rologie i - European Congress of Virology

5 th European Congress of VirologyIn M PMV CA two cysteines (C193 and C213) correspond to their positionsin HIV 1 but there is also one additional cysteine (C82) that is notpresent in CA HIV 1. Our goal was to study the effect of mutations C82,C193 and C213 on in vitro formation and morphology of VLPs assembledfrom M PMV Gag deletion mutant. These mutations prevent cysteinesfrom S S bond formation and thus could mimic the presence of reducingagents during assembly. Obtained VLPs were analyzed by transmissionelectron microscopy and ultracentrifugation in sucrose gradient, followedby SDS PAGE. Our findings could be important for futher understandingof retroviral particle assembly process.Financial support from specific university research MSMT No 20/2013and GACR P302/12/1895.1. Worthylake DK, et al. Acta Crystallogr Sect D Biol Crystallogr 1999;55: 85.The great diversity, wide distribution, and mobility of bats represent riskfactors for zoonotic transmission. SARS Coronavirus (CoV) and the novelhuman Betacoronavirus EMC are closely related to bat CoV from thegenus Betacoronavirus. A variety of batCoV belong to the genus Alphacoronavirus.However, no infectious CoV’s could be isolated from batsso far. To analyze coronaviral replication in chiropteran cells, the transmissiblegastroenteritis virus (TGEV) was used as model for the genusAlphacoronavirus. By Western blot analysis, reverse transcription PCRand immunofluorescence analysis virus entry, replication, and virus releasewas examined. Cells, transfected with the specific receptor for TGEV,became susceptible for infection which was shown by intracellular detectionof viral spike and membrane proteins. Release of viral RNA wasobserved for all tested cell lines. Successful release of infectious virus particlesfrom cell lines of African flying foxes (genus: Eidolon, Epomops, andHypsignathus), the common pipistrelle (Pipistrellus pipistrellus) and theshort tailed fruit bat (Tadarida brasiliensis) was revealed after subsequentviral replication in inoculated swine testis cells. In contrast, supernatantsobtained from Daubenton’s bat did not contain any infectious virus. Collectively,these data lead to the assumption that beside the viral receptor,cellular factors could play a crucial role for species restriction.REF 173Association of apolipoprotein B with Hepatitis C virus is involved inviral infectivity and is dependent on the liver enriched protein CidebChristophe RAMIÈRE 1,2,3 , Liviu Sorin ENACHE 4 , MarinePORCHEROT 2,3 , Olivier DIAZ 2,3 , Laure PERRIN COCON 2,3 , VincaICARD 1 , Caroline SCHOLTÈS 1,2,3 , Vincent LOTTEAU 2,3 , PatriceANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, FRANCE; 4 University of Medicineand Pharmacy, TârguMure, ROMANIAIn patients’ blood, Hepatitis C Virus (HCV) circulates as lipo viral particles(LVP), hybrid particles associating viral and lipoprotein components, inparticular apolipoprotein B (apoB). Contrary to natural LVPs, HCV particlesproduced in vitro in Huh7.5 cells (HCVcc) are characterized bypoor association with apoB and low specific infectivity. We thus hypothesizedthat apoB could be involved in HCV infectivity and examinedthe mechanisms responsible for its association with HCV virions. Wefirst demonstrated by neutralization experiments that 2 monoclonal antiapoB antibodies, recognizing epitopes in the Low–Density LipoproteinsReceptor binding domain of apoB, were able to strongly neutralize (ca80 to 90%) infection of Huh7.5 cells by HCVcc, contrary to antibodiestargeting epitopes outside this domain. We also identified Cideb, a liverenriched protein involved in lipoprotein assembly, as an essential factorfor association of HCV with apoB. We showed that Cideb directly interactedwith HCV E2 envelope glycoprotein. We also observed that Cidebexpression was low in Huh7.5 cells and that Cideb overexpression in thiscell line was sufficient to induce secretion of E2 in association with apoB.Moreover, differentiation of Huh7.5 cells in presence of human serumand DMSO led to a dramatic increase of Cideb expression and furthersecretion of HCV particles characterized by higher association with apoBand improved specific infectivity. Together these results suggest that apoBplays an important role in HCV entry and that Cideb is essential for HCVassociation with apoB.REF 174Production of infectious virus particles of Alphacoronaviruses differsin a variety of bat speciesAnna Theresa RUEDIGER, Christel SCHWEGMANN WEELSUniversity of veterinary medicine, Hannover, GERMANYREF 175The Epizootic Hemorrhagic Disease Virus (EHDV) Induces andBenefits from Cell Stress, Autophagy and ApoptosisBen SHAI 1 , Eran SCHMUKLER 1 , Roy YANIV 1 , Naomi ZIV 1 , GalitHORN 1 , Velizar BUMBAROV 2 , Hagai YADIN 2 , Nechama I.SMORODINSKY 1 , Eran BACHARACH 1 , Ronit PINKASKRAMARSKI 1 , Marcelo EHRLICH 1Tel Aviv University, Tel Aviv, ISRAEL; Kimron Veterinary Institute, BeitDagan, ISRAELThe mode and timing of virally induced cell death withhold the potentialof regulating viral yield, viral transmission and the severity of virally induceddisease. Orbiviruses such as the Epizootic Hemorrhagic Disease Virus(EHDV) are non enveloped and cytolytic. To date, the death of cells infectedwith EHDV, the signal transduction pathways involved in this processand the consequence of their inhibition have yet to be characterized. Here,we report that two EHDV isolates (EHDV2 Ibaraki and an Israeli isolate ofEHDV7) induce protein kinase R (PKR) activation, protein synthesis inhibition,c Jun N terminal kinase (JNK) activation, cell rounding, autophagyand apoptosis. The timing of these phenomena and the effects of specificchemical inhibitors of PKR, JNK and autophagy suggest they are interconnectedand sequential. Moreover, these inhibitors and a specific caspaseinhibitor reduced the yield of infectious virions, suggesting that the activitiesof PKR and JNK and the induction of autophagy and apoptosis arebeneficial to EHDV replication. Moreover, through live cell microscopy,we identified a mode of EHDV induced cell death involving the ruptureof a bubble like membrane protrusion, which results in spillage of thecellular cytoplasmic contents. This mode of cytolysis may have potentialimplications in viral spread and in the host response to EHDV infection.REF 176RhoB is important for Human Cytomegalovirus lytic replicationGeorge SOURVINOS, Demetrios SPANDIDOS, Nektaria GOULIDAKIMedical School, University of Crete, Heraklion, GREECEHuman Cytomegalovirus (HCMV), a ubiquitous herpesvirus, is a significantpathogen causing medically severe diseases to immunocompromisedindividuals. HCMV primary lytic infection is followed by long terms oflatency. During productive infection, HCMV has evolved a variety of interactionswith proteins of the host, facilitating its successful replication andspread. RhoB belongs to the family of Rho GTPases, which regulatesdiverse cellular processes. Rho proteins are implicated in the entry andegress from the host cell of mainly and herpesviruses, whereas herpesvirusesare the least studied in this regard. Here we set out to studythe role of RhoB GTPase during HCMV lytic infection. We examined thecellular distribution of RhoB during the course of the wild type HCMVAD169 virus infection in fixed cells and of the recombinant UL32 EGFPVirologie, Vol 17, supplément 2, septembre 2013S167