF 

and the poorly characterized PilM, PilN, PilO and PilP proteins. in order to improve our understanding of the pilus assembly machinery, 

we employed a bacterial two-hybrid system to identify the interactions between eleven Pil proteins, including almost all of the assembly 

proteins. This uncovered many novel protein-protein interactions, that have been quantified, giving a better view of the system. This 

showed that PilM, PilN and PilO form an inner membrane sub-machinery that interacts with PilE. Further characterization of this submachinery, 

which is underway, is expected to shed light on the molecular mechanisms of pilus assembly. 

HA05/06 Escherichia coli K1 invasion of human brain microvascular endothelial cells via a dynamin-independent pathway 

liP NAM lOH1 , Naveed A. Khan2 , Theresa Ward1 1 2 London School of Hygiene and Tropical Medicine, London; University of Nottingham, Sutton Bonington 

Escherichia coli (E. coli) K1 is one of the most common Gram negative bacteria that cause neonatal bacterial meningitis. Previous studies 

have extensively investigated bacterial entry pathways into primary human brain microvascular endothelial cells (HBMEC) to gain insight 

into how the bacteria cross the blood brain barrier in vivo. Some evidence suggests that the bacterium invades, and possibly transcytoses, 

HBMEC by exploiting caveolin-mediated uptake, a clathrin-independent endocytic pathway. Dynamin, a large GTPase, has been implicated 

in the membrane fission of caveolae buds and in this study its role in bacterial entry into HBMEC was investigated. We found that 

overexpression of green fluorescent protein (GFP) tagged dominant negative dynamin 2 [Dyn2(aa)K44A] did not affect the bacterial 

invasion based on quantitative microscopy scoring. Our immunofluorescence staining additionally found that flotillin, a lipid raft marker 

associated with clathrin-independent endocytosis, accumulated around invading bacteria as well as intracellular bacteria. We would like to 

propose that E. coli K1 is able to invade HBMEC via a dynamin-independent endocytic route, which requires flotillin. The role of flotillin in 

transcytosis of the bacteria is being further explored with mutant flotillin constructs. 

HA05/07 Neisseria meningitidis isolates that do not express factor H-binding protein retain their ability to cause invasive 

meningococcal disease 

liONEl TAN1 , Jay lucidarme2 , rachel Exley1 , Jamie Findlow2 , ray Borrow2 , Christoph Tang1 1 2 Imperial College London, London; Health Protection Agency, Manchester 

Neisseria meningitidis recruits the negative complement regulator, factor H (fH), to its surface via factor H binding protein (fHbp), enabling 

it to evade the immune system and cause disease. fHbp is a component of two vaccines currently in clinical trials. We have identified 

strains from individuals with invasive meningococcal disease in which fhbp either contained a frame shift mutation or was entirely absent. 




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HA05 Cont. 

We confirmed that these strains do not express fHbp by whole cell EliSA and Western analysis. Furthermore, we demonstrate that 

these strains have reduced capacity to bind fH by far Western analysis. in spite of the lack of expression of this important virulence factor 

and vaccine antigen, these meningococcal isolates retain their ability to cause invasive disease. This suggests that expression of fHbp is not 

essential for virulence in these strains. Furthermore, the lack of expression of fHbp may also have implications for fHbp containing vaccines, 

with a lack of coverage of fHbp deficient strains. 

HA05/08 Tetanus toxoid as a carrier protein for saccharide-protein conjugate vaccines 

KAY lOCKYEr, Fang Gao, Angela Martino, Karena Burkin, Barbara Bolgiano 

National Institute for Biological Standards and Control, Potters Bar 

A comparison of structural features of tetanus toxoid (TT) used as a carrier protein in numerous different polysaccharide-protein conjugate 

vaccines to protect against Haemophilus influenzae type b, Neiserria meningitidis serogroups A, C, W-135 and Y, as well as serotype 18C 

of Streptococcus pneumoniae has been made. The aim of the study is to better understand the range of tetanus toxoid conjugates being 

used, and ultimately to elucidate structure-immunogenicity relationships. The TT starting materials used for conjugation were all of similar 

quality, but formed conjugates with wide size ranges. All the conjugates had two chromatographic peaks, and these were analysed further 

by SEC-MAllS for more precise sizing and PS-to-protein loading estimates. The conformation of the TT starting materials showed some 

slight differences, but in general, the toxoid conformation was preserved following conjugation, as judged by fluorescence spectroscopy and 

immunogenicity of the carrier protein. 

Conclusions: The presence of novel clones in South East Asia underlines the need for further studies to determine the molecular 

epidemiology of circulating pneumococci from carriage and disease. Data from this study has directly informed policy regarding 

pneumococcal surveillance in Singapore; MlST is now being performed on all Singaporean iPD isolates. 

HA05/09 Multilocus sequence typing of Cronobacter sakazakii reveals genetic signature for meningitic strains 

SuSAN JOSEPH, Stephen Forsythe 

Nottingham Trent University, Nottingham 

Background: Cronobacter (previously Enterobacter sakazakii) is a diverse genus in the family Enterobacteriaceae, consisting of 

pathogens associated with human infections, like meningitis, septicaemia and necrotizing enterocolitis, especially among infants and 

immunocompromised adults. A 7-loci Multilocus Sequence Typing (MlST) scheme has now been established for the genus (http://www. 

pubMlST.org/cronobacter), which has provided a robust and reliable characterization tool for the organism. it is based on the analysis of 

the nucleotide sequences of seven protein coding genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 nt). 

Methods: The Cronobacter MlST scheme was applied to 168 diverse strains (temporally, geographically and source defined). 

Results: 54 defined Sequence Types (ST) have been obtained for the entire genus. Of these, Cronobacter sakazakii, the type species of 

the genus, has 23 unique STs to date. A clear pattern has been observed in certain STs with respect to the source of the isolates. Among 

these, ST4 has been of particular interest, because of its statistically significant dominance of isolates associated with a number of neonatal 

meningitis cases across the world, including some fatalities. Virulence of these strains is supported by in vitro tissue culture analysis. 

Conclusions: The infectivity of C. sakazakii appears to vary according to clonal source. 

HA05/10 Informing vaccine policy through the molecular characterization of pneumococci in Singapore 

EliTA JAuNEiKAiTE1 , Nicholas W.V. Churton1 , raymond lin2 , Martin l. Hibberd3 , Johanna Jefferies1,4,5 , Stuart C. Clarke1,4,5 1 2 Sir Henry Wellcome Laboratories, University of Southampton School of Medicine, Southampton; National Public Health Laboratory, 

Singapore; 3Infectious Diseases, Genome Institute of Singapore; 4Health Protection Agency, Southampton; 5Southampton NIHR 

Biomedical Research Unit in Respiratory Medicine, Southampton 

Background: Streptococcus pneumoniae is a major cause of sepsis, meningitis and respiratory disease worldwide. There is currently a lack of 

epidemiological data on invasive pneumococcal disease (iPD) from South East Asia. We present epidemiological data on iPD from the 

national surveillance scheme in Singapore. 

Methods: We characterized 159 iPD isolates (children 18 years: n=144) received by the 

National Public Health laboratory in Singapore from June 2009 to August 2010 using serotyping and MlST. 

Results: Commonly occurring serotypes in children


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HA05 Cont. 

whereas PCV10 and PCV13 – 51% and 74.8% of iPD respectively. There was a high level of diversity in sequence types (STs) among the 

pneumococcal serotypes. Thirty-eight new STs and 18 new alleles were identified. New STs were most prevalent amongst serotypes 3, 19F 

and 23F. 

Conclusion: Although pneumococci from this study were genetically diverse and included numerous new STs, the pneumococcal conjugate 

vaccines could be highly beneficial in preventing iPD in Singapore. 

HA05/11 Detection of pneumococcal carriage: a systematic review 

rEBECCA GlADSTONE1 , Stuart Clarke1,3 , Jo Jefferies1,3 , Saul Faust1,2 1 2 3 University of Southampton, Southampton; Wellcome Trust Clinical Research Facility, Southampton; Health Protection Agency, 

Southampton 

Detection of upper respiratory tract Streptococcus pneumoniae carriage is central to surveillance of pneumococci in the conjugate vaccine 

era, as carriage acts as a marker for changing epidemiology. Many variables exist for collection and processing which may influence 

detection, this can make inter-laboratory or international comparisons difficult. To address this problem a World Health Organisation 

working group published a standard method for detection of pneumococcal carriage. This Pubmed literature review sought to establish 

the exact methods used by carriage studies and determine to what extent carriage studies have adopted the standard method published 

by the WHO.The majority of studies did not follow a standard method for detection of pneumococcal carriage. The nasopharynx was 

the predominant sample collection site, however, the swab type and transport media varied more between studies. The preponderance 

of studies did not follow the WHO standard method, predominantly due to alternative swab and media choice.There is little published 

evidence to support a standard method, with a small number of studies investigating few variables. uptake of a standard method may be 

increased by evidence to support a change in protocol. An evidence based, standard method would increase the value of collected data for 

extrapolation to disease. 

HA05/12 Molecular analysis of Streptococcus pneumoniae isolates from South-east Asia: informing national vaccine policy 

JOHANNA JEFFEriES1,2 , Nancy Tee4 , N Kumar5 , M. Yasim Yusof5 , Shamala Devi Sekaran5 , Stuart Clarke1,2 1Sir Henry Wellcome Laboratories, Division of Infection, Inflammation & Immunity, University of Southampton School of Medicine, 

Southampton; 2Health Protection Agency, Southampton; 3Southampton NIHR Biomedical Research Unit in Respiratory Medicine, 

Southampton; 4Dept of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore; 5Dept of 

Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 

Background: Streptococcus pneumoniae remains a leading cause of serious infection. There is little published epidemiological data regarding 

invasive pneumococcal disease (iPD) in South East Asia. This is the first study to use multi-locus sequence typing (MlST) to investigate 

clonal relationships among Singaporean and Malaysian iPD isolates. 

Methods: We characterized 86 iPD isolate from Singaporean children and a further 30 iPD isolates from a large Malaysian teaching hospital 

using serotyping and MlST. 

Results: Serotypes 14, 6B, 19A and 19F accounted for 80% of Singaporean iPD cases. For Malaysian pneumococci, serotype 19F was 

the most common (n = 5, 16.7%) followed by 4, 19A, 23F and 6 (n= 3, 10%) each. We observed 50 sequence types (STs) from the 

86 Singaporean pneumococci and 26 from the 30 Malaysian isolates; a high proportion of STs from both study centres were novel STs. 

Serotype distribution was similar to that observed elsewhere. 

HA05/13 Enteroviral meningitis: diagnostic features and clinical follow-up 

NAOMi GADSBY1 , lizzie Elliott2 , Heli Harvala1 , Mae Wong3 , Kate Templeton1 1 2 Specialist Virology Centre, Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh; Royal Hospital for Sick Children, Edinburgh; 

3Neonatal Unit, Royal Infirmary of Edinburgh, Edinburgh 

Enterovirus is the most common cause of meningitis in the uK, but is not thought to lead to long-term neurological sequelae. in this study 

we investigated the diagnostic features and clinical follow-up of enteroviral meningitis cases diagnosed by our Virology laboratory. 

82/1274 (6.4%) CSFs tested between 2007 and 2008 were positive for enterovirus by PCr. Clinical presentation, CSF protein levels, WBC 

numbers and differential cell counts showed little difference between enterovirus positive cases and matched negative controls. Children 

under the age of 12 months were the most commonly affected, accounting for 46.3% of cases. Between 2003 and 2010, 87 children in 

this age group were diagnosed with enteroviral meningitis. Of these, no developmental concerns were noted for 45 patients seen either 

on formal follow-up or informally on subsequent presentation for unrelated reasons. Clinical follow-up was not planned for a further 18 




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patients. Data were incomplete for 20 patients, one who had deceased. One patient had severe neurological sequelae due to bacterial 

meningitis, and the remaining three patients were followed up for other reasons. Therefore, children less than 1 year were most commonly 

found positive for enteroviruses in CSF but there was no evidence of neurological sequelae. 

HA05/14 Contribution of pneumococcal NanA to the activation of brain endothelium 

BrANDON J. KiM, Anirban Banerjee, Kelly S. Doran 

Dept of Biology and Center for Microbial Sciences, San Diego State University, San Diego, CA, USA 

Meningitis is the main neurological complication of systemic infection with Streptococcus pneumoniae (pneumococcus). in order to invade 

the central nervous system SPN must penetrate human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises 

the blood-brain barrier (BBB). We have shown recently that the pneumococcal surface-anchored neuraminidase, NanA, promotes 

immune activation and BBB invasion; however the host cell receptor and signaling mechanisms leading to chemokine induction are not 

known. Previous studies using bacterial NanA mutants have mapped il-8 induction to the N-terminal lectin binding domain with limited 

contribution of the sialidase catatlytic activity. Here we further demonstrate that both recombinant NanA protein and a NanA protein that 

is deficient in enzymatic activity activate il-8 transcription and protein secretion from hBMEC. Our data also indicate that bacterial uptake 

and chemokine induction is significantly reduced in a Focal Adhesion Kinase (FAK) deficient cell line. This suggests a novel role for FAK 

and FAK signaling in host defence during pnuemococcal infection. As FAK is a non receptor protein tyrosine kinase involved in signaling 

downstream of integrins, continued studies seek to characterize the role of integrins and integrin signaling pathways modulated by NanA 

during the pathogenesis of pneumococcal meningitis. 

HA06 Guarding microbial diversity 

↑Contents 

HA06/01 Selective isolation and biosystematic studies of Nocardiae isolated from hay meadow soil 

YASHODHArA PATil, Michael Goodfellow 

Newcastle University, Newcastle UponTyne 

Actinomycetes provisionally assigned to the genus Nocardia on the basis of colonial and pigmentational properties were isolated from hay 

meadow plot 6 at the Cockle Park Experimental Farm, Morpeth, Northumberland using a basal medium supplemented with gentamicin and 

antifungal antibiotics. 13 representative strains representing the various colony types growing on the selective isolation plates were found 

to have chemotaxonomic and morphological properties typical of the genus Nocardia. 16S rrNA gene sequencing studies showed that the 

isolates formed a distinct subclade in the Nocardia gene tree that was most closely related to Nocardia acidivorans. The isolates were readily 

distinguished from their nearest phylogenetic neighbours using phenotypic properties and hence can be considered to represent one or 

more novel Nocardia species. representative strains were found to contain novel non-ribosomal peptide and polyketide synthatase genes. 

Thus these results are of both ecological and bacteriological value as they show that novel acidotolerant nocardiae are common in soil and 

are a prospective source of new bioactive compounds. 

HA06/02 Detecting the extent of dactylosporangial diversity in soil using culture-dependent and culture-independent 

procedures 

JENilEiMA DEVi KSHETriMAYuM, Byung-Yong Kim, Michael Goodfellow 

Newcastle University, Newcastle uponTyne 

A twin track approach was designed to determine the extent of dactylosporangial diversity in a hay meadow soil. Two basal media 

supplemented with gentamicin and antifungal antibiotics supported the growth of presumptive member of genus Dactylosporangium. 

representative isolates were found to be bona fide member of the genus as they gave the genus-specific amplification product using 

the genus-specific primer and produced whole-organism hydrolysates rich in either meso or meso hydroxy diaminopimelic acid. Twenty 

representative isolates formed a member of distinct phyletic lines in the 16S dactylosporangial gene tree, which were closely related to the 

Dactylosporangium luridium and Dactylosporangium luteum. Similarly, clones derived from community DNA isolated from the soil samples 

were assigned to phyletic lines which correspond to those found in the culture dependent study. Fourteen isolates were shown to posses’ 

non -ribosomal peptide and polyketide synthase gene. it can be concluded that novel dactylosporangea are not only widely distributed 

in the soil but also form a potentially rich source of novel metabolites. little interest has been shown in the member of the genus 

Dactylosporangium as they are difficult to isolate and grow and hence rarely featured screening studies despite the fact that they produce 

interesting novel secondary metabolites. 




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HA08 Microbial PAMPS 

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HA08 & HA09 

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HA08/01 A novel core genome-encoded superantigen contributes to lethality of community-associated MrSA necrotizing 

pneumonia 

GilliAN J. WilSON1 , Keun Seok Seo2 , Timothy Connelley1 , Olivia N. Chuang-Smith3 , Caitriona M. Guinane1 , 

robyn A. Cartwright1 , Joo Youn Park2 , Patrick M. Schlievert3 , Gregory A. Bohach2 , W. ivan Morrison1 , J. ross Fitzgerald1 1 2 3 University of Edinburgh, Edinburgh: Mississippi State University, Mississippi, United States: University of Minnesota Medical School, 

Minneapolis, USA 

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immunomodulation and severe systemic illnesses such as toxic 

shock syndrome. All known Staphylococcus aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of 

strains. We have identified a novel SAg (SElx) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains including 

the epidemic community-associated methicillin-resistant S. aureus (CA-MrSA) uSA300 clone. SElx has a unique predicted structure 

with a truncated B-domain, but exhibits the biological activities characteristic of a SAg including mitogenicity, pyrogenicity and endotoxin 

enhancement. SElx is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad 

role in disease pathogenesis. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus 

species, followed by diversification by point mutation and assortative recombination resulting in at least 17 different alleles. SElx variants 

made by human- or ruminant-specific clones demonstrated overlapping but distinct activation of human and bovine Vβ-specific T-cells, 

indicating functional diversification in different host species. importantly, SElx made by uSA300 contributed to lethality in a rabbit model of 

necrotizing pneumonia, revealing a novel virulence determinant of CA-MrSA disease pathogenesis. 

HA08/02 Host/microbial cross-talk in the gut: in-depth investigation of the symbiotic effects of Roseburia homini 

i.E. MulDEr, A. lan, V. Gaboriau-routhiau, K. Garden, E. logan, M.i. Delday, r.i. Aminov, N. Cerf-Bensussan, D. Kelly 

Rowett Institute of Nutrition and Health, University of Aberdeen, AB21 9SB; INSERM U793, Université Paris Descartes, Faculté de 

Médecine, Paris, France 

Gut immune education and homeostasis requires a complex dialogue between the host and the colonizing microbiota. Certain 

immunological functions are the prerogative of specific bacterial species, emphasizing the need for studies which dissect the contributions 

of individual species to host immunity. Roseburia hominis is a prevalent member of the human gut microbiota, and interactions with the 

mucosal immune system were investigated using germfree mice mono-associated with this anaerobic bacterium. FiSH revealed colonization 

of ileal and colonic tissues with highest bacterial numbers in the colon, closely associated with the gut mucosa. Full-genome sequencing of R. 

hominis enabled design of a microarray platform to uncover bacterial genes required for colonization and adaptation in the gut. Motility and 

chemotaxis, membrane transport and substrate metabolism were most affected. Host microarray analyses showed that R. hominis-induced 

gene expression was highest in the colon, with a prevalence of innate immune and barrier function genes 

HA09 Food biosecurity 

↑Contents 

HA09/01 How different are they? Next generation sequencing of Campylobacter bacteriophage genomes tells us more 

Natasha ritchie1 , JuSTiN PACHEBAT1,2 , Carolina Marcano3 , David Brown1 , Jim Ajioka1 1 2 3 University of Cambridge, Cambridge; University of Aberystwyth, Aberystwyth; Illumina UK Ltd, Little Chesterford 

Campylobacter jejuni is a Gram negative, microaerophilic bacteria found in the faeces of chickens. Broilers are chickens raised specifically 

for meat production, and contaminated broiler meat is considered to be the most common cause of gasteroenteritis (campylobacteriosis) 

in humans. Campylobacter species are infected by lytic bacteriophages, with the virulence of the phage specific to the strain of the host 

bacteria. There has been a resurgence of interest in the use of bacteriophages as therapeutic agents to reduce C. jejuni contamination 

of meat at the abattoir (1). Campylobacter bacteriophage genomes range in size from 130–320kbp. The bacteriophage DNA is highly 

methylated, making genome analysis difficult using standard techniques. in 2010, Timms et al. used 454 pyrosequencing and sanger 

sequencing to show that the genomes of two Group ii phage were highly conserved (2). in this study we report on the use of solexa 

sequencing to characterize Campylobacter bacteriophage genomes, and the development of a bacteriophage protein prediction and 

annotation pipeline. We also discuss the characteristics of these genomes with regard to bacteriophage genome stability, phage-host 

interactions, and the application of this information to phage therapy. 

References: (1) Carrillo et al., Applied and Environmental Microbiology, 2005. (2) Timms et al., BMC Genomics, 2010. 




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HA09 Cont. 

HA09/02 Acquisition of an 83 kDa conjugative plasmid in Salmonella enterica Enteritidis confers increased invasiveness and 

promotes the growth of S. Enteritidis in eggs 

lEANNE SAiT1 , Chris Coward2 , Tristan Cogan1 , Duncan Maskell2 , Thomas Humphrey3 1 2 3 University of Bristol, Bristol; University of Cambridge, Cambridge; Univeristy of Liverpool, Liverpool 

Since the mid-1980’s there has been an ongoing world-wide outbreak of non-typhoidal Salmonellosis, largely attributed to the consumption 

of eggs contaminated with Salmonella enterica serovar Enteritidis (SE). SE is more prevalent in eggs than S. Typhimurium (STm), the 

second most reported serovar, but STm grows to a high level (>106 CFu/egg) more often than SE when it is present. We have observed 

that strains of SE which carry an 83kDa plasmid, can grow to the levels normally seen with STm. High level growth, combined with the 

propensity for SE to contaminate eggs, represents a serious public health threat which could be exacerbated by the spread of this plasmid. 

Transfer of the 83kDa plasmid (pS1400_89) from SE S1400/94 into a plasmid-free strain increased the frequency of high-level growth in 

eggs. Genomic indexing and sequencing of pS1400_89 showed that it is related to the incl1 plasmid Col1b-P9. in addition, a pS1400_89 + 

strain of NCTC 13349 was more invasive of cultured epithelial cells than the plasmid-free strain. Deletion of the genes encoding SEF17 and 

SEF21 fimbriae in the pS1400_89 + strain reduced invasion, indicating that altered regulation of fimbriae may be involved in the enhanced 

invasion phenotype. 

HA09/03 LPS O-antigen structure and repeat length are important determinants for colonization of spleen, reproductive 

tract and eggs in chickens 

lEANNE SAiT1 , Chris Coward2 , Tristan Cogan1 , Penelope Gramiri2 , lisa Williams1 , Emma Trantham1 , Duncan Maskell1 , 

Thomas Humphrey3 1 2 3 University of Bristol, Bristol; University of Cambridge, Cambridge; University of Liverpool, Liverpool 

Salmonella Enteritidis(SE) is the predominant salmonella serovar associated with contamination of hens’ eggs. Others such as Salmonella 

Typhimurium(STm) can also establish infections in hens, however why SE represents a more a significant public health threat than other 

salmonella serovars, remains elusive. 

SE survives better in egg albumen than STm, and SE and STm have different lPS O-antigen (OAg) structures (STm:O4; SE:O9). We 

examined whether OAg structure was related to survival in albumen and infectivity in laying hens 

Mutants were constructed in OAg synthesis genes. Genes determining the OAg serotype were also exchanged between SE and STm to 

generate SE(O4) and STm(O9). rough(ΔwaaL) and semi-rough(Δwzy) mutants showed reduced survival in albumen, as did impairment in 

production of Very-long OAg(ΔfepE). Decreased production of long-OAg(Δwzz) did not result in a survival defect. SE(O4) had reduced 

albumen survival, while STm(O9) showed increased survival. 

In vivo, SE(O4) colonized the spleen in lower numbers than wild-type. No difference was observed in reproductive tissues. Both αfepE and 

αwzy mutants colonized the reproductive tracts at lower levels than wild-type and were not found in eggs. This could either be due to 

increased killing in albumen, reduced colonization of reproductive tract tissues or a combination of both. 

HA09/04 Is carriage of Campylobacter jejuni by broiler chickens affected by breed or husbandry factors? 

liSA WilliAMS1 , leanne Sait1 , Emma Trantham1 , Tristan Cogan1 , Tom Humphrey2 1 2 University of Bristol, Bristol; University of Liverpool, Liverpool 

Modern broiler chickens reared in intensive indoor systems have been selected to grow rapidly. recently, consumers have moved towards 

purchasing birds produced in enhanced welfare systems, which use a slower growing breed. These birds appear to have different levels of 

Campylobacter carriage in the field, but the reasons for this are unknown. 

Carriage of Campylobacter, weight, behaviour and health of a fast and a slow growing breed of chicken was measured in experimental 

flocks. Experiments were conducted to examine the effect of breed, diet and stocking density. At 21d, birds were either infected with 

Campylobacter or given a placebo. Cohorts of birds were euthanased at intervals and samples taken for examination for Campylobacter. 

in all experiments, Campylobacter was detected in the caeca and by enrichment from the liver and spleen from both breeds by 2 dpi. low 

level colonization persisted until 28 dpi. Breed and diet significantly affected the frequency of liver colonization, but had no effect on the 

frequency of colonization or numbers of Campylobacter in the caecum. 

The data indicate that it is a probably a combination of factors that leads to reduced colonization of the slower growing chickens under 

commercial conditions. 




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HA09 Cont. 

HA09/05 Investigation of the role of Salmonella enterica Enteritidis genomic islands in colonization of the chicken 

reproductive tract 

CHriS COWArD1 , leanne Sait2 , Tom Humphrey3 , Tristan Cogan2 , Duncan Maskell1 1 2 University of Cambridge, Dept of Veterinary Medicine, Cambridge; University of Bristol, Dept of Clinical Veterinary Science, Bristol; 

3University of Liverpool, Liverpool 

infection with Salmonella enterica serovars is a major cause of human gastrointestinal disease with S. Enteritidis (SE) being by far the most 

common serovar in the united States and European union accounting for over 50% of cases. Consumption of infected eggs and egg 

products has been the most commonly identified route of infection. The association of SE with eggs suggests that this serovar has specific 

traits that facilitate interaction with the reproductive organs of layers and/or entry to/survival in the egg. recent genome sequencing has 

revealed genomic islands in SE and the avian-adapted serovar Gallinarum that are not present in Typhimurium, the second most common 

serotype associated with human disease. To determine whether these loci have a role in colonization, we investigated the phenotype of 

deletion mutants following oral inoculation of chickens. 

None of the mutants were defective in colonization of reproductive tissues, and liver and caecal colonization was broadly unaffected. in 

contrast, colonization of the spleen was decreased in all mutants. invasion assays using the HD11 cell line showed that this decrease in 

splenic load did not correlate with a reduced ability to invade chicken macrophages. 

HA09/06 The potential use of ozone gas to reduce fungal post-harvest spoilage of fresh produce 

iAN SiNGlETON1 , Nikos Tzortzakis2 , Anne Borland1 , Jerry Barnes1 1 2 Newcastle University, Newcastle upon Tyne; Technological Education Institute of Crete, Heraklion, Greece 

large amounts of fresh produce can be lost to fungal spoilage during transport and storage and there is interest in alternatives to traditional 

post-harvest pesticides to reduce post-harvest loss. Ozone is a powerful anti-microbial agent and can be applied easily to fresh produce 

in gaseous form. This work demonstrates the potential use of ozone gas to inhibit fungal spoilage. Our results show that ozone gas at low 

concentrations e.g. 200 ppb (v/v) and lower, can inhibit fungal spoilage e.g. Botrytis cinerea on a variety of fruit, and reduces fungal spore 

production. This reduction in spore production would act to reduce the spread of disease during post harvest transit/storage. Ozone 

exposure also appears to ‘prime’ fruit tissue to resist subsequent fungal attack but the exact mechanism of action is not understood at 

present. Overall, it appears that ozone gas has significant commercial potential to reduce fungal spoilage of fresh produce. 

HA09/07 Analysis by pyrosequencing of microbial changes at weaning in pigs: a preliminary study 

EliZABETH KiNG1 , Christine Dodd1 , Alexander Marshall1 , Aziz Aboobaker1 , robin Spiller2 , Kenneth Mellits1 1 2 University of Nottingham, Nottingham; Nottingham Digestive Diseases Centre (University of Nottingham), Nottingham 

The post weaning period in pigs is accompanied by increased susceptibility to disease. limited use of antibiotics in water is appropriate on 

those farms with enteric disease issues in order to counteract possible outbreaks. To determine the microbiological ramifications of such 

treatment we investigated the changes that occur in microflora at weaning with the inclusion of antibiotics. in this preliminary study DNA 

was extracted from pig faecal samples from 2 litter mates before and after weaning. Microbial diversity was assessed using roche 454 

pyrosequencing. Pyrosequencing identified the presence of 854 species before and 695 species after weaning. Our results showed shifts 

in the balance of genera: the Clostridium genus decreased from being the dominant group (40% of identified species) to 17% of identified 

species in response to antibiotics and weaning; in contrast the proportion of Ruminococcus species identified increased from 12% to 40% 

and became the dominant group. lactic acid bacteria decreased and Gram negative species declined dramatically including the pathogens 

Salmonella, Campylobacter and E.coli, which are the targets of the antibiotic used (Colistin). Pyrosequencing thus provided an insight into the 

large diversity of microflora within the gut, and how it changed rapidly in response to weaning and antibiotics. 

HA09/08 Not presented 




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HA09 Cont. 

HA09/09 Proteome and genes expression in Campylobacter strains adapted to disinfectant treatment under chemostat 

culture conditions 

AliCJA BArCZYNSKA, Cyril V. Carroll 

Dept of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland 

Campylobacter spp. are Gram-negative spiral bacilli that is frequently associated with bacterial diarrhea. Cases of campylobacteriosis are 

mainly associated with handing raw poultry or eating raw or undercooked poultry meat. unlike other food-borne pathogens, Campylobacter 

spp. are apparently fragile organisms that are unable to multiply outside the animal host and are highly susceptible to a number of 

environmental conditions as they lack most of the stress response factors common in other enteric bacteria. 

This work used an in vitro chemostat model to study resistance development in Campylobacter to food industry disinfectants (Citric Acid, 

Acidify Sodium Chlorite). A number of different food derived C. jejuni were grown in chemostat culture over 40 generations and exposed 

to increasing increments of disinfectants. During the time course of exposure to the disinfectants representative samples were taken from 

the chemostat adapted culture and analysed using proteome (1D/2D gel electrophoresis) and genes expression technology (rT-PCr). 

in general, Campylobacter cells exposed to and adopted to increasing doses of disinfectant (CA, ASCh) produced less proteins overall and 

those that were produced, were at diminished levels. 

Campylobacter adapted to the disinfectant (CA) resulted in increased expression (~300%) of the CiaB (Campylobacter invasion antigen) and 

CmeA (multidrug efflux pump) genes. 

HA09/10 Differences in the biofilm properties of laboratory adapted and recent isolates of Salmonella enterica 

MArY COrCOrAN1 , Dearbhaile Morris1 , Niall De lappe2 , Juliette Ward2 , Jean O’ Connor2 , Ger Doran2 , Peter Dockery1 , 

Martin Cormican1,2 1 2 School of Medicine, Clinical Science Institute, National University of Ireland, Galway, Galway, Ireland; National Salmonella Reference 

Laboratory, Clinical Science Institute, National University of Ireland, Galway, Galway, Ireland 

Background and objective of work: Salmonella enterica biofilm formation in food processing environments may be important as a potential 

source of contamination. Well characterized strains such as S. Typhimurium lT2, Sl1344 and S. Agona Sl483 are regularly used as models 

in studying biofilm formation. We have assessed biofilm formation by these strains compared to recent clinical and food isolates. 

Methods: Biofilm growth was established using the CDC biofilm reactor (CBr). isolates studied were the 3 strains above and recent isolates 

of the same serovars. Glass, stainless steel, polycarbonate plastic, glazed tile and concrete were used as substrates for biofilm growth. The 

CBr was operated under batch-phase for 24 hours followed by continuous-flow phase for 24 hours. Biofilm density was assessed by 

electron microscopy and by removal viable counts of detached cells. 

Results: Biofilm density as assessed by viable counts were lower for laboratory adapted strains of S. Agona compared with recent isolates. 

EM suggested that biofilm detachment by sonication was less complete for S. Typhimurium lT2 than for recent isolates of S. Typhimurium. 

Conclusion: We suggest that caution may be required in extrapolating observations regarding biofilm biology from laboratory adapted strains 

to wild-type strains. 

HA09/11 Toxigenic Bacillus weihenstephanensis isolated from convenience food 

AGNiESZKA BEDNArCZYK-DrAG, Elzbieta Daczkowska-Kozon 

West Pomeranian University of Szczecin, Szczecin, Poland 

B.weihenstephanensis is a psychrotolerant species of Bacillus cereus group, potentially pathogenic to humans owing to enterotoxins 

production, mainly HBl and NHE. until recently one case of emetic B.weihenstephanensis isolated from soil, producing emetic toxin – 

cereulide, was reported. in this work the presence and toxigenicity of B.weihenstephanensis in convenience food requiring cold storage prior 

to consumption was determined. Food products tested were vegetable salads and packed vegetables. All the isolated strains were tested 

for the presence of B.cereus specific 16S rDNA fragment, emetic strains specific ces gene fragment, B.weihenstephanensis characteristic cspA 

gene sequence, psychrototolerant strains specific 16S rDNA fragment, HBl, NHE and CytK enterotoxins gene fragments (hblDAC, nheABC 

and cytK). Potentially emetic strains were subjected to boar sperm motility and BCET-rPlA assays. Six of isolated strains were confirmed 




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as B.weihenstephanensis. Two of those were carrying ces gene fragment and were positive in boar sperm motility assay actually producing 

cereulide. Both strains carried hblD, hblC, nheA and nheB gene fragments and none hblA and nheC ones. Only one strain carried cytK gene 

fragment and was positive in BCET-rPlA assay. in this study it was proved that convenience food such as vegetable salads can be a vehicle 

of emetic and enterotoxic 

B. weihenstephanensis. 

HA09/12 Genotypic and phenotypic diversity of emetic Bacillus cereus strains isolated from food 

AGNiESZKA BEDNArCZYK-DrAG, Elzbieta Daczkowska-Kozon 

West Pomeranian University of Szczecin, Szczecin, Poland 

The aim of the study was to compare a toxigenic ability and phenotypic and genetic profiles of B.cereus group strains of food origin. Based 

on 72 phenotypic features a phenotypic similarity of the strains was assessed using uPGMA method. Genotypic diversity of the strains was 

determined by testing the presence of 16 gene fragments, including those encoding for several virulence factors (two 16S rDNA fragments, 

cspA, hblD, hblA, hblC, nheA, nheB, nheC, cytK, piplC, pcplC, sph, hlyII, hlyIII, ces), and seven rAPD-PCr assays using different primers and their 

combinations. results showed that most of the emetic B.cereus strains tested differed phenotypically from non-emetic B.cereus strains of the 

group, while the presence of gene fragments encoding various virulence factors and several rAPD-PCr profiles do not distinguish emetic 

strains from other representatives of the group. 

HA10 Insect symbiosis 

↑Contents 

HA10/01 Screening a panel of tick cell lines for the detection and identification of endosymbiotic micro-organisms 

PilAr AlBErDi, Matthew Dalby, lesley Bell-Sakyi 

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh 

Ticks transmit numerous viral, bacterial and protozoan pathogens of humans and domestic animals. in addition, ticks harbour symbiotic 

viruses and bacteria which are not known to be pathogenic for vertebrates. Continuous cell lines derived from tick tissues are increasingly 

important research tools for isolation and study of tick-borne pathogens in vitro. The roslin Wellcome Trust Tick Cell Biobank houses the 

world’s largest collection of tick cell lines, currently numbering >40 lines derived from fifteen ixodid and two argasid species including most 

of the vectors of tick-borne diseases worldwide. Very few of these cell lines have so far been screened for chronic infection with symbiotic 

micro-organisms which could become pathogenic if introduced into a vertebrate host, vitally important when considering tick cell culture 

systems as sources of antigen for vaccine development. using PCr-based methods, we screened a representative panel of tick cell lines 

for the presence of viruses and bacteria. DNA and rNA extracted from the cells were, respectively, screened with pan-bacterial 16S 

primers, and used to generate cDNA which was screened using primers which amplify known tick-borne virus genera. PCr products were 

sequenced to aid identification, and infected cell lines were further characterized by electron microscopy. 

HA10/02 Isolation and characterization of symbiotic actinomycetes from bees as a roadmap to novel bioactive compounds 

BYuNG-YONG KiM1,2 , Niketh Mutylala1 , Avinash Bonda1 , Nick Allenby3 , Jeff Errington2,3 , Mike Goodfellow1 1 2 School of Biology, Newcastle University, Newcastle upon Tyne; Institute for Cell and Molecular Biosciences, Newcastle University, 

Newcastle upon Tyne; 3Demuris Ltd., Newcastle Biomedicine Bio-Incubators, Newcastle upon Tyne 

There is an urgent need to find new drugs, to control the spread of antibiotic resistant pathogens and to treat life-threatening diseases 

such as cancer. Amongst prokaryotes, actinomycetes remain a unique source of bioactive compounds, notably antibiotics. Actinomycetes 

which are found from neglected habitats have been explored in many studies and there are increasing evidences that novel actinomycetes 

can be discovered from unexplored areas including symbiotic relationship between actinomycetes and insects. in this study, 26 symbiotic 

actinomycetes were isolated from Bumble bee and identified on the basis of colony morphology, chemical markers and molecular 

characteristics. Most isolates were assigned to the genus streptomyces based on the 16S rrNA gene sequences. All isolates were tested for 

antibiotic production against different Gram negative and positive bacteria and it was found that 3 isolates inhibited E. coli K12 (12%) and 

17 isolates inhibited B. subtilis 168 (65%). One of most inhibitory isolates, isolate N16, which is closest to S. nojiriensis lMG 20313T (100%) 

was further investigated by fermentation and chemical method to identify the metabolite. The results provide promising evidence that 

the insects such as bee have diverse symbiotic actinomycetes especially streptomyces and hence could be rich sources for novel bioactive 

compounds. 




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HA12 Virus pathogenesis 

↑Contents 

HA12/01 The murid herpesvirus 4 entry complex undergoes post-endocytic conformation changes before engaging in viral 

membrane fusion 

DANiEl l. GlAuSEr, Philip G. Stevenson 

Division of Virology, Dept of Pathology, University of Cambridge, Cambridge 

The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells via endosomal uptake followed by fusion of the viral envelope with 

membranes of lAMP-1 + endosomes, the latter resulting in release of capsids into the cytoplasm. Here, we analyze conformation changes 

in the viral entry complex consisting of gB and gH/gl during virus entry by in situ probing of protein conformations with monoclonal 

antibodies. We dissect conformation changes into pH-dependent changes preceding fusion and fusion-related changes by blocking 

endosomal acidification with Concanamycin A or ammonium chloride and by blocking viral membrane fusion with a neutralizing gB-specific 

antibody. Our data show that gB undergoes a pH-dependent conformation change preceding fusion, as well as a strictly fusion-related 

conformation change. We also show that the gH/gl heterodimer dissociates in a pH-dependent manner upstream of fusion, while gH 

undergoes a conformation change during viral membrane fusion. While the conformation changes in gB and dissociation of gH/gl preceding 

fusion occur independently from each other, the fusion-related changes in gB and gH appear tightly linked. Together, our data support a 

model in which the post-endocytic, pH-dependent conformation change in gB and dissociation of gH/gl lead to fusion-competent forms of 

gB and gH which together mediate viral membrane fusion. 

HA12/02 Analyses of the extent of HSV-1 viral promoter activation that can precede the establishment of neuronal latency 

JOAO PrOENCA, Heather Coleman, Viv Connor, Stacey Efstathiou 


HSV-1 establishes life-long latency in sensory neurons and it is widely assumed that latency is the consequence of a failure to initiate 

virus immediate early (iE) gene expression. However using a Cre reporter mouse system in conjunction with Cre expressing HSV-1 

recombinants we have previously shown that activation of the iE iCP0 promoter can precede latency establishment in up to 30% of latently 

infected cells. During productive infection of non-neuronal cells iE promoter activation is largely dependent on the transactivator VP16 a 

late structural component of the virion. Of significance, VP16 has recently been shown to exhibit altered regulation in neurons: where its de 

novo synthesis is necessary for iE gene expression during both lytic infection and reactivation from latency. 

in the current study we utilized the reporter mouse model system to investigate whether VP16 promoter activation within neurons could 

be compatible with cell survival and latency. in contrast to the high frequency activation of representative iE promoters prior to latency 

establishment, no evidence of efficient cell marking was observed using a virus recombinant expressing Cre under VP16 promoter control. 

