Study On The Identification And Detection Methods Of Hirame Rhabdovirus (HRV)

A strain of rhabdovirus was isolated from cultured stone flounder (Kareius bisoloratus) and its biological and physico-chemical characteristics were studied. Focal areas of cytopathic effect (CPE), such as shrinking, rounding and detaching, was observed in 8 cell lines including FHM and EPC. The optimal replication temperature of the virus was 20℃. The virus was sensitive to chloroform, pH3, pH10 and heat treatment, the virus replication was not inhibited by IUDR. This indicated that the isolate was RNA virus with envelope. Typical bullet-shape particles with the size of (60-80) nm×(160-180) nm were observed in the cytoplasm of infected EPC cells. A 515 bp fragment was amplified from the viral glycoprotein gene by RT-PCR. The fragment was sequenced and it shared more than 99% homology with the Korea reference strain of HRV. Five protein bands were observed by SDS-PAGE with the molecular weights 200 kD, 56 kD, 42 kD, 29 kD and 24 kD respectively. These data indicated that the isolate strain SR080113, was hirame rhabdovirus and was named HRV-Shandong strain.The complete genome of the strain SR080113 was cloned and sequenced with RT-PCR and RACE. The length of the viral genome was 11,037 nt, similar to the HRV Korea strain (GenBank Acc. No. NC₀05093) with the identity of more than 95%. The content of G+C in viral RNA was 50.8%. Six open reading frames (ORFs) was induced and analyzed, coding nucleocapsid protein, Phosphoprotein, matrix protein, glycoprotein, non-virion protein and RNA-dependent RNA polymerase separately. The phylogenetic analysis of nucleotide sequences and amino acid sequences suggested that the isolate had the genomic characters of the genus Novirhabdovirus.Three molecular detection methods including RT-PCR, real-time RT-PCR, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were set up, with the sensitivity of 103.5TCID50/100μL, 100 copies per test, and 101.5TCID50/100μL, respectively. A fragment of viral RNA, containing target fragment, was transcripted in vitro which could be used as the reference positive control for quantitative analysisin the real-time RT-PCR test.A sandwich ELISA using monoclonal antibody was set up for the detection of HRV and the sensitivity was 102.5TCID50/100μL. The ELISA kits were prepared and the consistency and stability of the kits were evaluated. The coefficient of variation in one lot was 1.99%-9.51% and was 4.22%-8.70% among different lots.Two hundred and twenty-four samples of cultured marine fish were collected in the surveillance program of marine fish diseases in China, in which, 123 samples were detected by isolation of virus, RT-PCR, real-time RT-PCR, LAMP and ELISA. Thirteen samples from Yellow sea and Bohai sea were HRV positive. These fish includs Kareius bicoloratus L. and Scophthalmus maximu.The detection methods were evaluated during the assay and their diagnostic performance characteristics were analyzed.