Betanodavirus infection in Kuhlia rupestris and Ambassis marianus and isolation of betanodavirus from infected pond water.

This is the first report of betanodavirus infection in 2 species of finfish, Kuhlia rupestris (jungle perch) and Ambassis marianus (estuary perchlet). This report also describes isolation of betanodavirus from infected pond water using the SSN-1 cell line. Histopathology of K. rupestris larvae revealed vacuolation in the eye and brain, which was confirmed using betanodavirus-specific immunohistochemistry. The eye and brain from A. marianus and betanodavirus isolated from pond water were confirmed using real-time PCR and Sanger sequencing. High throughput sequencing was used to obtain betanodavirus sequences from paraffin blocks containing infected K. rupestris. The phylogenetic analysis of betanodavirus RNA1 and RNA2 sequences from all 3 sources were associated with the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The RNA1 nucleotide sequence from jungle perch showed 100% identity with the betanodavirus water isolate and 99.37% identity with A. marianus. Furthermore, we have demonstrated the usefulness of combining recovery of viable virus from environmental samples through fish cell line culture with PCR testing as a means of validating the efficacy of chlorination to eradicate betanodavirus from the pond environment.

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.