[Detection of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. Diagnostic validation].

Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen that attacks rainbow trout (Oncorhynchus mykiss) farming. The only diagnostic method recognised by the European community for VHS is based on the detection of the viral agent in cell culture followed by an immunological identification of the pathogen. Reverse transcription followed by a double amplification of the polymerase chain reaction (RT-PCR) for the gene encoding the viral glycoprotein G is proposed here as an alternative method to the virus assay in cell culture. The RT-PCR was found to have three advantages over the viral assay method. First, the RT-PCR was found to be more rapid than the virus assay method (24 h compared to 72-120 h). Second, this method was found to be more sensitive than the virus assay (2.10(-2) pfu of virus were detected per millilitre of viral suspension compared to 2.10(-1) pfu.mL-1 by inoculation of the cell lines EPC and RTG2). Third, the RT-PCR was shown to be specific towards the VHS virus assay (represented by its four serotypes VHS I, VHS II, VHS 23/75 and VHS IV). Moreover no amplification was obtained with the other rhabdoviruses used: infectious haematopoietic necrosis virus (American strain Amend 72, WRAC, RB, SRVC) and French reference strain 69/87, eel viruses, spring viraemia of carp virus and pike fry virus. The validation of this method was performed on organs removed from experimentally infected rainbow trout and ovarian fluid samples from farmed broodfish from D0 to D150. By using RT-PCR between D30 and D60, 13 samples from nine experimentally infected trout (ten kidney-spleen pools and three brains) tested positive, whereas only nine samples (four kidney-spleen pools and five brains) from six fish were positive at D30. The last positive response was obtained by RT-PCR at D60 for kidney-spleen pools from three fish. At D150, all the results were negative. From the 60 ovarian fluid samples tested, 28 were VHS positive by the RT-PCR versus 15 by the virus assay method. Eleven out of the 60 broodfish had neutralised anti-VHS, six were negative by RT-PCR and by the virus assay, four were positive by RT-PCR and negative by virus assay and one positive by both methods. The specificity, sensitivity and rapidity of the RT-PCR method makes it an attractive alternative to classical virological methods currently recommended by European Fish Health Surveillance Programmes.