We conclude that iE promoter activation preceding latency establishment is likely to occur through a VP16 independent mechanism. 

HA12/03 Detection and genetic characterization of enteroviruses circulating among wild populations of chimpanzees and 

gorillas in Cameroon; relationship with human and simian enteroviruses 

HEli HArVAlA1,2 , Colin Sharp2 , Martine Peeters3 , Peter Simmonds1 1 2 3 Clinical Virology Centre, Edinburgh; University of Edinburgh, Edinburgh; University of Montpellier, Montpellier, France 

Enteroviruses (EVs), members of the family Picornaviridae are a genetically and antigenically diverse range of viruses infecting humans and 

several Old World monkey (OWM) species. Despite their known wide distribution in primates, nothing is currently known about the 

occurrence, frequency and genetic diversity of enteroviruses infecting apes. To investigate this, 27 chimpanzee and 27 gorilla faecal samples 

collected from undisturbed jungle areas with minimal human contact in Cameroon were screened for EVs. Four chimpanzee samples but 

none of the gorillas were positive. Genetic characterization of the VP1, VP4 and VP2 genes, the 5’unstranslated region and partial 3Dpol 

sequences enabled chimpanzee-derived EVs to be identified as (a) the species A type, EV76, (b) a new species D type (assigned as EV111 

along with a previously described human isolate from the Congo by the iCTV) and (c) two as a new species B type (assigned as EV110) 

most closely related to the SA5 isolate recovered from a vervet monkey. The identification of EVs infecting chimpanzees related to those 

circulating in human and OWM populations provides evidence for cross-species transmission of EVs between primates. However, the 

direction of transfer and the existence of primate sources of enterovirus infections in humans require further investigations. 

HA12 

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HA12 Cont. 

HA12/04 Picorna-like virus pathogenesis in honey bees: the effects of virus diversity and the mite Varroa destructor on the 

virus–host interactions 

EuGENE rYABOV1 , Jonathan Moore2 , Aleksey Jironkin2 , James Bull1 , David Chandler1 , Nigel Burroughs2 , David Evans1 1 2 School of Life Sciences, University of Warwick, Coventry CV4 7AL; Warwick Systems Biology Centre, University of Warwick, Coventry 

CV4 7AL 

Picorna-like viruses related to Deformed wing virus (DWV) are widespread in honeybees in Europe, but usually these viruses cause 

symptomless infections and accumulate to low levels. Exposure of honeybees to the ectoparasitic mite Varroa destructor, which feeds 

on honeybee haemolymph, results in dramatic increase of the replication of DWV-like viruses and the development of virus-induced 

pathogenesis. Varroa may harbour DWV or closely related Varroa destructor virus-1 (VDV-1) which could be transmitted to honeybees. By 

using deep sequencing, we identified novel recombinants between DWV and VDV-1 which accumulated to higher levels than DWV in 

both honeybees and Varroa. These recombinants, which contained the structural protein-coding sequence derived from VDV-1 and the 

non-structural protein-coding sequence derived from DWV, were more efficiently transmitted between Varroa and honeybees than DWV. 

We have used microarray transcriptional profiling to identify honeybee genes, the expression of which is influenced by Varroa and/or by 

the viruses. This has allowed us to suggest the potential molecular mechanisms of the effect of Varroa on the virus-honeybee interactions. 

HA12/05 Are endogenous feline leukaemia viruses really endogenous? 

H. STEWArT, O. Jarrett, M. Hosie, B. Willett 

MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity & Imflammation, College of Medical, Veterinary 

& Life Sciences, University of Glasgow, Glasgow 

Feline leukaemia Virus is a feline pathogen in which the envelope glycoprotein (Env) determines subgroup and disease manifestation. 

FelV-B arises through recombination between FelV-A and endogenous FelV (enFelV) presumably after co-packaging of viral genomes 

within a virion; thus FelV-B infections are always found alongside FelV-A. Although most enFelV retroelements are defective, potentially 

functional enFelV genomes have been described. We hypothesised that viral packaging of these genomes in vivo could lead to enFelV 

transmission between cats. Such infections would appear phenotypically as subgroup B. 

After screening 300 FelV field isolates, two (2518 and 4314) typed as subgroup B only. rT-PCr for exogenous FelV revealed that 2518 

contained a truncated FelV-A genome, bearing a ~900kB deletion. 4314 virions did not contain exogenous genomes. However a fulllength 

env could be detected within both isolates using enFelV-specific primers. Sequence analysis revealed homology with enFelV env, 

with recombination breakpoints ~200bp from the Su/TM cleavage site. For both isolates, the majority of the TM domain and 3’ lTr were 

derived from a presumably functional exogenous FelV genome that was no longer present. We hypothesise that an exogenous lTr allows 

otherwise-endogenous viruses to achieve higher rates of transcription and allows them to outgrow the original exogenous virus. 

HA12/06 The signal peptide of the envelope of a recently integrated endogenous sheep betaretrovirus plays a major role in 

escaping gag-mediated late restriction induced by transdominant enjsrv proviruses 

AlESSiA ArMEZZANi1 , Frédérick Arnaud1,2 , Marco Caporale1 , Claudio Murgia1 , Massimo Palmarini1 1MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, 

Veterinary and Life Sciences, University of Glasgow, Glasgow; 2EPHE, Université de Lyon 1, INRA, UMR754, École Nationale 

Vétérinaire de Lyon, IFR 128, Lyon, France 

Background: The exogenous Jaagsiekte sheep retrovirus (JSrV) co-exists with highly related endogenous retroviruses (enJSrVs), stably 

integrated in the sheep genome. Two of the 27 enJSrV proviruses isolated to date, enJS56A1 and enJSrV-20, possess a defective Gag 

polyprotein that blocks the late replication steps of JSrV, as well as other enJSrVs, by a mechanism known as JSrV late restriction (Jlr). 

interestingly, a recently integrated provirus, enJSrV-26, is able to escape Jlr. 

Results: We demonstrate that Jlr escape by enJSrV-26 is due to a single amino acid substitution, which renders the signal peptide (SP) 

of its envelope glycoprotein not functional. indeed, the enJSrV-26 SP does not increase neither Gag expression nor viral particles release, 

unlike the functional SPs of JSrV and enJSrVs. We demonstrate that Jlr escape by enJSrV-26 depends on the presence of the functional 

enJS56A1 SP and on the ratio between enJSrV-26 and enJS56A1 Gag. interestingly, by qPCr analysis, we reveal that the domestic sheep 

has acquired, by genome amplification, several copies of the enJS56A1 provirus. 

Conclusions: These data reinforce the notion that transdominant enJSrV proviruses have been positively selected by domestic sheep, and 

that the co-evolution between endogenous and exogenous sheep betaretroviruses and their host is still occurring. 




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HA12 Cont. 

HA12/07 Morbillivirus V proteins show multiple methods to block interferon action 

SENTHil KuMAr CHiNNAKANNAN, Michael D Baron 

Institute for Animal Health, Pirbright, Surrey 

Morbilliviruses form a closely related group of viruses which from their P genes encode the P protein and three non-structural proteins 

V, W and C. Here we demonstrate that although the P, V and W proteins of rinderpest virus (rPV) could all bind STAT1 and block 

interferon (iFN)-induced gene transcription, only the V protein could effectively block the iFN-induced antiviral state. results from several 

assays suggest that the complete V protein is essential for the inhibition of this antiviral state as well as for the binding of STAT2 and the 

effective blocking of STAT2 phosphorylation. 

The V proteins of rPV, MeV (measles virus), PPrV (peste des petits ruminants virus) and CDV (canine distemper virus) were compared by 

co-immunoprecipitation, promoter-reporter and vesicular stomatitis virus cytopathic effect reduction assays. They showed varied abilities to 

bind STAT1 or STAT2 and to block iFNα (type i) and iFNγ (type ii) induced gene transcription. The ability to block Type ii iFN correllated 

with STAT1 binding, but there was no correlation between STAT1/STAT2 binding and the block of iFNα induced gene transcription or 

antiviral state. Our results suggest that morbillivirus V proteins have varied abilities to overcome type i and ii iFN action. 

HA12/08 Strong APObEC3H activity in wild felids contributes to intrinsic immunity to FIV 

iSABEllE DiETriCH1 , William McEwan2 , Margaret Hosie1 , Brian Willett1 1 2 University of Glasgow, Glasgow; MRC Laboratory of Molecular Biology, Cambridge 

Feline immunodeficiency virus (FiV) is a lentiviral pathogen associated with an acquired immunodeficiency syndrome (AiDS)-like illness in 

domestic cats. However, in non-domestic felids the pathogenesis is variable and infection may be inapparent. A potential reason for the 

differential effects of infection on distinct hosts is that long-term host-virus co-evolution has led to an enhanced ability of host restriction 

factors to limit viral growth. APOBEC3 proteins form part of the vertebrate intrinsic immune response, deaminating cytosines in nascent 

retroviral cDNA leading to a loss of viral genetic integrity, while simultaneously impeding reverse transcription. 

in this study, we amplified and cloned APOBEC3 cDNAs from cat (Felis catus) and lion (Panthera leo) peripheral blood lymphocytes. Their 

expression levels were determined by quantitative real-time PCr and their ability to modulate the infectivity of FiV virions produced in their 

presence was ascertained. We found that lion APOBEC3H (PleA3H) possessed a significantly higher ability to limit FiV replication than that 

of the domestic cat (FcaA3H). Systematic mutagenesis of PleA3H to FcaA3H revealed four amino acid residues in functionally important 

protein domains that were associated with a gain of function in PleA3H and may have arisen as an adaptation of lions to lentiviral infection. 

HA12/09 Identifying novel interaction partners for the HCV NS5A protein by screening a human SH3 domain phage display 

library 

ZSOFiA iGlOi1 , Arunas Kazlauskas2 , Kalle Saksela2 , Mark Harris1 1 2 University of Leeds, Leeds West Yorkshire; University of Helsinki, Helsinki, Finland 

Hepatitis C virus (HCV) can establish a persistent infection, leading to chronic liver disease and cirrhosis. in this context the HCV NS5A 

protein has been intensely studied as it contains a C-terminal polyproline motif (PxxPxr) that binds to the Src homology 3 (SH3) 

domains of a number of cellular proteins involved in signal transduction. Despite this interest, the precise role of these interactions in the 

HCV lifecycle remains largely unknown. To address this issue, we generated genotype 2a (JFH1) subgenomic replicons where a biotin 

tag was introduced into the NS5A coding region and screened a phage display library containing all 296 human SH3-domains for novel 

interactions. From the screen, amongst others we identified amphiphysin i, a protein involved in clathrin mediated endocytosis and CMS. 

interestingly CMS has been implicated in epidermal growth factor receptor trafficking; previously shown to be perturbed by NS5A in a 

PxxPxr dependent manner. These interactions were confirmed by immunofluorescence and immunoprecipitation experiments in a human 

hepatoma cell line permissive for HCV infection. 

HA12/10 So what would a dog lymphoma virus look like? Using genomic data to identify retroviruses 

rACHAEl TArliNTON1 , Hannah Barfoot1 , robert Gifford2 , richard Emes1 1 2 University of Nottingham, Leicestershire; Aaron Diamond Aids Research Centre, New York, USA 

Dogs are known to suffer from a high incidence of leukaemia and lymphoma. This has led to several attempts to identify a ‘dog lymphoma 

virus’ and there are several publications describing retroviral activity in canine cancers. However there is no published sequence data on dog 

retroviruses. We have taken the approach of characterizing the endogenous (inherited) viruses in the canine genome and have identified a 

group of gammaretroviruses that are restricted to dogs and closely related animals. None of the loci we have identified to date appear to 




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encode for fully functional retroviruses (all have deletions over the surface unit of the envelope gene), however they are transcriptionally 

active with pol gene rNA detectable in normal dog tissue and cell culture lines by real time PCr. retroviral loci polymorphism in different 

breeds of the same animal is well described in other species (such as pigs, mice and chickens) and we consider that functional loci may well 

be present in less inbred dogs than the one the genome build is derived from. We consider the gammaretroviral family we have identified 

to be the best candidates to date for a potential dog lymphoma virus. 

HA12/11 Interaction of human respiratory syncytial virus with the cell cycle 

WEiNiNG Wu, John Barr, Julian Hiscox 


Human respiratory syncytial virus (HrSV) is an important respiratory pathogen, and is a major health risk for the very young and elderly, 

and has significant association with the development of childhood asthma through airway remodelling. As with many viruses, there is 

a constant interplay between HrSV and the host cell. On the one hand HrSV is trying to generate conditions more favourable for 

virus replication whereas on the other, the cell is trying to induce the anti-viral state. An emerging area of rNA virus research is their 

involvement with the cell cycle and subversion of cellular growth to promote virus replication. The cell cycle describes the ordered growth 

and division of cells and is controlled by positive and negative regulators. Given the central role of the cell cycle in cell biology, we have 

defined the interaction of this process with HrSV and the functional implications for the virus and for the host cell. using a model airway 

cell line and primary airway epithelial cells infected with HrSV, we have shown that the abundance and localization of cyclins are altered, as 

well as the transcription of several key genes. This work and the functional implications of these findings will be discussed. 

HA12/12 Sequestering nucleolar proteins to virus replication centres during KSHV infection 

STuArT MACNAB1,2 , Adrian Whitehouse1,2 1 2 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire; Astbury Centre 

for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire 

The nucleolus is classically described as the site of ribosome biogenesis, but recent studies suggest it is implicated in many aspects of cell 

biology. The plurifunctional nature of the nucleolus has been highlighted by extensive proteomic analysis revealing that over 4000 cellular 

proteins localize in this sub-cellular domain. Multiple viruses encode proteins which localize to the nucleolus, however the implication of 

virus-nucleoli interaction is not fully understood. To elucidate the interplay between herpesviruses and the nucleolus, we has employed a 

quantitative proteomic SilAC approach to determine nucleolar proteome changes during the various stages of the KSHV lytic replication 

cycle. results indicate significant changes occur during lytic reactivation involved in: DNA synthesis, replication and repair, gene expression, 

rNA processing, synthesis and modification and protein synthesis and transport. Notably, multiple proteins involved in cellular DNA 

replication were displaced from the nucleolus during the early stages of KSHV replication; Specifically the complete mini-chromosome 

maintenance complex, uBF, rad50 and structural maintenance of chromosome proteins. We are currently investigating whether these 

displaced cellular proteins are required for efficient viral DNA replication and whether inhibiting the function of these displaced cellular 

proteins could be a possible novel antiviral strategy. 

HA12/13 Sequence and characterization of a novel gammaherpesvirus from field voles (Myodes glareolus) 

JAMES STEWArT1 , David Hughes1,2 , Anja Kipar1 , Neil Hall1 , Alistair Darby1 1 2 University of Liverpool, Liverpool; University of Leeds, Leeds 

We recently reported the isolated two novel gammaherpesviruses, one from a field vole (Microtus agrestis) and the other from wood mice 

(Apodemus sylvaticus). Here we report the sequence of the genome of field vole herpesvirus (FVHV). FVHV had similar genome structure 

as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68) having the core gammaherpesvirus genes. Homologs of the MuHV4 unique 

open-reading frames were not present but several trNA-like genes were. There were, however, a number of unique open-reading frames, 

one of which with homology to interleukin 13. Biological characterization of FVHV indicated that it grows poorly in cell culture and is highly 

cell-associated. After infection of laboratory mice, FVHV was found to replicate, albeit to low titres in lungs and form a persistent infection 

in the spleen. FVHV is therefore a novel member of the gammaherpesvirus family and the first example of a virus containing a homolog of 

interleukin-13 





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HA12/15 bst-2/Tetherin paralogues within the sheep genome possess different restriction properties 

liTA MurPHY1 , Sarah Black3 , Thomas E. Spencer3 , Frederick Arnaud2 , Massimo Palmarini1 1 2 3 MRC- University of Glasgow Centre for Virus Research, Glasgow; UMR754 UCBL INRA ENVL EPHE, Lyon, France; Laboratory for 

Uterine Biology and Pregnancy, Dept of Animal Science, Texas A&M University, Texas, USA 

The domestic sheep represents a unique model to investigate the interaction between retroviruses and their host. Several copies of 

endogenous betaretroviruses (enJSrVs), highly related to the exogenous and pathogenic jaagsiekte sheep retrovirus (JSrV), have colonized 

the sheep genome in the last 5–7 million years. enJSrVs play important roles in host physiology. We showed that the interplay between 

enJSrVs and host restriction factors such as bone marrow stromal cell antigen 2 (oBST-2) may have influenced the tropism of these viruses 

(Arnaud et al., J Virol 2010, 84: 4415). interestingly, oBst-2 is duplicated in the genomes of ruminants. The oBst-2 paralogs (oBst-2A and 

oBst-2B) appear to inhibit JSrV/enJSrVs exit with different mechanisms. oBST-2A and oBST2-B differ significantly in intracellular localization 

both in the presence or absence of viral proteins. in transfection assays, oBST-2A ‘tether’ viral particles to the cell membrane like other Bst- 

2 orthologoues. However, oBST-2B does not block efficiently JSrV/enJSrVs Gag release but reduces Env incorporation into viral particles. 

Thus, oBST-2B may offer yet another mechanism followed by cellular restriction factors to block the replication of retroviruses. 

HA12/16 Mapping the interaction domain for a vaccinia virus inhibitor of the Iκb kinase complex 

CAMillA BENFiElD1 , Daniel Mansur1 , Brian Ferguson1 , Mohammad Bahar2 , Stephen Graham2 , Asa Oldring2 , Jonathan Grimes2 , 

David Stuart2 , Geoffrey Smith1 1 2 Section of Virology, Dept of Medicine, Imperial College London; The Division of Structural Biology and Oxford Protein Production 

Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford 

The iκB kinase (iKK) complex regulates activation of NFκB, a critical transcription factor in mediating inflammatory and immune responses. 

Not surprisingly, therefore, many viruses seek to inhibit NF-κB activity. The vaccinia virus (VACV) B14 protein contributes to virus virulence 

by binding to the iKKβ subunit of the iKK complex and preventing NFκB activation in response to pro-inflammatory stimuli. B14 is a Bcl-2 

like protein that crystallises as a homo-dimer. Multi-angle light scattering, however, indicated that B14 is monomeric at lower concentration 

in solution. This observation suggested that the hydrophobic dimerization interface of B14 might mediate its interaction with iKKβ and 

this was addressed by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric, but still 

co-immunoprecipitated with iKKβ and blocked NFκB nuclear translocation and NFκB-dependent gene expression. Therefore, B14 homodimerization, 

and the Y35 residue, is non-essential for binding and inhibition of iKKβ by B14. in contrast, an F130K mutant neither bound 

iKKβ nor inhibited NFκB-dependent gene expression, demonstrating that this residue is required for the B14-iKKβ interaction. Mapping 

the iKKβ interaction domain of VACV B14, a viral inhibitor of the iKK complex, should prove valuable for the rational development of 

pharmacological iKK inhibitors. 

HA12/17 Signs of immunopathology in macaques infected with multiple simian retroviruses that exacerbates SrV-2 

infection 

JANE MiTCHEll, Simon Hood, Ghazi Auda, Neil Almond, Nicola rose 

National Institute for Biological Standards and Control, Hertfordshire 

in retroviral co-infection one or more of the viruses may display altered dynamics impacting on pathology and disease. To characterize 

the nature of such changes we studied cynomolgus macaques naturally infected with three retroviruses. DNA was isolated from eleven 

tissues from macaques infected with SFV-1 (4), SrV-2/SFV-1 (4), STlV-1/SFV-1 (3), SrV-2/STlV-1/SFV-1 (6), and one uninfected macaque. 




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Proviral load and distribution were evaluated by PCr. immunopathology in the MlN and spleen was assessed by H&E staining and 

immunohistochemistry, using antibodies against T-, B-lymphocytes and macrophages. 

Our data reveal that in co-infected macaques a significant increase in the SrV-2 proviral distribution and a trend towards an increase in the 

proviral load of SrV-2, but not STlV-1 or SFV-1, occurs. Pathological changes were enhanced in co-infected macaques, identified by the 

presence of nodular hyperplasia or, in extreme instances, follicular depletion. The greatest number of B-, T-lymphocytes and macrophages 

were seen in triple-infected macaques and an observed atypical distribution of the B- and T-cells is suggestive of altered immunopathology. 

Thus co-infection increases the distribution and proviral load of SrV-2 to create an atypical immunopathology within the lymphoid organs 

which may hinder the host’s ability to control SrV-2 infection and exacerbate pathology and overt disease. 

HA12/18 Assessment of new cell substrates for mumps virus growth and replication 

lAurEN PArKEr, Philip Minor, Silke Schepelmann 

National Institute for Biological Standards and Control, Hertfordshire 

Background: Suspected mumps cases are currently confirmed by PCr detection of mumps virus (MuV) nucleotide sequences followed 

by virus isolation from the sample using Vero cells. This cell culture system for clinical MuV isolation has come under scrutiny due to the 

high likelihood of lack of sensitivity and selecting for attenuated or adapted virus variants, which may result in false negative results and 

production of clinical isolates not suitable for research. Therefore, a highly sensitive cell culture isolation system would be of great use. 

Methods: A panel of 40 cell lines was screened using a GFP-expressing MuV, and those demonstrating high fluorescence and some evidence 

of cytopathic effects (CPE) were investigated further. The cell lines were infected with representative wild-type (Mu90) and vaccine (GSK) 

mumps strains in order to determine which can produce high yields of infectious progeny. Plaque assays were also performed on these cell 

lines using Mu90. 

Results and conclusion: Experimental findings have identified some interesting cell lines which may offer an alternative cell substrate for MuV 

isolation. Viral genomic stability studies with clinical samples will be carried out in the future to assess the potential of these cell lines for 

diagnostic and research use further. 

HA12/19 Characterization of immune cell population changes during GbV-b infection 

Simon Hood1 , EDWArD MEE1 , Helen Bright2 , Neil Berry1 , Nicola rose1 1 2 National Institute for Biological Standards and Control – Health Protection Agency, South Mimms; Pfizer Research and 

Development, Sandwich 

infection of red-bellied tamarins (Saguinus labiatus) with the hepacivirus GBV-B is a recognised surrogate model of HCV infection in man. 

Detailed characterization of immune cell populations in GBV-B-infected tamarins has hitherto not been reported. We have developed 

panels of antibodies for flow cytometry and immunohistochemistry and applied these in order to characterize populations of CD3+, 

CD4+, CD8+ and CD20+ cells prior to infection and at time of termination either 6 or 24 weeks post-infection. We have also determined 

the expression levels of Programmed Death 1 (PD-1), a marker of T-cell exhaustion, on CD4+ and CD8+ cells from infected animals. 

The data generated provide the first insight into the dynamics of immune cell populations in an important animal model of HCV infection. 

The developed antibody panels will be applicable to a range of studies in red-bellied tamarins. Further work will include the identification of 

CD4+ and CD8+ T cell epitopes targeted during GBV-B infection in order to determine those responses associated with viral clearance. 

HA12/20 Mutational studies reveal divergent evolutionary mechanisms maintaining conserved serine residues in hepatitis C 

virus envelope glycoproteins 

riCHArD BrOWN1 , George Koutsoudakis2 , richard urbanowicz1 , Deeman Mirza1 , Natalia Wysocka1 , louisa Price1 , 

Matthijs Backx1 , Patrick McClure1 , Anna Albecka3 , Jean Dubuisson3 , Sofia Perez-del-Pulgar 2 , xavier Forns2 , Alexander Tarr1 , 

Jonathan Ball1 1 2 3 The University of Nottingham, Nottingham; University of Barcelona, Barcelona, Spain; Institut Pasteur de Lille, Lille, France 

The Hepatitis C Virus (HCV) pandemic infects 170 million people, causing a chronic infection in 80% of cases. No vaccine is available and 

current therapies are only partially effective. The envelope glycoproteins E1 and E2 form heterodimers which are distributed over the 

surface of the viral envelope. These highly variable proteins facilitate cellular entry and are subject to host immune targeting. inspection 

of a diverse panel of HCV E1E2 genes revealed of a number of conserved serine residues which exhibit genotype/subtype-specific 

underlying codon structures. Serine is unique among amino acids: it is encoded by two divergent codon types (uCN and AGY) which are 

not interchangeable by a single nucleotide substitution. This unique property facilitated in vitro exploration of the evolutionary constraints 




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underlying residue conservation in a highly polymorphic viral protein. We employed a mutational approach, utilizing multiple parallel in vitro 

systems, to interrogate the phenomenon of serine codon switching in HCV E1E2. These data indicate maintenance of conserved serines 

occurs via divergent mechanisms, with functional constraints at both the protein and rNA level. This novel description of the differential 

constraints underlying residue conservation in highly variable viral proteins may inform development of new antiviral strategies. 

HA12/21 Studies on flavivirus pathogenesis 

KArEN MANSFiElD1,2 , Nicholas Johnson1 , Tom Solomon2 , Anthony Fooks1,2 1 2 Veterinary Laboratories Agency, Addlestone, Surrey; University of Liverpool, Liverpool 

Many flaviviruses are associated with encephalitic disease in animals or humans. louping ill virus (liV) causes encephalitic disease in sheep, 

whereas the closely-related Tick-borne encephalitis virus (TBEV) generally does not. 

To identify critical differences in the host immune responses between these two viruses that influence infection outcome, we compared 

viraemia and immune response in sheep (5 sheep per group, subcutaneously infected). Clinical signs and detection of virus in CNS tissues 

were observed with liV-infected sheep, but not TBEV-infected sheep. 

liV-infected sheep demonstrated detectable viraemia from day 4 to 7 post-inoculation (p.i.), correlating with a statistically significant 

increase in rectal temperature. in comparison, there was no detectable viraemia or temperature increase with TBEV-infected sheep. 

However, serum neutralizing antibodies were detected from day 4 p.i for both TBEV and liV-infected sheep. 

Post-mortem CSF samples from both liV- and TBEV-inoculated sheep demonstrated an increase in a number of pro-inflammatory 

cytokines/ chemokines associated with inflammatory disease (such as il-1a, il-6 and CCl5). Evidence for an increase in CSF antiinflammatory 

il-10 levels following TBEV infection suggests a possible mechanism by which the host can limit disease. This study may 

contribute to the development of novel or improved vaccines and therapies for flavivirus infections. 

HA12/22 Whole-genome sequencing of herpesviruses using Agilent SureSelect and Illumina next-generation sequencing 

technology 

ANNE PAlSEr1 , imogen Yi-Chun lai1,2 , Peter Ellis1 , Paul Kellam1,2 1 2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA; MRC Centre for Medical 

Molecular Virology, Dept of Infection, University College London. 46 Cleveland Street, London W1T 4JF 

little is known about genome diversity of herpesviruses and there are currently only 4 genome sequences for Epstein-Barr virus (EBV) and 

3 genome sequences for Kaposi’s sarcoma-associated herpesvirus (KSHV) in Genbank. Whole genome sequencing of large DNA viruses is 

complicated by the difficulty in seperating virus DNA from human DNA. We have addressed this issue using Agilent SureSelect technology 

to pull out virus sequence from host DNA and sequence the captured virus DNA using illumina sequencing. 

We designed 120mer probes that tile across the KSHV, EBV type 1 and EBV type 2 genomes. DNA from cell lines latently infected with 

either EBV or KSHV or both viruses was hybridized to the probes and the captured DNA was illumina sequenced. using this approach we 

have generated near full genome sequence of KSHV from two KSHV+ primary effusion lymphoma (PEl) cell lines (BCBl-1, BC-3) and EBV 

from two Burkitt’s lymphoma cell lines (Akata, Daudi). We also sequenced both viruses from three dual infected PEl cell lines (HBl-6, BC- 

1, JSC-1), including both the episome and virus released upon lytic reactivation. These data enable an initial estimation of genome diversity 

and highlight differences at the genetic level between EBV and KSHV from different cell lines. 

HA12/23 Deeper insights into HIV-1 sequence diversity in a cohort of recent seroconverters 

ASTriD GAll1 , Steve Kaye2 , David Bonsall2 , richard rance1 , Gregory J Baillie1 , Sarah J Fidler2 , SPArTAC Trial investigators2 , 

Jonathan N Weber2 , Myra O McClure2 , Paul Kellam1 1 2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA; Jefferiss Research laboratories, 

Faculty of Medicine, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG 

Dynamics of Human immunodeficiency Virus 1 sequence diversity and divergence are associated with immune control during primary 

infection and progression to AiDS. Next generation deep sequencing is a quantitative method which surpasses previous sequencing 

approaches with regard to depth of sequence analysis and throughput. We determined that at a 750-fold sequence depth HiV sequences 

present at 1.5% of the population were detected vastly in excess of error rates. Here we investigated the population sequence of the 

envelope V3 region as a surrogate for HiV-1 inter- and intrapatient genome diversity and to quantify minor variants. A cross-sectional 

study of 30 uK MSM antiretroviral-naive patients immediately following seroconversion to HiV-1 enrolled in a rCT (SPArTAC); where 

participants were randomized to 12 or 48 weeks immediate short pulse ArT or no therapy; revealed the composition of major and minor 




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variants varied considerably between individuals. Sequence population diversity measured by Shannon Entropy was independent of clinical 

markers (viral load, time from seroconversion, CD4 count) of infection. Serial sampling over 60 weeks from individuals defining three 

different diversity profiles showed that patterns of continuing diversification and divergence could be readily detected, as well as archived 

virus reemergence and evidence of initial infection by diverse variants. 


HA12/25 Systematic analysis of virus host interactions of HPV-16 protein 

rAMYA M GuNDurAO, Juergen Haas 

Division of pathway medicine, University of Edinburgh 

Cervical cancer is caused by persistent infection with certain Human Papilloma Virus (HPV) types. HPV-16 and 18 are responsible for about 

70% cervical cancers. HPV-16 codes for 8 viral proteins and the interactions of these proteins with cellular partners play a pivotal role in its 

pathogenesis; two such well studied interactions are E6 with p53 and E7 with prB. However, there is a need to identify more interactions 

to fully understand the functioning of the virus and develop a cure for its malignancies. To this regard, a systematic analysis of virus host 

interactions was done by screening the viral proteins against testis epithelial cell library using yeast-two-hybrid (Y2H) assay. A library of 

HPV-16 proteins in Y2H bait and prey vectors was generated using invitrogen® Gateway cloning system and used for the assay. The assay 

has identified several interesting and previously unknown interactions which may have a biological significance. These interactions are being 

validated by mammalian pull down assay, luMiEr assay which will be followed by other biochemical approaches. Novel interactions will 

provide a better insight and develop our understanding of the biology of the virus. 

HA13 Intracellular life 

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HA13/01 Genetic and biochemical analysis of bacterial protein Cas3: r-loop formation as a step in CrISPr immunity 

JAMiESON HOWArD1 , Elisa Noll1 , ivana ivancic-Bace1 , Edward Bolt1 1 2 University of Nottingham, Nottingham; University of Zagreb, Zagreb, Croatia 

We have been investigating the CriSPr immunity protein Cas3 using genetics and biochemistry in bacteria and archaea. Cas3 functions 

in a system called CriSPr immunity, which is hypothesised to silence invading nucleic acids through their targeting by host CriSPr rNA 

(crrNA) via an unknown mechanism. We report that purified bacterial (E. coli) and archaeal (Methanothermobacter) Cas3 proteins catalyse 

base pairing of rNA, including crrNA, with homologous DNA by forming r-loops: rNA-DNA hybrids invaded into duplex DNA. We 

describe the basic biochemistry of this reaction and how it might direct recognition of invading homologous DNA by crrNA to explain 

the absolute requirement of Cas3 for CriSPr immunity in E. coli. Putative helicase functions of Cas3 were also investigated and will be 

described in detail. Genetic analysis of cas3 in E. coli supports a role of cas3 in promoting or stabilizing r-loop formation in cells, including an 

intriguing interaction with a DNA topoisomerase. 




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HA13/02 The role of mitochondria in regulating hypervirulence in the fungal human pathogen C. gattii 

KErSTiN VOElZ, robin C. May 

University of Birmingham, School of Biosciences, Birmingham 

Cryptococcosis is a fatal fungal disease caused by the facultative intracellular pathogens Cryptococcus neoformans and C. gattii. Of 

particular interest is a hypervirulent C. gattii subgroup that is causing ongoing outbreaks in Canada and the northwestern uSA. We 

recently demonstrated that the outbreak isolates share an increased ability to proliferate within phagocytes. This ability correlates with 

the expression of mitochondrial genes and a characteristic tubular morphology of cryptococcal mitochondria. We are now investigating 

the role of mitochondria in adapting to the intracellular environment within macrophages. Our data suggest that outbreak strains 

initiate mitochondrial tubularization in response to host specific hypoxic, oxidative and/or nitrosative stresses, shortly after phagocytosis, 

whereas non-outbreak isolates fail to form tubular mitochondria even after 24 hours. in parallel, we are undertaking a comparative 

study of cryptococcal mitochondrial genomes to try and determine the underlying molecular mechanism for tubularization. in summary, 

mitochondria and/or mitochondrial functions seem to play a critical role in regulating hypervirulence in C. gattii and potentially other fungal 

pathogens. 

HA13/03 The intracellular survival of Streptococcus agalactiae in macrophages 

lEANNE SMiTH, Nicola Cumley, robin May 


Streptococcus agalactiae, otherwise known as group B streptococcus (GBS), is able to cause serious invasive infections in human newborns. 

Asymptomatic colonization of the genito-urinary tract of approximately one third of the female adult population provides a stable 

environment for GBS existence within humans. up to 70% of the children born to colonized women acquire GBS in utero or during 

birth. Although the majority will not develop serious infection, in rare cases newborns are unable to control GBS colonization, resulting in 

dissemination and meningitis. GBS can survive for prolonged periods following phagocytosis, but the extent to which this trait varies and/ 

or correlates with clinical outcome is not known. To address this, we have screened 50 clinical GBS isolates from uK blood samples for 

intracellular survival in J774 macrophage-like cells. We are also focusing on the molecular basis for this intracellular persistence and the use 

of polarised THP1 cells to better model GBS interactions with primary monocyte-derived macrophages. in addition, we are now extending 

this approach to use transposon mutagenesis to identify GBS virulence factors that are required for intracellular survival. 

HA13/04 An integrative approach to study intracellular bacterial infection dynamics 

OliViEr rESTiF, Andrew J. Grant, Yun Shan Goh, Trevelyan J. McKinley, Pietro Mastroeni 

University of Cambridge, Dept of Veterinary Medicine, Cambridge 

Studying the dynamics of bacterial infections at the cellular level remains a huge challenge because of the limitations of direct observation, 

especially in vivo. Changes in bacterial numbers over time during infection can result from a combination of basic processes, such as division, 

death, phagocytosis or cell lysis, which can vary in time and space. Disentangling the relative importance of those mechanisms is an essential 

step to improve our understanding of infection dynamics and immunity, but this often requires very sophisticated experimental setups. 

Mathematical models provide a flexible and powerful method to determine the likely contribution of unobserved mechanisms based on 

experimental data. Salmonella enterica Typhimurium is a well-established biological model for typhoid in mice, but also an important human 

pathogen responsible for enteric infections as well as septicaemia. We investigated the intracellular dynamics of infection both in vitro and 

in vivo using a combination of fluorescence microscopy and mathematical modelling. We show that it is possible to start reconstructing the 

dynamics of infection and make inference on fundamental but unobserved mechanisms of infection from just two time points. in particular, 

our model predicts heterogeneities within phagocytic cell populations that have important implications for our understanding of immunity. 

HA13/05 Genes required for colonization of food animals by Salmonella Typhimurium 

rOY CHAuDHuri1 , Eirwen Morgan2 , Sarah Peters1 , Stephen Pleasance1 , Debra Hudson2 , Holly Davies2 , Jinhong Wang1 , 

Pauline van Diemen2 , Anthony Buckley2 , Alison Bowen2 , Gillian Pullinger2 , Daniel Turner3 , Gemma langridge3 , Keith Turner3 , 

Julian Parkhill3 , ian Charles4 , Duncan Maskell1 , Mark Stevens2 1 2 3 Dept of Veterinary Medicine, University of Cambridge, Cambridge; Institute for Animal Health, Compton; The Wellcome Trust 

Sanger Institute, Hinxton; 4The ithree Institute, University of Technology Sydney, Sydney, Australia 

Chickens, pigs and cattle are major reservoirs of Salmonella enterica, a food-borne pathogen of worldwide importance. little is known about 

Salmonella genes required to colonize the intestines of these animals. We screened a library of 8550 random insertion mutants of 




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S. enterica serovar Typhimurium in parallel through each of these reservoir hosts. After oral inoculation, the genotype and relative fitness of 

7702 mutants in each host were assessed by massively-parallel sequencing of the regions flanking inserts. A core set of genes is required for 

infection of all three host species, and smaller sets of genes are specific for infection of each host. Defined null mutants were obtained for 

several candidate genes identified in the screen, and these were confirmed as attenuated in chickens. By assigning roles to 2721 different 

genes in highly-relevant hosts our data facilitate systems approaches to understand Salmonella pathogenesis, and the rational design of novel 

cross-protective vaccines. 

HA13/06 replication dynamics of Salmonella during systemic disease 

KATHrYN WATSON, Sophie Helaine, David Holden 


Salmonella enterica serovar Typhimurium (Salmonella) causes a systemic disease in susceptible mouse strains that is widely used as a 

model of human typhoid fever. We developed a reporter system based on fluorescence dilution (FD) that directly measures intracellular 

bacterial replication at both the population and single cell level. in an effort to understand how Salmonella colonizes host tissues during 

disease, we applied FD to study Salmonella replication during systemic infection of mice following oral inoculation. Bacteria that had not 

undergone a single division were found in the Peyer’s patches (PPs), mesenteric lymph nodes (MlN) and spleen. Hence, replication is not 

a prerequisite for Salmonella to traverse the intestinal wall and reach deeper tissues. We found that bacteria underwent replication very 

rapidly after invasion of PPs and that PPs represent a more permissive site for bacterial replication than the MlN. We are currently using 

FD to determine the contribution of the Salmonella-encoded pathogenicity island-2 type 3 secretion system (SPi-2 T3SS) to replication of 

Salmonella in vivo. 

HA13/07 Identification of effectors involved in intracellular replication of Salmonella Typhimurium 

riTA FiGuEirA, Sophie Helaine, David Holden 


Salmonella enterica serovar Typhimurium (S. Typhimurium) is able to replicate inside mouse macrophages via the activity of a SPi-2-encoded 

type-three secretion system (T3SS), but the repertoire of effectors involved in this process has not been defined. using Fluorescence 

Dilution (a dual-fluorescence reporter system that allows direct measurement of intracellular replication) we investigated the contribution 

to replication of individual effectors delivered by the SPi-2 T3SS. in a screen conducted in mouse bone marrow-derived macrophages, 

we measured and compared the replication kinetics of S. Typhimurium deletion mutants for all known SPi-2 effectors. Several mutant 

strains with replication defects were identified, thereby revealing that intracellular replication is the result of the contribution of numerous 

effectors. Of these mutants, only a minority were already known to display poor replication and many of the corresponding effectors are of 

unknown function; these are currently being investigated in more detail. This work offers further insights into the requirements for successful 

replication inside cells, a hallmark of Salmonella infection. 

