Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat protein-based ELISA and RT-PCR

The coat protein encoded by the nodavirus RNA2 gene originally isolated fro m greasy grouper, Epinephelus tauvina, was cloned, expressed as a recombina nt polyhistidine-tailed fusion protein and characterized by immunoblot anal ysis. The purified recombinant protein was used to develop an indirect enzy me-linked immunosorbent assay (ELISA) to detect body exudate and plasma ant ibodies against the coat protein in both experimentally infected and commer cial barramundi. In addition, the nucleotide sequence was employed to devel op a RT-PCR detection assay based on the T4 region. The results showed that the virus could be detected as early as 3 days post-infection by RT-PCR wh ile antibodies against the recombinant coat protein were detectable on day 6 post-infection. Among 112 commercial barramundi samples collected from Oc tober 1999 to April 2000, 9% showed positive ELISA results which were furth er verified by Western blot.