A genetic probe for identification of the turbot aquareovirus in infected cell cultures

Abstract

A nucleic acid hybridization assay has been developed that can detect small amounts of the turbot aquareovirus (TRV) in infected cells. Complementary DNAs were synthesized from the dsRNA genome segments of TRV and used to generate a large number of recombinant plasmids. Plasmids corresponding to genomic segments 1 through 8 were identified by Northern blot analysis. A randomly selected clone, Clone 66, hybridizing to genome Segment 8, showed high specificity hybridizing only RNA from TRV, and not with the RNAs of 4 other aquareoviruses, or with RNA extracted from infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV) or with uninfected cells. A super(32)P-labeled probe was able to detect as little as 50 ng of TRV purified virus dsRNA. Time course experiments indicated that TRV RNA can be detected in infected cells 96 h post-inoculation, a time when cytopathic effect is still not evident. These results indicate that the dot blot assay described here could be used as an effective diagnostic tool for the detection of TRV infections.