HA13/08 Next-generation transcriptomics reveals ppGpp as a global regulator of non-coding rNA and virulence gene 

expression in Salmonella Typhimurium 

ViNOY rAMACHANDrAN1 , Neil Shearer1 , Cynthia Sharma2 , Jörg Vogel2 , Jay Hinton3 , Arthur Thompson1 1 2 3 Institute of Food Research, Norwich; Institute for Molecular Infection Biology, Würzburg, Germany; The Moyne Institute of Preventive 

Medicine, Trinity College, Dublin 2, Ireland 

To facilitate successful infection, S. Typhimurium undergoes a rearrangement of its transcriptome in response to host environmental 

stimuli. The bacterial alarmone guanosine-3’,5’-bisdiphosphate (ppGpp) plays a major role in mediating the environmental regulation of the 

intracellular (STiN ) and extracellular (STEx ) virulence gene expression programmes of S. Typhimurium (1). in order to further characterize 

the ppGpp-dependent transcriptome, we performed strand specific differential rNA-seq of S. Typhimurium wild-type and ppGpp0 strains 

grown under conditions that model the STEx programme (2). We identified primary transcription start sites for 1952 OrFs (44% of total 

OrFs). We made the novel discovery that 45% of published srNAs of S. Typhimurium Sl1344 are ppGpp-dependent under this growth 

condition, compared to 19% of the total gene expression. We also discovered extensive antisense transcription, 20% of which is also 

ppGpp-dependent. Our data shows that ppGpp plays a much more universal regulatory role than previously recognised. We propose that 

this facilitates the rapid remodelling of the Salmonella transcriptional programme in response to infection-relevant environmental stimuli. 

References: (1) Thompson, A. et al., J Biol Chem, 2006. 281(40): 30112–30121; (2) Sharma, CM. et al., Nature, 2010. 464 (7286): 250–255 




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HA13/10 In vitro respiratory response of K562 cells on Neospora caninum infection 

HANY ElSHEiKHA1 , Paul Smith2 1 2 School of Veterinary Medicine & Science, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD; Nottingham Medical 

School, Queen’s Medical Centre, Nottingham NG7 2UH 

Neospora caninum is an intracellular protozoan parasite that is a major cause of abortion in cattle and neurological disorders in canines. An 

insight into the bioenergetic aspects of parasite infection on host cells may elucidate possible new treatments for neosporosis. To investigate 

the effect of N. caninum on the oxidative respiration of host cells, the O consumption rate (OCr) of K562 cells was measured using Clark 

2 

O electrodes using either 10 mM glucose or NNN’,N’tetramethylphenylenediamine/ascorbate (TMPD/asc) as energy sources. The OCr 

2 

of K562 cells stimulated by TMPD/asc (–22±2.1 nmol O /10 2 6 cell/min, n=12) was significantly increased 2 h post N. caninum infection 

compared to controls (–14±1.3 nmol O /10 2 6 cell/min, n=11; p50% of disease cases. in the uK, cervical disease management is largely performed through organized liquid based cytology 

(lBC) screening although HPV DNA testing is being introduced to enhance the effectiveness of the screening programme. There are 

several detection tests for HPV DNA, however, detecting virus mrNAs by reverse transcriptase PCr may give a better indication of viral 

activity – and by implication – clinical relevance. 

A novel qrT-PCr assay was developed to investigate association of HPV16 l1 mrNA expression with disease status in 223 cervical lBC 

samples. While the assay successfully detected l1 mrNA, we found that there was no correlation between clinical outcome and late 

mrNA status and that samples positive for l1 also expressed E6/E7 oncogene mrNA. The mechanism(s) for HPV-induced cervical disease 

appears to be much more complex than currently appreciated. These data will sharpen focus for relevant rNA-based detection protocols 

in future. 




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HA14/02 In vitro adaptation of foot-and-mouth disease virus Type O to guinea pig cell lines 

AMiN ASFOr, David Paton, Donald King, Nick Knowles, Mana Mahapatra 

Institute for Animal Health Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF 

Adaptation of foot and mouth disease virus to cell culture involves multiple virulence determinants. The aim of this study is to determine 

which part of the virus genome is important for virus adaptation to guinea pig cell lines. 

FMDV type O1 Kaufbeuren wild type (O1K WT) showed no evident infectivity on three different guinea pig derived cell lines namely, fetal 

fibroblasts (Crl-1405), colon epithelial cells (CCl-242) and lung fibroblasts (CCl-158). Two of the cell lines, (Crl-1405 and CCl-158), 

showed cytopathic effect reaching to 100% and 40% respectively at the second passage whereas CCl-242 cell line did not show any signs 

of infectivity even after the third passage. interestingly after passage of WT-CCl-158-P1 into Crl-1405 cells the virus was able to infect 

the non-permissive cell line (CCl-242). Capsid sequence analysis of these viruses revealed 1 substitution in VP2 (VP2-130), 4 in VP3 (VP3- 

68, 85, 123 and 173) and 1 in VP1 (VP1-133) at passage 2. Two more amino acids substitutions were detected in VP3 (VP3-56 and 60) 

after passage 3. Full genome sequencing followed by a reverse genetics approach will be taken to find out which amino acid/s mediate the 

process of adaptation. 

HA14/03 Genetic modification of the heparin-binding exosite of the factor X serine protease domain inhibits Ad5/FX 

complex binding to hepatocytes 

MArGArET DuFFY1 , Angela Bradshaw1 , John McVey2 , Alan Parker1 , Andrew Baker1 1 2 BHF Glasgow Cardiovascular Research Centre, Glasgow; Thrombosis Research Institute, London 

Adenovirus 5 binds factor x in the bloodstream. Fx bridges the Ad5/Fx complex to heparan sulfate proteoglycans on the cell surface. 

Pharmacological blockade of the heparin-binding exosite in the serine protease domain of Fx prevents cell binding and gene transfer 

mediated through Fx1 . Previous studies have indicated that the residues Arg-93, lys-96, Arg-125, Arg-165, lys-169, lys-236 and Arg-240 

constitute this exosite2 . The aim of this study was to identify the specific residues in the SP domain mediating Ad5 attachment to HSPGs. A 

human Fx plasmid construct with mutations at all seven residues was generated. Stable cell lines were generated to constitutively produce 

the wildtype and mutant recombinant Fx in the presence of vitamin K. The rFx generated was biologically active and had the predicted 

molecular weight. Surface plasmon resonance analysis showed the SP mutations had no effect on rFx binding to Ad5 hexon thus any effect 

on gene transfer was due to inefficient cell binding. In vitro assays demonstrated that the exosite mutations blocked the ability of Fx to 

enhance cell binding (1.75±0.25x104 SP mutant vs. 10.28±0.276x104 WT vector genomes, p


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of other picornaviruses, especially FMDV. This virus is capable of extremely rapid replication and can also adopt a persistent state where 

little replication occurs. To gain insight into these mechanisms, we carried out in vitro selection to produce rNA aptamers to 3D pol for use 

as molecular tools. Our previous selections identified the first rNA aptamers to 3D pol , one of these (termed 47tr) inhibited 3D pol in an 

elongation assay, with an iC 50 of 20 nM. We have demonstrated that 3D pol forms higher order structures (fibers and tubes) but only when 

all components of an elongation assay are present. These higher order structures are reminiscent of, but differ from, structures formed by 

poliovirus 3D pol . interestingly, these structures do not form in the presence of 47tr. in addition, we have shown that the FMDV 3Dpol has 

terminal transferase activity, adding nucleotides to rNA templates in a primer-independent manner. We are currently investigating the roles 

of these features of 3D pol to suggest a model for FMDV replication 

HA14/06 Engineering a replication competent bluetongue virus expressing reporter genes 

ANDrEW SHAW1 , Maxime ratinier1 , Franziska Kaup1 , Marco Caporale1 , Frazer rixon1 , Eva Veronesi2 , Peter Mertens2 , 

Massimo Palmarini1 1MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity & Inflammation, College of Medical, 

Veterinary & Life Sciences, University of Glasgow, Glasgow; 2Vector-Borne Disease Programme, Institute for Animal Health, Pirbright, 

Woking 

Bluetongue virus (BTV) is an arbovirus and the causative agent of Bluetongue, an economically important disease of ruminants. The use of 

reporter genes incorporated into viral genomes allows the real time monitoring and specific localization of virus infection. using reverse 

genetics, we have rescued a BTV-1 mutant expressing the coding sequences of either enhanced green fluorescent protein (BTV-eGFP) 

or mCherry fluorescent protein (BTV-mCherry). We inserted the reporter genes within segment 10 of the BTV genome which encodes 

the NS3/3A protein. Confocal and western blot analysis confirmed the expression of modified NS3/3A proteins in virus-infected cells. 

Furthermore, the intracellular distribution of the fluorescent proteins was similar to the wild-type NS3/3A protein. Virus growth curve 

experiments showed that BTV-eGFP and BTV-mCherry were replication competent but reached lower titres than wild type BTV in 

mammalian cells. in mammalian cells the BTV-mCherry mutant reached higher titres than BTV-eGFP and was shown to possess reduced 

pathogenicity (relative to wild type BTV-1) in adult mice deficient in type i iFN receptor, but was shown to be replication competent in 

Culicoides midges. Thus, BTV-mCherry represents a useful tool with which we can further investigate some aspects of infection, spread and 

replication of BTV. 

HA14/07 Understanding adenovirus replication in pancreatic cancer using an organotypic culture system 

NATAliE FOx1 , reem El-Ghazawi2 , Valerie Speirs1 , Andrew Smith2 , Alan Melcher1 , Caroline Verbeke2 , Eric Blair1 1 2 University of Leeds, Leeds; St James’s University Hospital, Leeds 

Human adenoviruses (Ads) have shown promise for use in oncolytic viral therapy. Currently, most vectors are derived from Ad5 (species 

C) which utilises the Coxsackie and Adenovirus receptor (CAr) for cellular attachment. However, many tumours display low levels of 

CAr, limiting the transduction efficiency of vectors based on Ad5. in contrast, the species B Ad do not interact with CAr and instead 

interact with CD46 and/or Desmoglein 2, which are expressed in many cancers. Therefore species B Ads may offer a useful alternative for 

development as oncolytic vectors. 

To test species B Ads for pancreatic cancer therapy, an organotypic model of pancreatic cancer has been developed. This model assesses 

the ability of different Ad serotypes to enter and replicate in pancreatic cancer cells. Pancreatic cancer and normal tissue has been sectioned 

into 250μm slices and shown to remain viable in culture for several days, retaining normal histology. Entry of Ad vectors expressing EGFP 

were detected after 24 hours and wild-type Ad replication (as judged by late protein expression) can be detected after 24 hours and for up 

to three days. 

HA14/08 Functional and biophysical analysis of human respiratory syncytial virus M2-1 protein 

SiAN TANNEr1 , Thomas Edwards1 , Brian Dove2 , Julian Hiscox1 , John Barr1 1 2 University of Leeds, Leeds; Health Protection Agency, Porton Down 

Human respiratory Syncytial Virus (HrSV) is a negative-sense rNA virus responsible for globally significant levels of severe respiratory 

tract disease in young infants, the elderly, and the immunocompromised. No vaccine is available and treatment is supportive. M2-1 protein 

is an HrSV-encoded co-factor of the viral polymerase that acts as a transcriptional anti-terminator. it is essential for the virus’ sequential 

mode of transcription, making M2-1 a potential target for anti-viral therapeutics. However, there is little confirmed biophysical data for 

the protein and its mechanism of action is unknown. M2-1 requires phosphorylation to function, and has been shown to bind rNA with 




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debated specificity. Data shown here confirms, by mass spectrometry, that M2-1 protein expressed in baculovirus-infected insect cells is 

phosphorylated at the same two sites previously identified for mammalian-expressed protein: serine 58 and serine 61. The affinity of rNA 

binding to M2-1 can be investigated using fluorescence polarization techniques, with both viral and non-viral rNAs; this may help determine 

the specificity of this interaction and contribute to the currently incomplete model of M2-1 function in HrSV infection. 

HA14/09 Cellular membrane rearrangements triggered by infectious bronchitis virus 

HElENA MAiEr1 , Eleanor Cottam2 , Pippa Hawes3 , Jennifer Simpson3 , Tom Wileman2 , Paul Monaghan3 , Paul Britton1 1 2 3 Institute for Animal Health, Compton, Berkshire; University of East Anglia, Norwich; Institute for Animal Health, Pribright, Surrey 

infectious Bronchitis Virus (iBV), a Coronavirus, is the most economically important infectious disease of domestic fowl in the uK, causing 

losses in both meat-type and commercial egg layer birds. We observed previously that iBV, like other Coronaviruses, induces double 

membrane vesicles (DMVs) in the cytoplasm of infected cells, as well as other novel structures. rearranged cellular membranes are 

hypothesised to act as a scaffold for assembly of the viral replication complex. DMVs resemble cellular autophagosomes, involved in the 

targeting of cytoplasmic contents for degradation. However, it has been suggested that autophagy may play a role in innate immunity to 

invading microbes. Therefore, the role of DMVs in iBV replication remains unclear. Preliminary electron tomography has been performed 

to determine how the rearranged membranes in iBV infected cells relate to one another in 3 dimensions. Furthermore, the cellular location 

of viral replicase protein nsp4, thought to anchor the viral replication complex to cellular membranes, has been studied in infected cells by 

confocal microscopy and has shown that there is only a small degree of colocalistion with a replicative intermediate, dsrNA. Finally, the 

time-course for the onset of viral rNA and protein synthesis, and progeny particle release has been determined. 

HA14/10 Disruption of subnuclear structures in adenovirus infection and cell transformation 

lAurA WHiTE, Nicky James, Gareth Howell, Eric Blair 

Universi0ty of Leeds, Leeds 

Adenoviruses (Ads) are non-enveloped, icosahedral viruses with double stranded DNA genomes of around 36kbp in length. it has 

previously been shown that infection of the host cell with Ad5 induces rearrangement of several sub-nuclear structures. We have previously 

shown that in Ad5-infected human cells, there is rearrangement of Cajal bodies (CBs) from typically 1–5 punctate domains per cell to 

numerous microfoci arranged into clusters termed ‘rosettes’. However, it is unknown how Ad infection induces this change in CB structure. 

Confocal microscopy using the CB marker protein coilin revealed five distribution patterns of CBs during Ad infection; diffuse, punctate 

perinucleolar, punctate nucleolar, speckles and rosettes. Cells transformed with the E1 region of the Ad genome (containing early region 

genes E1A and E1B) also appear to exhibit these CB morphologies excluding rosettes. in addition, during Ad5 and Ad12 infection there is 

partial co-localization of coilin and Ad E1A protein, and this co-localization is also evident in E1-transformed cells. However, inhibition of 

protein synthesis in infected cells using cytosine arabinoside revealed viral replication is required for formation of rosettes. 

HA14/11 Withdrawn 

HA14/12 Investigations into the role of p12 during early retroviral infection 

DArrEN WiGHT, Virginie Boucherit, Kate Bishop 

National Institute for Medical Research, London 

retroviruses like Moloney murine leukemia virus (MoMlV) encode three main structural proteins in their gag gene, matrix (MA), capsid 

(CA) and nucleocapsid (NC). Most retroviruses also code for a small proline-rich protein between MA and CA of unknown function. in 

MoMlV this protein is p12, it contains the late-domain for MoMlV and has also been shown to function before nuclear entry. Changing 

stretches of 4–6 amino acids (aa) to alanine revealed two regions of p12 required for early infection, one at the N-terminus and one at the 

C-terminus. We have mutated all 35 aa that make up these regions to finely map the role of each residue during infection. The infectivity of 

MoMlV virus like particles (VlPs) containing the mutated p12 was tested in D17 cells. From 35 mutant VlPs 19 were less infectious than 

the wild-type VlP with infectivity reduced 2- to 575-fold. The most significant reductions in infectivity were seen when C-terminal aa were 

altered. interestingly, individual aa changes towards the N-terminus of p12 had a less significant effect on infectivity than changing 4–6 aa 

to alanine together. We will discuss these results in light of our previous work suggesting that p12 has two domains with different 

functions. 




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HA14/13 retroviral restriction factors: studies of the Fv1 promoter 

ViCTOriA FElTON 

NIMR, London 

The Fv1 gene, located on mouse chromosome 4, is hypothesized to originate from the gag region of an endogenous retrovirus. it appears 

to have been co-opted by mice for the purpose of preventing infection by different classes of murine leukemia virus at a stage after entry 

into the target cell, but before integration into the host genome. Two major alleles are present in inbred mice, Fv1n and Fv1b , where Fv1n confers resistance to B-tropic MlV and Fv1b to N-tropic MlV. in the 1970s the murine cell line SC-1 was described as being dually sensitive 

to both N-and B-tropic MlV. We have identified the presence of a functional Fv1n allele in this cell line and have shown by Sodium Bisulfite 

sequencing that methylation of the region upstream of Fv1 is the cause for the loss of Fv1 expression. While the exact mechanism of 

Fv1 restriction is not understood, it is known that in restricting cells lines, Fv1 is expressed at very low levels. To better characterize the 

sequences controlling Fv1 expression we have begun a more extensive study of the region upstream of Fv1, which from our studies with 

SC-1 is predicted to contain the Fv1 promoter. 

HA14/14 Examining the role of γ-herpesvirus exonucleases in mediating loss of cellular mrNA 

ViCTOriA SHEriDAN1 , James Stewart2 , Bernadette Dutia3 , Bahram Ebrahimi1 1 2 Institute of Integrative Biology, Liverpool University, Liverpool; Institute of Infection and Global Health, Liverpool University, Liverpool; 

3Centre for Infectious Diseases, The Roslin Institute and Royal School of Veterinary Sciences, University of Edinburgh, Edinburgh; 

4Institute of Integrative Biology, Liverpool University, Liverpool 

Eukaryotic viruses can down-regulate and/or inhibit cellular gene expression within their host, a phenomenon known as host shut-off. it is 

thought this mechanism affords an important biological advantage for pathogens at key stages post infection. in γ-herpesviruses, the gene 

responsible for host shutoff has been shown to be the DNA exonuclease. However, the precise mechanisms by which host shut-off is 

mediated by viral exonucleases remains unclear, and how this affects viral pathogenesis are yet to be determined. The aim of this study 

was to investigate the role of viral DNA exonuclease in the context of virus infection and how it may contribute to viral pathogenesis using 

the murine model, MHV-68. using BAC technology, we constructed a stop mutant and its corresponding revertant in the MHV-68. We 

used global and gene-specific assays to determine the role of viral exonuclease in host shutoff at both transcription and translation levels. 

We found virus mediated host shutoff to be cell type dependent. We also present data as to how virus infection causes cellular rNA 

degradation which provides an insight into the molecular mechanism of host shutoff. The implications of these findings in the context of 

natural virus infection in vivo are discussed. 

HA14/15 Proteasomal degradation of bunyamwera orthobunyavirus NSs protein 

iNGEBOrG VAN KNiPPENBErG, richard M. Elliott 

University of St Andrews, St Andrews, Fife 

During Bunyamwera virus (BuNV) infection of cultured mammalian cells, the viral protein NSs is actively degraded after reaching peak 

levels at 12hpi. We show that this is the result of lysine-dependent proteasomal degradation, by using proteasome inhibitors as well as 

using a mutant virus (rBuN-NSs4Kr) in which all four lysines in NSs are replaced by arginines. Although not all orthobunyaviruses have 

lysine residues in NSs (or indeed an NSs protein at all), one of the four lysines is completely conserved in the Bunyamwera serogroup 

viruses. The plaque phenotype of rBuN-NSs4Kr was similar to wt BuNV, but there was a difference in growth in interferon-competent 

cells (A549): whereas wt virus grows to slightly lower titres at 37°C than at 33°C, the mutant virus grows to the same titres at both 

temperatures. We interpret this as being the result of the continued presence of NSs, the interferon antagonist, throughout infection with 

rBuN-NSs4Kr. The effect of this mutation on infection of Balb/c mice, the possible mechanism of targeting NSs for degradation, and the 

potential reasons for degradation during wt infection will be discussed. 

HA14/16 bluetongue virus NS1 specifically upregulates viral gene expression by binding to the sequence which is conserved 

at the 3ʹ end of all ten viral transcripts 

MArK BOYCE, Polly roy 

London School of Hygiene and Tropical Medicine, London WC1E 7HT (Email: mark.boyce@lshtm.ac.uk) 

Bluetongue virus (BTV) is the prototype member of the Orbivirus genus of the reoviridae. A characteristic of Orbivirus infections is 

the production of large amounts of cytoplasmic tubular structures composed of non-structural protein 1 (NS1). NS1 is the most highly 

expressed viral protein, but its functions remain unknown.The fusion of BTV genome segment 10 sequences to GFP resulted in the 




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increased abundance of GFP when all BTV proteins were co-expressed, and a screen of BTV proteins revealed that NS1 is sufficient 

to produce this effect. using a renilla luciferase reporter assay the sequence in segment 10 required for the upregulation was mapped 

to a short non-coding sequence which is conserved at the 3’ end of all the BTV mrNAs. NS1-dependent upregulation was confirmed 

during virus replication using a reporter virus containing a segment 10-renilla luciferase fusion generated using reverse genetics. An rNA 

binding assay showed that NS1 binds to single stranded rNA and has specificity for the conserved 3’ end sequence, but does not bind 

polyadenylated substrates. We conclude that NS1 is an upregulator of viral gene expression, and that it discriminates between viral and 

cellular mrNAs through specificity for the conserved 3’ end present in all BTV mrNAs. 

HA14/17 Analysis of the role of the eIF4F complex in calicivirus translation 

AZiMAH ABDul WAHAB1 , Elizabeth royall1 , Nicolas locker1 , ian Goodfellow2 , lisa O. roberts1 1 2 University of Surrey, Surrey; Imperial College London, London 

Caliciviruses cause important disease of humans and animals; noroviruses are responsible for outbreaks of gastroenteritis in man, and feline 

calicivirus (FCV) causes an urT infection in cats. We have previously shown that translation initiation on calicivirus mrNA is dependent on 

the presence of a protein covalently linked to the 5’ end of the viral genome, VPg, thats interacts with the cap-binding protein eiF4E. We 

are currently carrying out further analysis of the exact requirements for the components of the eiF4F cap-binding complex for translation 

initiation on FCV and murine norovirus (MNV) mrNAs. using specific small molecule inhibitors, we have shown that FCV and MNV have 

different requirements for eiF4E and eiF4G, as inhibitors of the eiF4E:eiF4G interaction inhibit FCV, but not MNV, replication. We have also 

observed changes in the phosphorylation status of eiF4E and its regulating protein, 4E-BP1, during infection. We see an increase in eiF4E 

phosphorylation coupled with dephosphorylation of 4E-BP1; these changes would inhibit cap-dependent translation and may also inhibit 

VPg-dependent translation. We are currently dissecting the signalling pathways involved in these changes. Our hypothesis is that these 

changes may mediate the switch from translation of the viral rNA to replication by inhibiting calicivirus protein synthesis. 

HA14/18 replication of temperature-sensitive mouse hepatitis virus mutants 

luKE PAYNE, lauren Turrel, Hawaa Al-Mulla, Benjamin Neuman 


Viral replication is affected by a number of intra- and extracellular factors, one of which is temperature. This study looks at the effect of 

temperature on the replication of several temperature-sensitive mouse hepatitis virus (MHV) mutants to better understand how viral 

factors modify intracellular membranes and how specific cistrons affect viral growth. recovery assays were performed to discriminate 

between severe and total defects in rNA synthesis. Analysis by plaque assay showed the ability of some mutants to recuperate after being 

returned to the permissive temperature. The rate of the recovery and final titre of the virus varied depending on the non-structural protein 

carrying the temperature sensitive lesion. Electron microscopy was used to analyse the number and the morphology of the intracellular 

double-membrane vesicle (DMV) structures formed, allowing correlation of DMV phenotype with the final virus titre. These assays will be 

useful in rapidly determining the rNA phenotype of new temperature sensitive mutants. The characterization of the effect of temperature 

on MHV replication could elucidate pathways providing useful targets for novel treatments. 

HA14/19 Not received 

HA14/20 role of segment 2 gene products in A/WSN/33 virus growth and pathogenesis in mice 

SANDrA VATEr1 , Bernadette M. Dutia2 , Yvonne ligertwood2 , richard M. Elliott1 1 2 University of St Andrews, St Andrews, Fife; University of Edinburgh, Edinburgh, Fife 

influenza A viruses express up to 12 proteins. Genome segment 2 encodes three proteins, the largest being the polymerase subunit PB1. 

An N-terminally truncated version of PB1 (N40) was described in 2009. The 3rd protein is PB1-F2. This protein was shown to have 

different functions such as induction of apoptosis, regulation of the viral polymerase activity and enhancement of secondary bacterial 

infections. Previous work on PB1-F2 was mainly done by using mutant viruses with the PB1-F2 start codon deleted, which leads to an 

increase in the expression level of N40. in this study we used recombinant WSN viruses lacking the PB1-F2, N40 or both OrFs, and 

examined the effects of these mutations on virus growth, viral replication, apoptosis and virulence. Whereas a PB1-F2 deletant strain with 

increased N40 levels showed a small plaque phenotype, delayed growth, and reduced virulence in a mouse model, PB1-F2 deletant viruses 

with normal N40 levels showed a wild type phenotype. A double mutant lacking both PB1-F2 and N40 also was not attenuated in vitro and 

in vivo, suggesting that neither PB1-F2 nor N40 are important for the WSN viral life cycle. However, an elevated level of N40 seems to be 

detrimental for the virus. 




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HA14/21 Not received 

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HA14/22 Illumina sequencing, assembly and analysis of influenza A H1N1 (2009) 

Gregory Baillie1 , Monica Galiano2 , EVE COulTEr1 , rachael Wash1 , Astrid Gall1 , Anne Palser1 , Simon Watson1 , Andrew 

rambaut3 , Oliver Pybus4 , Maria Zambon2 , Paul Kellam1 1 2 3 Wellcome Trust Sanger Institute, Cambridge; Centre for Infections, Health Protection Agency, London; Institute of Evolutionary 

Biology, Edinburgh; 4Dept of Zoology, University of Oxford, Oxford 

Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly-evolving viruses. With complete 

genome data, it’s possible to identify and trace individual transmission chains of viruses, like influenza during the course of an epidemic. We 

have developed methods for next generation sequencing of influenza A and sequenced 26 influenza A H1N1 genomes from the 2009 

pandemic. 

rNA from clinical samples was rT-PCr amplified using an 8-segment PCr method. PCr products were multiplex by sequencing 12 

samples per lane on an illumina GAii; reads were assembled against a reference sequence (A/California/04/2009 genome) and assemblies 

were evaluated for coverage and quality. 

By comparing to other influenza A H1N1 (2009) genomes from the uK, we demonstrate that the uK epidemic is composed of many cocirculating 

lineages, of which at least 12 were exclusively or predominantly uK clusters. The estimated divergence times of two clusters predate 

the detection of H1N1pdm in the uK, suggesting H1N1pdm was circulating in the uK before the first clinical case. Crucially, 3 clusters 

contain isolates from the second wave of infections in the uK, two of which represent chains of transmission that have persisted within the 

uK between the first and second waves. 

HA15 Osmotic & oxidative stress responses 

↑Contents 

HA15/01 Thioredoxins and hydrogen peroxide signalling in the fungal pathogen Candida albicans 

AlESSANDrA DA SilVA DANTAS, Miranda J. Patterson, Deborah A. Smith, Brian A. Morgan, Janet Quinn 

Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne 

Candida albicans is the major systemic fungal pathogen of humans. Hence, it is important to understand how this organism evades the 

fungicidal mechanisms mounted by the host’s immune system such as the generation of toxic reactive oxygen species (rOS). Exposure 

of C. albicans to H O results in a significant remodelling of the transcriptome and proteome. However, although the ability of C. albicans 

2 2 

to sense and respond to rOS is essential for virulence, the intracellular signalling mechanisms underlying such responses are poorly 

understood. redox-sensitive antioxidant proteins, such as thioredoxins, have recently been demonstrated in model yeasts and mammalian 

cells to have oxidative stress signalling functions. C. albicans has three open reading frames annotated as thioredoxin-related proteins; Trx1, 

Trx2 and Txl1. We have recently shown that Trx1 plays a central role in H O -signalling in C. albicans by regulating both the rOS-induced 

2 2 

activation of the AP-1 like transcription factor Cap1 and the Hog1 SAPK. Moreover, Trx1 regulates H O -induced hyperpolarised bud 

2 2 

growth in this fungal pathogen via regulation of the checkpoint kinase rad53. We have now extended this analysis to investigate the roles 

of Trx2 and Txl1 in H O -signalling in C. albicans, and data pertaining to this will be presented 

2 2 

HA15/02 Identification of cellular targets for the Hog1 SAPK in Candida albicans 

DEBOrAH SMiTH1 , David Stead2 , Zhikang Yin2 , Alistair Brown2 , Janet Quinn1 1 2 Newcastle University, Newcastle upon Tyne; University of Aberdeen, Aberdeen 

The major fungal pathogen of humans, Candida albicans, encounters a variety of environmental stresses during disease establishment and 

progression within the host. Consequently, stress responses are intimately associated with the virulence of this medically relevant fungus. 

The Hog1 Stress Activated Protein Kinase (SAPK) plays a central role in C. albicans stress responses and is essential for virulence. However, 

little is known about either the upstream regulators of the Hog1 SAPK module, or the downstream targets of this kinase. in an attempt 

to identify uncharacterized upstream regulators and cellular targets of the SAPK, tandem affinity purification (TAP) and peptide mass 

fingerprinting were employed to identify proteins that physically associate with C. albicans Hog1. Ten proteins were identified as potential 

Hog1-interacting proteins using this approach. One such protein was the previously characterized Hog1 substrate, the serine/threonine 

protein kinase rck2. in addition, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gap1) was found to co-purify with 

Hog1, and subsequent co-precipitation experiments confirmed that Hog1 and Gap1 form a complex in C. albicans. Evidence is also 

presented that Hog1 plays a role in Gap1 modification following exposure of C. albicans to oxidative stress. 




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HA15/03 The response of Bacillus anthracis to oxidative stress 

SuSANNE POHl, Wang Yung Tu, Kevin Waldron, Colin Harwood 

Newcastle University, Newcastle upon Tyne 

The endospore-forming Gram-positive pathogen Bacillus anthracis is responsible for the usually fatal disease, inhalational anthrax. The 

success of this pathogen is dependent on its ability to subvert elements of the innate immune system of its animal hosts. B. anthracis spores, 

which are the main infective agent, are engulfed by and are able to germinate in patrolling alveoli macrophages. in order for the infection 

to progress, the resulting vegetative cells must resist the antimicrobial oxidative burst mounted by the host NADPH oxidase complex. 

The response of B. anthracis to such macrophage-related stresses is therefore of major importance to this pathogen, and consequently we 

have analysed the superoxide and peroxide stress stimulons of B. anthracis strain uM23C1-2 by means of a combined transcriptomics and 

proteomics approach. The results show distinct patterns of expression in response to paraquat (endogenous superoxide) and hydrogen 

peroxide stress. While the main response to paraquat is the induction of iron uptake pathways, the response to peroxide predominantly 

involves the induction of DNA repair metabolism. Comparisons between the responses of B. anthracis and related soil bacterium, B. subtilis, 

reveal differences that are likely to be relevant to their respective environments. 

HA15/04 regulation of oxidative stress responses in the fission yeast S. pombe by the 2-Cys peroxiredoxin Tpx1 

AliSON DAY, Sarah Taylor, Jonathon Brown, Jonathan rand, Brian Morgan, Elizabeth Veal 


Peroxiredoxins (Prx) are ubiquitous, abundant peroxidases with important roles in protection against cancer and ageing. Although the 

thioredoxin peroxidase activity of typical 2-Cys Prx protects against peroxide-induced cellular damage, Prx have additional activities in rOS 

responses as signalling proteins and chaperones. indeed, it has been proposed that the sensitivity of eukaryotic Prx to reversible inhibition 

of their thioredoxin peroxidase activity may be important for regulating biological responses to hydrogen peroxide. However, it is not 

clear how each of their different activities contribute to the role of Prx in the rOS response. Previous work in our lab has shown that the 

single 2-Cys peroxiredoxin in S. pombe Tpx1 is essential for the transcriptional response to a range of hydrogen peroxide concentrations. 

reversible oxidation of Tpx1 acts as a molecular switch regulating the activation of two signalling pathways; the stress-activated p38/ 

JNK-related MAPK Sty1 and the AP-1-like transcription factor Pap1. We have utilised Tpx1 mutants affecting specific activities of Tpx1 to 

elucidate the underlying mechanisms by which Tpx1 controls responses to peroxide. These data provide further indication of how the cell 

tailors its response to varying concentrations of peroxide and indicate that Tpx1 has multiple functions in regulation of the Sty1 MAPK. 

HA15/05 Detection of nitrosylated metalloproteins using infrared and other spectroscopic methods: yes or NO? 

JOHN TYSON MuNNOCH1 , Martin Walsh2 , Neil Hunt3 , Nicholas Tucker1 1University of Strathclyde, Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, Hamnett Wing, 

161 Cathedral Street, Glasgow G4 0RE; 2Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, 

Didcot, Oxfordshire OX11 0DE; 3University of Strathclyde, Dept of Physics, University of Strathclyde, SUPA 107 Rottenrow East, 

Glasgow G4 0NG 

Many intracellular bacterial pathogens are capable of survival inside macrophages because of their ability to prevent nitric oxide (NO) induced 

cell death, as such elucidating the molecular mechanisms of sensing and detoxifying NO is of key interest. Two bacterial proteins Nsrr 

(Streptomyces coelicolor) and catalase (Corynebacterium glutamicum; Cg) were chosen to determine if an iron NO (Fe-NO) interaction could 

be measured spectroscopically primarily using Fourier Transform infra-red (FTir) but also uV-Visible (uV-Vis) Spectroscopy. Nsrr 

is a transcriptional regulator and has been shown to directly sense NO utilizing a [2Fe-2S] cluster and is thought to regulate NO detoxification 

machinery. Catalase, a protein containing a haem group, is capable of detoxifying peroxynitrite. The uV-Vis data for catalase with and without 

NO present indicated a shift in the Soret peak (~400nm–~440nm) in addition to relative appearance of Alpha and Beta peaks (~540nm and 

580nm respectively). FTir analysis detected a sustainable signalat pH7, long enough to run a 2-Dimensional-ir experiment tofurther elucidate 

the dynamics of theinteraction. Nsrr has been entered into crystallization trials for structural determination along with Cg catalase with 

promising results which are indicative that Fe-NO interactions can be measured using dynamic spectroscopic techniques. 

HA15/06 Unique conservation of the albaflavenone biosynthetic pathway in Streptomyces 

SuZY C. MOODY1 , Bin Zhao2 , Michael Waterman2 , Steve Kelly1 , David C. lamb1 1 2 Institute of Life Science, School of Medicine, Swansea University, Swansea; Depts of Biochemistry & Center for Structural Biology, 

Vanderbilt University School of Medicine, Nashville; USA 




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The Gram positive, filamentous bacterial genus Streptomyces is characterized by its complex lifecycle and by the ability to produce a wide 

variety of secondary metabolites, including many antibiotics. usually soil dwelling, osmotic, oxidative and heat stress are all commonly 

encountered, and the bacterial cells have intricate biochemical processes for survival. recently, we have demonstrated that S. coelicolor 

makes albaflavenone, biosynthesized by the products of a two gene operon consisting of a sesquiterpene cyclase and a cytochrome P450 

enzyme. 

in the present work, bioinformatic analysis revealed this operon to be uniquely conserved within the genus. Extraction and subsequent mass 

spectrometry analysis show that the pathway is active and albaflavenone produced in at least five diverse species. The unusual conservation 

suggests an important role for this antibiotic, beyond antibiosis. Further investigation of S. coelicolor suggests that albaflavenone production 

may be highly susceptible to a stressful environment. Our current work is further examining the relationship between environmental stress 

and albaflavenone production. 

HA15/07 Inhibition of MrSA by british honeys 

STEFAN KliMACH, leighton Jenkins, rose Cooper 

University of Wales Institute Cardiff, Cardiff 

Medical grade manuka honey from New Zealand has been shown to interrupt cell division in MrSA, but the effect of British monofloral 

honeys on MrSA is unknown. Nineteen samples of British honey produced between 2009 and 2010 were collected and investigated for 

their authenticity and floral origin. Most honey samples were from a single nectar source although one British honey was found to contain 

mixed grains with manuka pollen. Agar well diffusion bioassays with Staphylococcus aureus NCTC 6571 and the determination of MiCs with 

MrSA showed that all of the British honey samples had lower antibacterial activity than a sample of a medical grade manuka honey. Some 

British honeys generated hydrogen peroxide on dilution, unlike manuka honey. However, the British honey sample that contained manuka 

pollen and a sample of ivy honey each exhibited non-peroxide activity. Five of the British honeys with highest levels of antibacterial activity 

were tested for their effect of the structural integrity of MrSA by transmission electron microscopy. Significant effects on cell division in 

MrSA were not observed. This suggests that different honeys have differing inhibitory effects on Gram positive bacterial cells and that those 

honeys selected for clinical use must be carefully chosen. 

HA15/08 The involvement of rNA polymerase sigma factors in the response of Corynebacterium pseudotuberculosis to in vitro 

oxidative stress 

THiAGO luiZ DE PAulA CASTrO1,4 , luis Gustavo Carvalho Pacheco2 , Fernanda Militão1 , Caroline Pereira Domingueti1 , 

Bianca Mendes Souza1 , Núbia Seyffert1 , Anne Cybelle Pinto1 , Wanderson Marques Silva1 , Fernanda Alves Dorella1 , Artur Silva3 , 

Anderson Miyoshi1 , Christopher Dowson4 , Vasco Azevedo1 1 2 Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazi; Federal University of Bahia, Salvador, Bahia, Brazil; 

3 4 Federal University of Pará, Belém, Pará, Brazil; University of Warwick, Coventry, West Midlands 

Corynebacterium pseudotuberculosis is the aethiological agent of Caseous lymphadenitis, disease that seriously affects sheep and goats in 

England and other countries. Capable to persist and replicate inside macrophages, C. pseudotuberculosis overcomes the effects caused by 

reactive oxygen/nitrogen species from the host. Considering that the bacterial resistance to such condition is promoted by the activation 

of specific sets of genes, which may be still related to pathogenicity and virulence, the present work aimed to investigate the involvement 

of different transcriptional regulators, belonging to the sigma factors class, in the response to oxidative stress generated in vitro. Performed 

kinetic curves indicate a significant slowing of bacterial growth when cultures in initial exponential phase, OD =0.2, are submitted to 

(600nm) 

40mM hydrogen peroxide or 15μM plumbagin. After 15, 60, 180 and 270 minutes of treatment, total rNA was extracted and analysed by 

rT-qPCr. Among the 8 sigma factors genes annotated, 5 presented enhanced transcriptional levels under oxidative conditions: sigB, sigH, 

sigM, sigC and sigK, while the last two were not previously described as involved in this kind of adaptation for other Gram-positives. in the 

next step we will evaluate the stress resistance and infectivity of already obtained mutant strains for the listed sigma factors genes. 

HA15/09 Investigation of factors influencing sigma b (σb)-dependent gene expression in Listeria monocytogenes: growth 

phase and osmotic stimulation of σb activity 

MArTA uTrATNA, Conor O’Byrne 

Bacterial Stress Response Group, National University of Ireland Galway, Galway, Ireland 

Listeria monocytogenes is ubiquitous in nature. it can be transmitted via food and causes listeriosis, characterized by a 30% mortality 

rate in humans. Transcriptional reprogramming achieved with the polymerase alternative sigma factor (σB ) plays an essential role in L. 

monocytogenes virulence and stress tolerance. The mechanisms underlying regulation are unknown in L. monocytogenes but they are likely 




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HA15 Cont. 

to involve posttranslational regulation. To investigate the modulation of σ B activity, transcript levels of sigB and three genes, previously 

identified as σ B -dependent (opuCA, lmo2085, lmo2230) were established in the exponential phase of growth with real-time rT-PCr in 

L. monocytogenes EGD and in a corresponding mutant lacking functional σ B . A dramatic and proportional increase in the transcript levels 

occurred in case of the four genes selected as the level of osmotic stress was gradually increased from 0 to 0.9 M NaCl. A rapid but short 

induction of transcription of each of the four genes at the 15 min time interval was recorded in wild type but not observed in ΔsigB strain 

following osmotic upshock. in conclusion, σ B in L. monocytogenes is activated rapidly and transiently after sudden exposure to osmotic stress 

and its activity can be fine-tuned according to the environment encountered. 

