A combined cell culture and RT-PCR method for rapid detection of piscine nodaviruses

A rapid method to detect piscine nodaviruses, the causal agents of viral nervous necrosis (VNN), at low virus levels is described. The reverse transcription-polymerase chain reaction (RT-PCR) technique required 10(4)-10(5) TCID50 for detection of four strains representing different genotypes of piscine nodavirus: striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV) and barfin flounder nervous necrosis virus (BFNNV). All the genotypic variants were isolated at the lowest titre (10 degrees TCID50) by the E-11 cell line, a clone derived from the SSN-1 cell line, but it took approximately 10 days until the cytopathic effect (CPE) appeared in cells inoculated at low virus levels. When the virus was inoculated at various concentrations in E-ll cells and incubated at 20 or 25 degreesC, cells inoculated previously with virus at a dose of 10 degrees TCID50 became positive within 48 h incubation in the RT-PCR test. The present combined procedure of cell-culture and RT-PCR techniques will be useful as a rapid and convenient method to detect infective viral particles from asymptomatic carriers or samples with low virus levels.