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. Plasrnids 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 32~-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.

Cloning, sequence analysis and expression of the major outer capsid protein gene of an aquareovirus.

“…The VP7 protein, encoded by genome segment 10, has been shown to be glycosylated and is the major outer capsid protein (Samal et al, 1990 ;Subramanian et al, 1994 ;Shaw et al, 1996). In addition, using reciprocal RNA-RNA hybridization studies, it has been shown that genome segment 10 is the most variable gene between genogroups A and B (Lupiani et al, 1993 a). The nucleotide sequences of the major outer capsid proteins of other members of the family Reoviridae have been reported (Both et al, 1982 ;Purdy et al, 1985 ;Bassel-Duby et al, 1985 ;Chen et al, 1990 ;Qian et al, 1991).…”

“…Like rotaviruses, aquareoviruses have a genome composed of 11 segments of doublestranded RNA (dsRNA ; Lupiani et al, 1995). On the basis of cross-hybridization studies using total genomic RNA probes, aquareoviruses have been classified into five genogroups (A-E ; Lupiani et al, 1993 a). Among these, genogroup A was found to be the most prevalent.…”

“…2 (b), SBR and recombinant VP7 proteins reacted equally with the monospecific serum. In aquareoviruses, five genogroups (A-E) have been identified using reciprocal RNA-RNA cross-hybridization (Lupiani et al, 1993 a). Therefore, studies were conducted to examine whether recombinant VP7-monospecific serum could be used to differentiate between strains of the different aquareovirus genogroups.…”

“…Therefore, studies were conducted to examine whether recombinant VP7-monospecific serum could be used to differentiate between strains of the different aquareovirus genogroups. In order to do this, two representative strains of genogroup A and one of each one of the other four genogroups (Lupiani et al, 1993 a) were purified and analysed by Western blot. As shown in Fig.…”

“…SBR genome segment 10 dsRNA was cloned into the BamHI site of the pGEM-7Zf(j) plasmid vector (Promega) as previously described (Lupiani et al, 1993 b). Sequence analysis of overlapping cDNA clones specific for viral segment 10 was carried out by the dideoxynucleotide chain termination method using the Sequenase 2.0 sequencing kit (US Biochemical).…”