HA15/10 Expression analysis reveals diverse functions for genes encoding flavodiiron proteins in Anabaena PCC 7120 

MAriA ErMAKOVA, Yagut Allahverdiyeva, Natalia Battchikova, Eva-Mari Aro 

University of Turku, Turku, Finland 

Flavodiiron proteins (FDP), involved in detoxification of O or NO in anaerobic Bacteria and Archaea, were found in all so far sequenced 

2 

cyanobacteria. Genome of Synechocystis PCC 6803 contains four genes encoding FDPs. The Flv1 and Flv3 proteins function in Mehler 

reaction on the reducing side of Photosystem i. The Flv2 and Flv4 proteins participate in photoprotection of Photosystem ii. 

Filamentous N -fixing cyanobacteria Anabaena sp. PCC 7120 contains six genes encoding FDPs. There are two copies of both flv1 and flv3 

2 

genes corresponding to those in Synechocystis. in order to determine the reasons for their duplication we analysed the expression profiles 

under different environmental conditions. 

The expression level of the ‘b’ genes increased upon a shift from high to ambient CO level and also during the high light treatment 

2 

independently of the presence of combined nitrogen in the growth medium. Contrary, the transcription level of the ‘a’ genes depended 

only on the availability of combined nitrogen in the growth medium, with strong induction upon nitrogen deprivation. We suggest that the 

‘extra’ copies of the flv genes in Anabaena, the flv1a and flv3a genes are related to the nitrogen-fixing metabolism by performing ‘heterocystspecific’ 

Mehler reaction. 

HA15/11 Delay of cellular cycle by the inhibition of FtsZ by 8-oxo-GTP 

AlExANDrO rODríGuEZ rOJAS, Paolo Natale, Miguel Vicente, Jesús Blázquez 

National Centre for Biotechnology, Madrid, Spain 

Exposure to oxygen radicals generates several modified nucleic bases. The 8-oxo-7, 8-dihydroguanine (8-oxoG) is one of the most 

abundantoxidative lesions, and seems to play a critical role in mutagenesis and carcinogenesis. However, little attention has been paid to 

the epigeneticconsequences of 8-oxoG formation. in this study, we assess whether inactivation of mutT, the gene whose product sanitizes 

the nucleotide pool by hydrolyzing the 8-oxoG, has any consequence for the cellular cycle. The lower growth rate in minimal media or in 

oxidative conditions of a mutTdefective strain in comparison to its wild-type parental counterpart in Escherichia coli, suggest that the cell 

cycle may be delayed by the formation of 8-guanine nucleotides, a result that was supported in vitro by the inhibition of the GTPase activity 

of the central division protein FtsZ by 8-oxo-GTP. 

HA15/12 beyond bottlenecks in membrane production 

ravi K.r. Marreddy 

University of Groningen, The Netherlands 

The structural and functional analysis of complex multi-domain (membrane) proteins is hampered in large part due to problems associated 

with their overproduction in a functional state. The bacterium Lactococcus lactis is a suitable host for overexpression of membrane 

proteins. Although many pro- and eukaryotic proteins are expressed well in L. lactis, other proteins are difficult to overproduce. in order to 

understand ‘why certain membrane proteins are well expressed and others not’, we used a combined proteomics-transcriptomic approach 

to identify potential expression bottlenecks. The overproduction of membrane proteins in L. lactis invoked a general stress response 

(upregulation of various chaperones) and a severe metabolic burden. Also, we detected a specific cell envelop stress response, which is 

mediated by a two-component system CesSr, indicative of limitations in membrane protein folding and turnover of mis-folded protein. 

Furthermore, deletion of cesSR genes, in particular ftsH, oxaA2 and rmaB lowered the expression levels of target membrane proteins. In 

trans complementation of these knock-out strains, using either low or high-copy plasmids, restored the production of target membrane 

protein. in fact, modulating the expression of CesSr benefited the production yields of membrane proteins from different origins, initial 

successes in improving the biosynthesis of medically-important membrane proteins have been obtained. 




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HA15/13 Organization of the electron transfer pathways to oxygen and nitrite in the obligate human pathogen, Neisseria 

gonorrhoeae: how are electrons transferred to the outer membrane? 

AMANDA HOPPEr, Jeffrey Cole 

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K. 

Neisseria gonorrhoeae inhabits an oxygen-limited environment. Two of its eight c-type cytochromes, cytochromes c and c , form a 

4 5 

bifurcated electron transfer pathway to the single cytochrome oxidase, cbb (1). When oxygen is limiting, the gonococcus reduces nitrite to 

3 

NO catalysed by the copper-containing nitrite reductase, AniA, which is an outer membrane lipoprotein. Mutants defective in cytochromes 

c , c , c c and c , or c and c all reduce nitrite, though more slowly than the parent, implying the existence of multiple electron transfer 

2 4 5, 2 4 2 5 

pathways to AniA. CcoP is one of four subunits constituting the cytochrome cbb terminal oxidase. Surprisingly, unlike CcoP in many other 

3 

bacteria, CcoP from gonococci and commensal neisseria contains three rather than two haem-binding motifs. Mutants defective in this 

motif reduce nitrite at only 50% of the rate of the parent, providing the first published report implicating a cytochrome oxidase subunit 

in electron transfer to a nitrite reductase (2). Three other outer membrane redox proteins are the gonococcal azurin, laz, cytochrome c 

peroxidase, Ccp, and the NO-binding protein, cytochrome c’. unlike azurins in other bacteria, gonococcal laz is not thought to transfer 

electrons to AniA. A model of how electrons might be transferred to these outer membrane redox proteins from the electron transfer 

chain in the cytoplasmic membrane will be presented. 

References: 1. li et al. (2010) Journal of Bacteriology 192: 2395–2406. 2. Hopper et al. (2009) FEMS Microbiology letters 301: 232–240. 

Keywords: outer membrane lipoproteins; electron transfer; oxygen toxicity; nitrite reduction; gonococcal cytochromes 

HA16 Virus structure & assembly 

↑Contents 

HA16/01 role of phospholipase D2 in influenza virus assembly and budding 

AMANDA STuArT, Maria Amorim, Emily Bruce, Paul Digard, David Brown 

Virology Division, Dept of Pathology, University of Cambridge, Cambridge 

The cellular pathways involved in influenza A virus assembly and egress are poorly understood. We have previously shown that influenza 

virus budding is dependent upon the cellular rab11 recycling pathway. Phospholipase D2 (PlD2) has been shown to be involved in 

membrane trafficking and recycling pathways and we therefore decided to investigate a potential role for PlD2 in influenza virus infection. 

Cells depleted of PlD2 showed up to 3 logs reduction in virus titre clearly demonstrating PlD2 is required during infection. The depletion 

did not affect the ability of the virus to enter and express its genome, but instead affected later stages of the virus life cycle. in PlD2depleted 

cells, both rab11 and viral genome accumulated in a large perinuclear structure and failed to traffic to the plasma membrane as 

analysed by live imaging of GFP-tagged rNPs, immunofluorescence and fluorescence in situ hybridization. Thin section electron microscopy 

of the plasma membrane revealed abnormal budding events similar to that we had observed previously with rab11 depleted cells. These 

data further support our findings on the importance of cellular membrane recycling pathways during iAV assembly and demonstrate a role 

for PlD2 in regulating these pathways. 

HA16/02 HIV-2 genome dimerisation and viral infectivity: a critical role for the packaging signal and stem-loop 1 

ANNE l’HErNAulT1 , Jane Greatorex2 , Andrew lever1 1 2 University of Cambridge, Cambridge; Health Protection Agency, Cambridge 

The rNA packaging signal (Psi) of Human immunodeficiency Virus type-2 (HiV-2) is present on all viral rNA species and a co-translational 

mechanism and limited availability of the Gag protein have been shown to confer specificity of genomic rNA encapsidation. We have 

demonstrated previously that the viral rNA genome is dimeric within the virion particles and that motifs within the encapsidation signal 

are critical for this process. However, the exact relationship between genome dimerization and encapsidation, as well as how these two 

processes relate to HiV-2 infectivity, remains unclear. Here, we designed a series of mutants to investigate rNA motifs within HiV-2 Psi and 

the associated Sl-1 structure. Wild type and mutant viruses were evaluated for their ability to replicate in T-cells; the genomes of WT and 

mutant HiV-2 were analysed by native northern blot and their packaging efficiencies were determined. Our results confirm the existence of 

a tight relationship between dimerization and encapsidation of HiV-2 genomic rNA. Mutant viruses that failed to dimerise also showed a 

packaging defect. Furthermore, a GGAG motif within HiV-2 Psi appears critical for viral infectivity. Ongoing experiments will help determine 

what exact role this purine-rich sequence plays in the late stages of HiV-2 replication cycle. 




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HA16/03 A conserved residue in HCV core important for infectious virus assembly 

Allan Angus1 , Antoine loquet2 , Francois Penin2 , ArViND PATEl1 1 2 Centre for Virus Research, University of Glasgow, Glasgow; Institut de Biologie et Chimie des Proteines, CNRS and University of Lyon, 

Lyon, France 

using the HCV cell culture system, we identified a single residue (G33) within domain i of the viral core protein that when changed to 

alanine created a mutant virus with an interesting phenotype. Following transfection into Huh-7 cells, the G33A rNA replicated efficiently 

yet the extracellular and intracellular infectious virus titres were reduced by 2 logs, indicating that this residue is important for infectious 

virus assembly. During serial passaging of cells replicating this mutant rNA, we reproducibly observed the reversion of virus infectivity to 

WT levels. Moreover, this increase in infectivity coincided with compensatory mutations arising in close proximity to the original G33A 

substitution. Examination of core protein [residues 2–45] structure (PDB entry 1CWx) indicates that the residue G33 is in close contact 

with residue F24, and that the G33A mutation induces a steric clash with F24 which is reversed by the observed compensatory mutations 

bringing them spatially close to F24. Molecular simulations revealed that these mutations disturb the distribution of core conformational 

preferences, which is likely related to the modulation of virus production. Together, these data highlight the plasticity of core domain i 

conformation and illustrate the relationship between its structural tolerance to mutations and virus assembly. 

HA16/04 Characterization of the membrane binding properties of the respiratory syncytial virus matrix protein in vitro 

rOBErT YEO, Helen McPhee, Victoria Money, John Sanderson 

Durham University, Durham 

rSV is an enveloped rNA virus that acquires its membrane by budding from the host cell. internally, the membrane is associated with a 

proteinaceous layer comprised of M protein that directs assembly. Characteristically the regions of cell membrane upon which the virus 

assembles and buds are detergent resistant membranes (DrMs) rich in sphingolipids and cholesterol. To determine if M had an inherent 

propensity to bind to DrMs we artificially reconstituted membranes with varying lipid mixtures, some of which would mimic DrMs. By 

using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force 

microscopy on monolayers transferred to OTS-treated silicon wafers enabled factors that influence the disposition of the protein onto 

the lipid interface to be characterized. in the absence of sphingomyelin, rSV M protein penetrates monolayers composed of mixtures of 

phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. in mixtures containing sphingomyelin, 1,2-dioleoylsn-glycero-3-phosphocholine, 

and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface 

of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Notably, upon prolonged incubation with lipids M 

spontaneously self assembled into uniform helical structures similar to filamentous virus previously observed by EM. 

HA16/05 Visualization of a calicivirus–receptor interaction at sub-nanometer resolution 

DAViD BHEllA1 , ian Goodfellow2 1 2 Medical Research Council and University of Glasgow Centre for Virus Research, Glasgow; Imperial College London, London 

The Caliciviridae family comprises positive-sense rNA viruses of medical and veterinary significance. in humans, caliciviruses are a major 

cause of acute gastroenteritis; while in animals they cause respiratory illness, conjunctivitis, stomatitis and haemorrhagic disease. unlike many 

Caliciviruses, Feline Calicivirus (FCV) may be propagated in cell culture, providing a tractable model for studies of virus-host interactions. 

We have previously shown that Feline Calicivirus binds to its receptor Junctional Adhesion Molecule 1 (JAM-1) at the outer face of the 

P2 domain of its capsid protein VP1. Cryo electron microscopy was used to solve the structure of the complex at 18Å resolution. JAM- 

1 binding induces conformational changes in the viral capsid that we hypothesise may represent the early stages of viral uncoating. We 

have now extended the resolution of this study to 9Å resolution. The reconstruction reveals flexibility in the P domain, induced by JAM-1 

binding, that compromises the resolution in this region. A revised quasi-atomic resolution model of the complex is presented based on the 

higher resolution data and the recently published crystal structure of FCV. 





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HA16/07 Structural analysis of Core-E1 signal peptide and analysis of the requirements for cleavage of the genotype 3a 

signal sequence by signal peptide peptidase 

VErENA OEHlEr1 , Ana Filipe1 , roland Montserret2 , François Penin2 , John Mclauchlan1 1 2 MRC-University of Glasgow Centre for Virus Research, Glasgow; Institut de Biologie et de Chimie des Protéines, Lyon, France 

Cleavage by signal peptide peptidase (SPP) at the core-E1 signal peptide is necessary to generate mature core protein that attaches to lipid 

droplets. Few substrates for SPP have been identified and little is known about recognition of motifs within signal peptides by the protease. 

in this study, we have determined the structure of a synthetic peptide based on the core-E1 signal peptide sequence of HCV strain JFH1. in 

addition, we have extended mutational analysis of the sequences required for efficient SPP cleavage to include the core-E1 signal peptide 

from genotype 3a (GT3a) strains, which typically encode Phe at position 182 and His at position 187. Alteration of the JFH1 core-E1 

signal peptide sequence to GT3a had no effect on virus production. Mutation of previously identified residues Ala180, Ser183, Cys184 in 

the GT3a signal sequence had only a modest effect on SPP cleavage but did lower virus production. However, introducing an additional 

mutation at His187 severely impaired processing of core and led to a substantial decrease in production of infectious HCV. Together, our 

structural studies and mutational analysis provide deeper insight into the mechanism for cleavage of the core-E1 signal peptide by SPP and 

its importance to virus assembly. 

HA16/08 biochemical analysis of the transmembrane domains of NS2 

BArNABAS KiNG1 , Stephen Griffin2 , Mark Harris1 1 2 University of Leeds, Leeds; Leeds Institute for Molecular Medicine, Leeds 

The non-structural 2 (NS2) protein of hepatitis C virus has been shown to play a key role within the life cycle. it participates in multiple 

protein-protein interactions to facilitate virion assembly. Despite our growing knowledge of the interactions between NS2 and other 

proteins, it remains unclear as to how NS2 interacts with membranes. NS2 is thought to assume a topology with three transmembrane 

domains (TMD) but there is as yet no unambiguous biochemical data to support this model. To address this we have generated a panel of 

NS2 mutants and GFP-fusion proteins and analysed their membrane interactions by a combination of immunofluorescence and membrane 

fractionation. Our data are consistent with the proposed model. 

HA16/09 Characterization of a novel viroporin encoded by the human papillomavirus E5 oncoprotein 

lAurA F. WETHErill1 , Kris K. Holmes1 , Mark Verow1 , Toshana l. Foster1 , Elizabeth Atkins1 , Sayaka Sato1 , rebecca ross1 , 

Mark Harris1 , G. Eric Blair1 , Stephen Griffin2 , Andrew Macdonald1 1 2 Faculty of Biological Sciences, University of Leeds, Leeds; LIMM, St James Medical School, University of Leeds, Leeds 

E5 is the least characterized of the three oncoproteins encoded by Human Papillomavirus 16, as efforts to study the biochemical properties 

of the protein have been hampered by its hydrophobic nature. The 83 amino acid protein contains three transmembrane regions 

and oligomerises in cells. Here, we have refined a bacterial expression system that circumvents the problems of hydrophobic protein 

purification and the limitations of chemical synthesis. We present the first TEM images of E5 and demonstrate that E5 is oligomeric and 

can insert into lipid membranes. Furthermore, we show that E5 possesses channel activity and facilitates release of carboxyfluorescein from 

liposomes. Due to paucity in structural data, we have used in silico methodology to design a model of an E5 oligomer. Moreover, we have 

adopted a rational approach to E5-specific inhibitor design that has successfully identified the first E5 inhibitor, representing a breakthrough 

in rational drug design. Furthermore we demonstrate that this compound does not prevent oligomerization or insertion of E5 into 

liposomes. in summary we present a novel function for the E5 oncoprotein as a viroporin and demonstrate the first in silico designed E5 

inhibitor. These findings may lead to the first of a novel class of anti-HPV therapeutics. 




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HA16/10 Studying disassembly and re-assembly of ssrNA viruses using analytical ultracentrifugation 

AMY M. BArKEr, P.G. Stockley 


ssrNA viruses are major pathogens in every kingdom of life. Their assembly and disassembly pathways are potential drug targets but remain 

unexploited to date. This project is concerned with how viruses and bacteriophages package genomic rNA into their coat protein shells. 

in solution ssrNA free is highly complex allowing for base pairing and stacking keeping the favoured minimum energy conformation. 

Measuring the hydrodynamic radius of complete virus shells is easily done using many techniques, this project utilises sedimentation velocity 

analytical ultracentrifugation and electron microscopy as the primary tools for investigating virus size.The ssrNA genome of bacteriophage 

MS2 is known to occupy a larger volume in solution than the interior volume of the capsid. svAuC has been used to investigate fragments 

of the MS2 genome to see how buffer component affect hydrodynamic properties of the sedimenting material. Other work is directed at 

understanding the assembly of simple plant viruses using the model systems Satellite Tobacco Necrosis Virus and Turnip Crinkle Virus. We 

have compared in vitro reassembly reactions of STNV coat protein with 30-mer rNA aptamer B3 and solution studies with TCV virions to 

compare their hydrodynamic radius under different buffer components and pH. 

HA16/11 Investigating assembly mechanisms of a T=1 virus-like particle 

rOBErT FOrD, Thomas Wilkop, roman Tuma, Peter Stockley 

University of Leeds,, Leeds, 

using a recombinant expression system, a T =1 Satellite Tobacco Necrosis Virus (STNV)-like particle was expressed in E. coli and purified. 

Conditions for disassembly and reassembly have been defined in vitro, making it possible to investigate capsid assembly using various 

biophysical techniques. VlP assembly is dependent on the presence of the coat protein N-terminal arm and rNA, where in E. coli the rNA 

that gets packaged is the STNV coat protein mrNA. By immobilizing coat protein whilst in the presence of free coat protein subunits under 

reassembly conditions, partially assembled capsids were prepared for rNA aptamer selection against the rNA binding interfaces. Seventeen 

clones were isolated, with one aptamer termed B3 containing the most significant sequence similarity to the STNV-1 genome, which 

includes a stretch of 10/10 nucleotides. These 10 nucleotides fold to form a stem loop displaying the motif ACAA, and it is this region 

which triggers virus assembly using purified STNV coat protein monomer. This work will discuss how the biophysical techniques of analytical 

ultracentrifugation (AuC) and single molecule fluorescence correlated spectroscopy (smFCS) can yield useful information on stoichiometry 

and kinetics of recombinant STNV capsid assembly using this system. 

HA16/12 Comparative stoichiometric analysis of two bunyaviruses: oligomeric formation of the N protein and specificity 

of rNA binding. 

STEPHEN CArTEr1 , Mark Kainz2 , Julian Hiscox1 , John Barr1 1 2 The University of Leeds; Ripon College, Wisconsin, USA 

The Bunyaviridae family is one of the largest virus families and is classified into the Orthobunyavirus, Toposvirus, Nairovirus, Phlebovirus 

and Hantavirus genera, which together contains at least 350 known species. These viruses are frequently listed as emerging or reemerging 

viruses, and among the most medically and agronomically important. Bunyavirus pathogenesis depends on formation of the 

ribonucleoprotein (rNP) complex, in which the bunyavirus genome is entirely encapsidated by the virus-encoded nucleocapsid (N) protein. 

Only in the form of the rNP is the genome replicated, transcribed and packaged into new progeny particles. We have previously described 

important structural and functional features of the prototype Bunyamwera virus (Orthobunyaviridae) N protein, including tetramer formation, 

rNA length, rNA specificity and the role of the conserved amino acids in these activities. Of interest when comparing the relationships 

between the N protein sequences of all the five genera is the close relationship between the orthobunyaviruses and tospoviruses. For 

this reason analysis of the Tomato spotted wilt virus N protein has been undertaken, which has revealed the stoichiometric characteristics 

of oligomer formation and rNA binding. We have paired this information with our previous functional analysis findings to reveal similar 

structure-function relationship of the N protein for both viruses. 

HA16/13 resurrecting SW1, from sequence to pseudovirion 

CHriSTOPHEr HArTE, Benjamin Neuman 


The novel gamma coronavirus SW1 was detected in 2008 by microarray analysis of diseased tissue harvested from a captive whale 

deceased as the result of infection (Mihindukulasuriya et al., 2008). Subsequent attempts to rescue live virus failed and whilst enough genetic 




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HA16 Cont. 

material was recovered for the compete sequence of SW1 to be elucidated the virus remains uncharacterized beyond sequence analysis. 

Failure to recover live virus suggests the tropism of SW1 is severely restricted making traditional in vitro analysis with current cell lines 

impossible. Therefore, the approach chosen for this research is recombinant expression of SW1 structural proteins in cell culture, facilitating 

the spontaneous assembly of pseudovirions which may be tracked from site of assembly through to cell egression and therein elucidating 

processes and factors involved in viral assembly and budding. Host cells will be transduced to express viral proteins by lipofection of 

plasmids delivering viral gene sequences downstream of a chicken α-actin promoter, chosen for constitutive moderate level expression. 

This demonstrates the ability of synthetic biology in overcoming previously insurmountable obstacles. Here, expression of SW1 structural 

proteins will allow the mechanistic comparison of a rare, novel coronavirus with well-characterized strains in spite of the absence of a live 

virus sample. 

HA16/14 Characterization of the VP1-2 ubiquitin-specific protease domain 

MAriANNE BOlSTAD1 , Fernando Abaitua1 , Colin Crump2 , Peter O’Hare1 1 2 Imperial College London; Cambridge University, Cambridge 

HSV-1 VP1-2, essential for virus entry and assembly, is proteolytically cleaved resulting in production of a 55 kDa N-terminal peptide, an 

event linked to successful infection. The N-terminus encompasses a conserved ubiquitin specific protease (uSP) domain, whose biological 

role remains unclear. We established cell lines with different forms of the N-terminal cleavage product that would either be targeted or not 

to the nucleus, and either retain uSP activity or not, due to a single substitution in the catalytic triad. We examined whether expression of 

the uSP domain could rescue the known early defect of the tsB7 mutant virus which is blocked in entry and does not undergo cleavage. 

The isolated domain was unable to complement the tsB7 entry defect. However deletion of the uSP motif abolished complementation 

of a VP1-2 –ve virus. Despite this, a mutation in the active site, demonstrated to abrogate uSP function, showed significant rescue of the 

mutant virus. While to date no substrate specificity has been established, we now show using the mutant VP1-2 uSP, that the uSP domain 

is itself ubiquitinated and rapidly deubiquitinated by its own action in cis. A virus with a specific mutation in the catalytic triad is currently 

being characterized 

HA16/15 recombinant picornavirus capsid protein VP4: multimerization and permeability in model membranes 

ANuSHA PANJWANi1 , Nicola Stonehouse2 , Dave rowlands2 , Toby Tuthill1 1 2 Institute for Animal Health, Pirbright; University of Leeds, Leeds 

Enveloped viruses employ well characterized fusion mechanisms to penetrate the membrane during cell entry. Non-enveloped viruses lack 

a lipid envelope and must penetrate the membrane by alternative mechanisms which remain poorly understood. The picornavirus family 

includes pathogens responsible for significant diseases including poliomyelitis, foot-and-mouth disease and the common cold. These viruses 

are amongst the smallest and simplest of mammalian viruses and therefore make suitable models for studying non-enveloped entry. 

During picornavirus cell entry, the small internal capsid protein VP4, is released from the virus, interacts with the cell membrane and is 

implicated in the formation of a membrane pore through which the viral rNA genome is delivered into the cytoplasm for the initiation 

of replication. We previously demonstrated membrane permeability is induced by recombinant rhinovirus VP4-GST fusion protein. in this 

study we have produced recombinant VP4 lacking a fusion partner and optimised methods for protein purification. recombinant VP4 has 

been shown to multimerize into higher order structures in the presence of model membranes and induce membrane permeability using 

a liposome dye release assay. This membrane permeability is similar to that observed with virus or native VP4 isolated from virus, and 

consistent with previous reports of VP4 function. 



HA16/18 Quasi-lattices in virology – the encasing forms of protein capsids 

DAViD SAlTHOuSE, reidun Twarock 


Much work has been done to comprehend the structures of icosahedral virus capsids. Caspar and Klug theory was the first to predict the 

surface structures of these viruses via tesselations by subdividing each face of an icosahedron into T triangles. later on, the crystallographic 

structures of viral capsids have been studied by Janner. Here we are using non-crystallographic tessellations (quasi-lattices) instead to predict 

the 3-dimensional structure as well as the encasing forms of the viral capsid proteins of the CCMV virus. 




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HA16/19 Virology and carbon chemistry: symmetry as a common thread? 

JESS WArDMAN, Tom Keef, reidun Twarock 


A substantial number of viruses exhibit icosahedral symmetry. Their 3D structures, including important features such as their epitopes or 

rNA organization, can be modelled via mathematical tools that describe how icosahedral symmetry should be organized at multiple radial 

levels. Carbon atoms can form fullerene shells that also show icosahedral symmetry, and these fullerenes can form ‘onions’, where several of 

these fullerene shells are nested inside one another. This nesting results in a structure with overall icosahedral symmetry that has important 

structural features at multiple specific radial levels. We show here how the same mathematics explains the positions of these structural 

features in both viruses and carbon onions – in particular, the onion of C60, C240 and C540. 

HA16/20 In vitro assessment of targeting peptide incorporation within the knob domain of human adenovirus type 

48 fibre 

lynda Coughlan1 , HANNi uuSi-KErTTulA1 , Alan l. Parker1 , Jerome Custers2 , Stuart A. Nicklin1 , Andrew H. Baker1 1Institute of Cardiovascular & Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow; 

2Crucell, 2301 CA Leiden, Netherlands 

Human Adenovirus type-5 (Ad5) is a commonly used vector for gene therapy. However, its clinical utility is potentially limited by high 

seroprevalence rates and off-target liver sequestration and toxicity. Therefore, the use of rare species adenoviruses (eg. species D; Ad48) 

as vectors is appealing. Targeted viral delivery to defined tissues (eg. tumours) can be achieved through the incorporation of peptide ligands 

within virus structural proteins and such strategies have been reported extensively for Ad5. 

We used predictive structural modelling (SWiSS-MODEl) to assess the DG and Hi loop domains of the Ad48 fiber knob, for the 

incorporation of a 20aa peptide, A20FMDV2 (A20). This peptide targets ανβ6 integrin, which is overexpressed in human carcinomas. 

recombinant knob proteins (Knob48, Knob48-DG-A20 and Knob48-Hi-A20) were purified following expression in E.coli. Trimerization 

of the fiber knob domain is critical for viral assembly. We confirmed that Knob48 successfully formed a trimer however Ad48-DG-A20 

did not. Knob48-Hi-A20 also formed a trimer, however we obtained very low protein yield. Preliminary results indicate that Knob-Hi-A20 

forms insoluble inclusion bodies in E.coli. However, this does not necessarily preclude it from adequate assembly in mammalian cells 

following infection. Therefore, the Hi loop still represents a suitable site for peptide incorporation. 

HA17 Cell-to-cell transmission of viruses 

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HA17/01 Focal adhesion kinase inhibitors enhance cell-to-cell spread of herpes simplex virus type-1 

AMY ZHOu, Helena Browne 


Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase known to be involved in the entry process of some herpesviruses including 

herpes simplex virus type-1 (HSV-1). Studies using sirNA show that FAK depletion can significantly inhibit growth of HSV-1 (Cheshenko 

et al., 2005). in this study two different FAK inhibitors were employed to determine the effect of blocking FAK autophosphorylation and 

subsequent activation on HSV-1 infection. 

Contrary to expectations, both inhibitors induced a significant increase in HSV-1 plaque size and viral yields in a dose-dependent manner 

when cells were infected at low multiplicity, yet had no effect on virus growth following high multiplicity infection and did not impact 

on HSV-1 entry. An increase in plaque size was also observed when assays were carried out on drug-treated cells in the presence of a 

neutralizing antibody, suggesting that these inhibitors may increase cell-to-cell spread. Consistent with this view was the finding that these 

compounds increased cell adhesion. Together, these results imply that FAK inhibitors can increase the spread of HSV-1 through cell-cell 

contacts. 




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HA18/01 Feline immunodeficiency virus (FIV): a model for human immunodeficiency virus-1 (HIV-1) latency? 

CHi NGAi CHAN, Elizabeth l. McMonagle, Brian J. Willett 


Feline immunodeficiency Virus (FiV) induces an immunodeficiency in cats that is similar to AiDS in HiV-infected humans. The growth of FiV 

in CD4 + T-cells is dependent on the activation state of the cells. Here, we ask whether parallels can be drawn between the establishment 

of a latent infection state in HiV-1 and FiV infections of CD4 + T-cells. using the interleukin-2 (il-2)-dependent primary feline T-cell line 

Mya-i as our in-vitro model system, we discovered that when il-2 was removed from the cells prior to infection with FiV, a state of latency 

was established. This latency could be reversed by adding T-cell activating phorbol esters PMA and Prostratin. Further investigations showed 

that in the presence of il-2, PMA and Prostratin both inhibited virus production, and that the different Protein kinase C (PKC) isoforms 

were vital mediators of the dual effects of the phorbol esters, suggesting a complex role played by PKC in virus production under different 

cellular environments. This discovery may lead to the use of Mya-i cells as an in vitro model for testing FiV and HiV-1 latency-reversing 

compounds and FiV infection of the domestic cat as a small animal model for HiV-1. 

HA18/02 Efficacy of monovalent whole inactivated equine influenza virus vaccines containing the OIE recommended strains 

for 2010 against challenge with a Florida sublineage clade 2 virus 

NEil BrYANT, Adam rash, leah Prowse-Davis, liz Medcalf, Alana Woodward, Fernando Montesso, romain Paillot, 

richard Newton, Debra Elton 

Animal Health Trust, Kentford, Newmarket, Suffolk 

in response to the circulation of antigenically distinct equine influenza viruses (EiV) of the Florida sub-lineage with an 8-fold difference in 

Haemagglutination inhibition assay (Hi) titres, the World organization for animal health (OiE) recommended that EiV vaccines should 

contain a virus from both clades 1 and 2. The likely protection that would be provided by monovalent inactivated vaccines containing these 

strains (A/eq/South Africa/4/03 or A/eq/richmond/1/07) with a carbomer adjuvant was determined. Ponies were challenged with A/eq/ 

richmond/1/2007, a member of the Florida sub-lineage clade 2. unvaccinated control ponies all showed clinical signs of infection together 

with high levels of virus shedding. Although there was a range of different antibody levels at the time of challenge, protection achieved by 

both vaccines was characterized by significantly reduced signs of disease when compared with unvaccinated control ponies and reduced 

virus shedding. There were no significant differences between the vaccinated groups with the caveat that the study was conducted on 

small groups of ten ponies per vaccine. in summary, we demonstrated that vaccination with either currently recommended vaccine strain 

significantly protected against infection with A/eq/richmond/1/2007 despite the differences observed in antigenicity as determined by Hi 

assay using ferret sera. 

HA18/03 Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance 

STEPHEN HArE1 , Ann Vos2 , reginald Clayton2 , Jan Thuring2 , Maxwell Cummings2 , Peter Cherepanov1 1 2 Imperial College London, London; Tibotec, Beerse, Belgium 

The development of HiV integrase (iN) strand transfer inhibitors (iNSTis) and our understanding of viral resistance to these molecules 

have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) 

intasome with raltegravir and elvitegravir, establishing the general iNSTi binding mode. We now present an expanded set of cocrystal 

structures containing PFV intasomes complexed with first- and second-generation iNSTis at resolutions of up to 2.5 Å. importantly, 

the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the iNSTi- bound 

intasome active site. We show that like the Q148H/G140S and N155H HiV-1 iN variants, the analogous S217H and N224H PFV iNs 

display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in iNSTi-free and -bound forms revealed 

that the amino acid substitutions necessitate considerable conformational rearrangements within the iN active site to accommodate an 

iNSTi, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an 

interaction between HiV-1 iN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/ A mutations in 

drug-resistant viral strains. 

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HA18/04 Identification of further antigenic sites of serotype O foot-and-mouth disease virus using polyclonal sera and 

non-neutralizing monoclonal antibodies (mAbs) 

DArYl BOrlEY1,2 , David Paton1 , Terry Jackson1 , Mana Mahapatra1 1 2 Institute for Animal Health, Pirbright, Surrey; Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of 

Oxford, Oxford, Oxfordshire 

Currently 5 neutralizing antigenic sites have been identified on the surface of serotype O foot and mouth disease (FMD) virus. However 

work conducted by Dunn and colleagues at iAH, Pirbright, uK suggested that other unidentified epitopes may play a role in FMD-vaccine 

induced protection. The main aim of this work is to identify these additional sites and elucidate their significance. For this purpose an 

antibody pull-down technique was used to identify epitopes recognised by polyclonal bovine vaccinate serum and non-neutralizing mAbs. 

Viruses that ‘escaped’ antibody binding were sequenced and the significance of any changes determined by EliSA. 

The results have demonstrated that this technique is a useful tool in generating escape mutants from polyclonal serum and non-neutralizing 

mAbs, so far identifying five new residues (VP2 23, 98, 132, 188 and VP3 30). The results also confirmed the presence of antibodies to 

serotype O antigenic sites 3 and 4, identified using mAbs, in bovine vaccinate serum. 

HA18/05 Production of novel monoclonal antibodies to C clade isolates of HIV-1 

MArK HASSAll1 , Mark Page1 , Mark robinson3 , Mike Seaman2 , Simon Jeffs3 , Neil Almond1 1 2 3 NIBSC, Hertfordshire; Harvard, Cambridge, USA; Imperial College London 

The native HiV-1 envelope spike exists as trimers on the virion surface. Therefore antibodies elicited following immunization with trimeric 

envelope vaccines may be directed against epitopes found on functional envelope spikes. 

We produced novel monoclonal antibodies by priming mice with plasmid DNA expressing either CN54 or ZM96 gp140 sequences, and 

boosting with CN54 trimeric rgp140. Spleen cells were fused with NS-0 cells and hybridomas were screened for production of antibodies 

which bound trimeric rgp140. 

18 monoclonal antibodies were isolated from 16 colonies, which were characterized by binding in EliSA and western blotting, 

neutralization of pseudotyped viruses and isotype. Specificity for the V3 region was detected in 6 of these antibodies, suggesting that the 

trimeric protein is not altering the main focus of the response compared to monomeric protein. 3 were identified as having neutralizing 

capability in a pseudotype assay, all of which were mapped to the V3 region. Although cross clade binding of antibodies was detected the 

neutralization appeared to be C clade specific. 

From this work a number of useful monoclonal antibodies have been produced, however the there appears to have been little benefit of 

using trimeric rgp140 as an immunogen rather than monomeric rgp120. 

HA18/06 A multiplex assay for the evaluation of avian influenza H5 and H7 neutralizing antibodies directed against 

haemagglutinin 

NiGEl TEMPErTON1,3 , Giovanni Cattoli2 , Edward Wright3 1 2 Viral Pseudotype Unit, School of Pharmacy, University of Kent, Chatham Maritime, Kent ME4 4TB; FAO, OIE and National 

Reference Laboratory for Newcastle Disease and Avian Influenza, 35020 Legnaro, Italy, 3MRC/UCL Centre for Medical Molecular 

Virology, University College London, London W1T 4JF 

The continuous rapid evolution of lPAi and HPAi H5 and H7 influenza viruses in domestic poultry has major public and animal health 

implications. We have described a modular multiplex assay for the study of neutralizing antibodies directed against influenza H5 and H7. 

This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HA envelopes in the same serum/ 

plasma sample. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, a bivalent H7N1/H5N9 vaccine, or infected with 

an H7N3 virus were evaluated in this assay and the results correlated strongly with Hi data. For the sera obtained from chickens vaccinated 

with a bivalent vaccine the neutralization assay was performed with a single pseudotype virus (H5 or H7) as a monoplex assay or with the 




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two viruses mixed 1:1 (H5 and H7) as a multiplex assay and the neutralizing antibody profiles were found to be equivalent. This multiplex 

assay is undergoing further evaluation as a routine screening method for the detection of antibodies against notifiable H5 and H7 infections 

in birds. 

HA18/07 Changes in antenatal rubella susceptibility rates in Cwm Taf (South) NHS Trust 

linda A. Matthews1,2 , Selena Gray1 , Debra Gray1 , lYNNE M. lAWrANCE1 1 2 Faculty of Health & Life Sciences, University of the West of England, Bristol; Virology Laboratory, Pathology, Royal Glamorgan 

Hospital, Llantrisant 

Congenital rubella Syndrome (CrS) can affect the children of women infected because they were susceptible to rubella during pregnancy. 

Therefore, vaccination of schoolgirls was introduced and then replaced by the MMr vaccine in 1988. CrS incidence remains low currently 

but the vaccination policy changes may yet have an impact, and the media driven controversy around the MMr has affected uptake rates. 

This study reports data, drawn form ante-natal screening samples, for one Welsh NHS Trust area over a six-year period (2005–2010). 

The level of rubella susceptibility (defined by an antibody level of


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CMM Clinical & medical microbiology 

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CMM/01 Comparative metagenomic analysis of plasmid-encoded functions in the human gut microbiome 

BriAN JONES1 , Julian Marchesi2 1 2 University of Brighton, Brighton; University of Cardiff, Cardiff 

little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. using the culture independent 

TrACA system in conjunction with a comparative metagenomic approach, we have recently explored the pool of plasmids associated 

with the human gut mobile metagenome. This revealed that some plasmids or plasmid families are present in the gut microbiomes of 

geographically isolated human hosts with a broad global distribution (America, Japan and Europe), and are potentially unique to the human 

gut microbiome. Functions encoded by the most widely distributed plasmid (pTrACA22) were found to be enriched in the human gut 

microbiome when compared to microbial communities from other environments, and of particular interest was the increased prevalence of 

a putative relBE toxin-antitoxin addiction module (TAM). Subsequent analysis revealed that this was most closely related to putative TAMs 

from gut associated bacteria belonging to the Firmicutes, but homologues of the relE toxin were associated with all major bacterial divisions 

comprising the human gut microbiota. Comparative metagenomic analysis of a further 124 individuals comprising the METAHiT dataset 

highlighted large inter-individual variation in incidence and relative abundance of pTrACA22 type TAMs among healthy individuals, and 

indicated potential differences in health and disease. 

CMM/02 Evaluation of the Cepheid Xpert TM MrSA/SA blood culture assay and its potential clinical impact 

lASANTHA rATNAYAKE, William Olver 

Dept of Medical Microbiology, Ninewells Hospital, Dundee 

Objective: Preclinical evaluation of the Cepheid xpert TM MrSA/SA blood culture assay and its potential clinical impact in the management of 

patients with Staphylococcus aureus bacteraemia in Ninewells hospital, Dundee, uK. 

Methods: One hundred and thirty blood culture bottles with staphylococci from 99 patients were analysed using the Cepheid xpert TM 

MrSA/SA blood culture assay. Comparison was made with routine culture and sensitivity. The potential clinical impact of this assay was 

evaluated retrospectively by case note review to determine the percentage of meticillin-sensitive S. aureus (MSSA) and meticillin-resistant S. 

aureus (MrSA) patients in whom appropriate treatment could have been started earlier. 

