rologie i - European Congress of Virology

5 th European Congress of VirologyThis study describes the preparation of recombinant proteins E and NS1of WNV and USUV and the characterization of Monoclonal antibodies(MAbs) against WNV, USUV and TBEV in order to evaluate their applicabilityin virological identification and serological diagnosis. Viruses usedas immunogen in mice for MAb production were WNV reference and fieldstrains and USUV and TBEV field strain. MAb reactivity was evaluatedby different methods:1 indirect immunofluorescence (IFI) using referenceand field flaviviruses isolated from Europe and Africa,2 Virus neutralizationtest,3 Indirect ELISAs using recombinant E and NS1 proteins ofWNV lineages 1 2 and USUV in Baculovirus expression system,4 CompetitiveELISAs performed to analyze the competition between selectedMAbs and experimental sera obtained from different species. Flaviviruscross reactive MAbs proved to be useful for viral detection in pathologicalspecimens or in infected cell cultures. MAbs specific to immunogenicepitopes have been used for the development of competitive ELISAs forWNV, USUV and TBEV antibody detection. In addition, a double antibodysandwich ELISA based on NS1 specific Mabs able to differentiateWNV L1,L2 and USUV has been developed.This research has receivedfunding from the EU 7th Framework Programm EUROWESTNILE.REF 276Virulence determinants of West Nile Virus Strains circulating inEuropeKhaled ALSALEH 1 , Céline BAHUON 2 , Ana VAZQUEZGONZALEZ 3 , Miguel Angel JIMÉNEZ CLAVERO 4 , AntonioTENORIO 3 , Maha DRIDI 5 , Bénédicte LAMBRECHT 5 , SylvieLECOLLINET 2 , Philippe DESPRÈS 1 , Nathalie PARDIGON 11 Institut Pasteur, Unité des Interactions Moléculaires Flavivirus Hôtes,Paris, FRANCE; 2 Agence Nationale de Sécurité Sanitaire (ANSES), Laboratoirede Santé Animale, Maisons Alfort, FRANCE; 3 Laboratory ofArboviruses and Viral Imported Diseases, National Center of Microbiology,Institute of Health “Carlos III”, Majada, Madrid, SPAIN; 4 Centrode Investigación en Sanidad Animal (CISA) INIA, Ctra, Algete El Casars/n, 28130 Valdeolmos, Madrid, SPAIN; 5 Operational Direction of ViralDiseases, Veterinary and Agrochemical Research Center (CODA CERVAVAR), 99 Groeselenberg, 1180 Br, Brussels, BELGIUMWest Nile virus (WNV) is a neurotropic, mosquito borne enveloped RNAflavivirus. Zoonotic transmission of WNV occurs between avian hosts andornithophilic mosquito vectors, but can cause disease in horse and human,that are incidental dead end hosts. Although Mediterranean WNV strainswere considered as less virulent, recently, several outbreaks and epizooticshave been reported in Eastern and Southern Europe. To investigatethe viral factors involved in the virulence of European strains, we comparedthe highly pathogenic isolate Israel 98 (IS98) and an Italy strainisolated from a magpie in 2008 (IT08). Fifteen amino acid changes and25 nucleotide differences in the 3 ′ UTR were found between the 2 isolates.When introduced separately or combined in an IS98 infectious clone, thesemutations and differences had no effect on viral replication and virus productionin vitro. Moreover chimeras of the 2 isolates did not show anysignificant difference in virus virulence in newborn mice. However, IT08was less pathogenic than IS98 in one day old SPF chickens. Interestingly,IT08 strain but not IS98 was able to induce the fusion of C6/36 cellssuggesting that the isolates are not disseminated in the same manner inmosquitoes. In order to identify viral determinants responsible for thesedifferences, we are currently studying chimeras between the 2 isolates inone day old SPF chickens and in Culex pipiens mosquitoes. In summary,our results indicate that mice might not be discriminant enough to evaluateWNV virulence and the vector might be a virulence limiting factor.REF 277Excretion of West Nile virus in urine during acute infectionLuisa BARZON 1,2 , Monia PACENTI 2 , Elisa FRANCHIN 1,2 , SilvanaPAGNI 1,2 , Thomas MARTELLO 2 , Margherita CATTAI 2 , RiccardoCUSINATO 2 , Giorgio PALÙ 1,21 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALYDetection of West Nile virus (WNV) RNA in urine has been anecdotallydescribed and proposed for the diagnosis of WNV infection. This studyreports the application of real time RT PCR for the detection of WNVRNA in urine as a routine procedure in support to the diagnosis of WNVinfection during the large outbreak that occurred in North Eastern Italy in2012. Fourteen out of 32 (43.8%) patients with symptomatic WNV infection,i.e., neuroinvasive disease (WNND) and fever (WNF), had detectableWNV RNA in urine at the time of diagnosis, at a higher rate and load andfor longer time than detection of WNV RNA in blood. At variance, detectionof WNV RNA in urine was less frequent (2 out of 14, 14.2%) inblood donors in whom WNV infection was identified by WNV NAATscreening. Different patterns of WNV viraemia and viruria were observedin patients with WNND, WNF, and in blood donors with subclinicalinfection, with typically both viraemia and viruria observed in patientswith WNND, viruria but no viraemia in patients with WNF, and viraemiabut no viruria in asymptomatic blood donors. Infectious virus wasisolated from urine of a patient with WNND and high WNV RNA loadin urine. In conclusion, this study demonstrated the diagnostic utility ofWNV RNA detection in urine by real time RT PCR for case confirmationin patients with WNND and WNF. The virus was isolated from the urineof a patient with WNND, indicating that WNV excreted in urine may beinfectious.REF 278The Tick Cell Biobank facilitates tick borne arbovirus researchLesley BELL SAKYIThe Pirbright Institute, Pirbright, UNITED KINGDOMThe Tick Cell Biobank is the world’s largest collection of ixodid and argasidtick cell lines. Many tick species of medical and veterinary importanceare represented, including Ixodes ricinus, Hyalomma anatolicum and Ornithodorusmoubata, and attempts to establish new cell lines from EuropeanDermacentor species are ongoing. By supplying tick cell lines and trainingin their maintenance to scientists all over the world, the Tick Cell Biobankunderpins global research into ticks and the many diseases they transmit.Tick cell lines are particularly useful for propagation and study of arbovirusescausing zoonoses including tick borne encephalitis and CrimeanCongo haemorrhagic fever, and livestock diseases such as African swinefever and Nairobi sheep disease, providing a practical alternative to useof live ticks particularly for viruses requiring high level containment. Forexample, material for large scale transcriptomics and proteomics can beproduced in vitro under controlled conditions, and silencing of host cell andvirus genes by RNAi facilitates functional genomics studies. Most existingtick cell lines harbour apparently endogenous viruses, about whichalmost nothing is yet known. It is possible that these putative tick virusesmay play a role in modulation of the tick cell innate immune response,allowing the development of a low level persistent arbovirus infection inthe absence of obvious cytopathic effect that is characteristic of tick celllines.Virologie, Vol 17, supplément 2, septembre 2013S197