rologie i - European Congress of Virology

5 th European Congress of Virology28. GASTROINTESTINAL VIRALINFECTIONSPosters: REF 578 to REF 589REF 578Human Enteric Adenoviruses 40 and 41 in 293 and Caco 2 cellsCasandra MANGROO 1 , Dr. Martha BROWN 1,21 Department of Laboratory Medicine & Pathobiology, University ofToronto, Toronto, CANADA; 2 Department of Molecular Genetics, Universityof Toronto, Toronto, CANADAFastidious human enteric adenoviruses, serotypes 40 and 41 (HAdV 40/41;species F), are important agents of paediatric enteritis. Unlike other adenoviruses,they grow to high titre in the small intestine but typically donot cause disease elsewhere in the body. In cell culture, infectivity is poor,even in 293 cells, whose endogenous HAdV 5 E1 sequences complementthe low level E1 expression of HAdV 40/41. Recent work in this laboratoryidentified a major, though incomplete, block in virion uptake in 293and A549 (lung epithelial) cells. A comparable block in intestinal epithelial(Caco 2) cells, grown under conditions optimized for susceptibility toHAdV 40/41, was unexpected. Caco 2 cells are less susceptible to infectionas cells become differentiated with time, and are most susceptible insub confluent undifferentiated cultures, in particular, when the edges areexposed. Recent experiments using HAdV 41 EGFP and 293 cells haveaddressed the hypothesis that virions are stable during passage through thestomach, but require modification by intestinal conditions for successfulentry/genome delivery. Consistent with the hypothesis, virion infectivitywas increased by exposure to synthetic gastric fluid followed by syntheticintestinal fluid, as determined by flow cytometric analysis of infectedcultures. The unexpected finding was that exposure to gastric fluid alonewas sufficient for the increased infectivity. Subsequent experiments willaddress structural modifications induced by gastric conditions and stepsin the entry pathway facilitated by these modifications.REF 579Validation and performance of an internally controlled multiplexedreal time PCR run on the BDMAX for the detection of Norovirus(NoV) in 7 day working systemKate TEMPLETON, Juliet KENICER, Julie WHITE, PeterMCCULLOCHNHS Lothian, Edinburgh, UNITED KINGDOMThe study aim was to assess performance of a multiplexed NoV GI/GIIPCR with EAV internal control on the BDMAX. A secondary objectivewas to assess BDMAX as a suitable platform for routine testing by nonprofessionally registered staff.The initial validation phase collected 337 samples in January and February2012. 312 samples were faecal specimens and 25 were vomit; all patientshad diarrhoea or vomiting or both. 1 ml suspensions were made by adding25 mg of solid sample or two drops of liquid sample to H2O containingEAV, spun down and extracted both on the easyMAG (Biomerieux) andthe BDMAX (Becton Dickinson). PCR multiplex was performed and analysedon the ABi 7500 or the BDMAX. Following the validation phase theassay was run on the BDMAX for routine diagnosis and then switched backto the easyMAG and ABi to compare performance. The BDMAX NoVmultiplex was 94% sensitive (96/102) and 99% specific (233/235.) 8.6%of positives were GI and the rest were GII. The assay on the BDMAX had2% inhibition and 10.6% inhibition on the EasyMAG/ABi. 1414 sampleswere tested routinely: 361 on the EasyMAG/ABi 7500 and 1053 on theBDMAX. Mean turnaround time was 3 Hrs 47mins on EasyMAG/ABiand 3 Hrs 55mins on BDMAX. During 2011/12, only 29% of NoV testsamples were achieving 15:15 cut off time for resulting. Over the sameperiod during 2012/13 this improved to 75%. The BDMAX could be runmore frequently with minimal hands on time and uncomplicated ease ofuse. The validated NoV Multiplex PCR performed well on the BDMAXand brought significant practical advantages including walk away functionality,minimal hands on time and low level of technical ability requiredto operate the machine. This allowed routine NoV diagnosis to be carriedout 7 days by non professionally registered staff and helped minimise theimpact of NoV sample numbers on other molecular tests.REF 580New Sydney 2012 strain of norovirus detected in patients with acutegastroenteritis in PortugalInês COSTA 1 , João MESQUITA 3 , Elisabete VEIGA 1 , MónicaOLEASTRO 1 , Maria SÃO JOSÉ NASCIMENTO 21 Laboratório Nacional de Referência das Infeções Gastrintestinais,Departamento de Doenças Infeciosas, do Instituto Nacional de S, Lisboa,PORTUGAL; 2 Laboratório de Microbiologia, Departamento de CiênciasBiológicas, Faculdade de Farmácia da Universidade do Porto, Porto,PORTUGAL; 3 Escola Superior Agrária, Instituto Politécnico de Viseu,Viseu, PORTUGALNorovirus (NoV) is the leading cause of diarrheal disease across all agegroups as well as half of all gastroenteritis outbreaks worldwide. Surveillancesystems showed an increase in NoV activity in late 2012 relatedto the emergence of a new NoV genotype II.4 variant, termed Sydney 2012,with the first report coming from Australia in March 2012. Until date, Sydneyvariant circulation in Portugal has not been reported. Aim: To studythe genetic variation of NoVs strains circulating in Portugal in patientswith acute gastroenteritis from May 2011 to December 2012. Methods:A total of 36 NoVs positive samples were studied. Viral RNA was extractedusing NucliSens ® easyMAG TM (bioMérieux) and tested with primerstargeting the POL and the VP1 major capsid genes with Qiagen One StepRT PCR Kit. PCR products were sequenced with Big Dye Terminator v1.1in the Sequencer Analyser ABI Prism 3130 xl (PE Applied Biosystems).NoVs sequences were analysed using the Food borne Viruses in Europe(FBVE) network NoV genotyping tool and NCBI Blast. Results: Overall,14 (39%) samples were positive for the Sydney GII.4. variant. Theremaining 22 (61%) were also identified as GII.4 with a predominance ofthe New Orleans 2009 variant (16/22, 72.7%). Sydney variant was onlydetected from samples dating from June 2012.Conclusion: This is the first report of the circulation of NoV GII.4 Sydneyvariant in Portugal.REF 581Molecular dating in the evolution of primate bocavirusesIgor BABKIN 1 , Irina BABKINA 1 , Aleksandr TUMENTSEV 2 , ArtemTIKUNOV 1,2 , Aleksander KURILSHIKOV 1 , Nina TIKUNOVA 11 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIA; 2 Novosibirsk State University, Novosibirsk, RUSSIAHuman bocavirus (HBoV) is associated with acute gastroenteritis inhumans, occurring mostly in young children and elderly people. Fourbocavirus genotypes have been found so far. Since there were no data onthe contribution of HBoV to gastroenteritis in Russia, 1781 fecal samplescollected from infants hospitalized with acute gastroenteritis in Novosibirsk,Russia during one year were tested for the presence of nucleicacids from HBoV and three major gastrointestinal viruses (rotavirus A,norovirus II, and astrovirus). HBoV was detected only in 1.9% of thesamples. Complete genome sequencing of three Novosibirsk isolates wasperformed. An evolutionary analysis of these sequences and the availableVirologie, Vol 17, supplément 2, septembre 2013S281