Results: The assay performed with 96.3% and 100% sensitivity and 100% and 98.9% specificity for MSSA and MrSA, respectively. Negative 

predictive value was 98.9% and 100% for MSSA and MrSA respectively. use of the Cepheid xpert TM MrSA/SA blood culture assay result 

would have meant almost 50% of patients with MSSA receiving earlier appropriate antibiotics. 

Conclusion: This assay is an easy to perform, automated PCr test with a very high sensitivity and specificity for MSSA and MrSA. This would 

enable earlier initiation of appropriate therapy in patients resulting in better clinical and economic outcomes. 

CMM/03 Optimizing plasmid transformation, and large-scale random transposon mutagenesis in the probiotic Escherichia coli 

Nissle 1917 

JONATHAN NZAKiZWANAYO, Wendy McFarlane, Brian Jones 

University of Brighton, Brighton 

Our aim was to optimise protocols for rapid, efficient transformation of probiotic Escherichia coli strain Nissle 1917 (EcN) based on 

standard chemical transformation, and test the functionality of two mini-Tn5 transposon mutagenesis systems. 

Chemically competent cells (CCCs) were prepared from cultures grown at a range of temperatures, to a range of cell densities. The effect 

of heat shock duration was also tested. Transformation efficiencies were compared using puC19 and calculated as transformats/μg puC19 

DNA. Cell density and duration of heat shock were found to contribute to transformation efficiency, but physiological status of cells had the 

greatest influence, with growth at room temperature (rT) producing a 22-fold increase in transformation efficiency compared to growth at 

37°C. Greatest efficiencies were achieved when CCCs were prepared from rT cultures at 0.5 OD and a heat shock of 180s. 

600, 

Delivery of mini-Tn5 suicide vectors prl27 and puTKm using optimised transformation parameters yielded 310 and 0 mutants/μg DNA 

respectively, indicating the hyperactive transposase encoded by the prl27 system is required for EcN mutagenesis. prl27 derived mutants 

were confirmed to possess single random insertions by Southern hybridization. A bank of 11,328 mutants has now been generated and 

high-throughput screens for genes involved in host-microbe interaction implemented. 

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CMM/04 Helicobacter pylori and TNF-α-induced ADAM17 phosphorylation in gastric epithelial cells 

urSZulA BurSKA, Philip robinson, Jean Crabtree 

Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds 

Helicobacter pylori transactivates the epidermal growth factor receptor (EGFr) stimulating heparin-binding epidermal growth factor 

shedding by membrane-bound ADAM17. Previous studies identified that H. pylori upregulates ADAM17 in gastric epithelial cells and 

ADAM17 is over expressed in gastric cancer. How H. pylori regulates ADAM17 cell surface expression and enzymatic activity in gastric 

epithelial cells is unknown. ADAM17 has three phosphorylation sites, Threonine 735 and Serines 791 and 819. The aims were to identify 

the importance of these kinase phosphorylation sites in H. pylori-induced ADAM17 activation and signalling. Cellular expression and 

localization of ADAM17 and its phosphorylated isoforms before, and after, H. pylori and TNF-α stimulation in AGS gastric epithelial cells was 

examined by confocal microscopy and Western blotting. ADAM17 was localized mainly in the cytoplasm and the AG/Er system. ADAM17 

was phosphorylated at Thr735 by H. pylori and TNF-α stimulation, while Ser791 and Ser819 were dephosphorylated. Phosphorylation did 

not affect the localization of ADAM17. ADAM17 showed the same patter of phosphorylation after stimulation with both TNF-α (15 mins) 

and H. pylori (90 mins). Discovery of the mechanism of ADAM17 activation could identify new therapeutic targets for the treatment of H. 

pylori infection and gastric cancer. 

CMM/05 Complete genome sequencing and annotation of two human gut specific bacteriophages infecting Bacteriodes sp. 

Gb124 

lESlEY OGilViE, David Diston, James Ebdon, Huw Taylor, Jon Caplin, Brian Jones 


The human gut harbours a diverse microbial community which in turn plays host to mobile genetic elements and bacteriophages, forming 

the gut mobile metagenome. in particular, the role of bacteriophages in the development and function of the gut microbial community 

remains largely unexplored. We isolated and sequenced the genomes of two human gut specific phages infecting Bacteriodes sp. GB-124. 

Genome analysis of phage A (36,254 bp; GC content 46.56%) and B (47, 159 bp; GC content 38.75%) revealed 43 and 68 putative 

open reading frames, respectively. Putative genes in both phages exist on the same DNA strand and are organized into functional 

clusters encoding lysis, replication and regulation, and packaging and structural proteins. Phage B shares a high degree of similarity with the 

Bacteriodes sp. phage B40-8, whereas Phage A lacks any significant homology. These data add to the growing number of human gut phage 

genome sequences and augments our understanding of a microbial ecosystem with a major impact on our development and health. 

CMM/06 Evaluation of functional roles of mannose-binding protein from Acanthamoeba T4 to human brain microvascular 

endothelial cells by chemical mutagenesis 

ABDul MATiN1 , Muhammad ismail1 , Khalid Mehmood2 , Suk-Yul Jung3 1 2 Institute of Biomedical and Genetic Engineering, PO Box: 2891, Sector: G-9/1, Islamabad, Pakistan; Dept of Surgery, Army Medical 

Corps, Mangala, Pakistan; 3Dept of Biomedical Laboratory Science, Namseoul University, 21 Maeju-ri, Seonghwan-eup, Seobuk-gu, 

Cheonan-city, Choongnam, Republic of Korea, 

Acanthamoeba is a protozoan pathogen that can cause a blinding keratitis and fatal granulomatous encephalitis, however the pathogenic 

mechanisms of these organisms remain incompletely understood. Adhesion of amoebae to the host cells is thought to be a primary step 

in the pathogenesis of Acanthamoeba infections and is mediated by a mannose-binding protein expressed on the surface of Acanthamoeba. 

in support, our recent studies have shown that clinical isolates of Acanthamoeba exhibit significantly higher binding to the human brain 

microvascular endothelia cells, which constitute the blood-brain barrier and the human corneal epithelial cells in a mannose-binding protein 

dependent manner. in an attempt to further determine the precise properties of a mannose-binding protein, carbohydrate selection 

approaches are employed to produce Acanthamoeba expressing decreased levels of mannose-binding protein and their ability to exhibit 

host cell binding and produce host cell cytotoxicity determined using in vitro assays. The results demonstrating the precise role of mannosebinding 

protein in the pathogenesis of Acanthamoeba are presented. 




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CMM/07 Understanding the contribution of biofim formation and urease production to blockage of urethral catheters by 

Proteus mirabilis 

NiNA HOlliNG, Danielle lednor, Cinzia Dedi, Geoffrey Hanlon, Bhavik Patel, Brian Jones 


Proteus mirabilis is a major cause of catheter associated urinary tract infections, and forms extensive crystalline biofilms on catheter surfaces. 

To identify genes involved in P. mirabilis biofilm formation ~2000 mini-Tn5 mutants were screened to identify biofilm enhanced (BFE) or 

deficient (BFD) mutants. 125 BFD and 117 BFE mutants were recovered and a sub-set of each characterized further. Genes disrupted 

in selected mutants were identified, and their ability to encrust all-silicone urethral catheters was tested using an in vitro model of the 

catheterised urinary tract. Crystalline biofilm on blocked catheters was quantified by measuring concentration of calcium along the length of 

catheters using flame photometry. The ability of mutants to elevate urinary pH, and persist in bladder models was comparable to the wild 

type, and no significant differences in the time taken for catheters to become blocked were observed. However, levels of encrustation along 

the length of the catheters varied between mutants and the wild type, with the first 8cm of the catheter (eye-hole onwards) exhibiting the 

greatest concentrations of crystalline material in all cases. These results demonstrate urease production and elevation of urinary pH, rather 

than ability to form biofilms, is the key factor in catheter blockage. 

CMM/08 Phospholipases activities assessment of the clinical and the environmental isolates of Acanthamoeba 

ABDul MATiN1 , Salik Nawaz2 , Muhammad ismail1 , Suk-Yul Jung3 1 2 Institute of Biomedical & Genetic Engineering, PO Box: 2891, Sector: G-9/1, Islamabad, Pakistan; Dept of Pharmaceutical Sciences, 

Punjab University, Lahore, Pakistan; 3Dept of Biomedical Laboratory Science, Namseoul University, 21 Maeju-ri, Seonghwan-eup, 

Seobuk-gu, Cheonan-city, Choongnam, Republic of Korea 

Acanthamoeba is an opportunistic protozoan parasite, which can cause fatal granulomatous encephalitis and a blinding keratitis. However, 

the pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Aims of the present study were (i) 

to determine phospholipase activities in Acanthamoeba and (ii) to determine their role in the pathogenesis of Acanthamoeba. using an 

encephalitis isolate (belonging to T1 genotype), a keratitis isolate (belonging to T4 genotype) and an environmental isolate (belonging to 

T7), we demonstrated that Acanthamoeba exhibited phospholipase A2 and phospholipase D activities in a spectrophotometric-based 

assay. interestingly, encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, 

but environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PlD activities 

compared with the PlA2. The functional role of phospholipases was determined in in vitro assays using human brain microvascular 

endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that compound 48/80 partially inhibited Acanthamoeba 

encephalitis isolate cytotoxicity of the host cells, while PlA2 inhibitor, i.e., cytidine 5’-diphosphocholine had no effect on parasite-mediated 

HBMEC cytotoxicity. Further studies in phospholipases should determine their roles in the biology and pathogenesis of Acanthamoeba, and 

may also prove useful in the differentiation of Acanthamoeba genotypes. 

CMM/09 Characterization of the MtrCDE multidrug efflux pump from Neisseria gonorrhoeae 

THAMArAi JANGANAN1 , li Zhang1 , Vassiliy Bavro3 , Dijana Vinkovic2 , Nelson Barrera2 , Carol robinson4 , Maria ines Walmsley1 , 

Adrian Walmsley1 1 2 School of Biological & Biomedical Sciences, University of Durham, Durham; Dept of Chemistry, University of Cambridge, Cambridge; 

3 4 Dept of Physics, University of Oxford, Oxford; Dept of Chemistry, Physical & Theoretical Chemistry, University of Oxford, Oxford 

Gram-negative bacteria utilise tripartite efflux pumps that span the inner and outer membranes to actively extrude drugs. These pumps 

are assembled from inner (iMP) and outer (OMP) membrane proteins that are bridged by a periplasmic membrane fusion protein (MFP). 

There is evidence to suggest that the assembly of these pumps is accompanied by an induced fit of the OMP, from a sealed to an open 

conformational state which is driven by the accommodation of the MFP into an intra-protomer groove on its surface. However, structural 

analysis of the OMPs reveals that each protomer forms a structural repeat, so that there is a pair of grooves for each protomer: one intraprotomer 

and one between the protomers, each of which could act as an MFP interaction site. We have investigated the assembly and 

function of the Neisseria gonorrhoeae MtrCDE-pump and established that the MFP MtrC binds to the OMP MtrE at both the intra- and 

inter-protomer grooves. inserting cysteines in the interprotomer-groove of MtrE allows MtrE-MtrC complexes to be trapped by crosslinking 

with a Mr consistent with that expected for MtrE MtrC . Our studies suggest that the pump is assembled with a stoichiometry of 

3 6 

3:6:3. 




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CMM/10 Metabolic investigation of the gender differences in energy metabolism of C3H/Orl mice in response to their 

microbial status 

rENAuD MESTDAGH1 , Sandrine P. Claus1,2 , Jeremy K. Nicholson1 , Elaine Holmes1 1 2 Imperial College London, London; Reading University, Reading 

The gut microbiota play an essential role in the host-metabolism. intestinal bacterial species are engaged in various metabolic aspects, and 

disregulation of bacterial metabolism trigger metabolic disorders and diseases (i.e. obesity, diabetes, irritable bowel syndrome, etc). 

To better understand this complex interplay, the metabolic phenotype of germ-free (n=20) and conventional (n=20) C3H/Orl mice was 

characterized using a NMr-based metabolic profiling approach, with a focus on gender differences (20 males, 20 females) and energy 

metabolism in urine and brown adipose tissue (BAT). Physiological data of age-matched germ-free and conventional C3H/Orl mice showed 

that male animals had a higher weight than females in both groups. in addition, conventional males had a significantly higher total body fat 

content compared to conventional females while this sexual difference disappeared in GF animals. 

GF animals displayed a higher level of D-3-hydroxybutyrate which is the signature of the activation of B-oxidation and a lower level of 

lactate in hydrophilic phase of BAT extracts when compared to their conventional counterpart. Altogether, these results indicate that the 

gut microbiota impact severely the energy metabolism in male mice suggesting different mechanisms of regulation of pyruvate metabolism 

and biosynthesis of ketone bodies according to the microbial status of C3H/Orl mice. 

CMM/11 Activity of protein antibiotics against multidrug-resistant bacterial biofilms 

KArEN SMiTH1 , Angela rinaldi1 , Gordon ramage2 , Daniel Walker1 1 2 University of Glasgow, Glasgow; University of Glasgow Dental School, Glasgow 

Chronic infection of the lower respiratory tract of cystic fibrosis (CF) sufferers with P. aeruginosa (PA) leads to a continuous degradation of 

lung tissue and ultimately respiratory failure. Current antibiotic therapies fail to completely eliminate PA due to its natural ability to grow in 

biofilms and form drug-tolerant small colony variants (SCV). in this study we produced and purified two highly potent protein antibiotics, 

pyocins S2 and S5. The activity of these bacteriocins was evaluated in vitro using mucoid and non-mucoid clinical isolates of PA from 

children with CF, grown in surface-adhered biofilms and as SCVs, and compared to the activity of antibiotics routinely used in the clinical 

setting. The in vivo activity of pyocin S2 was also assessed in an insect model of PA infection (Galleria mellonella larvae). As assessed by CFu 

counts and fluorescent microscopy, the pyocins had superior antimicrobial activity to the commonly-used antibiotics aztreonam, ceftazidime 

and tobramycin against biofilm-associated PA and SCVs. Pyocin S2 also successfully resolved PA infection in an insect model and was found 

to be non-toxic to human lung epithelial cells (A549). These results suggest that the S-pyocins could potentially play an important role in 

the treatment of PA biofilm-associated infections in CF. 

CMM/12 Lower weights of stool sample used in nucleic acid extraction allows for efficient PCr-DGGE analysis of microbial 

communities 

CHriSTOPHEr STEWArT1 , John Perry2 , Janet Berrington3 , Manjusha Narayanan3 , Stephen Cummings1 1 2 3 Northumbria University, Newcastle upon Tyne; Freeman Hospital, Newcastle upon Tyne; Royal Victoria Infirmary, Newcastle upon 

Tyne 

Nucleic acid extraction is fundamental to the success of downstream processes, thus obtaining optimum DNA yield is essential. When 

analysing neonatal stool, such as investigating necrotizing entercolitis (NEC), it must be appreciated that there may be insufficient sample to 

adhere to the recommended weight outlined by the manufacturer of the kit. 

Aim: Evaluate how the weight of neonatal stool used in nucleic acid extraction affects the DGGE community profile. 

Methods: DNA and rNA were extracted from the samples and PCr-DGGE was used to analyse the bacterial communities using universal 

V3 primers. 

Results: using lower sample weights generates community profiles in DGGE resembling that of the larger weight samples. The intensity of 

bands is generally higher in the larger weights but smearing is also intensified. in the rNA profile, the lower weights in some cases show 

bands more intensely than the respective band at higher weights. 

Conclusion: lower weights of sample, compared to those outlined by the manufactures, can be used for comparison of microbial DGGE 

profiles. An exclusion criterion of 100mg per sample, compared with 250mg suggested in the manufacture protocol, can be used for nucleic 

acid extraction. This will result in the inclusion of more samples in the study. 




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CMM/13 Undiagnosed chronic hepatitis b is prevalent in the british–Chinese community of the north-east of England 

Stuart McPherson1,2 , MANOJ VAlAPPil3 , Samuel Moses3 , Gary Eltringham3 , Carolyn Miller1 , Kerry Baxter1 , Benjamin Brown4 , 

Amanda Chan5 , Paul Klapper4 , Mark Hudson1,2 , Margaret Bassendine1,2 1 2 Liver Unit, Freeman Hospital, Newcastle upon Tyne; Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne; 

3 4 5 Health Protection Agency, Newcastle upon Tyne; Health Protection Agency, Manchester, United Kingdom, Newcastle Chinese 

Healthy Living Centre, Newcastle upon Tyne 

Background: Chronic hepatitis B(HBV) is highly prevalent in China and the Far East(>8% seroprevalence). Whilst AASlD 

guidelines recommend HBV screening and vaccination in subjects born in high(>8%) or intermediate(2–7%) endemic areas, there are no 

current uK guidelines. 

Methods: Case finding was undertaken in collaboration with the North-East Chinese community. Dried blood spots from finger-pricks were 

tested for HBsAg, HBcAb(Abbott ArCHiTECT®). HBsAg positive individuals were to undergo confirmatory testing and be referred for 

specialist assessment. 

Results: Of 315 subjects, 31(10%) were HBsAg positive, of whom 7 were previously diagnosed with HBV but were not under followup. 

Excluding previously diagnosed individuals, HBsAg prevalence was 8%. 50(16%) subjects were HBcAb alone positive. HBsAg positive 

individuals were significantly younger than HBcAb and negative subjects (44±12 vs 59±15 vs 52±19 years, p


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gene PCr-DGGE of DNA isolated from the same samples. in all environments molecular techniques revealed a greater diversity than 

culture and, in some environments, where the number of micro-organisms was low, culture may have significantly underestimated the level 

of microbial diversity (e.g. worktops). For other environments (e.g. sinks) a similar level of diversity was observed using both techniques 

but the different species were detected. The development of these methods, and their application in the hospital environment, will aid 

our understanding of the role of the hospital environment in HAi, of the effectiveness of cleaning, and the interpretation of microbial 

environmental monitoring data. 

CMM/16 Analysis of the microbial community in a 29-patient cystic fibrosis cohort across a 20 month period 

ANDrEW NElSON1 , Steven Bourke2 , Anthony De Soyza3,4 , John Perry5 , Stephen Cummings1 1 2 University of Northumbria, Newcastle upon Tyne; Adult Cystic Fibrosis Unit, Royal Victoria Hospital, Newcastle upon Tyne; 

3 4 Transplantation & Immunobiology Group, Newcastle University, Newcastle upon Tyne; Respiratory Medicine, Freeman Hospital, 

Newcastle upon Tyne; 5Dept of Microbiology, Freeman Hospital, Newcastle upon Tyne 

Introduction: The microbial communities present in CF sputum are polymicrobial and consist of bacteria, viruses and fungi. Although stratified 

studies have demonstrated a change in the CF bacterial microbiota with increasing age, individual patients have not been followed for a long 

period of time. 

Aim: The aim of this study was to follow 29 CF patients over a period of up to 20 months to determine how the bacterial and fungal 

communities differ over this period and to see if a shift in the microbiota could be linked with acute pulmonary exacerbations. 

Methods: 29 adult CF patients were recruited and spontaneously expectorated sputum samples were collected. DNA was extracted from 

the samples and PCr-DGGE was used to analyse the bacterial and fungal communities using universal primer sets. 

Results: Samples were collected and analysed by PCr-DGGE. Factors that affected microbial communities between patients included 

sex, genotype and being culture positive for P. aeruginosa. Both bacterial and fungal communities varied between sample dates but fungal 

communities were more stable than those of bacteria. 

Conclusion: The bacterial and fungal communities both varied between patients and over the time course we investigated. Further 

investigation is required to determine the cause of these changes. 

CMM/17 Disease-associated XMrV sequences explained by PCr contamination 

Stéphane Hué1 , ElEANOr GrAY1 , Astrid Gall2 , Aris Katzourakis3 , Choon Ping Tan1 , Charlotte Houldcroft2 , Stuart Mclaren2 , 

Deenan Pillay1 , Andy Futreal2 , Jeremy Garson1 , Oliver Pybus3 , Paul Kellam1,2 , Greg Towers1 1 2 3 University College London, London; Wellcome Trust Sanger Institute, Cambridge; University of Oxford, Oxford 

xenotropic murine leukaemia virus-related virus (xMrV) is a retrovirus that has been the subject of intense debate since its detection in 

prostate cancer and chronic fatigue syndrome patient samples. Here we demonstrate that Taqman PCr primers previously described as 

xMrV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient 

samples and confound specific xMrV detection. To consider the provenance of xMrV we sequenced xMrV from the cell line 22rv1, 

which is infected with an MlV-x indistinguishable from patient derived xMrV. Bayesian phylogenies show that xMrV sequences derived 

from unlinked patients form a monophyletic clade with interspersed 22rv1 clones (p>0.99). The cell line-derived sequences are ancestral 

to the patient-derived sequences (p>0.99). We also show that xMrV sequences acquire diversity in the cell line but not in patient samples. 

We conclude that xMrV detected by PCr methods in patient samples is the likely result of PCr contamination, and the described xMrV 

sequences arose from the tumour cell line 22rv1, which was probably infected with xMrV during xenografting in mice. 

CMM/18 Molecular and functional analysis of Haemophilus influenzae porin P2 and investigation into its role in host– 

pathogen interactions 

MAHDE ASSAFi, Neil Oldfield, Jafar Mahdavi, Karl Wooldridge, Dlawer Ala’Aldeen 


Haemophilus influenzae type b (Hib) is a major world-wide cause of bacterial meningitis. Despite the use of highly effective capsular-based 

conjugate vaccines in some countries, significant levels of Hib disease remain in countries that have not implemented the vaccine. We have 

previously shown that an important step in H. influenzae binding to microvascular endothelial cells of the blood-brain barrier is through 




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interaction of the outer membrane porin OmpP2 with the human laminin receptor (lr). Here, we investigated this interaction further 

by expressing and purifying specific regions of OmpP2 and testing the ability of these fragments to bind to lr using EliSA. The region 

corresponding to extracellular loop 2 (amino acids 71–79) was required for optimal binding to lr. Furthermore, H. influenzae mutants 

lacking OmpP2 loop 2 and a whole gene deletion mutant were created. Whole cell pull-down assays and flow cytometry analysis showed 

that binding of recombinant lr to both mutant strains was decreased compared to the wild-type, with the whole gene deletion mutant 

showing the largest reduction. This confirms that OmpP2 is required for binding to lr and that the surface accessible loop 2 is important 

for this interaction. 

CMM/19 Analysing interactions between Chlamydia trachomatis and human spermatozoa 

SuSAN ANDrEW1 , Navin Kumar2 , Maud Dumoux1 , Maria O’Donovan4 , Christopher Carne3 , Hamid Jalal2 , 

richard Hayward1 1 2 Institute of Structural & Molecular Biology, University College London and Birkbeck, London; Clinical Microbiology & Public Health 

Laboratory, Addenbrooke’s Hospital, Cambridge; 3Dept of Genitourinary Medicine, Addenbrooke’s Hospital, Cambridge; 4Dept of 

Histopathology, Addenbrooke’s Hospital, Cambridge 

Male factor infertility (MFi) has a complex aetiology, yet remains a significant problem, as 22% of infertility in developed countries is 

caused by a male factor alone. A substantial proportion (~40%) of women infected with Chlamydia trachomatis experience pelvic 

inflammatory disease, of whom 20% become infertile. However, while some derogatory effects are observed in vitro, the case for a 

link between C.trachomatis infection and MFi remains unresolved. Here we investigate whether urethral C.trachomatis infection adversely 

affects semen quality and how C.trachomatis interact at a cellular level in vivo. 239 semen samples were screened for common genital 

pathogens by qPCr. 13% were infected with varying load of C.trachomatis, often in combination with other coinfecting pathogens. 

Cytological parameters (sperm vitality, concentration, motility) were assayed, and the effect of infection investigated. C.trachomatis 

positive samples were observed by fluorescence microscopy, and bacteria observed in association with sperm heads, tails and midpieces, 

often at multiple loci on single cells. These data provide new insight into the prevalence, effects and nature of the interaction between 

C.trachomatis and sperm in vivo. 

CMM/20 The interaction of Streptococcus pneumoniae with human pleural mesothelial cells defines an in vitro model of 

empyema 

ClAirE HEATH1 , Saul Faust2 , John Heckels1 , Myron Christodoulides1 1 2 University of Southampton, Southampton; University of Southampton Wellcome Trust Clinical Research Facility, 

Southampton 

Introduction: The clinical incidence of bacterial empyema in children caused by Streptococcus pneumoniae is increasing worldwide. Empyema 

is defined as the fibropurulent stage of pleural disease, characterized by the presence of pus in the pleural cavity following bacterial infection 

and is often a complication of pneumonia. We tested the hypothesis that disease-causing isolates induce an innate inflammatory response 

from pleural cells following bacterial adhesion and invasion. 

Methods: An in vitro model of empyema, using a monoculture of human pleural (Met-5A) cells was infected with both laboratory and clinical 

strains of pneumococci of different serotypes, and also with mutants deficient in various virulence genes. The capacity of each strain to 

adhere to, invade and cause cytotoxicity to Met-5As and to induce an innate inflammatory response was investigated. 

Results: Significant differences in the abilities of different pneumococcal strains to adhere to and invade Met-5A cells were observed. in 

particular, clinical empyema isolates interacted poorly with pleural cells. Whole bacteria failed to induce an innate immune response in 

Met-5A cells, whereas bacterial lysates did elicit a cytokine response. 

Conclusions: interaction of pneumococci with pleural cells is influenced by virulence factor expression and the pathogen may employ a 

suppressive mechanism to evade immune detection. 




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CMM/21 In situ rickettsial diagnostics 

VErA PADEr, Sally Cutler 

University of East London, London 

Application of molecular diagnostics has resulted in an exponential increase in detection of Rickettsia. Molecular techniques based upon 

typing variable surface proteins and multi-spacer typing have been proven as valuable in deciphering complicated taxonomic relationships 

among this group. We applied a multi-locus sequence typing approach to characterize conserved house-keeping genes to a diverse range 

of arthropods. initial screening with the genus specific citrate gene was followed by MlST analysis. 145 pools of Ornithodoros moubata ticks 

from Tanzania, 45 pools of Argas persicus ticks from Ethiopia; 81 pools of various species of ticks from israel; and 49 pools of Amblyomma 

variegatum ticks from Kenya were analysed. Amplicons from soft ticks (O. moubata and A. persicus), showed greatest homology with 

Rickettsia felis, R. akari or R. hoogstraalii. Whether this is a pathogen or endosymbiont within these ticks remains to be resolved. These tick 

species bite humans, thus potentially resulting in positive serology or infection. Kenyan ticks showed presence of R. africae in 4 samples and 

R. hoogstraalii-like Rickettsia in 3 samples. Ticks from israel yielded 18 positives 15 of which were sequenced revealing 12 as R. massiliae and 

3 R. aeschlimannii. Different loci gave variable success the merits of which will be discussed. 

CMM/22 Withdrawn 

CMM/23 The effect of the uniform policy on hospital-acquired MrSA at the Princess royal University Hospital 

MANOrEET SiHErA1 , Andrew Cai2 , M. Atta3 , Mandy Clement3 1 2 3 King’s College London, School of Medicine, London; King’s College Hospital, London; Princess Royal University Hospital, Farnborough 

Hospital acquired infections (HAi) with MrSA are a major concern to all health organizations in the uK. Poor hand hygiene is a contributing 

factor to transmission and as such it has been proposed that unsatisfactory hand-washing could be due to clothing and jewellery around 

the lower arms of healthcare workers. The DoH’s ‘bare below the elbows’ decree has attempted to address this issue. However, further 

evidence is required to support it. This paper examines whether a restrictive uniform policy reduces the rates of HA-MrSA. 

Compliance with the uniform policy and case numbers of HA-MrSA were audited over two 3-month periods, before and after the policy 

became compulsory. 

The audit demonstrated good policy compliance in areas of short sleeves and no ties but poor compliance with removal of wristwatches 

and rings. The numbers of new HA-MrSA cases were 140 before and 49 cases after the policy change; providing rates of 140/3972 

(3.50%) and 49/4,598 (1.06%) respectively. 

Following the introduction of the uniform policy a three-fold reduction in the rate of HA-MrSA was noted. Whilst confounding factors 

such as MrSA screening and community exposure to MrSA could have played a role, compliance with the uniform policy is clearly an 

important contributory factor. 


CMM/25 The role of phagocytes in controlling Pseudomonas aeruginosa infection 

SONAli SiNGH, Paul Williams, Miguel Cámara, luisa Martínez-Pomares 


Pseudomonas aeruginosa is an opportunistic pathogen capable of causing life-threatening infections in susceptible individuals, particularly 

patients with cystic fibrosis. As the innate immune system not only contains infection, but also triggers and shapes the ensuing adaptive 

response, understanding its role in the defence against P. aeruginosa is vital. This study aims to identify the conditions required for P. 

aeruginosa clearance / inhibition and cellular recruitment by two of the major innate immune players in bacterial infections – macrophages 

(Mf) and neutrophils. 

Our work so far demonstrates that under non-opsonic conditions M-CSF-induced human monocyte-derived Mf express TNF-a, il-6, 

il-1b, il-18, il-8, MCP-1 and MiP-1a in response to viable P. aeruginosa PA01 (multiplicity of infection = 1). These Mf were ineffective 

at containing bacterial growth, however, and were instead killed over the course of infection. Priming Mf with 100u/ml iFN-g shifted the 

cytokine profile to a more proinflammatory one, but did not improve bacterial clearance. Supplementation of iFN-g with inflammation- 




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promoting GM-CSF instead of homeostasis-promoting M-CSF may boost Mf antibacterial activity. Alternatively, addition of immunoglobulin 

or neutrophils to cultures may be required. unravelling the mechanisms by which phagocytes tackle P. aeruginosa could help us understand 

how and why infection is established. 


CMM/27 The development of a rapid diagnostic system for Mycobacterium sp. mounted on the glass slides by combining 

laser capture microscopy and nested PCr 

BAlKiS A.TAliP, Colm lowery, James Dooley 

University of Ulster, Coleraine 

Tuberculosis remains a highly prevalent disease in much of the world, especially in less developed countries of Africa and Asia. Conventional 

detection by sputum smear microscopy is insensitive, requires a skilled practitioner and does not even provide unequivocal proof of 

infection. The gold standard for diagnosis of Mycobacterium tuberculosis involves the use of microscopy followed by cultivation which 

requires up to 12 weeks for confirmation. We developed a system to obtain confirmatory diagnostic information from a stained sputum 

samples. We combined laser capture microscopy (lCM) with a nested polymerase chain reaction (PCr) assay, employing species-specific 

rpoB primers. Non-pathogenic Mycobacterium smegmatis was used as a model system with smears prepared and stained by the Ziehl- 

Neelsen method. Several DNA extraction methods were compared to determine the optimum system for recovery of amplifiable nucleic 

acid. We consistently detected low numbers of extracted cells, typically between 50 to 1000 cells per assay. DNA sequencing of the 

amplified material confirmed it as the rpoB gene.These results show it is possible to isolate bacterial cells from glass slides and to apply 

downstream molecular assays for DNA detection. This may offer a platform of rapid diagnostic techniques for M. tuberculosis and other 

difficult to culture pathogens. 


CMM/29 Characterizing the proteomic changes of Pseudomonas aeruginosa in response to treatment with manuka honey 

AlED rOBErTS, Sarah Maddocks, rose Cooper 

University of Wales Institute, Cardiff, Cardiff 

Background: Manuka honey is gaining widespread acceptance in clinical settings as a viable alternative to topical based treatments of surface 

wound infections. its broad spectrum coupled with high antibacterial efficacy that has yet to see resistance develop makes manuka honey a 

viable treatment option. However clinical uptake remains low due to a lack of understanding surrounding the mode of action. Here we use 

molecular methods to identify changes induced by sub-lethal concentrations of manuka honey. 

Methods: Pseudomonas aeruginosa NCiMB 8626 was exposed to sub-lethal concentrations of manuka honey for 3hr. Whole cell proteins 

were extracted then visualised using 2D gel electrophoresis, and compared to untreated cells. Proteomic changes were identified using 

PDQuest and MAlDi-TOF Mass spectroscopy. 

Results: 2D gel electrophoresis identified multiple changes in the proteome of P. aeruginosa following exposure to sub-lethal levels of 

manuka honey. Production of DnaK, (a major heat shock protein) was impeded following exposure to manuka honey, where as the 

expression of FliC (flagellin) was seen to increase. 

Conclusions: Manuka honey induces significant changes in protein expression in P.aeruginosa, which are likely to be detrimental to its survival. 

This offers an insight into the mode of antibacterial action of Manuka honey. 





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CMM/31 Selection of DNA aptamers against Proteus mirabilis using whole-cell SELEX 

NASA SAVOrY1,2 , Koji Sode2 , Kazunori ikebukuro2 , Brian Jones1 1 2 University of Brighton, Brighton; Tokyo University of Agriculture and Technology, Tokyo, Japan 

Aptamers are oligonucleotides that fold into unique structures and bind to a variety of target molecules. Due to their high affinity and 

specificity, aptamers have emerged as macromolecules that rival antibodies in diagnostic and therapeutic applications. Aptamers are 

generally selected from an oligonucleotide pool of random sequences through in vitro selection known as Systematic Evolution of ligands 

by Exponential enrichment (SElEx). using whole cells and the SElEx approach we have identified DNA aptamers against Proteus 

mirabilis B4 after six rounds of selection. P. mirabilis is a prominent cause of catheter-associated urinary tract infections (CAuTis), among 

patients undergoing long-term bladder catheterization. Although isolated aptamers showed broad specificity for other bacterial species, 

those aptamers displayed high affinity for P. mirabilis cells with dissociation constant (K ) values in the low-nanomolar range. While further 

D 

characterization and improvement of specificity are required, these aptamers will ultimately constitute useful tools for diagnosis and/or 

treatment of P. mirabilis CAuTi, as well as a powerful research tool for investigation of bacterial structure and function. Evaluation of these 

aptamers for treatment or prevention of CAuTi is currently underway in our laboratory. 


CMM/33 real-time PCr reveals the presence of three distinct Brachyspira species in human spirochaetosis 

laurens J. Westerman1 , Herbert V. Stel2 , Marguerite E.i. Schipper3 , leendert J. Bakker4 , Edwin C.H. Boel1 , 

Jan H.M. van den Brande5 , Peter D. Siersema6 , JOHANNES G. KuSTErS1 1 2 Dept of Medical Microbiology, University Medical Centre Utrecht, Utrecht, Netherlands; Dept of Pathology, Tergooiziekenhuizen, 

Blaricum, Netherlands; 3Dept of Pathology, University Medical Centre Utrecht, Utrecht, Netherlands; 4Central Laboratory for 

Bacteriology & Serology, Tergooiziekenhuizen, Hilversum, Netherlands; 5Dept of Internal Medicine Tergooiziekenhuizen, Hilversum, 

Netherlands; 6Gastroenterology & Hepatology University Medical Centre Utrecht, Utrecht, Netherlands 

Introduction: While the impact of infections with Brachyspira species has been studied extensively in veterinary medicine, little is known 

regarding human infections. Two species are thought to infect humans: Brachyspira aalborgi and Brachyspira pilosicoli. Prevalence rates are 

estimated between 1–32% in the general population and up to 63% in homosexual males. Diagnosis is based upon histology. 

Methods: Fifty colon-biopsies from 25 histologically confirmed spirochaete positive patients and 24 non-spirochaete colon-biopsies were 

identified. A primerset was designed against a 136bp region of the rrNA that is both specific for and conserved within all Brachyspira 

species. 

Results: All 25 spirochaete-positive patients showed amplification of the predicted product, whereas all 24 controls remained negative. 

SYBr-green melt-curves suggested a third species and this was confirmed by sequence analysis. 15/25 patients were infected with B. 

aalborgi, 2/25 with B. pilosicoli, and 4/25 with the novel Brachyspira species. in addition, four double infections were present. 

Conclusions: To our knowledge this is the first real-time PCr that allows for simultaneous detection and species discrimination of Brachyspira 

in routine biopsy materials. We conclude that a surprisingly high number of double infections is present and that a thus far unknown 

Brachyspira species is involved. 

CMM/34 Frequency and antibiotic susceptibility of Gram-positive bacteria in Makkah hospitals – Saudi Arabia 

ATiF ASGHAr 

Umm Al-Qura University, Makkah, Saudi Arabia 

The objective of this study was to estimate the frequencies and resistance rates of Gram-positive pathogens isolated from hospitals in 

Makkah, Saudi Arabia. 

This prospective study included clinical isolates from 1087 patients with Gram-positive bacterial infection at three Makkah hospitals- Saudi 

Arabia in 2008–2009. The patients’ demographic and laboratory data were collected. Standard microbiological methods were used to 

identify the organisms and test antimicrobial susceptibility. The results were interpreted according to the Clinical laboratory Standards 

institute (ClSi) guidelines. 




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Gram-positive pathogens infected all age groups but had no gender predominance. Staphylococcus aureus was the most common cause 

of wound infection and accounted for more than half of the clinical isolates (688 cases). Coagulase-negative Staphylococcus (CONS) was 

a common isolate from blood cultures. Wounds were the most common site of infection (37.6%). Enterococcus spp. and Streptococcus 

agalactiae were the second most common bacteria (26%). The resistance rates of S. aureus and CONS isolates were 39.4% and 82.4% for 

oxacillin, respectively. Among the streptococci, the resistance rates of Streptococcus pneumoniae were 21.1% and 16.7% for ampicillin and 

erythromycin, respectively. 

S. aureus infections were very common in the Makkah hospitals. Continuous monitoring for antibiotic susceptibility is necessary to reduce 

these and other nosocomial infections 

ENV Environment 

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ENV/01 Characterization of the prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment 

research biomarker 

VJESMiNE liM, Stephen Cummings, Amanda Jones, John Woodward, Takeshi Nakagawa 

Northumbria University, Newcastle upon Tyne 

lake Suigetsu is a small tectonic lake in central Japan containing annually laminated sediment. A recent study combined more than 300 

radiocarbon ages on terrestrial leaf macrofossils with varve ages from the core generated a quasi-continuous radiocarbon calibration model 

to the limit of radiocarbon dating (c.50,000BP). This core chronology provides an ideal basis for quantitative studies of climate change and 

palaeoenvironmental reconstruction. The present study aimed to identify and elucidate the diversity of the microbial communities down the 

sediment core to develop novel biomarkers for past climate change, using both culture-dependent and independent (molecular) techniques. 

Here we report the analysis of eighteen samples of the lake sediment sampled at every 4m of the 73.5m core covering the past 150,000 

years using PCr-DGGE for 16S rrNA genes from Eubacteria and Archaea. in addition, two of the eighteen sediment samples, indicative of 

a salinity shift between 10911BP (freshwater) to 6860BP (brackish) were subjected to a Dispersion and Differential Centrifugation (DDC) 

culture-based approach. A total of 183 taxa were isolated from freshwater sediment samples and 71 taxa from sediments after the saline 

influx, respectively. This suggests that environmental shifts impose an effect over the diversity of microbial communities, endorsing their 

potential as biomarkers. 

ENV/02 Determining the concentration of F+rNA and somatic bacteriophages from a range of livestock derived matter 

KATE lENTHAll1,2 , Chris Hodgson2 , Dave Chadwick2 1 2 University of Exeter, Exeter, Devon; Rothamsted Research, North Wyke, Okehampton, Devon 

Aim: The use of faecal indicator organisms as a means of assessing the potential presence of water-borne pathogens has been paramount 

to protecting public health for over a century. Two microbial parameters are now required to be examined; intestinal enterococci and 

Escherichia coli. The issue facing the legislators today is how to determine the source of that faecal pollution, e.g. human, ruminant or avian. 

Microbial source tracking methods are being developed to aid source identification. in this paper we present the preliminary data from an 

approach utilizing the typing of F + rNA bacteriophage. 

