Isolation and identification of a new strain Micropterus salmoides rhabdovirus (MSRV) from largemouth bass Micropterus salmoides in China

As an important farmed fish worldwide, largemouth bass (Micropterus salmoides) is suffering a severely disease caused by rhabdovirus, which has resulted to a massive economic loss. In the present study, a new Micropterus salmoides rhabdovirus (MSRV) strain named MSRV-NY01 was isolated from largemouth bass juvenile from a fish farm in Nanyang, Henan province, China. Cell culture, histopathological examination, reverse transcription-polymerase chain reaction (RT-PCR), plaque purification, phylogenetic analysis, transmission electron micrograph (TEM) observation, experimental infection, real-time quantitative polymerase chain reaction (RT-qPCR) were used for virus isolation and identification. The diseased fish exhibited abnormal symptoms with anorexia, exophthalmia, bloated abdomen, spiral swimming, hemorrhage on body surfaces, and fluid accumulation in the abdominal cavity. Histopathological examination showed obvious hemorrhage, inflammatory cell infiltration, and widespread cell degeneration and necrosis were presented in the liver, spleen, intestine and stomach of diseased fish. Obvious cytopathic effects (CPE) were induced in epithelioma papillosum cyprini (EPC), grouper spleen (GS) and grouper fin (GF-1) cell lines after inoculation of mixed liver and kidney homogenates from disease fish. RT-PCR, DNA sequencing and phylogenetic tree analysis showed that the virus belongs to rhabdovirus with the highest similar to the MSRV-FJ985 strain. Through five sequential rounds of plaque purification assay, a MSRV plaque isolate was derived and was confirmed by RT-PCR and DNA sequencing. TEM observation showed that numerous virions with typical bullet shape (100–150 nm in length and 70–90 nm in diameter) were present in infected GS cells. Thus, this newly isolated virus from diseased Micropterus salmoides was tentatively named as MSRV-NY01. Regression infection experiment indicated that MSRV-NY01 was highly pathogenic to Micropterus salmoides juvenile and the cumulative mortality of fish injected with 5 × 104, 104, 2 × 103, 4 × 102, 2 × 102, 102 and 50 PFU of virus were 100%, 100%, 100%, 96.7%, 96.7%, 93.3% and 90%, respectively. Moreover, RT-qPCR showed that MSRV was present in all 9 tested tissues of infected fish, and the virus loads of the spleen and heart were significantly higher than that in other tissues tested. In conclusion, we isolated and identified a new MSRV strain from diseased largemouth bass, and our results will provide new material and information for the prevention and control strategy for MSRV.