Detection of nodavirus in barramundi, <i>Lates calcarifer</i> (Bloch), using recombinant coat protein‐based ELISA and RT–PCR

Abstract

The coat protein encoded by the nodavirus RNA2 gene originally isolated from greasy grouper, Epinephelus tauvina , was cloned, expressed as a recombinant polyhistidine‐tailed fusion protein and characterized by immunoblot analysis. The purified recombinant protein was used to develop an indirect enzyme‐linked immunosorbent assay (ELISA) to detect body exudate and plasma antibodies against the coat protein in both experimentally infected and commercial barramundi. In addition, the nucleotide sequence was employed to develop 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 while antibodies against the recombinant coat protein were detectable on day 6 post‐infection. Among 112 commercial barramundi samples collected from October 1999 to April 2000, 9% showed positive ELISA results which were further verified by Western blot.