Establishment of Pyrosequencing Technology for Detecting Viral Hemorrhagic Septicemia Virus (VHSV)

[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.

α-Lipoic Acid Exerts Its Antiviral Effect against Viral Hemorrhagic Septicemia Virus(VHSV) by Promoting Upregulation of Antiviral Genes and Suppressing VHSV-Induced Oxidative Stress

Viral hemorrhagic septicemia virus(VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Currently, no effective vaccines, Food and Drug Administration-approved inhibitors, or other therapeutic intervention options are available against VHSV. α-Lipoic Acid(LA), a potent antioxidant, has been proposed to have antiviral effects against different viruses. In this study, LA(CC_(50)= 472.6 lmol/L) was repurposed to exhibit antiviral activity against VHSV. In fathead minnow cells,LA significantly increased the cell viability post-VHSV infection(EC_(50)= 42.7 lmol/L), and exerted a dose-dependent inhibitory effect on VHSV induced-plaque, cytopathic effects, and VHSV glycoprotein expression. The time-of-addition assay suggested that the antiviral activity of LA occurred at viral replication stage. Survival assay revealed that LA could significantly upregulated the survival rate of VHSV-infected largemouth bass in both co-injection(38.095% vs. 1.887%,P < 0.01) and post-injection manner(38.813% vs. 8.696%, P < 0.01) compared with the control group. Additional comparative transcriptome and q RT-PCR analysis revealed LA treatment upregulated the expression of several antiviral genes, such as IRF7, Viperin, and ISG15. Moreover, LA treatment reduced VHSV-induced reactive oxygen species production in addition to Nrf2 and SOD1 expression. Taken together, these data demonstrated that LA suppressed VHSV replication by inducing antiviral genes expression and reducing VHSV-induced oxidative stress. These results suggest a new direction in the development of potential antiviral candidate drugs against VHSV infection.

Establishment and Characterization of a Fin Cell Line Derived from the Atlantic Salmon Salmo salar and Its Application to Fish Virology Study

Atlantic salmon(Salmo salar)is an important economic fish that is seriously threatened by various viruses.A cell line designated as ASF derived from the caudal fin tissue of Atlantic salmon was established and characterized in this study.ASF cells grew well in Dulbecco’s modified Eagle’s medium(DMEM)containing 20%fetal bovine serum at 20℃.DNA sequencing and comparative analysis of the cytochrome B gene verified that the ASF cell line originated from Atlantic salmon.Chromosome analysis indicated that the modal chromosome number of ASF cells was 58.Viral susceptibility test showed that ASF cells were susceptive to two important fish viruses,viral hemorrhagic septicemia virus(VHSV)and red-spotted grouper nervous necrosis virus(RGNNV).Viral replication in ASF cells was further confirmed by qRT-PCR and transmission electron microscopy.Moreover,VHSV and RGNNV infections could induce the cellular responses in ASF cells,as indicated by the differential expression of cellular antiviral response-related genes,interferon-1 and Mx-1.In conclusion,the newly established ASF cell line can be applied as an in vitro tool in fish virology and immunity studies.