Isolation and identification of a new strain of hirame rhabdovirus (HIRRV) from Japanese flounder Paralichthys olivaceus in China

Abstract

Background: Hirame rhabdovirus virus (HIRRV) is a rhabdovirus that causes acute hemorrhage disease in fish culture, resulting in a great economic loss in parts of Asia and Europe.

Methods: In this study, we isolated a virus strain named as CNPo2015 from cultured Japanese flounder in Shandong province, China. Cell isolation, electron microscopic observation, RT-PCR detection and phylogenetic analysis were used for virus identification. Further, artificial infection experiment was conducted for virulence testing.

Results: The gross signs included abdominal distension, fin reddening and yellow ascitic fluid in the abdominal cavity. Histopathological examination revealed marked cell degeneration and necrosis in the kidney. The tissue homogenates induced obvious cytopathic effects in EPC, FHM and FG cell lines. Electron microscopic observation showed the virus had a bullet-like shape with a capsule membrane. RT-PCR and sequencing analysis revealed that CNPo2015 belonged to the HIRRV with high sequence identity to HIRRV isolates. Infection experiment confirmed that the HIRRV CNPo2015 strain was virulent to flounder juveniles with a LD₅₀ value of 1.0 × 10^5.9 TCID₅₀/fish.

Conclusion: In conclusion, we described the first isolation and characterization of a HIRRV from Japanese flounder in China. This will provide a candidate material for further research on the infection mechanism and preventive strategies of HIRRV.

Keywords: Hirame rhabdovirus; Identification; Paralichthys olivaceus; Pathogenicity.

Fig. 1

Clinical signs and pathologic changes of diseased Paralichthys olivaceus. a Symptoms on the surface of diseased fish showed congested fins, expanded abdomen and dark body color. b Gross appearance of visceral of diseased fish displayed signs including dark red spleen, congested intestine, and yellow ascitic fluid. c Kidney section of healthy fish. d Kidney section of diseased fish displayed pathologic changes including cell degeneration and necrosis (arrow), karyopyknosis with pieces of broken nucleus. Scale bars = 20 μm

Fig. 2

Virus isolation on four fish cell lines. Compared with uninfected fish cell lines, the infected EPC cell line showed typical CPE with cell rounding, detachment and dead cells. The infected FHM and FG cell lines were also observed with CPE. No CPE were observed in CAR cell line. Magnification, 30 ×

Fig. 3

Morphology of the virions under electron microscope. a A large amount of virions aggregated on the surface of the cell. b Virions were wrapped by vesicles in the cytoplasm. Scale bars = 200 nm

Fig. 4

RT-PCR analysis. a RT-PCR amplification from infected EPC cells using specific primers of HIRRV, VHSV, IHNV, VNNV and LCDV. b The flounder β-actin was used as an internal control gene

Fig. 5

Phylogenetic analysis performed using P genes from six HIRRV isolates. The P gene sequence of the HIRRV CNPo2015 is in red color. The scale bar at the bottom indicates 0.002 nucleotide substitutions per site

Fig. 6

Cumulative mortality rates of flounder juveniles after challenge with different doses of the HIRRV CNPo2015 through day 14 post-infection. Each group contained 30 fish