Methods and results: Concentrations of E.coli and F + rNA and somatic bacteriophage were determined from freshly voided sheep, cattle, 

pig, and chicken faecal material. Decimal dilutions of the faecal material were prepared in ¼ strength ringers solution. Bacteriophages were 

enumerated following the double agar overlay method of Adams (1959) and E.coli following standard methods (EA 2002). F + rNA and 

somatic bacteriophage were successfully enumerated from chicken faeces, F + rNA phage were enumerated in pig faeces, although in an age 

dependant manner. No Bacteriophage were detected in faecal matter from sheep or cattle. E.coli concentrations were positively correlated 

to the dry matter content of the faecal matter from all livestock types. 




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ENV/03 reduction of hexavalent chromium using Gram-negative environemtal bacteria 

MAriAM iSMAEl, Phillip Gardiner, Tom Smith 

Sheffield Hallam University, Sheffield, South Yorkshire 

Chromium is widely used in diverse industries including metal finishing, petroleum refining, leather tanning and textile manufacturing, 

resulting in large quantities being discharged into the environment. The oxidation states of chromium range from -2 to +6, but it primarily 

exists in +3 or +6 state under environmental conditions. Chromium exists in industrial wastes primarily in hexavalent form, which is highly 

soluble in water and extremely toxic and harmful to humans and environment. 

Microbial metal bioremediation is an attractive strategy due to its low cost and eco-friendly nature. A comparative study between four 

strains of Gram-negative bacteria (Escherichia coli, Enterobacter cloacae, Proteus mirabilis and Pseudomonas aeruginosa) for remediation 

of hexavalent chromium was performed. The bioremediation process was conducted with and without sodium azide, which was used 

as inhibitor of oxidative phosphorylation. The reduction activity was monitored using the colorimetric diphenylcarbazide assay during 

incubation times of up to 120 hours. Pseudomonas aeruginosa recorded the highest reduction rate of Cr (Vi), followed by Proteus mirabilis. 

The analysis of total chromium was measured using inductively coupled plasma-optical emission spectrometry (iCP-OES). The reduction 

reaction mediated by these strains was not substantially inhibited by sodium azide, indicating that oxidative phosphorylation enzymes are 

unlikely to be involved. 

ENV/04 Withdrawn 

ENV/05 The relationship between antiseptics/disinfectants and MrSA in Kerbala hospitals 

MuHSiN JABAr 

Microbiology Dept, Faculity of Science, Kerbala University, Kerbala City, Iraq 

There is no information available on Methicillin resistant Staphylococcus aureus (MrSA) in the main hospitals of Kerbala Ciyt. Therefore, a 

screening programme was done during the period from October, 2008 to November, 2009 to asses the MrSA level in those hospitals. 

Among 637 samples taken, MrSA distribution in both patient samples and nursing staff was 18%.The hospital equipments samples were 

having only 12% MrSA. The mutagenic effect of the sub-lethal doses of some hygienic materials on antibiotic-sensitive staphylococcus aureus 

(MSSA) was also detected. MSSA stains were exposed to sub-lethal doses of ethanol, formaldehyde, alcoholic, iodine, iodine hexane, 

and chlorine. More than 80% of these strains were converted to MrSA, among which 41% were resistant to three or more antibiotics 

simultaneously. This phenomenon reflects a mutagenic event caused by the above antiseptic/disinfectant agents which are mainly used in 

the measuring control methods of thecross infections inside the hospitals. The above procedures have also revealed a new data on the 

Minimum inhibition Concentrations (MiC) of the above disinfectants/antiseptics materials that are usually used for hygienic procedures 

inside the hospitals. in conclusion, the hospitals of Kerbala City are in an urgent need for implementation of an effective preventive 

measures against MrSA. 

ENV/06 Effect of soil structure on bacterial community dynamics in a PAH-contaminated artificial soil 

SEAN STOrEY, Nicholas Clipson, Evelyn Doyle 

University College Dublin, Dublin, Ireland 

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds, consisting of at least two benzene rings in various configurations. PAHs are 

produced during incomplete combustion of fossil fuels and are found in soils surrounding creosote manufacturing facilities and manufactured 

gas plants. Micro-organisms capable of degrading PAHs can be detected in most contaminated environments and strategies stimulating in 

situ microbial degradation have been used, with varying degrees of success, to remediate PAH contaminated sites. However, it is difficult to 

predict the outcome of bioremediation, as results vary from site to site, due in part to differences in soil properties. The aim of this study 

was to determine the effect of soil composition on bacterial communities in PAH-contaminated soils. An artificial soil system was designed 

in which soil composition (% organic matter, clay, sand) was varied. Microbial communities were established in artificial soils by amending 

them with soil from a former wood treatment facility. Phenanthrene (500 mg kg –1 ) was removed from all soils examined, and the rate of 




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degradation was highest (5.24 mg kg –1 day –1 ) in soil containing no organic matter, 20% clay and 70% sand. Molecular fingerprinting was used 

to examine the effect of soil composition on bacterial community dynamics. 

ENV/07 The novel thiocyanate hydrolase enzyme of environmental strain THI201 

ADEEBA HuSSAiN1 , Maki Saito2 , Toshiaki Sekine3 , Yoko Katayama1 1 2 3 Tokyo University of Agriculture & Technology, Tokyo, Japan; Japan Food Research Laboratories, Tokyo, Japan; Kohjinbio, Saitama, 

Japan 

Thiocyanate is a constituent of thioglucocides found in plant tissues especially in Brassica species, and thus widely distributed in nature. in 

addition, it is one of the major constituents of industrial waste water where the untreated thiocyanate often contributes to an increase in 

COD. A novel bacterium strain THi201 was isolated from lake water; it contains the enzyme thiocyanate hydrolase (SCNase) and can 

degrade thiocyanate to carbonyl sulfide. The SCNase was purified and single band of 53.1kDa was obtained by SDS-PAGE. Though the 

enzyme activity was found in purified fraction but it was quite unstable. in the genomic DNA of THi201, the gene of enzyme was found 

to be present in single copy by Southern hybridization technique. Cloning and sequencing of SCNase gene was performed and found that 

the OrF of SCNase is 82% identical with a protein containing twin-arginine translocation pathway signal of Thiobacillus denitrificans ATCC 

25259. This result indicates that the SCNase of THi201 is a novel enzyme and need to be purified abundantly for further characterization. 

Therefore subcloning in to expression vector and transformation of E. coli cell was performed which enabled the successful purification of 

SCNase. 

ENV/08 Withdrawn 

ENV/09 bioremediation ability of atrazine-degrading bacteria isolated from different soils in Awka, Anambra State, Nigeria 

CHiBuZO uMEH, Obianuju Obiefuna 

Nnamdi Azikiwe University, Awka, Anambra State, Nigeria 

Four bacterial strains capable of utilizing the herbicide atrazine as a sole source of carbon and nitrogen source were isolated from different 

soils in Awka, Nigeria by selected enrichment on mineral salt medium (MSM) containing the herbicide. Species of Serratia, Citrobacter, 

Proteus and Yersinia were isolated. Growth response studies carried out at intervals (0, 2, 4 6 and 24h) showed that the organisms utilized 

atrazine to grow in MSM containing different concentrations of the herbicide at 0.5g/l, 1.0g/l, 1.5g/l and 2.0g/l quantities. Optimum range 

of concentration that supported bacterial growth over the 24h incubation period was 0.5–1.5g/l. it was observed that MSM supplemented 

with glucose supported optimum bacterial growth up to the 24th hr; same medium supplemented with glucose and ammonium sulphate 

showed a decline in bacterial growth. Gas chromatography mass spectrophotometer (GCMS) readings showed that Citrobacter sp., Proteus 

sp. completely broke down 0.1g of the herbicide in 5.0 ml portion of MSM within 14 days but for Serratia sp. and Yersinia sp. there was an 

incomplete breakdown. The mixed bacteria culture synergistically degraded atrazine. The 4 bacterial species can grow in the presence of 

added herbicide and can be used in the bioremediation of atrazine-contaminated soils. 

ENV/10 Not presented 




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Fb Fermentation & bioprocessing 

FB/01 A novel enzymatic composition effective for prion degradation 

EMEKA OKOrOMA 1 , Diane Purchase 1 , Hemda Garelick 1 , Oduola Abiola 2 

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1 Dept of Natural Sciences, Middlesex University, The Burroughs, London NW4 4BT; 2 PAP Rashidah Sa’adatul Bolkiah Institute of 

Health Sciences, Universiti Brunei Darussalam, Gadong, BE1410, Brunei Darussalam 

Background and objective of investigation: The infective prion agent (PrPsc) is resistant to common proteases and conventional sterilization 

processes. Complete destruction of this agent by incineration and alkaline hydrolysis precludes applications such as decontamination of 

reusable medical instruments, laboratory equipments and specified risk materials potentially exposed to prions. Enzymatic method of prion 

degradation is a viable alternative but current methods require heat pre-treatment of prion material, addition of chemical surfactants, high 

temperature incubation and extended digestion time. This study investigates the efficacy of an enzymatic composition in the degradation of 

scrapie prion. 

Methods: ME7 scrapie prion was digested with an enzymatic composition and the digest evaluated for residual PrPsc in vitro by western 

blotting analysis. 

Result: Time-course degradation experiment revealed a consistent pattern of PrPsc disintegration with significant loss of PrPsc 

immunodetection in 2 h incubated at 50°C. Complete loss of PrPsc signal to undetectable levels as determined by western blotting was 

achieved in 10 min at 65°C. 

Conclusion: This novel enzymatic composition method overcomes key limitations of some of the reported methods and is potentially useful 

for decontamination of reusable medical and laboratory devices and other applications. 

FB/02 Molecular characterization of the multi-species methanogenic gene pool underpinning multi-phasic anaerobic digester 

treatment of combined municipal solid waste/sewage sludge 

STEVEN MOlONEY1 , David Jones1 , Komang ralebitso-Senior 1 , Eric Senior2 1 2 Teesside University, Middlesbrough; CLEMANCE, Middlesbrough 

Despite the intermittent 160-year exploitation of biogas methane, definitive fundamental studies of the underpinning complex microbial 

associations (multi-species gene pools) have been limited by `experimental uncontrollability`. laboratory models in the 80s gained insights 

of the two distinct catabolic populations, free-living and surface-attached, but low species culturability proved problematic. Fortunately, 

molecular approaches now enable accurate ‘fingerprinting’ as a prerequisite of anaerobic digestion optimization. 

Awareness of Britain`s abrogation of `One Planet living` grows so it is incumbent to consider past excesses such as treatment capacity 

overdesign by providing microbial evidence for greatly reduced (size/carbon) footprints. Thus, the goals of this eight-week SGM-sponsored 

summer studentship were to determine whether a four-stage bioreactor was capable of separating spatially (exclusive habitat domains), 

through dilution rate control, key catabolic species with retention of the constraints of the corporate association (overlapping activity 

domains) to provide the drivers to develop a single-stage but multi-phase bioreactor. 

DNA extraction preceded PCr/DGGE analysis with ‘universal’ 16S rrNA gene, sulphate-reducing bacteria (SrB) and archaea primers. 

Comparable population profiles characterized each vessel with a feed of primary settled sewage sludge/refuse organic fraction. These 

contrasted distinct profiles, particularly SrB, in response to a model feed. 




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GM/01 Genotypic analysis of deletion strains of an ‘essential’ mycobacterial two-component regulatory system 

ZEESHAAN-ul HASAN1 , Jade James1 , Amanda Brown1 , T loerger2 , J Sacchettini2 , Tanya Parish1 1 2 Barts and The London, School of Medicine & Dentistry, London, England; Texas A&M University, College Station, Texas, USA 

Mycobacterium tuberculosis is one of the most hazardous infectious agents in the world and is responsible for the deaths of over one million 

people each year. With the outbreak of drug resistant strains of M. tuberculosis and lengthy and complicated treatment regimens there is a 

necessity to develop new drugs. Senx3-regx3 is a key two-component regulatory system which plays a role in virulence in M. tuberculosis 

and regulates the uptake of phosphate in Mycobacterium smegmatis. The Senx3-regx3 system is essential in M. smegmatis, however, using 

a sophisticated genetic methodology we were able to construct deletion strains of this system in M. smegmatis. We hypothesized that these 

strains had compensatory mutations in members of the regulon. Whole genome and confirmatory sequencing of these deletion strains 

identified several potential mutations which could compensate for the deletion. Potential suppressors include a hypothetical toxin-antitoxin 

system and an ion-proton anti-porter. 

GM/02 SadA, a trimeric autotransporter from Salmonella enterica serovar Typhimurium, can promote biofilm formation 

and provides limited protection against infection 

Dhaarini raghunathan1 , Timothy J. Wells1 , FAYE C. MOrriS1 , robert K. Shaw2 , Saeeda Bobat1 , Sarah E. Peters3 , 

Gavin K. Paterson3 , Karina Tveen Jensen1 , Denisse l. leyton1 , Jessica r. Hitchcock1 , Duncan J. Maskell3 , Mark A. Webber1 , 

robin C. May1 , Calman A. Maclennan1 , laura J. Piddock1 , Adam F. Cunningham1 , ian r. Henderson1 1 2 3 University of Birmingham, Birmingham; Imperial College London, London; University of Cambridge, Cambridge 

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan 

Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, 

they can contribute to adherence, colonization and virulence and are frequently targets of antibody-mediated immunity. 

in this study, we have examined the properties of the Salmonella enterica serovar Typhimurium protein SadA, a purported trimeric 

autotransporter adhesin secreted via the Type V pathway. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro 

and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation and increased adhesion to human 

intestinal Caco-2 epithelial cells. immunization of mice with folded, full-length, purified SadA elicited an igG response which provided limited 

protection against bacterial challenge. When anti-SadA igG titres were enhanced by administering alum-precipitating the protein a modest 

additional protection was afforded. Therefore, despite SadA having pleiotropic functions it is not a dominant, protective antigen 

for antibody-mediated protection against Salmonella. 

GM/03 Insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria 

D.F. BrOWNiNG1 , T.J. Knowles2 , M. Overduin2 , i.r. Henderson1 1 2 School of Immunity & Infection, School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham 

The outer membranes of Gram-negative bacteria function as a barrier to protect cells from toxic compounds such as antibiotics and 

detergents. They are composed of phospholipids, lipopolysaccharide and two major classes of proteins, lipoproteins and β-barrel containing 

integral outer membrane proteins (OMPs). insertion of OMPs into the outer membrane of E. coli is achieved by the multi-protein BAM 

complex. An important component of this complex is the BamA protein, which is an essential protein that binds to unfolded β-barrel 

precursors via the polypeptide transport-associated (POTrA) domains at its N-terminus. The C-terminus of BamA contains a β-barrel 

domain, which tethers BamA to the outer membrane and is thought to be involved in OMP insertion. As BamA orthologues are found in 

all Gram-negative bacteria, we examined whether orthologues from a wide range of bacteria could functionally replace E. coli BamA. We 

demonstrate that only the closely related Salmonella enterica serovar Typhimurium BamA orthologue could function in E. coli. Experiments 

with chimeric protein fusions demonstrate that the β-barrel domains of many of the BamA orthologues can functionally replace that of 

E. coli but that their POTrA domains cannot. 

GM/04 Identification and analysis of a biofilm-associated gene PA3572 in Pseudomonas aeruginosa 

SANAYA PATEll, Martin Welch 

University of Cambridge, Dept of Biochemistry, Cambridge 




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The definition of a biofilm in the clinical setting remains broad and complicated due to the limited number of biofilm-specific markers 

identified. The aim of this study is to define a core set of biofilm-associated genes across different growth and nutrient conditions. A metaanalysis 

has been performed across three independent microarray studies comparing gene expression profiles of Pseudomonas aeruginosa 

biofilms vs. planktonic stationary phase cells. A putative ‘core’ biofilm-associated transcriptome has been defined based on the overlap 

between the datasets. At a modulation threshold of ≥ 1.4-fold, we identified 44 up-regulated genes and 31 down-regulated genes in 

biofilms compared with PS cultures. The ‘core’ biofilm is therefore comprised of < 80 gene products at this modulation threshold. 

Two hypothetical genes have been selected for further study. Of these two, PA3572 is one of the most highly-modulated transcripts. 

Predicted to encode a protein of 58 amino acids, PA3572 is located between mmsR, a regulator of the mmsAB operon (catabolism of 

branched chain amino acids) and a probable MFS (Major Facilitator Superfamily) transporter. We show that a mutant in PA3572 exhibits 

several biofilm-associated phenotypes, e.g., reduced attachment and motility. The possible role of PA3572 in biofilm physiology is currently 

being investigated. 

GM/05 The ytfMNP operon encodes integral outer-membrane proteins that contribute to the virulence of Salmonella 

Typhimurium 

TiMOTHY WEllS1 , Faye Morris1 , Khédidja Mosbahi3 , Joel Selkrig2 , Daniel Walker3 , Trevor lithgow2 , ian Henderson1 1 2 3 University of Birmingham, Birmingham; Monash University, Melbourne, Australia; University of Glasgow, Glasgow 

Salmonella enterica Typhimurium is a major cause of morbidity worldwide and mortality in children in sub-Saharan Africa. Outer membrane 

proteins (OMP) of Salmonella are of significance because they are at the interface between pathogen and host. recently, an integral OMP, 

YtfM, was described in E. coli. YtfM bears homology with the essential BamA protein and the TpsB proteins of the virulence-associated 

Two-partner secretion systems. Analysis of genome sequences shows that ytfM is present not only in S. Typhimurium but is ubiquitous in 

proteobacteria. in almost all cases ytfM is predicted to be in an operon with two other genes, ytfN and ytfP. Despite widespread distribution 

the function of the ytf locus proteins remains elusive. Here we use antiserum raised to YtfM, N and P to determine the subcellular locations 

of each protein. YtfM was confirmed as an OMP of S. Typhimurium. YtfN was found to be associated with the cell envelope, and YtfP was 

detected in the cytoplasm. Mutants created in the ytfMNP operon of S. Typhimurium were more sensitive to human serum, had increased 

motility and reduced virulence in a murine model of Salmonella disease. Our results suggest that the ytfMNP operon contributes to the 

virulence of S. Typhimurium. 

GM/06 Metabolic regulator linked to virulence in Pectobacterium atrosepticum through bypass of the rsm system 

MAriON CuBiTT1,2 , ian Toth2 , George Salmond1 1 2 University of Cambridge, Cambridge; Scottish Crop Research Institute, Dundee 

Pectobacterium atrosepticum is an agriculturally significant Gram-negative phytopathogen which exclusively infects potatoes causing soft 

rotting of tubers and blackleg disease of stems. The pathology of these diseases is principally caused by secretion of exoenzymes by the 

bacteria including pectate lysases, proteases and a carboxymethylcellulase, among other proteins. Virulence factors are produced in a coordinated 

fashion via regulatory networks, including the quorum sensing and rsm (regulator of secondary metabolite) systems. The rsm 

system involves a small, non-coding rNA, rsmB, and a global post-transcriptional ‘repressor’ protein, rsmA. rsmA binds to target transcripts 

preventing their translation, but this effect is counteracted by rsmB which sequesters the repressor protein away from mrNAs. Therefore in 

an rsmB mutant, rsmA is free to repress translation of specific transcripts, such as those coding for exoenzymes – including protease. 

in this study, a random transposon mutagenesis was carried out on an rsmB mutant strain, and mutants displaying restored protease 

production were identified. Three independent insertions restoring protease production to wild type levels were identified in a gene coding 

for a metabolic transcriptional regulator not previously linked to virulence. Further investigation has also shown that disruption of this gene 

leads to precocious production of the quorum sensing molecule OHHl. 

GM/07 The oxidation of norepinephrine affects the growth of Campylobacter jejuni in vitro 

EMMA TrANTHAM1 , David Tourigny2 , Tristan Cogan1 1 2 University of Bristol, Bristol; University of Leicester, Leicester 

Norepinephrine (NE) has been shown to augment the growth of many bacterial species under iron-restricted conditions in vitro, possibly 

by reducing iron(iii) to iron(ii), so increasing its availability. When NE reduces iron(iii) it is itself oxidised. The product of this oxidation can 

also reduce iron(iii), being oxidised itself in turn, and if enough iron(iii) is available oxidation can occur until melanin is formed. The speed at 

which this occurs, and the effect it has on bacterial growth in vitro is not fully known. 




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The optical density of Minimum Essential Medium Alpha (MEMα) was measured after the addition of NE and melanin formation was 

observed. An oxidation process was confirmed by measuring the reduction of resazurin. NE was added to MEMα at different time points 

prior to inoculation with C. jejuni. Pretreatment with NE up to 24h before bacterial inoculation resulted in a reduction in the lag phase of C. 

jejuni growth, but pretreatment for 48h (producing obvious melanin formation) inhibited the growth of C. jejuni. 

The results show that when NE is added to MEMα it is oxidised and the stage of oxidation of NE at the time of bacterial inoculation will 

affect the growth of C. jejuni. 

GM/08 Novel method for the assessment of entry of molluscum contagiosum virus into host cells and subsequent innate 

immune responses 

NiAMH BlYTHE, Subuhi Sherwani, laura Farleigh, Asif Nizam, Amanda Tonks, Joachim Jakob Bugert 

Cardiff University School of Medicine, Dept of Infection, Immunity and Biochemistry, Cardiff, Wales 

Molluscum contagiosum virus (MCV), a poxvirus pathogenic for humans, replicates well in the human skin in vivo, but not in vitro in standard 

monolayer cell culture. in order to determine the nature of the replication deficiency in vitro, the MCV infection process in standard cell 

culture has to be studied step by step. The method described uses luciferase reporter constructs to measure poxviral mrNA transcription 

activity in MCV infected cells in standard culture. Briefly, MCV isolated from human tissue specimen was integrity checked, quantitated 

by PCr, and used to infect a series of epithelial and fibroblast type cell lines of human and animal origin. The cells were subsequently 

transfected with a reporter plasmid encoding firefly luciferase gene under the control of a synthetic early/late poxviral promoter, a iFN 

beta reporter plasmid and a transfection control plasmid. After 16 hours cells were harvested and tested for MCV infection, interferon 

production and luciferase expression. The results obtained indicate that MCV can infect both human and animal cells and induces tissue 

type interferon in the process. The method can be used to optimize MCV infection in more complex eucaryotic cell culture models. 

GM/09 Type III secretion system expression in Pseudomonas aeruginosa biofilms and chronic infection 

JADE CHuNG1 , Olena rzhepishevska2 , Juliet Foweraker3 , Arne rietsch4 , Madeleine ramstedt2 , Martin Welch1 1 2 3 4 University of Cambridge, Cambridge; Umeå University, Umeå, Sweden; Papworth Hospital, Cambridge; Case Western Reserve 

University, Ohio, USA 

Pseudomonas aeruginosa is an opportunistic human pathogen and common cause of chronic infections in individuals with cystic fibrosis 

(CF). Chronic infections are associated with the biofilm growth mode, whereas acute infections are associated with increased virulence 

and expression of a toxin-secreting machinery called the Type iii secretion system (T3SS). Our previous study revealed that the T3SS was 

expressed and active in PAO1 biofilms, even though a two-component system, retS/ladS, has been proposed to reciprocally regulate 

these phenotypes. Preliminary data suggested that this expression is correlated with the development of anaerobic conditions within 

biofilms. using planktonic PAO1 cultures as a model, we showed that microaerophilic growth conditions can significantly enhance T3SS 

expression and activity. Moreover, analysis of a GFP-reporter for the T3 effector gene, exoS, by laser-scanning confocal microscopy revealed 

localization to the centre of continuous-flow PAO1 biofilms, confirming that T3SS expression occurred where cells are most oxygenlimited. 

importantly, T3SS structural components and effectors were also detected in CF sputa – a typically microaerobic environment – 

from individuals with chronic P. aeruginosa infections. Together, these data indicate that T3SS expression may, contrary to current thinking, 

play a role in chronic P. aeruginosa infections of the CF lung. 

GM/10 Iron uptake in Burkholderia pseudomallei 

lAurA MArSHAll1 , Philip ireland1 , Mitali Sarkar-Tyson1 , rick Titball2 1 2 Biomedical Sciences Dept, DSTL Porton Down,, Salisbury, Wiltshire SP4 0JQ; University of Exeter, Geoffrey Pope Building, Stocker 

Road, Exeter EX4 4QD 

A bioinformatic search for high affinity iron uptake systems in the human pathogen Burkholderia pseudomallei revealed that the 

genome encodes systems for the uptake of both ferric and ferrous iron. tonB, which powers ferric iron uptake is encoded on the small 

chromosome, alongside homologs of tonB complex genes exbB and exbD.A B. pseudomallei tonB deletion strain was constructed and was 

shown to havesignificantly reduced growth in luria-Bertani broth which could be restored by addition of iron salts to growth media. The 

growth rate was also restored by complementation of the mutant.Balb/C mice challenged with αtonB B.pseudomallei had a reduced time to 

death compared to the parental strain K96243, though the infection was not cleared from organs, indicating that whilst tonB is important 

for virulence of the bacteria, it is not required for survival within the host. Galleria mellonella challenged with the mutant strain also had a 

reduced time to death compared to those challenged with K96243. 

© Crown copyright. Dstl 2011 




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GM/11 Structural and functional characterization of the toxin–antitoxin and abortive infection system ToxIN 

FrANCESCA SHOrT, Tim Blower, xue-Yuan Pei, Ben luisi, George Salmond 


Bacteria have evolved a range of mechanisms to counteract the threat of viral infection. These include the abortive infection systems, which 

act to stop the spread of bacteriophages through a culture by causing the precocious death of infected bacterial cells, before the phage can 

replicate. ToxiN is an abortive infection system from Pectobacterium atrosepticum SCri1039 which consists of a protein toxin (ToxN) and 

an rNA antitoxin (Toxi). ToxN is an endoribonuclease which cleaves the repetitive Toxi precursor into individual 36-nucleotide repeats. 

The structure of the ToxiN complex was recently solved, and shows that the Toxi rNA forms a pseudoknot which binds ToxN over two 

extended surfaces to generate a triangular, trimeric complex held together exclusively through rNA-protein interactions. Site-directed 

mutagenesis of both the ToxN and Toxi components was performed to identify residues required for ToxN endoribonuclease activity, and 

for the in vivo inhibition of ToxN by Toxi. The ToxN recognition sequence was also determined. We are performing further biochemical 

and structural analysis of ToxiN and its homologues to better understand the principles behind the formation of the trimeric ToxiN 

complex, and the mechanism by which it mediates resistance to phage. 

GM/12 The deep phylogeny of influenza viruses 

Derek Gatherer 


influenza viruses are a major cause of mortality and morbidity through both pandemics of novel subtypes occurring every few decades, 

and subsequent annual seasonal outbreaks. However, the evolutionary relationships of influenza viruses to other members of the family 

Orthomyxoviridae remain largely unresolved. recent advances in molecular phylogenetic analysis using Bayesian statistical methods provide 

an opportunity to clarify this situation. The most conserved protein, encoding polymerase PB1, of 32 members of the Orthomyxoviridae 

(15 strains of influenza A, 10 of B, 2 of C and 5 other orthomyxoviruses) was analysed using BEAST. The most recent common ancestor 

(MrCA) of all orthomyxovirus PB1 proteins was estimated to have been in existence around 640 years ago (mean of probability 

distribution). The MrCA for influenza A, B and C PB1 proteins is dated to around 330 years ago. Since the MrCA for influenza A 

haemagglutinin protein has previously been estimated in two independent publications at 950 or 1110 years ago, this result implies either: 

1) that there may have been a period in orthomyxovirus evolution when there was reassortment between different species of virus, or 2) 

that substitution rates have fluctuated markedly within the Orthomyxoviridae, possibly as a consequence of changes in host. 

GM/13 Characterization of two putative c-di-GMP phosphodiesterases in Eca1043 

Hui TAN1 , ian Toth2 , George Salmond1 1 2 University of Cambridge, Cambridge; Scottish Crop Research Institute, Dundee 

The bacterium Erwinia carotovora subsp. atroseptica (Eca), a.k.a. Pectobacterium atrosepticum, is an economically important Gram-negative 

plant pathogen causing soft rot in potato tubers and black rot in potato stems. 

in the last few years, cyclic di-GMP, an intracellular signaling molecule, has been found to control virulence phenotypes in other gammaproteobacteria 

pathogens. 

intracellular c-di-GMP concentrations are modulated by two classes of proteins. Diguanylate cyclases (DGCs) containing a consensus 

GGD(E)EF domain, and phosphodiesterases (PDEs) that contain either an EAl or a HD-GYP domain, synthesise and degrade the molecule 

respectively. 

in Eca1043, genes ECA3548 and ECA3549 are divergently transcribed and encode putative PDEs. ECA3548 is the only gene in Eca1043 

predicted to encode an HD-GYP protein and ECA3549 is predicted to encode an EAl domain. 

The literature consensus shows that increasing concentrations of c-di-GMP (by over-expression of GGDEF-, or mutation of EAl/HD-GYP 

proteins) lead to an overall decrease in virulence and motility. 

This study demonstrates that ECA3548 and ECA3549 impose phenotypes on Eca1043 that do not agree with the consensus. Analysis of 

ECA3548 and ECA3549 mutants also show that expression of a number of GGDEF genes is modulated by ECA3549, implying a regulatory 

role for these two genes in the Eca1043 c-di-GMP network. 




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GM/14 In vitro analysis of bluetongue virus reassortants 

ANDrEW SHAW1 , Maxime ratinier1 , Kathryn Allan1 , Salvatore Gulletta1 , Peter Mertens2 , Massimo Palmarini1 1MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary 

& Life Sciences, University of Glasgow, Glasgow; 2Vector-Borne Disease Programme, Institute for Animal Health, Pirbright, Woking 

Bluetongue virus (BTV) is a segmented dsrNA virus. There are at least 24/25 serotypes of BTV circulating worldwide. Since 1998, Europe 

has been experiencing continuous outbreaks of bluetongue involving multiple serotypes. Sequence analyses of isolates collected from 

the Mediterranean basin, the Balkans and Northern Europe have all revealed examples of reassortment between different serotypes. 

The reasons underpinning the emergence of specific BTV reassortants are not completely clear. Here, we assessed whether all genomic 

segments can potentially reassort between distinct serotypes and we evaluated the replication fitness of the resulting monoreassortants. 

We used BTV-1 and BTV-8 as they are two of the main serotypes that have been circulating in these recent years in Europe. We found 

that every combination of BTV-1/ BTV-8 monoreassortants could be rescued by reverse genetics, although not necessarily with the 

same efficiency. For example, plaque assay and growth curve assays indicate that segment-1, encoding the viral rNA-dependent rNA 

polymerase, is largely responsible for the differences in growth and cytopathogenicity observed between the two strains of BTV-1 and BTV- 

8 that we used. The data obtained in this study will be useful to form the experimental framework necessary to understand the emergence 

of specific BTV serotypes in the field. 

GM/15 Withdrawn 

GM/16 Human neutrophils secrete elevated levels of matrix metalloproteinases 8 and 9 in the presence of Campylobacter 

jejuni 

AMANDA MACCAlluM, Eleanor lowe, Aileen Sherry, Sarah MacDonald, Paul Everest 


Neutrophils are likely to play a major role in the pathogenesis of Campylobacter jejuni infections. Activation of neutrophils results in the 

release of inflammatory molecules including matrix metalloproteinases (MMPs). MMPs are key enzymes functioning in the breakdown of 

ECM especially during pathological processes such as inflammation. Neutrophils were isolated from human blood and cultured with a C. 

jejuni strain or alone as a control. Four C. jejuni strains were used 11168, 81-176, 81116 and 81116 kpsm(capsule deficient mutant) and the 

time points in culture were 0, 2, 4, 6, 24 and 48 hours. At the end of each time point supernatants were collected and analysed for MMP-8 

and MMP-9 levels by EliSA. Neutrophils cultured with all four C.jejuni strains demonstrated significantly increased levels of MMP-9 secretion 

at 2, 4, 6 and 24 hours. MMP-8 secretion was also increased in all four strains but slightly later at 4, 6 and 24 hours. The findings suggest 

that C. jejuni results in an increased release of matrix metalloproteinases 8 and 9 from human neutrophils. MMPs play a key role in ECM 

breakdown, which if excessive or prolonged may result in tissue damage itself. We suggest that elevated levels of MMPs may be involved 

in the inflammatory events associated with C jejuni pathogenesis. MMP activity is regulated by tissue specific inhibitors of metalloproteinases 

(TiMPs). 

GM/17 The phage-shock protein response as a broad-spectrum therapeutic target 

STEPHANiE riCHArDS1 , Timothy Milne1 , Mitali Sarkar-Tyson1 , Ali Tavassoli2 , Petra Oyston1 1 2 Defence Science and Technology Laboratory, Biomedical Sciences Dept, Porton Down, Salisbury SP4 0JQ; University of Southampton, 

School of Chemistry, Southampton SO17 1BJ 

The Phage-shock protein (Psp) system is an extracytoplasmic stress response in bacteria that functions to maintain the cell membrane 

integrity during stress. it is thought to have an important physiological role in maintaining the PMF across the inner membrane. The Psp 

system is important in survival and virulence in several species of bacteria. For example, a psp mutant in Yersinia enterocolitica has a severe 

growth defect when the type iii secretion system is active and is attenuated in a mouse model of infection.Burkholderia pseudomallei is a 

Gram-negative bacterium and the causative agent of the disease melioidosis. Treatment of B. pseudomallei infection is problematic due to 

its inherent antibiotic resistance and therefore new antimicrobial targets are required. The project aims to evaluate the Psp system in B. 

pseudomallei as a therapeutic target using an innovative platform technology to generate novel cyclic peptide inhibitor compounds. rT-PCr 

analysis has indicated that a pspA homologue is up-regulated during growth under certain stress conditions. A pspA deletion mutant will be 

used to fully characterize the Psp system in B. pseudomallei in order to evaluate its potential as a therapeutic target. The Psp response will 

be investigated further using inhibitor compounds to disrupt the system. 




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GM/18 Insertional mutagenesis of the UV-sensitizing integrative conjugative element r391: generation of a complete 

mutational map and optimization of double mutational insertions 

PATriCiA ArMSHAW, J. Tony Pembroke 

University of Limerick, Limerick, Ireland 

The integrative conjugative element (iCE) r391 is a prototype of the SxT/r391 group of enteric iCE elements found extensively in Vibrio 

cholerae. r391 is 89kb and has been fully sequenced. it contains 96 open reading frames (orfs), including genes involved in mutagenic 

DNA repair however many of its gene are functionally cryptic. using the insertional mutagenesis protocol of Murphy et al. (2000) we have 

improved the length of deletion generated to up to 18kb allowing rapid knockout of iCE sequences. in addition we have utilised a mobile 

knockout cassette which allows knockouts to be constructed in particular hosts which facilitates functional analysis particularly for transfer 

deficient knockouts. using these protocols an extensive library of over 70 r391 knockout mutants has been generated to examine the uV 

sensitization effect and other specific functions associated with the r391 iCE. Knockout analysis have illustrated that deletion of orfs 90/91 

of r391 abolishes the uV sensitization effect. Orf 90/91 are homologues of flhC/flhD, transcriptional regulators of flagella synthesis and are 

considered global regulators of the r391 element. Orf 90/91 have been cloned and confirmed to cause the uV sensitization effect in a 

∆9091 mutant. 

GM/19 bapC autotransporter protein is a virulence determinant of Bordetella pertussis 

MOJTABA NOOFEli, John Coote, roger Parton 

Glasgow University, Glasgow 

A protein designated Bap-5 or BapC has been identified as a member of the B. pertussis autotransporter family and the present work 

suggests that this protein, like the characterized BrkA, is a Bvg-regulated serum-resistance factor and virulence determinant. B. pertussis bapC 

and brkA, bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent 

strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence 

of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA, bapC mutants, like the brkA mutant, were found to be more 

sensitive to the antimicrobial peptide, cecropin P1, than the parent strains. in the genome sequence of B. pertussis strain Tohama, bapC 

is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5’ end of the gene which creates a truncated BapC 

protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and 

amino acid polymorphisms but it appeared that all had an OrF that would be able to produce BapC. 

GM/20 Survey of the genome of Opitutus terrae Pb90-1 for biomass-converting enzymes 

MAriA BAWN1 , Meng Zhang1 , ruth lloyd2 , Simon Charnock2 , Gary Black1 1 2 University of Northumbria, Newcastle upon Tyne; Prozomix Limited, Northumberland 

The development of alternative energy resources is vital due to the inevitable depletion of the world’s fuel reserves and rising prices of 

crude oil. With this in mind, biomass is a maintainable prosperity in the future of renewable energy and in terms of a bio-based economy, 

the idea of degrading lignocellulose biomass into its constituent sugars has emerged to seek several innovative synthetic biofuels such as 

bioethanol by fermentation. Bioconversion of lignocellulosic biomass is initiated primarily by micro-organisms such as fungi and bacteria. 

in this study, genes encoding an endo-xylanase and β-xylosidase from Opitutus terrae PB90-1, an anaerobic soil bacterium with known 

genomic sequence, were isolated and expressed in Escherichia coli. The annotated endo-xylanase showed activity on oat spelt xylan 

whilst the β-xylosidase hydrolysed the resulting xylo-oligosaccahride products, mainly xylobiose. The genomic sequence of Opitutus terrae 

PB90-1 shows that these two enzymes are situated in the same gene cluster suggesting relatedness in their modes of action against 

biomass substrates. 

GM/21 Identification of bacteriocins in Pseudomonas syringae and Pectobacterium carotovorum/atrospectium 

rHYS GriNTEr, Karen Smith, Khedidja Mosbahi, Daniel Waker 

Glasgow University, Glasgow 

We are currently identifying and cloning bacteriocins from the economically important plant pathogens Pseudomonas syringae and 

Pectobacterium carotovorum/atrosepticum. These bacteriocins are of the colicin/pyocin class, being enzymatic, antimicrobial compounds, 

with a narrow spectrum of activity (activity is generally restricted to strain of the same species, or closely related bacterial species). Their 

enzymatic nature and narrow spectrum of activity, makes them promising targets, as a new generation of highly targeted bactericidal 

compounds. The bacteriocins can be readily indentified via bioinformatic analysis of available genome sequences of these organisms. 




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Bioinformatic analysis has yielded a significant number of homologues in the genomes of these organisms. recombinant expression and 

purification of a number of these putative bacteriocins, has yielded bacteriocin molecules with enzymatic activity in vitro and a significant 

killing spectrum against indicator strains in vivo. Characterised of bacteriocins from these species have numerous applications in plant disease 

control including; large scale recombinant production of protein for direct application to crops, engineering of attenuated ‘biocontrol strains’ 

expressing the bacteriocins, or creation of transgenic plants expressing one or a combination of the identified proteins. 

GM/22 Comparative analysis of ITS sequences between Dermanyssus gallinae mites from pigeon nests and layer hen farms in 

Southern Italy 

Domenico Galante1 , lucia Potenza2 , Maria Assunta Cafiero1 , luigi Cucchiarini2 , Cinzia Calcabrini2 , OliViEr SPArAGANO3 1 2 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Foggia, Puglia, Italy; Università degli Studi di Urbino, Dipartimento 

di Scienze Biomolecolari, Urbino, Marche, Italy; 3Northumbria University, School of Life Sciences, Ellison Building, Newcastle upon Tyne 

The red mite, Dermanyssus gallinae (Acarina: Mesostigmata), is a haematophagus ectoparasite affecting domestic and wild birds worldwide. it 

can also attack humans causing a highly pruritic dermatitis. Molecular tools in addition to the traditional methods have also been developed 

for the identification of this mite. The ribosomal internal transcribed regions such as iTS1, 5.8S ribosomal DNA and iTS2 are among 

the most studied regions in the phylogenetic analysis. We performed a comparative analysis of 30 DNA sequences of mites using the 

amplification of the ribosomal internal transcribed spacer regions. The mites were collected from 6 caged laying hen poultry farms and 7 

urban pigeon nests in Southern italy and identified as D. gallinae by morphological keys Our results show a genetic diversity between the 

two considered groups. The DNAs of mites from pigeon nests result to be highly conserved, while those from layer hens in farms are more 

variable, especially in the iTS1 region. These intra-species variations are probably determined by the massive use of acaricides observed 

in these poultry farms and may represent the possible evolution in the mite populations living under strong chemical pressure. We are 

working on the geo-distribution and some environmental parameters to investigate further such variation. 

