rologie i - European Congress of Virology

5 th European Congress of VirologyRift Valley fever virus (RVFV) is mosquito borne and highly virulent tohumans. The virus is able to infect many different animals and mosquitoesas well as various cell types in vitro but not much is known about the entrymechanisms.In this study we have used Rift Valley fever virus like particles (VLPs)containing a reporter gene to study the virus cell interaction. Cells orRVF VLPs were treated with various compounds to characterize theireffect on RVFV binding to its host cell. We also analysed the net surfacecharge of the RVFV glycoproteins since this can affect the cellularbinding.Binding of RVF VLPs to cells was dependent on charge and divalentions. Binding was inhibited by the highly negatively charged heparin.The RVFV glycoprotein Gn had a predicted isoelectric point (pI) of 7.6and a net positive charge of +1.7 at pH 7.4, suggesting interaction betweenthe Gn ectodomain and the negatively charged cell surface. RVFVGc on the other hand, was highly negatively charged, 14.2 at pH 7.4,most probably reflecting that Gc is not exposed until after binding. Sialicacid, 3 integrins and heparan sulfate was not important for RVF VLPbinding.Conclusions: The binding of RVFV to host cells was dependent on chargeinteractions between the cell surface and, most probably, the ectodomainof the RVFV Gn glycoprotein. Divalent ions were also important, whilesialic acid, 3 integrins and heparan sulfate had no involvement in theRVFV cellular binding.REF O125Functional comparison of the SARS CoV and bat borne SARS likeCoronavirus spike glycoproteins: entry process and interplay withchiropteran cellsMarkus HOFFMANN 1 , Tim GÜTZKOW 1 , Nadine KRÜGER 1 ,Christoph LOSEMANN 1 , Marcel Alexander MÜLLER 2 , Jan FelixDREXLER 2 , Karl Heinz ESSER 3 , Christian DROSTEN 2 , GeorgHERRLER 11 Institute of Virology/University of Veterinary Medicine Hannover, Hannover,GERMANY; 2 Institute of Virology/Bonn Medical Centre, Bonn,GERMANY; 3 Institute of Zoology/University of Veterinary Medicine Hannover,Hannover, GERMANYThe severe acute respiratory syndrome associated coronavirus (SARSCoV) uses the angiotensin converting enzyme 2 (ACE2) as a receptormolecule and horseshoe bats (genus Rhinolophus) are believed to serveas a natural reservoir. The detection of SARS CoV reactive antibodies inAfrican bats and RNA of SARS like coronaviruses (SL CoV) worldwide,lead to the question whether another zoonotic spillover can occur, but thefailure to isolate such a SL CoV makes it necessary to use alternativemethods to answer this question.We employed a pseudotype infection system, based on the vesicular stomatitisvirus (VSV) to analyze whether SL CoV S can mediate entry intomammalian (chiropteran) cell lines and established a cell based fusionapproach involving co expression of CoV S and human or chiropteranACE2. Furthermore, we used soluble CoV S to elucidate the presence ofan appropriate receptor molecule.VSV pseudotyped with SL CoV S was unable to infect any of the tested celllines; parallel infection experiments with filoviral glycoproteins, as well asinfections by paramyxo and influenza viruses confirmed the susceptibilityof chiropteran cells to viral infection. SARS CoV S mediated entry intochiropteran cell lines required human ACE2 expression in trans and thefusion assay revealed that only trypsin activated SARS CoV S was capableof syncytia formation. Finally, we show that soluble SL CoV S can bindto cells expressing human ACE2, leading us to the assumption that a postbinding step is responsible for the observed restrictions.REF O126Efficient replication of the novel human betacoronavirus EMC onprimary human epithelium highlights its zoonotic potentialRonald DIJKMAN 1 , Eveline KINDLER 1 , Hulda JONSDOTTIR 1 ,Doreen MUTH 2 , Ole HAMMING 3 , Rune HARTMANN 3 , ReguloRODRIGUEZ 4 , Robert GEFFERS 5 , Ron FOUCHIER 6 , ChristianDROSTEN 2 , Marcel MÜLLER 2 , Volker THIEL 1,71 Inst. of Immunobiology, Kantonal Hospital St.Gallen, St. Gallen, SWIT-ZERLAND; 2 Inst. of Virology, Univ. of Bonn Medical Center, Bonn,GERMANY; 3 Centre for Structural Biology, Univ. of Aarhus, Aarhus,DENMARK; 4 Inst. of Pathology, Kantonal Hospital St.Gallen, St. Gallen,SWITZERLAND; 5 Helmholtz Center for Infection Research, Braunschweig,GERMANY; 6 Viroscience Lab, Erasmus Medical Center, Rotterdam,THE NETHERLANDS; 7 Vetsuisse Faculty, Univ. of Zürich, Zürich, SWIT-ZERLANDThe recent emergence of a novel human coronavirus (HCoV EMC) in theMiddle East raised considerable concerns as it is associated with severeacute pneumonia, renal failure and fatal outcome, and thus resembles theclinical presentation of severe acute respiratory syndrome (SARS) observedin 2002/2003. Like SARS CoV, HCoV EMC is of zoonotic origin andclosely related to bat coronaviruses. The human airway epithelium (HAE)represents the entry point and primary target tissue for respiratory virusesand is highly relevant for assessing the zoonotic potential of emergingrespiratory viruses, such as HCoV EMC. Here we show that pseudostratifiedHAE cultures derived from different donors are highly permissiveto HCoV EMC infection, and by using RT PCR and RNAseq data weexperimentally determined the identity of seven HCoV EMC subgenomicmRNAs. Although, the HAE cells were readily responsive to type I andtype III interferon (IFN), we observed neither a pronounced inflammatorycytokine nor any detectable IFN responses following HCoV EMC, SARSCoV, or HCoV 229E infection, suggesting that innate immune evasionmechanisms and putative IFN antagonists of HCoV EMC are operationalin the new host. Importantly, however, we demonstrate that both type Iand type III IFN can efficiently reduce HCoV EMC replication in HAEcultures, providing a possible treatment option in cases of suspected HCoVEMC infection.REF O127Identification of cellular factors involved in HEV replication using anin vivo model of HEV infection in swine and a proteomic approachSophie ROGEE 1,2,3 , Jérome BOUQUET 1,2,3 , Elodie BARNAUD 1,2,3 ,Marine DUMAREST 1,2,3 , Philippe CHAFFEY 4,5,6 , Nicole PAVIO 1,2,31 UMR 1161 INRA, Maisons Alfort, FRANCE; 2 UMR virologie ANSES,Maisons Alfort, FRANCE; 3 ENVA, Maisons Alfort, FRANCE; 4 Institutcochin Inserm U1016, Paris, FRANCE; 5 Institut cochin CNRS UMR8104,Paris, FRANCE; 6 Université Paris Descarte, Paris, FRANCEHepatitis E virus (HEV) is a zoonotic virus and in industrialized countriespigs are the major source of HEV infections. In France, strains of HEVpresent in human and swine populations are of the same genotype andbelong to subtype 3f, 3e and 3c. Mechanisms involved in replication andvirulence of HEV are still unknown. To characterize the pathogenesis andvirulence effectors of different HEV subtypes, a study of the cellular factorsmodulated during HEV infection was carried out using a differentialproteomic approach. This study was performed in a model of experimentalinfection of pigs, with the three subtypes of HEV present in France.The kinetics of HEV fecal excretion and viral RNA detection in the liverand bile of each infected animal did not show significant difference in termsof duration or quantity, whatever the strain used. Differentially expressedVirologie, Vol 17, supplément 2, septembre 2013S93