5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>REF 125Resveratrol Inhibition <strong>of</strong> Human Rhinovirus ReplicationChiara NARDIS 1 , Chiara NARDIS 1 , Elena MATTIA 1 , Alessandra DELEO 1 , Antonio FRANCIOSO 2 , Luciana MOSCA 2 , PaolaMASTROMARINO 11 Department <strong>of</strong> Public Health and Infectious Diseases, Sapienza University,Rome, ITALY; 2 Department <strong>of</strong> Biochemical Sciences, SapienzaUniversity, Rome, ITALYRhinoviruses are the major etiological agents <strong>of</strong> common cold and asthmaexacerbations. However, no specific therapy is available. Resveratrol (RV)shows protective properties against several viral infections. We evaluatedthe antiviral effect <strong>of</strong> RV towards human rhinovirus 16 (HRV 16) in two differentcellular models: cell culture monolayer and human nasal epithelium,composed by basal, ciliated and mucus cells. Infection was established assingle or multiple cycles <strong>of</strong> virus replication. Viral yield in the presence<strong>of</strong> different non cytopathic concentrations <strong>of</strong> RV was assessed by plaqueassay. In both cellular systems, RV showed a strong inhibitory effect onHRV 16 infective particles production, that appeared to be dose and timedependent. It has been reported that HRV 16 induces activation <strong>of</strong> NF kBwhich in turn promotes the transcription <strong>of</strong> ICAM 1, the major receptor forHRVs. We examined the effect <strong>of</strong> RV on the HRV 16 induced expression<strong>of</strong> ICAM 1 and viral capsid proteins by immun<strong>of</strong>luorescence and westernblot assay. RV treatment inhibited viral protein synthesis and the HRV 16induced increase <strong>of</strong> ICAM 1 level. Since HRV 16 replication induces theproduction <strong>of</strong> pro inflammatory cytokines, we also evaluated the effects <strong>of</strong>RV on IL 6, IL 8, and RANTES levels in human nasal epithelia by ELISAassay. HRV 16 induced increase <strong>of</strong> proinflammatory cytokines was reversedby RV treatment. These results suggest that RV may have a possibleclinical application in HRV infections, by reducing viral replication andthe related inflammatory symptoms.REF 126Antiviral Drug Design Platform (AD2P): screening <strong>of</strong> dengue virusinhibitorsJustine LETIENNE 1 , Cécilia EYDOUX 1 , Sabine MILHAS 1,3 , KarineALVAREZ 1 , Barbara SELISKO 1 , Xavier MORELLI 3 , GillesQUERAT 2 , Bruno CANARD 1 , Jean Claude GUILLEMOT 11 CNRS, AFMB, UMR 7257, ESIL, Marseille, FRANCE; 2 UMR 190,Unité des Virus Emergents, Faculté de Médecine de Marseille, Marseille,FRANCE; 3 Centre de Recherche en Cancé<strong>rologie</strong> de Marseille (CRCM),CNRS UMR7258/INSERM U1068, Marseille, FRANCEWorldwide resurgence <strong>of</strong> dengue virus (DENV) infections is a majorconcern and few therapeutic treatments are available. The virus groupconsists <strong>of</strong> 4 serotypes and can induce similar symptoms ranging frommild febrile illness to a life threatening dengue hemorrhagic fever. Antiviralscreens have proved useful for the identification <strong>of</strong> novel humanemerging virus inhibitors. A screening workstation has been developed totest chemical compounds, based on the dynamic interface between threeplatforms: Interactions, Dynamics and Drug design platform (INT 3D);Screening platform Marseille Luminy (PCML) and <strong>Virology</strong> screeningplatform Marseille Timone (PCVMT). Our team (PCML) combines biochemicaland cell based screening assays to select efficient compounds(chemically synthesized or natural extracts). Our approach consists infocusing on non structural proteins <strong>of</strong> DENV that are essentials for viralreplication. A first strategy is to target non structural proteins NS5 and NS3to address the impact <strong>of</strong> drugs on enzymatic