GM/23 Characterization of a novel, genomic island-borne, accessory type 1-like fimbrial operon in Klebsiella pneumoniae 

JON VAN AArTSEN1 , Jasti Subbarao1 , Steen Stahlhut2 , Ewan Harrison1 , Hong-Yu Ou3 , Karen Krogfelt2 , Carsten Struve2 , 

Kumar rajakumar1 1 2 Dept of Infection, Immunity & Inflammation, University of Leicester, Leicester; Dept of Microbiological Surveillance & Research, 

Statens Serum Institut, Copenhagen, Denmark; 3Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, 

Shanghai Jiaotong University, Shanghai, China 

Klebsiella pneumoniae (Kp) is an increasingly important nosocomial pathogen. Members of this species possess the ability to manufacture 

several distinct repertoires of fimbriae that aid surface adhesion and niche colonization. This work describes a novel accessory fimbrial 

operon (fim2) located on a genomic island downstream of a trNA locus in Kp strain Kr116 (fim-positive, fim2-positive). fim2 has similar 

gene organization and predicted products to the species-conserved fim operon, but the two bear no nucleotide similarity. interestingly, 

similar to the Kp fim operon but unlike the Escherichia coli fim operon, fim2 encodes an EAl domain-bearing putative phosphodiesterase 

which may influence cyclic-di-GMP levels, virulence factor expression, motility and biofilm formation. To evaluate the role of fim2 in 

virulence, a fim2-minus mutant was constructed. The mutant was as effective as the wildtype in colonizing the murine gastrointestinal tract 

and did not display differential adhesion to HCT-8 ileocaecal cells. Similarly, no virulence attenuation was identified using lung infection and 

urinary tract infection models, and the mutant exhibited wild-type adherence to 5637 bladder cells. Despite the lack of a demonstrable 

virulence phenotype, 15% of Kp strains surveyed possess fim2, highlighting a potentially important role for this fimbrial gene cluster in the 

lifestyle of Kp. 

GM/24 Interaction of antibiotics combined with manuka honey on MrSA-15 

rOWENA JENKiNS, Teckla Kazimoto, rose Cooper 

University of Wales Institute Cardiff, Cardiff 

Background: The continued prevalence of antibiotic resistant strains of Staphylococcus aureus demands innovative treatments. Manuka honey 

has been shown to inhibit MrSA as well as numerous of other pathogenic organisms. The study of manuka honey in combination with 

various antibiotics could provide an option for reducing reliance on antibiotics and potentially reducing the extent of resistance seen in 

organisms. 

Methods: To determine the MiC of each antimicrobial being tested, EMrSA-15 (NCTC 13142) was challenged using honey, oxacilin, 

gentamicin, vancomycin, tetracycline and ciprofloxacin by microtitre plate broth dilution method. These drugs were then tested in 




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combination with honey at varying concentrations of the antibiotics (256 μg/ml -0.0625 μg/ml) and different concentrations of honey by 

chequer board titration. The antibiotics and honey were also tested alone and in combination by E strip diffusion method. 

Results: After treatment with manuka honey and antibiotics a large reduction in the MiC/MBC values of Oxacillin were seen. There was also 

a small drop in the MiC value for vancomycin. The MiC for the other three antibiotics was not altered. 

Conclusion: Combinations of antibiotics and honey leads to altered MiC values for some antibiotics. Honey could potentially be used to 

increase efficacy of beta lactam antibiotics. 

GM/25 Characterization of the gp25-like protein HsiF of the Type VI secretion system in Pseudomonas aeruginosa 

NADiNE lOSSi, rana Dajani, Paul Freemont, Alain Filloux 


Bacterial pathogens use a wide range of protein secretion systems to successfully colonize their host, one of which is the recently 

discovered T6SS. However, little is known about the structure and function of the individual components of the T6SS apparatus. The 

intensely studied VgrG and Hcp components have been proposed to form the T6SS puncturing device resembling the apparatus used by 

bacteriophages to inject DNA into bacterial cells, based on their structural resemblance to gp5/gp27 and gp19, respectively. in addition, a 

conserved component of the T6SS was identified that showed similarity to the lysozyme gp25. The opportunistic pathogen Pseudomonas 

aeruginosa contains three T6SS gene clusters within its genome, all of which encode a member of the gp25 family, which we named HsiF. 

This study investigates structural similarities between HsiF and gp25, explores the possibility that HsiF acts as a lysozyme and shows that 

HsiF is crucial for a functional T6SS. 

GM/26 Not presented 

GM/27 Clostridium difficile and innate immunity 

ClAirE COlENuTT, Huynh A. Hong, Patima Permpoonpattana, Simon Cutting 

Royal Holloway, University of London, Surrey 

Clostridium difficile is the most common cause of noscomial antibiotic-associated diarrhea in developed countries. C. difficile spores are the 

primary cause of infection whether by transmission or by antibiotic-induced growth of resident C. difficile. Toxins (A and B) produced by 

vegetative cells of C. difficile are the primary cause of disease yet what’s less understood is how the spore survives in the Gi-tract. We have 

begun a study to investigate how C. difficile spores might interact with intestinal cells using in vitro approaches. 

Firstly, we examined the adhesion of C. difficile spores to intestinal cell lines (HT29 and Caco-2). Spores were shown to adhere efficiently to 

cultured cells whereas vegetative cells exhibited minimal binding. The surface properties of spores were examined and the surface charge 

(zeta-potential) and hydrophobicity determined at low, medium and high pH. under all conditions spores were shown to be negatively 

charged and hydrophobic. We next examined the ability of spores to induce expression of the toll-like receptor (Tlr) genes demonstrating 

that Tlr genes are induced by C. difficile spores. Tlrs are the pattern recognition receptors that interact with potential pathogens and 

induce innate immune responses. The preliminary results of these studies will be presented. 




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GM/30 The role of TFF1 in mediating Helicobacter pylori colonization of the adherent mucus layer of E12 cells 

JuliE NAuGHTON, Brendan Dolan, Nicole Tegtmeyer, Marguerite Clyne 

University College Dublin, Dublin, Ireland 

Helicobacter pylori colonization of the gastric mucosa of humans is often considered a paradigm for chronic infection of mucosal surfaces. 

We hypothesised that the interaction of H. pylori with TFF1 dimer, a member of the trefoil factor family present in gastric mucus, is 

mediated by H. pylori lipopolysaccharide and promotes mucus colonization. 

Polarised HT29-MTx-E12 cells produce an adherent mucus layer that contains TFF1 and the gastric mucin MuC5AC. H. pylori co-localized 

with TFF1 in the HT29-MTx-E12 cell adherent mucus layer following infection and in gastric biopsies from infected humans. 

Culture of HT29-MTx-E12 cells in the presence of copper, which increases TFF1 dimer formation, resulted in a significant increase in 

colonization by H. pylori. 

isogenic mutants of H. pylori with truncated lPS core structures were produced and their binding to TFF1 and ability to colonize adherent 

mucus determined. One of these isogenic mutants of H. pylori was unable to interact with TFF1, and colonization of HT29-MTx-E12 cells 

was reduced 100-fold as compared to the wild-type strain (p


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GM/33 Selective isolation, characterization and screening of streptomycetes from Atacama Desert soils 

rAKESH SANTHANAM, Chinyere Okoro, Alan Bull, Michael Goodfellow 


in our search for novel natural products that can be developed as natural resources for healthcare we have focussed on the isolation of 

filamentous actinomycetes from extreme and neglected environments, including hyper-arid soils from the Atacama Desert. in the present 

study putative neutrophilic streptomycetes were isolated from Atacama Desert soil samples using a range of selective isolation media, 

including starch casein and raffinose- histidine agars supplemented with antifungal antibiotics. Thirty five isolates taken to represent the 

different colony types growing on the selective isolation plates had chemical and morphological properties consistent with their classification 

in the genus Streptomyces. The isolates were assigned to four multi-membered and three single-membered groups based on their ability to 

produce pigments on oatmeal and peptone-yeast extract agars. Seven strains taken to represent the colour groups formed distinct phyletic 

lines in the 16S rrNA Streptomyces gene tree and showed a unique combination of phenotypic properties and gave hits in a series of 

antimicrobial plug assays. These results provided further evidence that arid desert soils contain novel streptomycetes that are a promising 

source of new bioactive natural compounds. 


GM/35 Analysis of Plcr-mediated gene regulation in Clostridium acetobutylicum 

ANN-KATHriN KOTTE, Katrin Schwarz, Nigel Minton, Klaus Winzer 


in the Bacillus cereus group, the regulator Plcr and its cognate autoinducing peptide Papr are part of a quorum sensing system that controls 

the expression of many genes including those encoding extracellular virulence factors. interestingly, the genome sequence of the solventproducing 

bacterium Clostridium acetobutylicum ATCC824 features ten putative plcR homologues, eight of which are followed by a small 

open reading frame that potentially encodes an autoinducing peptide. 

To study the function of the ten identified plcR homologues, we insertionally inactivated all of them separately, using Clostron technology. 

All mutants were analysed for growth, solvent production, sporulation, granulose accumulation, and colony morphology. 

inactivation of one particular plcR homologue completely abolished sporulation, butanol production and granulose accumulation, all of 

which are characteristic and important traits of the organism. Mutation of another homologue resulted in increased acetone formation 

and a distinct, slimy appearance upon entry into stationary phase. The effects on solvent production are noteworthy as C. acetobutylicum is 

considered a prime candidate for the large-scale production of bio-fuels. 


GM/37 The glutaminyl cyclase of Rhodococcus equi: a role in virulence 

JAMAl HAiDEr, iain Sutcliffe, lynn Dover 

Northumbria University, Newcastle upon Tyne 

rhodococcal pneumonia is responsible for 3% of all foal deaths. Human infections have been reported with most being associated with 

compromised immunity. A bioinformatic survey of potential lipoproteins encoded in the Rhodococcus equi 103S genome revealed a 

putative glutaminyl cyclase gene. its predicted product shared 40% amino acid identity with the characterized enzyme from Carica papaya 

and contained a classic secretion signal and lipobox. Glutaminyl cyclase catalyses formation of pyroglutamyl residues from glutamine at the 

N-terminus of peptides and proteins affording them resistance to degradation by aminopeptidase. We have cloned and expressed the 

gene in E. coli and the purified enzyme has been characterized in vitro. The enzyme liberates ammonium when incubated with a Glu-Gly 

dipeptide substrate consistent with the formation of cyclised product. Our studies have demonstrated that the enzyme is relatively stable at 

60°C and has a low micromolar Km for the model substrate. 

A bioinformatic survey of potentially secreted proteins from R. equi indicates 40 possible substrate proteins that might be modified by this 

enzyme in vivo. Our ongoing studies will focus on determining whether this enzyme activity might have any role in R. equi pathogenicity. 




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GM/38 A novel NADP+-dependent alcohol dehydrogenase from Euglena gracilis Z 

iQBAl MuNir1 , Ashfaq Ahmad1 , Zahoor A. Swati1 , Yoshihisa Nakano2 1 2 Institute of Biotechnology & Genetic Engineering, Khyber Pakhtunkhwa Agricultural University Peshawar 25000, Pakistan; Dept of 

Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, 

Osa, Japan 

Euglena gracilis accumulates wax esters under anoxia, which are neutral lipids with considerable importance for pharmaceutical, cosmetic, 

dietetic and technical applications. E. gracilis grown on 1-hexanol produced a novel NADP + -dependent alcohol dehydrogenase (EC 

1.1.1.2), which was involved in the wax ester metabolism. The novel enzyme was purified to homogeneity from Euglena gracilis Z grown 

on 1-hexanol. SDS-PAGE and molecular exclusion chromatography revealed a native tetrameric protein of 213 kDa. The optimal pH 

range of the purified enzyme for oxidation reaction was 7.8 to 9 at 55°C, while the isoelectric point (pI) of the enzyme was determined 

as 5.7. unlike the previously reported mitochondrial NAD + -dependent ADH, this enzyme was located in cytosol and oxidized mainly mid 

and long-chain primary aliphatic alcohols using NADP + as a cofactor. The highest reaction rate was observed when 1-hexanol was used 

as substrate and the Michaelis constants (K ) for NADP m + and 1-hexanol were found to be 44 μM and 5.6 mM, respectively. Accordingly, 

the NADP + -specific ADH of Euglena gracilis, which is located in the cytosol and active towards the mid and long-chain fatty alcohols, is 

suggested to participate in the assimilation of fatty alcohols formed from wax esters under aerobic conditions, which are synthesized in 

anoxia (wax ester fermentation). 

Keywords: Euglena gracilis Z; Fatty alcohol assimilation; NADP + -alcohol dehydrogenase; Cytosolic ADH; Wax ester fermentation. 

GM/39 Mutagenesis studies in Rhodococcus equi: propionate metabolism and the methylcitrate cycle 

PHiliPPA BurGESS1 , Ken Smith1 , Wim Meijer2 , Sharon Kendall1 1 2 Royal Veterinary College, University of London, Hatfield, Hertfordshire; University College Dublin, Belfield, Dublin, Ireland 

Rhodococcus equi causes significant economic detriment to the horse racing industry, resulting in fatality or loss of use of infected foals 

aged between one and six months. R. equi is an intracellular pathogen, which targets alveolar macrophages to cause pyogranulomatous 

pneumonia. R. equi is also opportunistically zoonotic, recently identified as the cause of various pathologies in immuno-compromised 

humans. The closely related bacterium, Mycobacterium tuberculosis, has been used as a guide for R. equi research as both species show 

pathogenic similarities; both survive within the phagosomes of macrophages, preventing phagolysosomal maturation. Bioinformatic analysis 

and comparison of the R. equi and M. tuberculosis genomes has highlighted potentially important pathways in R. equi. A recently developed 

R. equi mutagenesis strategy has been used to generate strains containing large unmarked deletions within genes. One such mutant strain 

carries a deletion within the prpC gene, which encodes a methylcitrate synthase, involved in the methylcitrate cycle for odd chain fatty acid 

metabolism. This mutant exhibits an increased lag phase when grown in vitro on propionate as a sole carbon source. Growth is delayed 

rather than prevented, probably due to the presence of the compensatory methylmalonyl pathway. 

GM/40 A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism 

Streptomyces coelicolor A3(2) 

lOrENA T. FErNÁNDEZ-MArTíNEZ 1,4 , ricardo Del Sol1 , Meirwyn C. Evans1 , Susan Fielding1 , Paul r. Herron2 , 

Govind Chandra3 , Paul J. Dyson1 1 2 Institute of Life Science, School of Medicine, Swansea University, Swansea; Strathclyde Institute of Pharmacy and Biomedical Science, 

University of Strathclyde, Glasgow; 3Dept of Molecular Microbiology, John Innes Centre, Norwich; 4Inbiotec, León, Spain 

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used 

to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also 

constructed. individual cosmids from both the existing and new libraries were disrupted using the Tn5-based minitransposon Tn5062. A 

total of 35358 insertions were sequenced resulting in the disruption of 6482 genes (83% of the predicted OrFs). Complete information 

for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB (http://strepdb. 

streptomyces.org.uk/). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene 

function. 




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GM/41 Manuka honey effectively inhibits growth of Streptococcus pyogenes biofilms and has an impact on the expression of 

surface adhesins 

SArAH MADDOCKS, rose Cooper 

University of Wales Institute Cardiff (UWIC), Cardiff 

Group A streptococci (GAS; S. pyogenes) is the causative agent of numerous superficial and life threatening infections. GAS is transmitted to 

wounds post surgery, following skin grafts or traumatic wounds. Wounds provide a route of entry to the host and damaged tissues display a 

matrix of ligands to which S. pyogenes adhere and persist as a biofilm. Biofilms are intrinsically resistant to treatment. 

Manuka honey has established antimicrobial activity against bacteria, fungi, protozoa and viruses. its use in the clinical setting is re-emerging 

as a consequence of the rise in antibiotic resistant bacteria. This study shows that manuka honey can be used to inhibit the growth of 

biofilms of S. pyogenes and has identified the differential regulation of surface adhesins in response to honey treatment. 

Biofilms of S. pyogenes grown in the presence of 10% (w/v) manuka honey showed a reduction in biomass of approximately 50%, and 

growth was completely abrogated using solutions exceeding 20% (w/v). Molecular studies indicated that three surface adhesins (Sof, 

pilin and Spy0843), were differentially expressed in response to treatment with sub-lethal doses of honey. Therefore inhibition of biofilm 

development by manuka honey may be the result of altered expression of surface adhesins. 


GM/43 Improve the specificity of pan-viral microarray by using genus-specific oligonucleotide and reducing the interference 

of host genomes 

Yongqiang Jiang, Hong liu, Yuchang li, Guohui Chang, YiNHui YANG 

Beijing Institute of Microbiology & Epidemiology, Beijing, China 

Background: High-density 60–70 mer oligonucleotide microarrays have been explored for broad detection of a large number of viruses. 

However, relatively low specificity and the complex analytical processes are the major limitations when pan-viral oligonucleotide 

microarrays are used to detect viral pathogens. in this study, genus-specific oligonucleotides were employed as probes and modified sample 

preparations were carried out to improve the specificity and accuracy of the pan-viral oligonucleotide microarray. 

Methods: Genus-specific 63-mer oligonucleotide probes were used for screening human pathogenic rNA viruses. Host genomes were 

removed by DNasei/rNaseT1 digestion before viral nucleic acids extraction, and non-ribosomal hexanucleotides were used for reverse 

transcription to minimize the interference of host genomes. 

Results: By using DNasei/rNaseT1 digestion before viral nucleic acids extraction and non-ribosomal hexanucleotides for reverse 

transcription, the specificity of the microarray was improved. Moreover, the analytical process of hybridization results was simplified. 

Conclusions: The specificity of pan-viral microarray could be improved by using genus-specific oligonucleotides as probes and by using 

non-ribosomal hexanucleotides for reverse transcription. Combined with subsequent degenerate rT-PCr and sequencing process, this 

improved genus-specific oligonucleotides microarray provides a relatively flexible st 

GM/44 Metagenomic analysis of gut microbiota 

JuSTiN PACHEBAT, Kirsty Dougal, Tim Snelling, Eric Pinloche, Alejandro Belanche, Hilary Wogan, Susan Girdwood, 

Jamie Newbold 

Microbial genomics Lab, IBERS, Aberystwyth University, Aberystwyth 

Traditional culture based methods will only isolate between 1–5% of the microbial species present in the microbiome. Molecular techniques 

that allow researchers to study previously unculturable microbes, have shown that microbial communities are far more complex than 

previously thought. The advent of next generation sequencing has further revolutionised the field of metagenomics and enables researchers 

to take a detailed look at complex microbial populations. 

Here we report on the use of small subunit 16S rrNA hypervariable region 454 pyrosequencing to determine microbial diversity and 

species present in animal gut microbiota. We also comment on the use of solexa sequencing to characterize the meta-genomes and 

meta-transcriptomes of gut microbiota in animals. 




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GM/45 In vitro solubilization of insoluble fluorides by selected fungi 

SulAiMAN AlHArBi 

Sulaiman Ali Alharbi Dept of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, PO Box 2455, 

Saudi Arabia 

The ability of three species of Penicillium, isolated from soil, and Fusarium solani to solubilize insoluble fluorides was studied. All three 

Penicillium species and F. solani were capable of solubilizing calcium fluoride and fluorite. A single species of Penicillium and F. solani also 

solubilized a range of insoluble fluoride compounds. The ability of F. solani to solubilize calcium fluoride varied with the type of carbon 

source used and increased with an increasing amount of added fluoride up to 2% (w/v). No growth occurred above this amount. The 

addition of 1.5% (w/v) calcium fluoride also inhibited the formation of the black spores that are typically produced by Aspergillus niger, 

although sporulation returned when the fungus was transferred to medium lacking calcium fluoride. 

Keywords: Fluoride solubilization, fungi, Penicillium, Fusarium solani, environmental mycology. 

GM/46 Not received 

GM/47 Investigating the interactions of the plant-beneficial Pseudomonas fluorescens F113 with soil invertebrates 

MAriA SANCHEZ-CONTrErAS, Mario rincon 

Universidad Autonoma de Madrid, Madrid, Spain 

Fluorescent pseudomonads are efficient colonizers of the rhizosphere and phyllosphere of various plant species, including crops. in this 

ecological niche, bacteria are exposed to predation by bacteriophagous invertebrates such as nemadotes and amoeba. Therefore, it is 

possible that rhizospheric bacteria have molecular mechanisms evolved to face this ecological pressure. Genes homologous to those 

encoding insecticidal proteins have been found in the genomes of the three P. fluorescens sequenced strains (SBW25, Pf0-i and Pf5). 

recently, it has been demonstrated that strains SBW25 and Pf5 cause oral toxicity to Drosophila melanogaster larvae. 

The genome P. fluorescens F113 has been sequenced and it is at the stage of gap closure. As in other P. fluorescens strains, F113 

possess predicted proteins similar to insecticidal proteins, such as homologues to the tc genes that encode the Toxin Complex in the 

entomopathogenic bacterium Photorhabdus luminescens. Preliminary experiments with F113 showed toxicity against the model nematode 

Caenorhabditis elegans and the amoeba Acanthamoeba polyphaga. Experiments to test insecticidal activity of F113 towards larvae of Diptera 

(D. melanogaster) and lepidoptera (Galleria mellonella) are underway. 

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SC/01 Systems-wide investigation of the global rNA binding proteins, Hfq and rsmA, in virulence and secondary metabolite 

production in Serratia sp. ATCC 39006 

NABil WilF1 , robert Kingsley2 , Adam reid2 , Nicholas Croucher2 , Heidi Hauser2 , laurent Gatto3 , Svenja Hester3 , Kathryn lilley3 , 

Gordon Dougan3 , George Salmond3 1 2 3 Dept of Biochemistry, University of Cambridge, Camridge; The Wellcome Trust Sanger Institute, Hinxton; Cambridge Centre for 

Proteomics, University of Cambridge, Cambridge 

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium virulent in plant (potato) and animal (Caenorhabditis elegans) models. 

it produces two secondary metabolite antibiotics, prodigiosin and a carbapenem. A complex regulatory network controls production of 

prodigiosin, including a quorum sensing (QS) system and two global regulatory rNA-binding proteins, Hfq and rsmA. Construction of an 

S39006 ∆hfq mutant showed that production of prodigiosin and carbapenem was abolished and virulence was attenuated while an S39006 

rsmA transposon mutant was increased for production of prodigiosin and virulence. in order to define the complete regulon of Hfq and 

rsmA, deep sequencing of strand-specific cDNAs (rNA-seq) was used to analyse the whole transcriptome of S39006 ∆hfq and rsmA::Tn. 

Moreover, we have investigated global changes in the proteome using an lC-MS/MS approach with iTrAQ. Analysis of differential gene 

expression shows that Hfq and rsmA directly or indirectly regulate 2% and 11%of the genome with some correlation between rNA and 

protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. 

using a combination of transcriptomics and proteomics this study provides a systems-level understanding of Hfq and rsmA regulation and 

identifies similarities and differences in the regulons of two major regulators. 




163 




SC/02 Withdrawn 

↑Contents 

sC Cont. 

SC/03 Microbial encapsulation in monodisperse hydrogel microspheres enables fast and sensitive phenotypic analyses using 

flow cytometers 

lidia Delgado1 , Gloria Jurado2 , Gema Galayo2 , Elena Ogallar2 , lourdes Moreno3 , Juan Carlos rodriguez-Aguilera 4 , Angel Cebolla5 , 

Carolina Sousa3 , Maria Flores2 , SEBASTiAN CHAVEZ1 1 2 3 Dept of Genetics, Universidad de Sevilla, Seville, Spain; Ingeniatrics Tecnologias SL, Seville, Spain; Dept of Microbiology & 

Parasitology, Universidad de Sevilla, Seville, Spain; 4Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, Seville, 

Spain; 5Biomedal SL, Seville, Spain 

Characterization of micro-organisms usually involves culture during more than 20 generations in order to achieve the formation of 

macrocolonies on solid media. Alternatively, microencapsulation allows the detection of microbial growth by monitoring the development 

of microcolonies from encapsulated individual cells. Microbial proliferation inside the microcapsules can be detected using flow cytometry, 

provided that the population of microparticles exhibits appropriate optical and mechanical properties and is monodisperse in size and 

shape. 

using a CellENA ® Flow Focusing ® microencapsulator, we managed to produce monodisperse alginate microparticles containing individual 

bacteria, yeast and human stem cells. Alginate particle sizes were reproducibly selected from less than 100 μm to over 600 μm, by replacing 

the disposable nozzle. Sterility was preserved during the microencapsulation procedure, preventing undesired contaminations. 

Microencapsulated micro-organisms were used for a variety of application: from characterizing secreted enzymes to detection of 

thermosensitive mutants. Proliferation inside the particles was monitored by flow cytometry without requiring fluorescent labelling. 

SC/04 UbaA is required for the activation of ubiquitin-like proteins in Archaea 

NATHANiEl HEPOWiT, Sivakumar uthandi, Hugo Miranda, Julie Maupin-Furlow 

Dept of Microbiology & Cell Science, University of Florida, Gainesville, Florida, USA 

Proteins in eukaryotes can be post-translationally modified by ubiquitin (ub) and ubiquitin-like (ubl) proteins, thereby extending the 

functional diversity of the proteome. With the recent reports that ubls known as small archaeal modifier proteins (SAMPs) are central to 

the archaeal lineage, it is proposed that their conjugation use enzymatic mechanisms similar to those of eukaryotic ub/ubl-modification 

pathways. Eukaryotic ub/ubls are activated by E1 enzymes through adenylation and thioester bond formation prior to E2 and E3 mediated 

ligation. Although E2 and E3 are not conserved in archaea, we report here that the ubl activating enzyme of archaea (ubaA) is the archaeal 

counterpart of E1 using the haloarchaeon Haloferax volcanii as a model system. Activation involves the formation of a ubaA~SAMP 

complex, which is proposed to be thioester-linked between C188 of ubaA and the C-terminal glycine of SAMP. Knockout and single-point 

mutation studies revealed that K87 and C188 are crucial for SAMP conjugation. Certain ubaA residues not required for SAMP conjugation 

were crucial for growth in anaerobic conditions with dimethyl sulfoxide as a terminal electron acceptor. Along with phylogenetic analysis, 

this finding suggests that ubaA provides link between its eukaryal-shared role in ubl conjugation and bacterial-shared role in molybdenum 

cofactor biosynthesis. 



