Natural outbreak of viral encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax: study by nested reverse transcriptase-polymerase chain reaction

Abstract

In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.