The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout.

BACKGROUND: Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence). METHODS: Overlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection. RESULTS: Complete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0-10% mortality). CONCLUSIONS: When chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.

shRNAs targeting either the glycoprotein or polymerase genes inhibit Viral haemorrhagic septicaemia virus replication in zebrafish ZF4 cells.

Viral haemorrhagic septicaemia virus (VHSV) represents an important disease of finfish. To explore the potential of shRNAs to combat this disease nucleotide sequences of either the VHSV glycoprotein (G) or polymerase (L) gene were targeted. To test their function, shRNAs were expressed in zebrafish epithelial ZF-4 cells utilizing the zebrafish U6-2 promoter. Five of the six shRNA molecules successfully reduced VHSV replication by between 2 and 4 logs in titre relative to an irrelevant control shRNA at all MOIs and also reduced viral CPE at the highest MOI. To ensure that observed reductions in viral titre were dependent on shRNA silencing, potential non-specific antiviral responses were assessed. Only the ineffective shRNA, which formed an improper hairpin when analysed in silico, induced an antiviral response as measured by induction of interferon (ifnphi1) and Mx (MxA) genes. These results represent an important preliminary step in the generation of transgenic zebrafish resistant to VHSV.

Interchange of L polymerase protein between two strains of viral hemorrhagic septicemia virus (VHSV) genotype IV alters temperature sensitivities in vitro.

Viral hemorrhagic septicemia virus (VHSV) has four genotypes (I-IV) and sub-lineages within genotype I and IV. Using a reverse genetics approach, we explored the importance of the L gene for growth characteristics at different temperatures following interchange of the L gene within genotype IV (IVa and IVb) strains. VHSV strains harboring heterologous L gene were recovered and we show that the L gene determines growth characteristics at different temperatures in permissive cell lines.

A single amino acid mutation (I1012F) of the RNA polymerase of marine viral hemorrhagic septicemia virus changes in vitro virulence to rainbow trout gill epithelial cells.

UNLABELLED: Viral hemorrhagic septicemia virus (VHSV) is separated into four different genotypes (I to IV) with different sublineages (K. Einer-Jensen, P. Ahrens, R. Forsberg, and N. Lorenzen, J. Gen. Virol. 85:1167-1179, 2004; K. Einer-Jensen, J. Winton, and N. Lorenzen, Vet. Microbiol. 106:167-178, 2005). European marine VHSV strains (of genotypes I to III) are, in general, nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge, and here we also show that genotype IVa is nonpathogenic to trout. Despite several attempts, it has not been possible to link genomic variation to in vivo virulence. In vitro virulence to gill epithelial cells (GECs) has been used as a proxy for in vivo virulence, and here we extend these studies further with the purpose of identifying residues associated with in vitro virulence. Genotype Ia (DK-3592B) and III (NO/650/07) isolates, which are pathogenic to rainbow trout (O. B. Dale, I. Orpetveit, T. M. Lyngstad, S. Kahns, H. F. Skall, N. J. Olesen, and B. H. Dannevig, Dis. Aquat. Organ. 85:93-103, 2009), were compared to two marine strains that are nonpathogenic to trout, genotypes Ib (strain 1p8 [H. F. Mortensen, O. E. Heuer, N. Lorenzen, L. Otte, and N. J. Olesen, Virus Res. 63:95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high- and low-virulence strains were identified, and reverse genetics was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able to replicate and induce a cytopathic effect in trout GECs. The other six mutated variants remained nonpathogenic. IMPORTANCE: This is the first study to clearly link in vitro virulence of viral hemorrhagic septicemia virus (VHSV) with an amino acid residue in the L protein, a site located in conserved region IV of the L protein. In vitro virulence is documented by induction of cytopathic effects and viability studies of gill epithelial cells, and the observed cellular responses to infection are associated with increased viral replication levels. There are no previous studies addressing the importance of the L protein or the RNA-dependent RNA polymerase for virus virulence in vitro or in vivo. Therefore, the findings reported here should broaden the search for pathogenicity traits in novirhabdoviruses, and there is a possibility that the polymerase participates in defining the host species virulence of various VHSV strains.