A.Talip, B., 144 

Abaitua, F., 79, 131 

Abbot, P., 49 

Abdul Wahab, A., 122 

Abiola, O., 149 

Aboobaker, A., 104 

Abouelhadid, S., 96 

Adams, D.J., 11 

Aebischer, T., 20, 61 

Ahmad, A., 160 

Ajioka, J., 102 

Ala’Aldeen, D., 141 

Albecka, A., 112 

Alberdi, P., 106 

Alharbi, S., 162 

Allahverdiyeva, Y., 126 

Allan, K., 154 

Allenby, N., 106 

Allers, T., 37 

Allnutt, J., 135 

Almond, N., 54, 85, 86, 111, 134 

Al-Mulla, H., 122 

Alonso, S., 44 

Alsam, S., 92 

Alshamaki, K., 140 

Altan-Bonnet, N., 7 

Alvarez, F.J., 42 

Alves Dorella, F., 125 

Aminov, r.i., 102 

Amorim, M.-J., 5, 67, 73, 127 

Amos, M., 11 

Andersson, S., 169 

Andrade-Junior, D.r., 117 

Andrew, P., 28 

Andrew, S., 142 

Angus, A., 65, 128 

Arabov, M., 25 

Arafah, S., 59, 62 

Aresté, C., 68 

Armesto, M., 86 

Armezzani, A., 108 

Armshaw, P., 155 

Arnaud, F., 108, 111 

Aro, E.-M., 126 

Arrand, J., 68 

Asfor, A., 118 

Asghar, A., 145 

Ashcroft, A., 81 

Ashman, r., 42 

Assafi, M., 141 

Assunta Cafiero, M., 156 

Atkins, E., 128, 129 

Atkins, T., 17, 95 

Atta, M., 143 


164 



AUTHOrS 

Auda, G., 111 

Azevedo, V., 125 

Bac Tran, H., 84 

Backx, M., 112 

Bacon, J., 135 

Bahar, M., 111 

Bailey, D., 66 

Baillie, G.J., 113, 123 

Bajwa-Joseph, M., 84 

Baker, A.H., 118, 132 

Bakker, l.J., 145 

Bakker, S., 77 

Balfe, P., 7, 82 

Ball, J., 112 

Baluchova, K., 67 

Banat, i., 94 

Banerjee, A., 101 

Bangham, C.r.M., 56, 81 

Banks, J., 69 

Barclay, W., 56, 69, 83, 87 

Barczynska, A., 105 

Barek Fatmi, M., 95 

Barfoot, H., 109 

Barke, J., 49 

Barker, A.M., 130 

Barker, G., 67 

Barnes, J., 104 

Baron, M.D., 58, 109 

Barr, J., 6, 56, 57, 67, 110, 119, 130 

Barras, F., 75 

Barras, S., 45 

Barrera, N., 138 

Baskcomb, l., 39 

Bassendine, M., 140 

Battchikova, N., 126 

Baums, C.G., 29 

Bavro, V., 138 

Bawn, M., 155 

Baxter, K., 140 

Baz Morelli, A., 21 

Bean, T., 6 

Beardmore, r., 12 

Becher, D., 21 

Bednarczyk-Drag, A., 105, 106 

Belanche, A., 161 

Bell, S., 79 

Bellais, S., 33 

Bell-Sakyi, l., 106 

Beltz, G., 21 

Belyaeva, T., 91 

Benfield, C., 111 

Bennett, M., 37 

Bentham, M., 118, 128 

Bentley, K., 86 

Berkhout, B., 54, 86 

Berrington, J., 139 

Berry, N., 54, 85, 86, 112 

Betts-Hampikian, H., 64 

Bhattacharya, B., 82 

Bhella, D., 76, 79, 128 

Bickerton, E., 82 

Bishop, K., 120 

Black, G., 155 

Black, S., 111 

Blackbourn, D.J., 68 

Blackford, A., 39 

Blagojevic, B., 44 

Blair, G.E., 55, 59, 91, 119, 120, 

129 

Blázquez, J., 126 

Blower, T., 153 

Blumenkrantz, D., 83 

Blythe, N., 152 

Bobat, S., 150 

Boel, E.C.H., 145 

Bohach, G.A., 102 

Bolgiano, B., 99 

Bolstad, M., 79, 131 

Bolt, E., 38, 114 

Bonda, A., 106 

Bonsall, D., 113 

Borland, A., 104 

Borley, D., 134 

Borodavka, A., 77 

Borrow, r., 33, 98 

Bosshard, r., 66 

Bouaboud, A., 33 

Boucherit, V., 120 

Boulant, S., 118 

Bourgeois, A.l., 20 

Bourke, S., 141 

Boutell, C., 4, 65, 118 

Bowater, r.P., 38 

Bowen, A., 115 

Bowie, A., 55 

Boyce, M., 121 

Bracegirdle, P., 36 

Bradshaw, A., 118 

Bradshaw, C., 140 

Brammer, K., 38 

Brehony, C., 17, 97 

Brennan, B., 71 

Breuer, J., 25, 55, 87 

Bridgen, A., 58 

Bright, H., 54, 112 

Briles, D.E., 28 

Brimacombe, C., 7, 82 

Britton, P., 82, 86, 120 

Brockhurst, M., 15 

Broderick, N.A., 50 

Brown, A., 150 

Brown, A.J.P., 42, 75, 123 

Brown, B., 140 

Brown, D., 102, 127 

Brown, G., 40 

Brown, J., 124 

Brown, J.W.S., 8 

Brown, l.E., 28 

Brown, N., 20 

Brown, r., 112 

Brown, S., 14, 92 

Browne, H., 132 

Browning, D.F., 150 

Brownlie, J., 58 

Bruce, E., 5, 67, 127 

Bryant, N., 88, 133 

Buchmeier, M.J., 76 

Buck, A., 73 

Buckley, A., 115 

Buckling, A., 15 

Bugert, J.J., 152 

Bull, A., 159 

Bull, J., 108 

Buonaurio, r., 95 

Burgess, P., 160 

Burkin, K., 99 

Burroughs, N., 108 

Burska, u., 137 

Butcher, S., 77 

Butt, A., 74 

Buttigieg, K., 23 

Cahill, G., 46 

Cai, A., 143 

Calabressi, S., 56 

Calcabrini, C., 156 

Callaghan, K., 45 

Cámara, M., 143 

Cambillau, C., 81 

Cantalupo-lima, C.B., 117 

Caplin, J., 137 

Caporale, M., 72, 108, 119 

Cardas, M., 92 

Care, r., 17, 97 

Carne, C., 142 

Carroll, C.V., 105 

Carroll, M., 23, 57 

Carter, S., 130 

Cartwright, r.A., 102 

Carvalho Pacheco, l.G., 125 

Cattoli, G., 134 

↑Contents 



Caws, M., 28 

Cebolla, A., 163 

Cehovin, A., 98 

Celma, C., 83 

Cerf-Bensussan, N., 102 

Chadwick, D., 146 

Chakraborty, T., 64 

Chamberlain, J.l., 19 

Chambers, G., 117 

Chan, A., 140 

Chan, C.N., 133 

Chandler, D., 108 

Chandra, G., 160 

Chang, G., 161 

Chapman, D., 90 

Chapman, r., 39 

Charalambous, B., 93 

Charles, i., 115 

Charleston, B., 26 

Charnock, S., 155 

Chaudhuri, r., 115 

Chavez, S., 163 

Cherepanov, P., 90, 133 

Cheung, W., 86 

Childs, K., 66 

Chinnakannan, S.K., 109 

Chotiyarnwon, P., 84 

Chovanec, M., 40 

Christodoulides, M., 142 

Chuang-Smith, O.N., 102 

Chung, J., 152 

Churton, N., 99 

Clark, D., 18 

Clarke, S., 99, 100 

Claus, S.P., 139 

Clayton, r., 133 

Clement, M., 143 

Clerc, i., 81 

Clipson, N., 147 

Clyne, M., 158 

Cogan, T., 103, 104, 151 

Cohan, F.M., 94 

Coldham, N.G., 44 

Cole, G.T., 18 

Cole, J., 127 

Coleman, H., 107 

Colenutt, C., 157 

Connelley, T., 102 

Connelly, S., 76 

Connerton, i.F., 45 

Connor, V., 52, 107 

Cook, J., 22 

Cooper, r., 125, 144, 156, 161 

Coote, J., 155 


165 



AUTHOrS 

Cope, l., 18 

Corcoran, M., 105 

Cormican, M., 105 

Corser, D., 17 

Cottam, E., 120 

Coughlan, l., 132 

Coulter, E., 123 

Coward, C., 103, 104 

Cowper, A., 84 

Cowton, V., 85 

Crabb, B., 62 

Crabtree, J., 137 

Cranage, M., 23, 54, 85, 86 

Crawford, C., 34 

Croucher, N., 162 

Crump, C.M., 4, 79, 131 

Crusz, S.A., 92 

Cubie, H., 117 

Cubitt, M., 151 

Cucchiarini, l., 156 

Cuccui, J., 95, 96 

Cuchet-lourenco, D., 4, 65 

Cumley, N., 64, 115 

Cummings, M., 133 

Cummings, S., 139, 140, 141, 146 

Cunningham, A.F., 19, 22, 150 

Cunningham, C., 67 

Cuschieri, K., 117 

Custers, J., 132 

Cutler, S., 143 

Cutting, S., 22, 157, 158 

da Silva Dantas, A., 123 

Daczkowska-Kozon, E., 105, 106 

Dadios, N., 44 

Daigaku, Y., 39 

Daikoku, T., 79 

Dajani, r., 157 

Dalby, M., 106 

Dale, J., 54 

Daly, J., 86 

Daniell, T., 47 

Darby, A., 110 

Darch, S.E., 92 

Dargan, D., 67 

Darsley, M.J., 20 

Das, A., 86 

Dasgupta, S., 89 

Datz, A., 54 

Davidson, A., 58, 68 

Davies, A.A., 39 

Davies (Helen), H., 47 

Davies (Holly), H., 115 

Davison, A., 67 

Dawson, K., 74 

Day, A., 124 

de Greeff, A., 29 

de Groot, r.J., 83 

de Koning-Ward, T., 62 

De lappe, N., 105 

de Paula Castro, T.l., 125 

De Soyza, A., 141 

de Wilde, A.H., 6 

Dean, P., 62 

Decorosi, F., 94 

Dedi, C., 138 

Dehghan, M., 104 

Dehio, C., 169 

Del Sol, r., 160 

Delday, M.i., 102 

Delgado, l., 163 

Delmas, S., 37 

Delury, C., 59 

Demontis, M.A., 56 

Depledge, D., 55, 87 

De-Soyza, A., 140 

Desra, A., 41 

Desselberger, u., 86 

Devescovi, G., 95 

Devi Kshetrimayum, J., 101 

Devi Sekaran, S., 100 

Di, Y., 74 

Diavatopoulos, D.A., 28 

Dickson, A., 10 

Dietrich, i., 84, 109 

Digard, P., 5, 6, 54, 67, 70, 73, 78, 

127 

Diggle, S.P., 92 

Dijksterhuis, J., 89 

Dillingham, M.S., 39 

Disson, O., 33 

Diston, D., 137 

Dittmar, M.T., 4 

Dixon, l., 90 

Dobbelaere, D., 63 

Dobner, T., 39 

Dobson, P., 5 

Doceul, V., 6 

Dockery, P., 105 

Dodd, C., 47, 104 

Dodding, M.P., 8, 9 

Dolan, B., 158 

Dooley, J., 144 

Doran, G., 105 

Doran, K.S., 29, 101 

Douce, G., 24 

Dougal, K., 161 

Dougan, G., 162 

Douglas, A.E., 48, 49 

Dove, B., 6, 119 

Dover, l., 159 

Dowson, C., 125 

Doyle, E., 147 

Doyle, N., 5 

Drane, D., 21 

Dreja, H., 23 

Droese, B., 76 

Dubuisson, J., 112 

Dudasova, Z., 40 

Duffy, M., 118 

Dumoux, M., 61, 142 

Dunn, l., 88 

Duprex, P., 25 

Dutia, B.M., 121, 122 

Dykeman, E., 77 

Dyson, P.J., 160 

Dziadek, J., 37 

Ebdon, J., 137 

Ebert, T., 18 

Ebrahimi, B., 65, 121 

Edwards, J., 16 

Edwards, T., 119 

Efstathiou, S., 52, 56, 107 

El ridi, r., 20 

El-Ghazawi, r., 119 

Elgueta Karstegl, C., 66 

Elliott, G., 80 

Elliott, l., 100 

Elliott, r.M., 71, 121, 122 

Ellis, P., 113 

Elsheikha, H., 117 

Elton, D., 88, 133 

Eltringham, G., 140 

Embley, T.M., 62 

Emes, r., 109 

Emmott, E., 110 

Erlwein, O., 55 

Ermakova, M., 126 

Errington, J., 106 

Evans, D.J., 53, 68, 108 

Evans, M.C., 160 

Evans, V., 67 

Everest, P., 154 

Everett, r., 4, 65 

Exley, r., 31, 98 

Fairweather, N., 22 

Fan, H., 18 

Fan, W.H., 79 

Farleigh, l., 152 

Farre, D., 72 

↑Contents 



Farrell, P.J., 66 

Fast, N.M., 89 

Faust, S., 100, 142 

Fazeli, M., 104 

Fazil Baksh, M., 76 

Feavers, i., 17, 97 

Felton, V., 121 

Feng, Q., 42 

Ferguson, B., 111 

Ferguson, D., 54, 86 

Fernández-Martínez, l.T., 160 

Ferrero, r.l., 41 

Fidler, S.J, 113 

Fielding, S., 160 

Fields, K., 64 

Fierens, K., 24 

Figueira, r., 116 

Filipe, A., 129 

Filloux, A., 157 

Findlow, J., 33, 98 

Firowicz-Krosko, J., 47 

Firth, A., 70 

Fishwick, C., 85 

Fitzgerald, J.r., 102 

Fitzgerald, P., 12 

Flack, D., 88 

Flanagan, A., 84 

Fleming, G., 51 

Flores, M., 163 

Fodor, E., 70 

Foeglein, A., 67 

Fooks, A.r., 52, 113 

Ford, r., 130 

Forns, x., 112 

Forrest, S., 118 

Forsythe, S., 99 

Foster, K., 13 

Foster, P.G., 62 

Foster, r., 6, 87 

Foster, T.l., 6, 87, 129 

Fothergill, J., 94 

Foweraker, J., 152 

Fox, N., 119 

Franz, S., 56 

Franzoni, G., 72 

Freemont, P., 157 

Freudenreich, C.H., 39 

Frosi, G., 33 

Früh, K., 3, 27 

Fukatsu, T., 48 

Fulde, M., 29 

Funnell, A., 45 

Futcher, B., 25 

Futreal, A., 141 


166 



AUTHOrS 

Gabius, H.-J., 31 

Gaboriau-routhiau, V., 102 

Gadsby, N., 100 

Galante, D., 156 

Galayo, G., 163 

Galbraith, J., 67 

Galiano, M., 123 

Gall, A., 113, 123, 141 

Gantier, M., 41 

Gao, F., 99 

García-Sastre, A., 43 

Garden, K., 102 

Gardiner, P., 147 

Gardner, A., 14, 15 

Garelick, H., 149 

Garson, J., 141 

Gatherer, D., 67, 153 

Gatto, l., 162 

Gay, N., 43 

Geijtenbeek, T.B.H., 43 

George, A.J., 96 

Georgiadou, M., 98 

Gerado, N., 48 

Gerhard, r., 29 

Gerondopoulos, A., 5 

Gerwig, G.J., 83 

Giese, S., 84 

Gifford, r., 109 

Gilbert, r., 80 

Gilchrist, S., 33 

Gill, M., 9 

Gillespie, S., 93 

Gilson, P., 62 

Girdwood, S., 161 

Gladstone, r., 100 

Glauser, D.l., 107 

Glover, B.J., 91 

Goethe, r., 29 

Gog, J., 70, 78, 91 

Goh, Y.S., 115 

González-roldán, N., 40 

Goodbourn, S., 44, 66 

Goodfellow, i., 66, 122, 128 

Goodfellow, M., 101, 106, 159 

Goss, r., 49 

Gottschalk, M., 28 

Goulding, D., 56 

Gow, N.A.r., 42, 75 

Graham, S., 111 

Graham, S.V., 71, 117 

Gramatiuk, S., 114 

Gramiri, P., 103 

Grand, r., 39 

Grant, A.J., 60, 115 

Grant, K., 65 

Gravel, S., 39 

Gray, D., 135 

Gray, E., 55, 87, 141 

Gray, r., 51 

Gray, S., 135 

Grayson, N., 77 

Greatorex, J., 127 

Greber, u., 3 

Greenhalgh, A., 94 

Greenhow, J., 54 

Gregorovic, G., 66 

Gregory, A., 62 

Griffin, A., 92 

Griffin, S., 6, 9, 87, 128, 129 

Griffiths, D., 55 

Griffiths, P., 25 

Grimes, J., 84, 111 

Grinter, r., 155 

Gritsun, T., 52 

Groppelli, E., 5 

Grove, J., 82 

Grüschow, S., 49 

Guarnaccia, C., 95 

Gudelj, i., 11 

Guého, A., 59 

Guerrero Alonso, A., 4 

Guinane, C.M., 102 

Gulletta, S., 72, 154 

Gundurao, r.M., 114 

Haas, J., 56, 114 

Haase, S., 62 

Habets, M., 15 

Hagedorn, M., 62 

Hague, C., 9 

Haider, J., 159 

Haider, M., 59 

Haimbach, r., 18 

Hall, N., 110 

Hall, r.A., 42 

Hall, Y., 23 

Ham, C., 85 

Hammad, H., 24 

Hanage, W.P., 27 

Hancox, l., 47 

Hanlon, G., 138 

Hannemann, H., 58, 68 

Hardham, J., 21 

Hare, S., 90, 133 

Harmer, N., 19 

Harris, H., 82 

Harris, M., 6, 9, 57, 72, 85, 109, 128, 

129 

Harrison, E., 156 

Harro, C., 20 

Harte, C., 130 

Hartsuiker, E., 37 

Harvala, H., 30, 100 

Harwood, C., 124 

Hasan, Z.-u., 150 

Hassall, M., 134 

Hatch, K., 135 

Hato, S., 42 

Hauser, H., 162 

Hawes, P., 120 

Haxton, B., 52 

Haynes, K., 75 

Hayward, r., 61, 142 

He, M., 23 

Heath, C., 142 

Heath, P., 32 

Heckels, J., 142 

Hecker, M., 76 

Hector, r., 67 

Heikkilä, O., 77 

Heinz, E., 62 

Helaine, S., 116 

Helenius, A., 3 

↑Contents 

Henderson, i.r., 19, 150, 151 

Henriques Norrmark, B., 30 

Hepowit, N., 163 

Hermann, M., 31 

Herod, M., 68 

Herron, P.r., 160 

Herzyk, P., 67 

Hester, S., 162 

Hewson, r., 57 

Hey, A., 97 

Hibberd, M., 97, 99 

Hicks, D., 52 

Hilburn, S., 56 

Hinson, T., 61 

Hinton, J., 116 

Hiscox, J., 6, 56, 57, 110, 119, 130 

Hitchcock, J.r., 150 

Hodgson, C., 146 

Holden, D., 60, 116 

Holden, N., 46, 47 

Holling, N., 138 

Hollinshead, M., 6 

Holmes, E., 139 

Holmes, K.K., 80, 118, 129 

Holst, O., 40 

Holzer, B., 58 

Homans, S., 6 

Hong, H.A., 157, 158 

Hood, D., 31 



Hood, S., 54, 111, 112 

Hope, G., 9 

Hopper, A., 127 

Horne, A., 117 

Hornsey, C., 53 

Horrocks, A.J., 13 

Hosie, M., 84, 108, 109 

Hosnil, T., 95 

Hosseinzadeh, S., 104 

Houldcroft, C., 141 

Howard, J., 114 

Howe, r.A., 44 

Howell, G., 120 

Hu, K., 7 

Huang, J.M., 22 

Hubscher, S., 7 

Hudson, D., 115 

Hudson, M., 140 

Hué, S., 141 

Hughes, D., 69, 110 

Hughes, M., 72 

Hughes, S., 90 

Huizinga, E.G., 83 

Humphrey, T., 103, 104 

Humphries, A., 8 

Hung, C.-Y., 18 

Hunt, N., 124 

Hurtgen, B., 18 

Hussain, A., 148 

Hussain, S., 88 

Hutchings, M., 49 

Hutchinson, E., 70, 78 

Huynh, H., 22 

Hyppiä, T., 77 

igloi, Z., 109 

ikebukuro, K., 145 

imhof, i., 87 

ireland, P., 152 

irving, A., 41 

islam, N.M., 89 

ismael, M., 147 

ismail, M., 137, 138 

ismail, r., 58 

ivancic-Bace, i., 114 

Jabar, M., 147 

Jackson, S., 39 

Jackson, T., 5, 134 

Jacobsen, M., 75 

Jagger, B., 54, 70 

Jagmann, N., 93 

Jalal, H., 142 

James, J., 150 


167 



AUTHOrS 

James, N., 120 

Janganan, T., 138 

Jarrett, O., 108 

Jauneikaite, E., 99 

Jefferies, J., 99, 100 

Jeffs, S., 134 

Jenkins, l., 125 

Jenkins, r., 156 

Jenkins, V., 90 

Jenner, r., 5 

Jiang, Y., 161 

Jironkin, A., 108 

Johnson, N., 52, 113 

Johnston, S.A., 63 

Jones, A.l., 36, 146 

Jones, B., 136, 137, 138, 145 

Jones, M., 87 

Jones, N., 74 

Jones (Daniel), D., 68 

Jones (David), D., 149 

Joseph, S., 99 

Joshi, A., 18 

Jourdan, S., 110 

Jung, S.-Y., 137, 138 

Jungck, J.r., 10 

Jurado, G., 163 

Kainz, M., 130 

Kalinina, N., 8 

Kaloriti, D., 75 

Kaltenpoth, M., 49 

Kalverda, A., 6 

Kamerling, J.P., 83 

Kämpfer, P., 35 

Kanda, r., 55, 87 

Kang, A., 23 

Kang, x., 157 

Kang, Z., 70 

Kanneganti, T.-D., 41 

Kaparakis-liaskos, M., 41 

Karayiannis, P., 54 

Kash, J., 54, 70 

Katayama, Y., 148 

Katzourakis, A., 141 

Kaup, F., 119 

Kaye, P., 20 

Kaye, S., 55, 113 

Kazimoto, T., 156 

Kazlauskas, A., 109 

Keef, T., 132 

Kellam, P., 5, 56, 72, 113, 123, 

141 

Kelly, D., 41, 102 

Kelly, K., 46 

Kelly, S., 124 

Kendall, S., 160 

Kenyon, D., 46 

Kenyon, J., 78 

Kerry, r., 140 

Khan, N.A., 31, 92, 98 

Khawaja, A., 70 

Killip, M., 66 

Kim, B.J., 101 

Kim, B.-Y., 101, 106 

Kim, S.H., 8 

King, B., 129 

King, D., 118 

King, E., 104 

Kingsley, r., 162 

Kipar, A., 110 

Kirsebom, l.A., 89 

Kiss, G., 76 

Klapper, P., 140 

Klasson, l., 169 

Klaus, J.P., 76 

Klimach, S., 125 

Knowles, N., 118 

Knowles, T.J., 150 

Koernig, S., 21 

Koethe, S., 82 

Kondracka, J., 30 

Kool, M., 24 

Kotte, A.-K., 159 

Koutsoudakis, G., 112 

Kowalewska, M., 46 

Kowarik, M., 95 

Kreft, J.-u., 15 

Krogfelt, K., 156 

Kroll, J.S., 97 

Kroschewski, H., 58 

Krstevska, K., 21 

Kucharski, A., 91 

Kudryavtseva, K., 78 

Kuhn, P., 76 

Kumar, N., 100 

Kumar (Navin), N., 142 

Kunding, A.H., 76 

Kunji, E.r., 62 

Kusters, J.G., 145 

l’Hernault, A., 127 

ladhani, S., 34 

laForce, F.M., 34 

lai, i.Y.-C., 113 

lamb, D.C., 124 

lambrecht, B.N., 24 

lan, A., 102 

langdon, r.H., 95, 96 

langereis, M.A., 83 

langford, P., 97 

langridge, G., 115 

laqtom, N., 73 

laurinmäki, P., 77 

lawrance, l.M., 135 

le Grice, S., 78 

lecuit, M., 33 

lednor, D., 138 

legiewicz, M., 78 

lenthall, K., 146 

lenzi, P., 81 

leProust, E., 55 

lereclus, D., 93 

leung, M., 93 

lever, A., 78, 86, 127 

levitz, S.M., 16 

leyton, D.l., 150 

li, M.-S., 97 

li (Yanwen), Y., 31 

li (Yuchang), Y., 157, 161 

lichière, J., 81 

lickorish, F., 46 

liefhebber, J., 9 

ligertwood, Y., 122 

lilley, K., 162 

lim, J., 146 

limpitikul, W., 84 

lin, F., 157 

lin, r., 99 

lithgow, P., 90 

lithgow, T., 151 

litt, D., 34 

liu, H., 161 

lloyd, r., 155 

locker, N., 122 

lockyer, K., 99 

loerger, T., 150 

logan, E., 102 

loh, l.N., 98 

lomonossoff, G., 81 

long, J., 69 

loquet, A., 128 

lossi, N., 157 

lotter, H., 40 

lourenco, S., 78 

lowe, E., 154 

lowery, C., 144 

lowry, K., 68 

lozach, P.-Y., 3 

lucchesi, D., 31 

lucidarme, J., 98 

luisi, B., 153 

lukashchuk, V., 65 

↑Contents 




168 



AUTHOrS 

Maccallum, A., 154 

Macdonald, A., 55, 59, 129 

MacDonald (Sandy), S., 49 

MacDonald (Sarah), S., 154 

MacFarlane, S., 8 

Maclennan , C.A., 19, 150 

Macnab, S., 110 

Maddocks, S., 144, 161 

Maertens, G., 90 

Mahapatra, M., 118, 134 

Mahdavi, J., 141 

Mahen, r., 67 

Maiden, M.C.J., 17, 27, 97 

Maier, H., 120 

Makino, S., 76 

Malasit, P., 84 

Mamczur, N., 85 

Mandic-Mulec, i., 94 

Mankouri, J., 9 

Mannering, S., 41 

Mansfield, K., 52, 113 

Manso, B., 65 

Mansur, D., 55, 111 

Maraskovsky, E., 21 

Marcano, C., 102 

Marchant, r., 94 

Marchesi, J., 136 

Maren-Ellegaard, K., 169 

Maringer, K., 80 

Markiv, A., 23 

Maroof, A., 20 

Marques Silva, W., 125 

Marreddy, r.K.r., 126 

Marriott, A., 135 

Marsh, M., 84 

Marsh, P., 135 

Marshall, A., 104 

Marshall, J.l., 19 

Marshall, l., 152 

Martínez-Pomares, l., 143 

Martino, A., 99 

Martin-Serrano, J., 79 

Marty, A., 85 

Maskell, D.J., 60, 103, 104, 115, 150 

Mason, G., 53 

Mastroeni, P., 60, 115 

Matin, A., 137, 138 

Matsuo, E., 69 

Matthews, D., 67 

Matthews, l.A., 135 

Mattiuzzo, G., 85 

Maupin-Furlow, J., 163 

May, r.C., 63, 64, 115, 150 

Mazel-Sanchez, B., 71 

McCauley, J., 88 

McClure, M.O., 55, 113 

McClure, P., 112 

McConvile, C., 68 

McCormick, C., 68 

McCowen, J., 23 

McCutcheon, J., 47 

McDonald, r., 148 

McEwan, W., 109 

McFarlane, M., 71 

McFarlane, W., 136 

McHugh, P.J., 40 

McHugh, T., 135 

Mcintyre, C.l., 30 

McKeating, J., 7, 82 

McKinley, T.J., 115 

McKnight, Á., 23 

Mclaren, S., 141 

Mclauchlan, J., 7, 9, 57, 68, 118, 129 

Mcleish, N., 30 

McMonagle, E.l., 84, 133 

McNeely, T., 18 

McNeill, J.A., 22 

McPhee, H., 80, 128 

McPherson, S., 140 

McShane, H., 19 

McVey, J., 118 

McWilliam leitch, E.C., 30 

Medcalf, l., 133 

Mee, E., 112 

Mehmood, K., 137 

Meier, r., 3 

Meijer, W., 160 

Meissner, M., 61 

Melcher, A., 119 

Mellits, K., 47, 104 

Mendes Souza, B., 125 

Mercer, J., 3 

Meredith, l., 82 

Mertens, P., 119, 154 

Mestdagh, r., 139 

Metzger, D.W., 22 

Midgley, C., 84 

Mifsud, J., 62 

Mifsud, K., 62 

Mika, J., 73 

Milho, r., 52 

Militão, F., 125 

Millan, D., 117 

Miller, C., 140 

Miller, J., 88 

Mills, D., 95 

Mills, K.H.G., 24 

Milne, l., 13 

Milne, T., 154 

Minor, P., 112 

Minton, N., 159 

Miranda, H., 163 

Mirza, D., 112 

Mitchell, J., 111 

Mitchell, S., 47 

Mitter, r., 8 

Miyoshi, A., 125 

Mohl, B.-P., 57 

Moloney, S., 149 

Monaghan, P., 5, 120 

Money, V., 80, 128 

Mongkolsapaya, J., 84 

Montesso, F., 133 

Montserret, r., 129 

Moody, S.C., 124 

Moore, J., 108 

Mora-Montes, H., 42 

Moreno, l., 163 

Moretti, C., 95 

Morgan, B.A., 123, 124 

Morgan, E., 115 

Morgan, F., 60 

Morris, D., 105 

Morris, F., 151 

Morris, F.C., 150 

Morrison, W.i., 102 

Mosbahi, K., 151, 155 

Moses, S., 140 

Mothes, W., 8 

Moule, M., 95 

Moye-rowley, W.S., 74 

Mrazek, J., 70 

Mueller, S., 25 

Mulder, i.E., 102 

Mumper, r.J., 22 

Munday, D., 6, 56 

Munir, i., 160 

Munnoch, J.T., 124 

Murgia, C., 108 

Murphy, l., 111 

Murray, J., 65 

Mutocheluh, M., 68 

Mutylala, N., 106 

Nair, V., 26 

Nakagawa, T., 146 

Nakano, Y., 160 

Narayanan, M., 139 

Natale, P., 126 

Naughton, J., 158 

Nawaz, S., 138 

Nejmeddine, M., 81 

↑Contents 

Nelson, A., 141 

Nelson, J., 148 

Netea, M.G., 42 

Neuman, B.W., 76, 122, 130 

Newbold, J., 161 

Newton, A.C., 46 

Newton, r., 133 

Ngugi, S., 17, 96 

Nicco Yu, Y.-T., 169 

Nicholson, J.K., 139 

Nicklin, S.A., 132 

Nicol, C., 91 

Nicoll, M., 52 

Nielsen-leroux, C., 93 

Nigsch, A., 44 

Nimnual, A., 25 

Nizam, A., 152 

Noerenberg, M., 71 

Noll, E., 114 

Noofeli, M., 155 

Norville, i., 19 

Nosanchuk, J.D., 62 

Nunez, A., 52 

Nzakizwanayo, J., 136 

O’Byrne, C., 76, 125 

O’Connor, J., 105 

O’Donovan, M., 142 

O’Hagan, D., 21 

O’Hare, P., 79, 131 

O’Shea, K., 19 

Obiefuna, O., 148 

Odds, F.C., 42 

Oehler, V., 129 

Ogallar, E., 163 

Ogilvie, l., 137 

Okoro, C., 159 

Okoroma, E., 149 

Oldfield, N., 141 

Oldring, A., 111 

Olver, W., 136 

Orr, A., 4, 65 

Ou, H.-Y., 156 

Overduin, M., 150 

Owsianka, A., 85 

Oyston, P., 154 

Pachebat, J., 102, 161 

Pader, V., 143 

Page, M., 54, 86, 134 

Paillot, r., 133 

Palmarini, M., 72, 108, 111, 119, 154 

Palser, A., 72, 113, 123 

Pancari, G., 18 




169 



AUTHOrS 

Panjwani, A., 131 

Pardieu, C., 5 

Parish, T., 150 

Park, A., 86 

Park, J.Y., 102 

Parker, A.l., 118, 132 

Parker, l., 112 

Parkhill, J., 115 

Parton, r., 155 

Pasdeloup, D., 79 

Patel, A., 57, 65, 85, 128 

Patel, B., 138 

Patell, S., 150 

Paterson, G.K., 150 

Patil, Y., 101 

Paton, D., 118, 134 

Patterson, M.J., 75, 123 

Payne, l., 122 

Pearson, A., 6, 77 

Peeters, M., 107 

Pei, x.-Y., 153 

Pelchen-Matthews, A., 84 

Pelicic, V., 98 

Pembroke, J.T., 155 

Penin, F., 128, 129 

Pereira, C., 73 

Pereira Domingueti, C., 125 

Perez-del-Pulgar, S., 112 

Perfect, J.r., 32 

Permpoonpattana, P., 22, 157, 158 

Perry, J., 139, 140, 141 

Peters, S.E., 115, 150 

Petito, J., 94 

Pettersson, B.M.F., 89 

Phetcharaburanin, J., 22 

Philipp, B., 93 

Phillip, P., 78 

Piddock, l.J., 150 

Pillay, D., 141 

Pinloche, E., 161 

Pinto, A.C., 125 

Pleasance, S., 115 

Pohl, S., 124 

Pollitt, E.J.G., 92 

Polo, S., 39 

Polonca, Š., 94 

Pongor, S., 95 

Poole, E., 53, 70 

Poolman, B., 73 

Popat, r., 92 

Pospisek, M., 70 

Potenza, l., 156 

Poyart, C., 33 

Price, J.T., 28 

Price, l., 112 

Prior, J., 17, 95, 96 

Proença, J., 52, 107 

Prowse-Davis, l., 133 

Pullinger, G., 115 

Purcell, P., 140 

Purchase, D., 149 

Pybus, O., 123, 141 

Quattroni, P., 31 

Quinn, J., 75, 123 

Qureshi, A., 53 

raghunathan, D., 150 

rahman, M., 96 

rajakumar, K., 156 

ralebitso-Senior, K., 149 

ramachandran, V., 116 

ramage, G., 139 

rambaut, A., 123 

ramstedt, M., 152 

rance, r., 113 

rand, J., 124 

randall, r., 66 

ranson, N., 77 

rash, A., 88, 133 

ratinier, M., 72, 119, 154 

ratnayake, l., 136 

rauch, S., 79 

raven, K., 70 

raymond, B., 93 

read, E., 5, 67, 73 

reece, S., 14 

reed, S., 37 

reed, S.J.F., 58 

reeves, M., 70 

reid, A., 162 

reid, C., 158 

ren, Y., 79 

restif, O., 115 

reynolds, G., 7 

richards, K., 55, 59 

richards, S., 154 

rietsch, A., 152 

rinaldi, A., 139 

rincon, M., 162 

risco, C., 9 

ritchie, N., 102 

rixon, F.J., 79, 119 

rizvi, T., 78 

robb, N., 70 

roberts, i.S., 96 

roberts, K., 83 

roberts, l.O., 5, 122 

roberts, S., 59 

roberts, A., 144 

roberts, A.P., 79 

robinson, C., 138 

robinson, M., 55, 57, 134 

robinson, P., 137 

rodríguez rojas, A., 126 

rodriguez-Aguilera, J.C., 163 

rogers, M., 110 

rohde, M., 29 

rose, N., 54, 85, 111, 112 

rosenthal, P., 88 

ross, r., 129 

ross-Thriepland, D., 72 

rowe, M., 10 

rowlands, D., 77, 80, 118, 131 

roy, P., 58, 69, 78, 82, 83, 121 

royall, E., 122 

roznovsky, l., 70 

rubin, S., 25 

rudden, M., 94 

rusch, A., 39 

ryabov, E., 8, 53, 108 

ryan, A., 158 

rzhepishevska, O., 152 

Sacchettini, J., 150 

Saddler, G., 46 

Sagot, i., 89 

Sait, l., 103, 104 

Saito, H., 74 

Saito, M., 148 

Saksela, K., 109 

Salmond, G., 151, 153, 162 

Salthouse, D., 131 

Sanchez-Contreras, M., 162 

Sanders, H., 17, 97 

Sanderson, J., 80, 128 

Santhakumar, D., 73 

Santhanam, r., 159 

Santos, S.A., 117 

Sarkar-Tyson, M., 19, 96, 152, 154 

Sasakawa, C., 64 

Sato, S., 129 

Sattler, N., 59 

Sauder, C., 25 

Sauerwein, r.W., 20 

Saugar, i., 39 

Saunders, K., 81 

Savory, N., 145 

Sawicki, S.G., 76 

Saxena, P., 81 

Schepelmann, S., 112 

Schiebler, M., 93 

↑Contents 

Schipper, M.E.i., 145 

Schlievert, P.M., 102 

Schmidt, F., 3 

Schneider-Schaulies, S., 82 

Schnurr, M., 21 

Schofield, l., 169 

Schregel, V., 118 

Schreier, H., 148 

Schroeder, J., 20 

Schroeter, A., 15 

Schubert-unkmeir, A., 30 

Schüller, C., 73 

Schuster, M., 14 

Schuster, S., 15 

Schwarz, K., 159 

Scott, A., 96 

Screaton, G., 84 

Seaman, M., 134 

Sedaghat Jahromi, S., 104 

Segura, M., 31 

Seipke, r., 49 

Seitsonen, J., 77 

Seitz, M., 29 

Sekine, T., 148 

Selkrig, J., 151 

Senior, E., 149 

Seok Seo, K., 102 

Seyffert, N., 125 

Shannon, T., 51 

Shannon-lowe, C., 10 

Sharma, C., 116 

Sharp, C., 107 

Shaw, A., 72, 119, 154 

Shaw, J., 8 

Shaw, r.K., 150 

Shearer, N., 116 

Shekarforoush, S.S., 104 

Shelton, H., 83 

Shepherd, D., 80, 81 

Sheridan, V., 121 

Sherry, A., 154 

Sherwani, S., 152 

Sheth, C.C., 42 

Short, F., 153 

Short, K.r., 28 

Siddell, S.G., 76 

Siersema, P.D., 145 

Siggins, M.K., 19 

Sihera, M., 143 

Sik Kim, K., 32 

Silva, A., 21 

Silva (Artur), A., 125 

Simmonds, P., 30, 53, 87, 107 

Simmons, C., 84 




170 



AUTHOrS 

Simpson, J., 120 

Sinclair, J., 53, 70 

Sinden, r.E., 18 

Singh, B., 89 

Singh, S., 143 

Singleton, i., 104 

Sinkovits, r., 77 

Skarnes, B., 56 

Skiena, S., 25 

Slack, M., 34 

Smith, A., 119 

Smith (David), D., 36 

Smith (Deborah), D., 20 

Smith, D.A., 123 

Smith, G., 6, 26, 55, 111 

Smith, H., 29 

Smith (Karen), K., 139, 155 

Smith (Ken), K., 160 

Smith, l., 115 

Smith, S., 18 

Smith, T., 140, 147 

Smith (Paul), P., 117 

Smith (Philippa), P., 39 

Snelling, T., 161 

Snijder, E.J., 6 

Snow, l.C., 44 

Sode, K., 145 

Sokoloski, K., 10 

Soldati, T., 59, 62 

Solomon, T., 113 

Sousa, C., 163 

Sparagano, O., 156 

SPArTAC Trial investigators, 113 

Speirs, V., 119 

Spencer, T.E., 111 

Spiller, r., 104 

Stack, S., 65 

Stackebrandt, E., 34 

Stahlhut, S., 156 

Stamou, D., 76 

Stanley, K., 140 

Starr, E., 76 

Stead, D., 123 

Stebbings, r., 54, 86 

Stel, H.V., 145 

Stella, M., 33 

Stevens, M., 115 

Stevenson, P.G., 9, 52, 107 

Stewart, C., 139 

Stewart, G., 39 

Stewart, H., 108 

Stewart, J., 65, 110, 121 

Stewart, M., 58 

Stockley, P.G., 77, 130 

Stokes, M., 44 

Stonehouse, N., 80, 81, 91, 118, 

131 

Stoodley, P., 11 

Storey, S., 147 

Strugnell, r.A., 28 

Struve, C., 156 

Stuart, A., 5, 67, 127 

Stuart, D., 111 

Suarez-Moreno, Z.r., 95 

Subbarao, J., 156 

Sumner, r., 55 

Sung, P., 68 

Surtees, r., 6, 57 

Susi, P., 77 

Sutcliffe, i.C., 35, 159 

Svobodova, S., 4 

Swati, Z.A., 160 

Sykes, A., 65 

Takamatsu, H., 90 

Taliansky, M., 8 

Tallima, H., 20 

Tan, C.P., 141 

Tan, H., 153 

Tan, l., 98 

Tanaka, K., 74 

Tanaka, Y., 81 

Tang, C., 31, 32, 98 

Tang, J., 23 

Tanner, S., 78, 119 

Tannich, E., 40 

Tardieux, i., 33 

Targett-Adams, P., 87 

Tariq, V., 12 

Tarlinton, r., 109 

Tarr, A., 112 

Tas, A., 6 

Tatebayashi, K., 74 

Tatineni, r., 87 

Taubenberger, J., 54, 70 

Tavassoli, A., 154 

Taylor, G.P., 56, 81 

Taylor, H., 137 

Taylor, M., 39 

Taylor, S., 124 

Tazi, A., 33 

Teale, C.J., 44 

Tedder, r., 55 

Tee, N., 100 

Tegtmeyer, N., 158 

Temperton, N., 134 

Templeton, K., 30, 100 

Teng, Y., 37 

Terra, V.S.A., 95, 96 

Tettmar, K., 55 

Thomas, C., 58 

Thomas, G., 49 

Thomas, r., 95 

Thompson, A., 116 

Thompson, C., 13 

Thompson, D., 33 

Thompson, E.P., 91 

Thompson, G., 6 

Thompson, J., 87 

Thorne, l., 66 

Thuenemann, E., 81 

Thuring, J., 133 

Tillmann, A., 75 

Tindall, B.J., 35 

Titball, r., 17, 19, 152 

Tolls, E., 158 

Tonks, A., 152 

Tötemeyer, S., 47 

Toth, i.K., 46, 151, 153 

Tourigny, D., 151 

Towers, G., 84, 85, 141 

Townsend, r., 140 

Trantham, E., 103, 151 

Travé, G., 91 

Trieu-Cuot, P., 33 

Tsao, E., 5 

Tu, W.Y., 124 

Tucker, N., 124 

Tuke, P., 55 

Tuma, r., 77, 130 

Tuomanen, E.i., 30 

Turnell, A., 39 

Turner, D., 115 

Turner, K., 115 

Turner, l., 41 

Turner, r., 9 

Turrel, l., 122 

Tuthill, T., 131 

Tveen Jensen, K., 150 

Twarock, r., 77, 131, 132 

Tyler, S., 87 

Tzortzakis, N., 104 

udakis, l., 51 

ulrich, H.D., 39 

umeh, C., 148 

unterholzner, l., 55 

urbanowicz, r., 112 

uthandi, S., 163 

utratna, M., 76, 125 

uusi-Kerttula, H., 132 

Valappil, M., 140 

Valdivia, r.H., 60 

Valentin-Weigand, P., 29 

van Aartsen, J., 156 

van den Brande, J.H.M., 145 

van der linden, l., 6 

van Diemen, P., 115 

van Doremalen, N., 83 

van Hemert, M.J., 6 

van Knippenberg, i., 121 

van Kuppeveld, F., 42 

van Nimwegen, M., 24 

Van Oijen, A., 3 

Vanni, E., 4 

Varani, G., 19 

Vasanawathana, S., 84 

Vater, S., 122 

Vavre, F., 50 

Veal, E., 124 

Veesler, D., 81 

Velicer, G.J., 169 

Venturi, V., 95 

Verbeke, C., 119 

Veronesi, E., 119 

Verow, M., 85, 129 

Vervoort, S., 83 

Vicente, M., 126 

Vickers, A., 34 

Vigašová, D., 40 

Vinkovic, D., 138 

Viti, C., 94 

Voelz, K., 115 

Vogel, J., 116 

Vopalensky, V., 70 

Vos, A., 133 

Wacker, M., 95 

Wagner, J., 42 

Waiyaiya, E., 84 

Wakelam, M., 9 

Waker, D., 155 

Waldron, K., 124 

Walker, D., 139, 151 

Walker, r., 20 

Walladge, B., 88 

Walmsley, A., 138 

Walmsley, M.i., 138 

Walsh, M., 124 

Walter, C., 57, 67 

Wang, J., 115 

↑Contents 




171 



AUTHOrS 

Ward, C., 25 

Ward, J., 105 

Ward, r., 58 

Ward, T., 98 

Ward, T.A., 40 

Wardman, J., 132 

Wash, r., 56, 123 

Waterman, M., 124 

Waters, r., 37 

Watson, K., 116 

Watson, r., 23 

Watson (Scott), S., 80 

Watson (Simon), S., 55, 123 

Watts, J., 148 

Way, M., 8, 9 

Wearing, H., 44 

Webb, J., 51 

Webber, M.A., 150 

Weber, J.N., 113 

Wei, W., 68 

Welch (Martin), M., 150, 152 

Welch (Matthew), M., 63 

Wells, C., 42 

Wells, T.J., 150, 151 

Werling, D., 90 

West, S.A., 92 

Westerman, l.J., 145 

Wetherill, l.F., 129 

Wheatly, r., 47 

Whelan, S., 4 

White, l., 120 

White, M.F., 38 

White, r.E., 66 

Whitehouse, A., 55, 69, 71, 110 

Whiteley, M., 15 

Wight, D., 120 

Wijburg, O.l., 28 

Wileman, T., 120 

Wilf, N., 162 

Wilkinson, C.r.M., 74 

Wilkinson, G., 67 

Wilkop, T., 77, 130 

Wilksch, J.J., 28 

Willart, M.M., 24 

Willenborg, J., 29 

Willett, B.J., 84, 108, 109, 133 

Williams, A., 23 

Williams, l., 103 

Williams, P., 143 

Williamson, D., 16 

Wills, B., 84 

Wills, M., 53 

Wilson, G., 7, 82 

Wilson, G.J., 102 

Wilson, i.A., 76 

Wilson, N., 21 

Wilson, S., 5 

Wilson (Joanna), J., 53 

Wilson (Julia), J., 50 

Wilusz, J., 10 

Wimmer, E., 25 

Winstanley, C., 94 

Winzer, K., 92, 159 

Wise, H., 6, 54, 70, 73, 78 

Wiseman, J., 47 

Wisniewska, K., 5 

Witteveldt, J., 30 

Wittman, M., 59 

Wogan, H., 161 

Wong, H.E.E., 97 

Wong, M., 100 

Wood, J., 69 

Woodman, i., 38 

Woodward, A., 88, 133 

Woodward, J., 146 

Wooldridge, K., 141 

Wooldridge, M., 46 

Wootton, M., 44 

Wren, B.W., 17, 95, 96 

Wright, F., 47 

Wright (Edward), E., 134 

Wright (Elli), E., 94 

Wu, W., 110 

Wysocka, N., 112 

Yamamoto, K., 74 

Yang, C., 25 

Yang, H.-Y., 74 

Yang, Y., 157, 161 

Yasim Yusof, M., 100 

Yates, l., 96 

Yeo, P., 80 

Yeo, r., 128 

Yin, Z., 123 

Yoksan, S., 84 

Young, D., 66 

Young, M., 88 

Yu, D., 49 

Yu, S., 37 

Yuan, x., 169 

Zambon, M., 123 

Zeng, Q., 83 

Zhang, l., 138 

Zhang, M., 155 

Zhang, Q., 9 

Zhang, x., 59 

Zhao, B., 124 

Zheng, S., 19 

Zhou, A., 132 

Zhou, l., 93 

Zitzmann, N., 88 

Zoll, J., 42 

↑Contents 




172 



ADDITIONAL AbSTrACTS 

HA03 A small rNA controls Myxococcus development and mediates a major social adaptation 

Yuen-Tsu Nicco Yu, xi Yuan, GrEGOrY J. VEliCEr 

Dept of Biology, Indiana University, Bloomington, IN 47405 USA 

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Non-coding small rNA (srNA) molecules regulate a vast array of processes in biology, but evidence for evolutionary adaptations 

mediated by mutation of srNA elements has been indirect. Here we identify a regulatory srNA in the fruiting myxobacteria, ‘Pxr’, and that 

negatively controls fruiting body development in the model species Myxococcus xanthus. We further show that a spontaneous mutation in 

Pxr abolished its regulatory function and thereby adaptively restored developmental proficiency to a socially defective cheater strain of M. 

xanthus. in wild-type M. xanthus, development is initiated only upon starvation, but deletion of the pxr gene allows development and spore 

production to proceed at high nutrient levels. Thus, Pxr serves as a major checkpoint controlling the transition from growth to development 

in the myxobacteria. Additionally, ongoing work utilizing the socially defective cheater as a powerful tool for identifying components of the 

Pxr regulatory pathway is described. 

HA08 Pathogen products in susceptibility and resistance to malaria 

louis Schofield 

Howard Hughes International Research Scholar, The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, 

Parkville 3050, Victoria, Australia 

Overall there is reasonable agreement from murine, simian and human infections on a framework for immune-mediated pathogenesis 

in malaria. The temporal outfolding of immune activation events are broadly as follows: Pathogen products (such as GPi or haemozoin) 

interact with Tlr2/4, 9 (and other lectins?), DCs and macrophages become activated to produce a wide range of regulatory cytokines 

with anti-parasite and disease promoting functions. Parasite isopentenyl-pyrophosphate interacts with gdTCr, and activated macrophages 

activate NK cells, leading to early/intermediate iFNγ output. Such responses appear largely protective. The parasite also produces specific 

ligands to counter-regulate the immunologically protective responses eg PfEMP-1. Nonetheless, immunological processes can contribute 

to acute febrile disease, the ‘cytokine storm’, cerebral malaria, placental malaria, and severe malarial anaemia. Chemokine networks play 

a critical role in the regulatory control of inflammation, immunity and disease. Genetic association data with clinical phenotypes in human 

case-control studies support these interpretations. recent extension to longitudinal cohort studies provides evidence for immunological 

variables in control of parasite densities. identification of specific pathogen products and their host response pathways has enabled the 

rational design of vaccine strategies to impart clinical immunity to malaria. 

HA10 recombination and gene transfer in Wolbachia infecting Drosophila 

SiV ANDErSSON, lisa Klasson, Kirsten Maren-Ellegaard 

University of Uppsala, Norbyvägen 18 C S-752 36 Uppsala, Sweden 

Here, we use the Rickettsiales for a discussion of the different evolutionary processes that have shaped the genomes of insect-associated 

bacteria. Whereas gene deterioration dominates the evolution of the Rickettsia genomes, Orientia has the highest fraction of repeated 

sequences observed in a bacterial genome. Our genomic comparison of Wolbachia wri that induces strong cytoplasmic incompatibility (Ci) 

in Drosophila simulans to Wolbachia wMel that infects Drosophila melanogaster has uncovered a highly recombining intracellular bacterial 

community. in effect, different genes support different strain relationships. We also present the genomes of the A-group strain wHa and the 

B-group strain wNo that induces weak Ci in Drosophila simulans, with a focus on gene transfers across strains. An interesting observation 

made previously is that the Wolbachia wri-like sequence reads detected in the genome assembly of Drosophila ananassae, strain Hawaii, 

are derived from inserts in the nuclear genome. A comparison to the Wolbachia sequences derived from a tetracycline treated line of 

D. ananassae from india suggests that the insertion happened once and that there are copy number variations in the inserted fragments. 

The different evolutionary trajectories reflect the occurrence of mobile elements in the endosymbiont population and the abundance and 

diversity of the host population. 

HA13 role of type IV secretion systems for the intracellular lifestyle of Bartonella 

Christoph Dehio 

Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland 

The arthropod-borne α-proteobacterial genus Bartonella comprises facultative intracellular pathogens that colonize endothelial cells and 

erythrocytes of their mammalian reservoir hosts, thereby causing long-lasting intraerythrocytic infections. The intracellular colonization of each 

of these two target host cell types critically depends on a distinct type iV secretion systems (T4SS). T4SSs are versatile transport machineries 

that translocate protein and/or DNA substrates across bacterial cell envelopes by a mechanism requiring direct contact with a target cell. 




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ADDITIONAL AbSTrACTS 

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The VirB/VirD4 T4SS translocates a cocktail of effector proteins (Beps) into endothelial cells that subvert multiple cellular functions leading 

to the establishment of chronic vascular infection. The effectors BepG or BepC and BepF trigger parallel signaling pathways leading to the 

internalization of a large bacterial aggregate of Bartonella henselae by human endothelial cells via the socalled invasome-mediated entry 

pathway. The Trw T4SS does not appear to translocate any known effector, but produces multiple variant pilus subunits critically involved 

in the host-specific adhesion to the erythrocyte surface as a prerequisite for the subsequent erythrocyte invasion process. The T4SSs of the 

bartonellae have thus adopted highly diverse functions in host cell colonization, highlighting their versatility as pathogenicity factors.

Spring Conference 2011 - Society for General Microbiology

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