Diagnosis of oyster herpes-like virus: development and validation of molecular immunological and cellular tools "VINO". Second periodic progress report: 4th January 2000 at 3rd January 2001

Type Contract report
Date 2001
Language English
Ref. FAIR-CT98-4334
Author(s) Renault Tristan1, Davison Andrew2, Xhonneux Florence3, Dorange Germaine4, Culloty Sarah5, Novoa Beatriz6, Dixon Peter7
Affiliation(s) 1 : IFREMER
2 : Institute of Virology, UK
3 : Eurogentec S.A., Belgium
4 : Université de Bretagne Occidentale
5 : University College Cork, Ireland
6 : lnstituto de Investigaciones Marinas (CSIC), Spain
Keyword(s) Pathology, Virology, Diagnosis, Herpes like virus, Validation molecular, Detection, Herpesviridae, OsHV-1, Diagnostic techniques, Oysters
Abstract The aim of the programme is to develop specific tools for diagnosing herpes-like virus infections in bivalves and to validate these reagents by using them in different European laboratories involved in shellfish epidemiological surveys. This will done using teclmiques developed by the patiners to characterise viruses by studying their genome and immunologically reactive proteins, to cultivate oyster cells and vertebrate cell lines and to perform epidemiological surveys among bivalves. The programme objectives are: 1 - Obtaining the complete oyster herpesvirus (OsHV-1) DNA sequence with determination of the genome structure. 2 - Comparing OsHV -1 with viruses belonging to the Herpesviridae family on the basis of sequence data and genome structure. 3 - Developing molecular tools for OsHV -1 detection. 4 - Developing immunological tools for OsHV -1 detection. 5 - Developing cellular tools for OsHV -1 detection using oyster prirnary cell cultures and vertebrate cell lines. 6 - Application of developed diagnostic tools for OsHV-1 detection in oyster samples from different geographical locations. Virus DNA cloning in cosmids and plasmids has been carried out in 1999 (Participant 2). The cloned viral DNA fragments have been used for characterising the virus genome and preparing specific diagnostic probes (Participants 1, 2 and 3). Identification of Îmmunogenic viral proteins provided information in 1999 that facilitated production of viral recombinant proteins in 2000 (Participant 3). Mice and rabbits have been immunised in 2000 using recombinant proteins in order to produce specific antibodies (Participant 3). A molecular biology workshop has been organised in May 2000 at the IFREMER laboratory (Partiicipant 1) in La Tremblade (Charente Maritime, France) with Participants 4, 5, 6 and 7, in order to ensure that common protocols are used for PCR and in situ hybridisation methods developed in 1999 by Participant 1. Following this workshop, preliminary PCR experiments on serially diluted viral genomic DNA furnished by Participant 1 have been carried out. Participants 1, 4, 5, 6 and 7 have performed the PCR analysis in their own laboratory using the OHV3/0HV 114 primer pair or the OHV1/OHV2 primer pair and the defined protocol. Positive and negative material (frozen larval samples and sections from fixed oysters) were also supplied by Participant 1. Frozen material has been analysed by PCR. Histological sections have been used in order to test two in situ hybridisation protocols by Participants 1, 4,5,6 and 7. A PCR procedure has also been developed in 2000 (Participant 1) and allowed to amplify short fragments of the OsHV -1 DNA after extraction of DNA from wax sections. Two primer pairs have been designed for this purpose: OH1/OH4 and IAP1/IAP2. Tests of oyster primary cell cultures and vertebrate cell lines have been pursued in 2000 in order to study the ability of the virus to replicate in vitro. The laboratories involved in mollusc epidemiological surveys (Paticipants 1, 5, 6 and 7) have taken bivalve samples during 1999 and 2000 in order to perform analyses to search herpes-like virus infections using the developed tools. In 2000, some samples have been analysed by PCR in order to diagnose herpes-like virus infections in bivalves.
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Renault Tristan, Davison Andrew, Xhonneux Florence, Dorange Germaine, Culloty Sarah, Novoa Beatriz, Dixon Peter (2001). Diagnosis of oyster herpes-like virus: development and validation of molecular immunological and cellular tools "VINO". Second periodic progress report: 4th January 2000 at 3rd January 2001. FAIR-CT98-4334. https://archimer.ifremer.fr/doc/00048/15947/