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.