The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout

Abstract

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout. IMPORTANCE Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout

“…Interestingly, this amino acid change was identified also in our analysis, where aa signatures R and S were significantly associated with the high and low virulent phenotype, respectively. Similarly, a recent study using a chimeric VHSV generated by reverse genetics suggested that positions N46, N82, N83, and P39, also identified in our study, were putative markers of virulence ( Vakharia et al, 2019 ), highlighting once again the potential of the analytical model herein presented for predicting virulence determinants. Indeed, reverse genetics is the most appropriate tool to verify the SAPs identified in this work.…”

“…Several studies have attempted to link VHSV virulence to specific molecular markers based on phenotype information and/or genetic data ( Betts and Stone, 2000 ; Campbell et al, 2009 ; Dale et al, 2009 ; Einer-Jensen et al, 2014 ; Kim et al, 2014 ; Ito et al, 2016 , 2018 ; Baillon et al, 2017 ; Vakharia et al, 2019 ; Yusuff et al, 2019 ). However, because most of these attempts have been based on the visual inspection of a restricted number of sequences, the mechanisms underlying virulence are still elusive and lack statistical support.…”

“…Clearly, their effect on the virulence to rainbow trout must be validated under experimental conditions. The putative virulence markers identified also include SAPs located in the N and P protein coding regions, which have recently been suggested to be major virulence determinants for VHSV in rainbow trout ( Ito et al, 2016 , 2018 ; Vakharia et al, 2019 ). On the other hand, our results are not fully in line with previous works that have ruled out the role of the glycoprotein, the non-virion protein and of the polymerase in determining VHSV virulence in rainbow trout.…”

“…In another study, the phenotype characterization of chimeric viruses resulting from the exchange of the N and P genes of viral clones with different pathogenicity revealed that the N gene plays an essential role in VHSV virulence to rainbow trout. More interestingly, the complete gain of function of chimeric viruses could have been achieved when N was combined with the P gene from the same ancestry ( Vakharia et al, 2019 ). One common denominator of some of the above mentioned studies aiming to identify virulence markers in VHSV genome, is that they are based on the comparison of only two phylogenetically distant isolates ( Vakharia et al, 2019 ; Yusuff et al, 2019 ).…”

“…More interestingly, the complete gain of function of chimeric viruses could have been achieved when N was combined with the P gene from the same ancestry ( Vakharia et al, 2019 ). One common denominator of some of the above mentioned studies aiming to identify virulence markers in VHSV genome, is that they are based on the comparison of only two phylogenetically distant isolates ( Vakharia et al, 2019 ; Yusuff et al, 2019 ). In addition, the virulence of chimeric VHSV was only evaluated in vivo by intra-peritoneal injection, which bypasses the natural infection barriers ( Campbell et al, 2009 ; Dale et al, 2009 ; Ito et al, 2018 ).…”