activities such as polymeraseor helicase activities. We are also interested in protein protein interactions(PPIs) virus network: viral (NS5/NS3) or cellular; in order to screenprotein protein interaction inhibitor (2P2I) library via HTRF or BRETassays. In addition, the data obtained in vitro are all validated in cellularcontext with a DENV replicon system and confirmed on cells infectedby dengue clinical isolates to assess inhibition effects and cytotoxicity <strong>of</strong>compounds. These strategies lead to the identification <strong>of</strong> promising DENVinhibitors.REF 127Polyclonal and monoclonal antibodies against human parechovirus 1and 3Brenda WESTERHUIS 1 , Ksm BENSCHOP 1 ,GKOEN 1 ,AqBAKKER 2 , Y CLAASSEN 2 , T BEAUMONT 2 , Kc WOLTHERS 11 Laboratory <strong>of</strong> Clinical <strong>Virology</strong>, Department <strong>of</strong> Medical Microbiology,Academic Medical Center, Amsterdam, THE NETHERLANDS; 2 AIMMTherapeutics, Academic Medical Center, Amsterdam, THE NETHER-LANDSThe immune response against picornaviruses is mainly humoral, andprotective neutralizing antibodies (Abs) are thought to be genotype specific.Human parechoviruses (HPeVs) are commonly detected picornavirusinfections in children. We observed that HPeV3 was difficult to neutralizein vitro, in disagreement with what is known for picornaviruses. The aimwas to study HPeV neutralization and develop neutralizing Abs againstHPeVs. Polyclonal Abs were obtained by rabbit immunization. Humanmonoclonal Abs were generated by generation <strong>of</strong> stable antibody producingB cells by genetic programming (AIMSelect). All Abs were testedfor neutralization in different cell lines. Polyclonal rabbit and monoclonalhuman antiHPeV1 Abs neutralized HPeV1 with high titers (>1:1024)and cross neutralization was found with HPeV2 (>512). In contrast, polyclonaland monoclonal aHPeV3 were not able to fully neutralize HPeV3infection in different cell lines. Although viral replication was inhibited tosome extent in the presence <strong>of</strong> Abs: the polyclonal aHPeV3 showed viralinhibition up to titer 1:256, and one monoclonal aHPeV3 inhibited replicationup to 1:32. In this study we generated highly neutralizing HPeV1Abs, unexpected both mAbs showed clear cross reactivity against HPeV2.Thus, cross neutralisation can occur in human picornaviruses, and therebypossibly cross protection in vivo as well. The generation <strong>of</strong> HPeV3 nAbswas difficult, and in line with our previous results. This finding needsfurther exploration.REF 128Mutation in the RNA-dependent RNA polymerase <strong>of</strong> Chikungunyavirus is responsible for resistance to T-705Nidya Segura GUERRERO 1,5 , Leen DEALNG 1 , Gilles QUERAT 2 ,Boris PASTORINO 2 , Mathy FROEYEN 6 , Kai DALLMEIER 1 , DirkJOCHMANS 1 , Felio BELLO 5 , Ali TAS 4 , Eric J. SNIJDER 4 , XavierDELAMBALLERIE 2 , Martina BRYON 3 , Johan NEYTS 1 , Martijn J.VAN HEMERT 4 , Pieter LEYSSEN 11 KU Leuven, Leuven, BELGIUM; 2 Université de la Méditerranée,Marseille, FRANCE; 3 Erasmus Medical Center, Rotterdam, THENETHERLANDS; 4 Leiden University Medical Center, Leiden, THENETHERLANDS; 5 Universidad del Rosario, Bogotá DC, COLOMBIAChikungunya virus (CHIKV) induced disease is an acute febrile illnesscharacterized by arthritis, arthralgia or severe joint pain that can persistfor several months up to years after the infection. There are no antiviraldrugs available for the treatment or prevention <strong>of</strong> CHIKV infections.Favipiravir, a nucleobase mimetic also known as T-705, is an effectiveand selective inhibitor <strong>of</strong> the replication <strong>of</strong> a broad spectrum <strong>of</strong> RNAviruses. We report here for the first time on the selective antiviral activity<strong>of</strong> T-705 on CHIKV replication and alphaviruses in general in tissueculture with median activities (EC50) in the range <strong>of</strong> 2-25 M observedfor lab as well as a panel <strong>of</strong> clinical strains <strong>of</strong> CHIKV. Additionally, firstpro<strong>of</strong>-<strong>of</strong>-concept <strong>of</strong> the anti-CHIKV effect <strong>of</strong> T-705 is demonstrated invivo. In the interferon receptor-deficient (AG129) mice oral administration<strong>of</strong> T-705 (300 mg/kg.day) reduced CHIKV induced mortality whenVi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S153
