rologie i - European Congress of Virology

5 th European Congress of Virologycells, and virus like particles of YFV was expressed in HEK 293T cells.Furthermore, we developed a panel of 31 hybridomas against the YFV,among which two monoclonal antibodies were well characterized. Withthese reagents it was possible to standardize two serologic immunoassays(sandwich ELISA) for the detection of IgG or IgM directed against YFVproteins in simian and human samples. Recombinant NS1 and VLPs showedto be good antigens for the detection of anti YFV immunoglobulins.And the ELISAs with these antigens showed high concordance in reactivityprofiles of samples when compared to a similar test using the nativeviral particle as antigen. Besides proving the usefulness of the developedtools for diagnosis of yellow fever, it was also possible to evidence the presenceof antibodies against NS1 protein in samples of primary vaccinationwith 17DD strain, in contrast with previous literature data.REF 444Real time label free measurement of virus infectivityAure SAULNIER 1 , Damien SOULET 1 , Cédric CHARRETIER 2 ,Nolwenn NOUGAREDE 11 Immunological and Microbiological Characterization Platform, AnalyticalR&D EU department, Sanofi Pasteur, Marcy l’Etoile, FRANCE;2 Virology and Immunology Platform, Analytical R&D EU department,Sanofi Pasteur, Marcy l’Etoile, FRANCEDifferent basics methods are commonly used to titer viral infectivity. Theseendpoint assays are time consuming and long lasting. Most of them arebased on direct examination of virus cythopathic effects and a specificvirus staining is often required to discriminate viral spreading. Real timecell analyzer (RTCA) system was developed to monitor dynamic label freeand non invasive cellular events using microelectronic biosensor technology.In this study, feasibility to use cell sensor technologies to substituteclassical limiting dilution method was evaluated. Large panel of virus families,known to be cytopathogenic, lytic or not, was investigated to have arepresentative view of the device performance. We compared in two celllines (Vero, MRC5) in parallel classical titration method to the real timeimpedance signal acquired with the xCELLigence RTCA (ACEA Biosciences).A comprehensive representation of the cell culture behavior wasobtained: the real time monitoring of the biological status of cells (cellularindex) showed a virus induced dose dependent impedance drop or changesfollowing infection. The time corresponding to 50% decrease/modulationin cell impedance was inversely proportional to virus infectious dose. Anextrapolation of final titers can be made sooner, few days post infection,based on the timing of the first impedance drop/modulation, for all virusfamilies tested on at least one cell substrate. We conclude that RTCA systemprovide a broad new tool, quantitative and high throughput, to realtime characterizing viral growth in cell culture.REF 445Utility of the QIAGEN artus PCR assays for the detection of humanherpes virus genomes in formalin fixed paraffin embedded clinicalsamplesOliver SCHILDGEN, Verena SCHILDGEN, Ramona Liza TILLMANN,Michael BROCKMANNKliniken der Stadt Köln gGmbH, Cologne, GERMANYObjectives: Qualitative and quantitative pathogen detection from formalinfixed paraffin embedded (FFPE) material is a major challenge in moleculardiagnostics, especially if no further unfixed clinical samples are availablefor microbiological or virological diagnostics. Thus the aim of the presentstudy was to analyse the utility of the artus real time PCR assays for thedetection of human herpes viruses (HSV1, HSV2, VZV, CMV, and EBV)in FFPE samples. Methods: Between April 2010 and September 2012we received requests for a total number of 91 clinical samples putativelypositive for human herpes viruses. FFPE material was subject to DNAextraction using the QIAGEN FFPE kit (QIAGEN GmbH, Hilden, Germany).PCRs for human herpesviruses were carried out using the artusassays for the detection of HSV1/2, EBV, CMV, and VZV according tothe manufacturer’s protocols. Results: In total, 27 samples were tested forHSV1 and HSV2, of those 3/27 (11.1%) were positive for HSV1 and 1/27samples was positive for HSV2. Of 50 samples tested for CMV, 6 werepositive (12%). 1/12 samples tested for VZV was positive for VZV and0/2 samples tested for EBV were positive. Conclusions: The PCR resultsshow that human herpes viruses can be detected from FFPE material withcommercial real time assays in an off label manner. These assays have theadvantage to detect small DNA fragments, which are a typical feature ofDNA extracted from FFPE material.REF 446Evaluation of a parechovirus PCR on cerebrospinal fluid samplespreviously considered as PCR negativeEvelyne SCHVOERER 1,2 , Aurélie VELAY 1,2 , Hélène JEULIN 1,2 , SibelBERGER 1 , Chantal FINANCE 1 , Véronique VENARD 11 Centre Hospitalier Universitaire Nancy Brabois, Laboratoire de Virologie,Vandoeuvre lès Nancy, FRANCE; 2“ Stress, IMmunité, PAthogènes”(SIMPA), EA 7300, Vandoeuvre lès Nancy, FRANCEBackground: It was admitted recently that parechoviruses can beresponsible for meningitis in young children, justifying the research ofparechoviruses in cerebrospinal fluid (CSF) to explore central nervoussystem syndromes. Aim: We are retrospectively evaluating a PARECHO-VIRUS R Gene PCR (bioMérieux, France), on CSF samples from childrenunder 5 year old, previously considered as negative for enteroviruses.Methods: The study consists of analyses performed on 50 CSF samplescollected from January 2012 August 2013. Until now, 36 CSF have beentested for parechovirus. After nucleic acid extraction on easyMAGTM(bioMérieux), PCR amplification (ABI 7500) has been realized with:ENTEROVIRUS R Gene kit, PARECHOVIRUS R Gene Kit, and theSuperscript III reverse transcriptase (RT) supplied with the PARE-CHOVIRUS R Gene Kit combined with the ENTEROVIRUS premix(PARECHOVIRUS R Gene template). Results: Two CSF samples out of36 were parechovirus positive. These parechoviruses have been detectedin children of less than 1 month old showing fever and typical signs ofmeningitis. Moreover, the combination of the ‘PARECHOVIRUS RT’ andthe ENTEROVIRUS premix has permitted the detection of enterovirusesin three out of 36 CSF samples. Conclusion: The RT supplied with thisPARECHOVIRUS kit allows us to save time when simultaneously usedwith enteroviruses and parechoviruses PCR detection kits. These preliminaryresults have highlighted that enteroviruses could be detected in threepreviously negative CSF samples, and parechoviruses in two additionalsamples.Virologie, Vol 17, supplément 2, septembre 2013S243