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

Abstract

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 ex...

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

“…Chimeric cDNA clones and recombinant VHSV. Construction of infectious clones of the VHSV MI03 and DK-3592B strains has been described previously, and the full-length clones were designated pVHSVmi and pVHSVdk, respectively (13,50). These two parental clones were used to construct VHSV chimeric clones.…”

“…The resulting eight chimeric recombinant viruses were viable in vitro and in vivo, but they did not exhibit any change in trout virulence relative to the parental clones. Therefore, we concluded that the VHSV G, NV, and L genes, comprising 70% of the viral genome, do not contain genetic determinants of trout virulence (50).…”

“…In contrast, the North American genotype IVb isolate MI03 from Michigan in the Great Lakes region (here noted as the "mi" strain) is avirulent in rainbow trout (41,42). We previously described generating infectious clones of these two VHSV strains (pVHSVdk and pVHSVmi, respectively) and demonstrating that the recombinant VHSV (rVHSV) produced by each clone had the anticipated contrasting virulence when tested in rainbow trout, referred to here as trout virulence (13,50). We use this qualified term because virulence in fish rhabdoviruses is host specific and there are many VHSV strains, including the MI03 strain used in this study, that are highly virulent in other fish species but have low virulence in rainbow trout (41,42).…”

“…We recently reported testing of rVHSVs generated from chimeric infectious clones in which the G, NV, G and NV, or G, NV, and L genes of the trout-avirulent pVHSVmi clone were replaced with the homologous genes from the trout-virulent pVHSVdk clone (50). Reciprocal chimeras were made using the pVHSVdk clone backbone and replacing the G, NV, G and NV, or G, NV, and L genes with the homologous genes from the pVHSVmi clone.…”

Genomic and immunogenic changes over the history of the Viral Hemorrhagic Septicemia (VHS-IVb) fish virus (=Piscine novirhabdovirus) in the Laurentian Great Lakes

“…It differed from 559 C03MU by a single AA in the L-gene, and was found in six different fish host species and the 560 sole two known invertebrate host species [99,100]. Yusuff et al [101] discerned no effect on 561 host specificity when swapping L-genes between C03MU and Ia's DK-3592B. L variants appear 562 to differ in optimal temperature range, since swapping L from IVa into IVb led to increased virus 563 at higher temperatures, than occurred for IVa [102].…”