rologie i - European Congress of Virology

5 th European Congress of Virologyisolated in 2010 in Lower Saxony, Germany in the same bat species. Theisolate [KC169985] showed 98.8% nucleotide identity with the GermanBBLV isolate [JF311903]. Several organs of the infected bat were investigatedby reference rabies diagnostic methods: fluorescent antibody test,cell culture inoculation test and qRT PCR. Antigen, infectious virus andhigh RNA levels were found in both the brain and salivary glands. Tracesof genomic RNA were also detected in the bladder, kidney, liver/spleen,heart and lung tissues. The results of Lyssavirus distribution investigationsuggest a possible transmission of the virus. To date, the BBLVstrain has never been found in Europe except in 2010 and in 2012 inGermany and France, in two isolated animals. The results of the passivesurveillance of bat rabies in France and the circumstances of discovery andthe results of Lyssavirus distribution investigations will be presented anddiscussed.REF 263Analysis of complete Puumala hantavirus genomes recovered from afatal case of nephropathia epidemica and bank voles (Myodes glareolus)from the site of infectionAngelina PLYUSNINA, Alexander PLYUSNINHaartman Institute, University of Helsinki, Helsinki, FINLANDPuumala virus (PUUV) is a major European hantavirus transmitted tohumans from bank voles and causing nephropathia epidemica. Herewe present the first complete PUUV genome directly recovered froma fatal case together with the results of a search for related geneticvariants in local bank voles. The findings (i) established genetic linkbetween the case and wild type PUUV strains, (ii) revealed that no mutationsaccumulated in the viral genome of PUUV during transmissionto the victim and the fatal infection that followed, and (iii) demonstratedthat the PUUV strain involved was neither unique nor rare geneticvariant.REF 264Optimization of a Commercial Hantavirus IgG Enzyme Immunoassayfor Human Use to Screen Infection Among Wild Rodents in Kirklareli,TurkeyCeylan POLAT 1 , Mehmet Ali OKTEM 1 , Ahmet KARATAS 2 , MustafaSÖZEN 3 , Ferhat MATUR 3 , Hakan ABACIOGLU 11 Dokuz Eylül University, Faculty of Medicine, Department of MedicalMicrobiology, Izmir, TURKEY; 2 Nigde University, Faculty of Arts andSciences, Department of Biology, Nigde, TURKEY; 3 Bülent Ecevit University,Faculty of Arts and Sciences, Department of Biology, Zonguldak,TURKEYCommercial assays for screening of specific Hantavirus antibodies inrodent sera do not exist. We therefore sought to optimize an enzymeimmunoassay (EIA) and an immunoblot assay (IBA) using commerciallyavailable Hantavirus antigens, Euroimmun Anti Hanta virus PoolELISA plates and Euroline Anti Hanta Profile 1 strips developed for usein human samples. The optimized assays were than used to screen 82rodent sera collected from Kirklareli Igneada region in 2009. The optimalserum dilutions were 1/50 and 1/100 for EIA and IBA, respectively. Theoptimal horseradish peroxidase (HRP) conjugated goat anti mouse IgGconcentration for EIA was 1/10,000, while 1/5,000 dilution of alkalinephosphatase (AP) conjugated goat anti mouse IgG dilutions was foundoptimal for IBA. We followed the manufacturer’s recommendations forsubstrate and incubation parameters. Twenty samples were initially positivefor anti Hantavirus IgG by EIA. Of these 16 were Dobrava virus(DOBV) and one was Puumala virus (PUUV) positive with IBA. Thenumber of DOBV positive samples from Apodemus flavicollis, Apodemusagrarius, Microtus guentheri, Apodemus sylvaticus and Apodemusiconus were 9, 3, 2, 1 and 1, respectively. One PUUV positive sample wasfrom Apodemus flavicollis. When immunoblot test was accepted as goldstandard, optimized EIA’s sensitivity and specificity was 100% and 93%,respectively.REF 265Human coronavirus EMC replication induces severe in vitro cytopathologyand is strongly inhibited by cyclosporin A or interferon alphatreatmentClara POSTHUMA 1 , Adriaan DE WILDE 1 , Stalin RAJ 2 , TheoBESTEBROER 2 , Stefan VAN NIEUWKOOP 2 , Monserrat BÁRCENA 1 ,Bart HAAGMANS 2 , Eric SNIJDER 1 , Bernadette VAN DEN HOOGEN 21 Leiden University Medical Center, Leiden, THE NETHERANDS;2 Erasmus MC, Rotterdam, THE NETHERLANDSCoronavirus (CoV) infections are associated with respiratory and entericdisease in humans and animals. The 2003 outbreak of severe acute respiratorysyndrome (SARS) highlighted the potentially lethal consequencesof CoV induced disease in humans. In 2012, a novel CoV (HCoV EMC)emerged, causing 16 human cases thus far, of which 9 had a fatal outcome.We characterized HCoV EMC replication and cytotoxicity in various celllines. Electron microscopy of infected cells revealed extensive membranerearrangements, including the formation of double membrane vesicles andconvoluted membranes, which were previously implicated in the RNAsynthesis of SARS CoV and other CoVs. Following infection, we observedrapidly increasing viral RNA synthesis and release of high titres ofinfectious progeny, followed by pronounced cytopathology. These characteristicswere used in an assay for antiviral compound screening in 96well format, by which cyclosporin A was found to inhibit HCoV EMCreplication in cell culture. Furthermore, HCoV EMC was found to be50 100 times more sensitive to interferon alpha (IFN a) treatment thanSARS CoV, an observation that may have implications for the treatmentof HCoV EMC infected patients. HCoV EMC infection did not prevent theIFN induced nuclear translocation of phosphorylated STAT1, in contrastto infection with SARS CoV where this block inhibits the expressionof antiviral genes. These findings highlight relevant differences betweenthese distantly related zoonotic CoVs in terms of their interaction with andevasion of the cellular innate immune response.REF 266Identification of viruses affecting Venezuelan batsFlor PUJOL 1 , Luzmir BOYER 2 , Mayra H HIDALGO 2 , DomingoJ. GARZARO 1 , Sara PAPO 31 IVIC, Caracas, VENEZUELA; 2 INIA, Maracay, VENEZUELA; 3 INSAI,Maracay, VENEZUELABat (Chiroptera) are reservoirs for zoonotic diseases with potential riskto human and animal health. The aim of this study was the molecularidentification of viruses affecting bats from various regions of Venezuela.Viral RNA or DNA was detected by PCR, using broad spectrum primerstargeting conserved regions of the viral genomes. A total of 54 bats (12vampires, 29 frugivores and 13 insectivores) were analyzed to identify:Lyssavirus (brain), Herpeviruses and Polyomaviruses (trachea and lung),Flaviviruses (heart and liver), and Astroviruses (intestines). Herpesviruses,subfamily Gammaherpesvirinae, were identified in 8 samples. Phylogeneticanalysis suggested their classification in the genus Rhadinovirus, butnot closely related to isolates from Old World bats. Four polyomaviruseswere identified, not closely related to the only bat isolate reported to date.One Astrovirus was identified, related to one clade comprising Old Worldbat and mouse viruses. Neither Flaviviruses nor Lyssaviruses were detectedin this sample group. However Rabies viruses isolated from differentVenezuelan animal species confirmed the circulation of Rabies genotypeVirologie, Vol 17, supplément 2, septembre 2013S193