Journal of Virological Methods 119 (2004) 65–72 Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation V. Barbosa-Solomieu a,b , L. Miossec a , R. Vázquez-Juárez b , F. Ascencio-Valle b , T. Renault a,∗ a Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France b Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Marine Pathology Unit, 23000 La Paz, B.C.S., Mexico Received 27 August 2003; received in revised form 11 February 2004; accepted 11 February 2004 Available online 30 April 2004 Abstract In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200 bp) were designed (C 9 /C 10 and B 4 /B 3 ). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C 9 /C 10 , which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data. © 2004 Elsevier B.V. All rights reserved. Keywords: Herpesvirus; Oyster; Crassostrea gigas; Ostrea edulis; In situ hybridisation; Polymerase chain reaction; Diagnosis; Archival samples 1. Introduction Since 1972 several viruses similar morphologically to members of the family Herpesviridae have been identified in various marine mollusc species around the world (Renault, 1998; Arzul et al., 2002). Viral detection was often associ- ated with high mortality rates in spat and larvae of farmed bivalves among which the Pacific oyster Crassostrea gi- gas (Nicolas et al., 1992; Hine et al., 1992; Renault et al., 1994a,b). More recently, herpes-like viruses were observed in adult Ostrea angasi (Hine and Thorne, 1997) and in larvae of Tiostrea chilensis (Hine et al., 1998), Ruditapes decus- satus (Renault and Arzul, 2001), Ruditapes philippinarum ∗ Corresponding author. Tel.: +33-546-36-98-36; fax: +33-546-36-37-51. E-mail address: trenault@ifremer.fr (T. Renault). (Renault et al., 2001) and Pecten maximus (Arzul et al., 2001a). The basic method to diagnose herpes-like virus infections has long been light microscopy, although this procedure appears to be poorly adapted to viral diseases. Additional techniques, such as transmission electron microscopy, need therefore to be used for confirmation of the diagnosis. Despite being time consuming and somewhat inadequate for epidemiological surveys, both techniques have been employed extensively because of the lack of alternative diagnosis procedures. In the case of bivalves, research into virus cytopathogenic effects in cell cultures is not practical owing to the absence of suitable cell lines. Furthermore, the development of serological methods is impeded by the absence of immunoglobulin production in molluscs. Within this context, the development of a protocol for the purification of viral particles from fresh infected larvae of C. gigas (Le Deuff and Renault, 1999) presented a way out 0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.02.007