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>used in both a pre- and post-exposure scenario. Currently, the precisemechanism <strong>of</strong> action <strong>of</strong> T-705 has still to be unraveled. We provide firstexperimental pro<strong>of</strong> that a single mutation in the RNA-dependent RNApolymerase (RdRP) is responsible for phenotypic resistance to T-705.To that end we generated CHIKV variants that were resistant to T-705and its analogue T-1105 and were found to bear mutations in viral nonstructuralproteins. Moreover, by reverse genetics a single K291R aminoacid change in the F1 motif <strong>of</strong> the viral RdRP could be shown to be responsiblefor full (∼2-fold) phenotypic resistance to the antiviral effect <strong>of</strong>T-705.This research was funded by the <strong>European</strong> Union (FP7/2007-2013 SIL-VER n o 260644).REF 129Anti-H5N1 Specific Polyclonal Immunoglobulins are Efficient to Neutralizeand to Block H5N1 Infection: A New Approach for the ClinicalManagement <strong>of</strong> H5N1 Infected PatientsC.H. HERBRETEAU 1 , P. BUCHY 2 , F. JACQUOT 3 , C. DURAND 1 ,S. RITH 2 , L. VACHER 1 , M. JOLIVET 1 , V. LOTTEAU 1 , H. RAOUL 3 ,V. DEUBEL 2 , J.F. SALUZZO 1 , B. LÉPINE 11 Fab’entech, Lyon, FRANCE; 2 Institut Pasteur in Cambodia, Phnom Penh,CAMBODIA; 3 INSERM Jean Mérieux BSL4 Laboratory, Lyon, FRANCEHighly pathogenic avian influenza virus (H5N1) remains a major globalhealth concern, with over 630 human cases reported since 2003.In this context, Fab’entech has developed an anti-H5N1 post-exposuretreatment based on the use <strong>of</strong> highly purified specific polyclonal immunoglobulinsfragments (Fabenflu ® ). Microneutralization in vitro assay wasused to investigate the neutralization activity <strong>of</strong> anti-H5N1 immunoglobulinfragments derived from horse plasma immunized against H5N1A/Vietnam/1194/04. 100 TCID50 <strong>of</strong> 21 H5N1 strains belonging to variousclades were incubated with a range <strong>of</strong> dilution <strong>of</strong> immunoglobulins andthen transferred onto MDCK cells for neutralization analysis. The resultsshowed a high level <strong>of</strong> neutralization <strong>of</strong> all the strains, isolated in differentpart <strong>of</strong> the world and representative <strong>of</strong> 10 years evolution <strong>of</strong> the avianH5N1 virus, thus confirming the excellent cross-neutralization activity<strong>of</strong> this product against a large panel <strong>of</strong> H5N1 strains. Efficacy was alsoinvestigated in multiple in vivo experiments in BALB/c mouse intranasallyinfected at Day 0 with 1LD50 or 10LD50 <strong>of</strong> A/Vietnam/1194/04 strain.An administration protocol consisting <strong>of</strong> 5 consecutive injections followingvirus inoculation (Day1 to Day 5) and an optimal dose <strong>of</strong> 10 IU/kgin mice was demonstrated to be efficient in reducing and delaying animalmortality. These data, associated with safety and pharmacokinetics data <strong>of</strong>a phase 1 clinical trial, demonstrate the excellent potential <strong>of</strong> Fabenflu ® tomarkedly reduce the mortality and morbidity associated with H5N1 avianinfluenza infection in human.S154 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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