Naturally Occurring Substitution In One Amino Acid In Vhsv Phosphoprotein Enhances Viral Virulence In Flounder

Author summaryViral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes huge economic losses to the fish culture industry throughout the world. Virulence among naturally occurring VHSV strains varies widely. However, little is known about the viral factors that determine VHSV virulence. Here, we identify a naturally-occurring, single-amino-acid substitution in the VHSV P protein that enhances VHSV virulence in flounder. This amino acid substitution in the P protein was detected only in highly virulent VHSV strains, and it enhances viral RNA synthesis and inhibits the interferon response of host cells early after virus infection. Recombinant VHSV containing this amino acid substitution caused increased mortality in flounder compared with the wild type. This is the first study to identify a naturally occurring amino acid substitution in VHSV that determines its virulence in flounder. We expect that our result can be applied to other fish species, and this finding will provide new opportunities to generate an effective VHSV vaccine.Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; P-P55L) that is specific to highly virulent strains. This P-P55L substitution was maintained stably after 30 cell passages. To investigate the effects of the P-P55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying P-P55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of P-P55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.