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 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.

Keywords: Fish; Glycoprotein; Non-virion protein; Polymerase protein; Rhabdovirus; VHSV; Virulence determinant.

Fig. 1

Construction of full-length cDNA clone of VHSV DK-3952B genome. Six overlapping cDNA fragments covering the entire VHSV genome were assembled by ligation into a pCI vector using the EcoRI, PvuII, EcoRV, KpnI, SalI, BclI, SmaI, StuI, BsrGI and NotI restriction sites. Assembly was carried out in 3 steps; (i) cloning of six fragments (F1-F6) separately in pGEM-T vectors (see methods), and addition of hammerhead ribozyme (HHRz) at the 5′-end of F1 and hepatitis delta ribozyme (HdvRz) at the 3′-end of F6; (ii) construction of A, B and C clones into the pCI vector; (iii) assembly of the full-length clone by ligating fragments from these three clones in the pCI vector. Restriction sites artificially created (*) and naturally present in the genome are indicated. Abbreviation: CMV, cytomegalovirus immediate-early enhancer and promoter

Fig. 2

Schematic presentation of full-length cDNA constructs of VHSV strains MI03 (mi) and DK-3592B (dk) and various chimeric VHSVs. A map of the VHSV genome is shown at the top of panels A and B, depicting coding regions of N, P, M, G, NV and L genes that are separated by the flanking regulatory untranslated and intergenic sequences (vertical bars). Selected restriction sites, important for the construction of chimeric cDNA clones, are shown. These unique sites are present in the intergenic regions of the clones, which does not affect the coding regions of viral proteins. All these constructs contain a cytomegalovirus promoter at the 3′-end. A). Chimeric cDNA clones derived by substituting VHSV DK-3592B gene(s) into the pVHSVmi full-length clone. B). Chimeric cDNA clones derived by substituting VHSV-MI03 gene(s) into the pVHSVdk full-length clone. Light green boxes depict the coding regions of VHSV strain MI03, whereas, the purple boxes represent the coding regions of VHSV strain DK-3592B

Fig. 3

Replication kinetics of parental rVHSVmi, rVHSVdk, and chimeric VHSVs in vitro. Monolayers of EPC cells were infected at an MOI of 0.01 with the chimeric viruses harboring substitutions of specific VHSV gene(s) in the parental rVHSVmi (panel A) or rVHSVdk (panel B) viruses derived from respective pVHSVmi or pVHSVdk plasmids. The viruses were harvested at the indicated time points, and virus titers were determined by plaque assay

Fig. 4

Virulence of parental rVHSVmi, rVHSVdk and chimeric rVHSVs in juvenile rainbow trout. Juvenile rainbow trout were challenged, as described in Table 3, by intraperitoneal injection with parental rVHSVmi or rVHSVdk, chimeric viruses with reciprocal gene exchanges, wild-type MI03 or DK-3592B viruses, or mock-challenged with PBS. Experiments 1 (circles) and 2 (squares) tested exchanges of the G, NV, G and NV, or G and NV and L genes. Mortality of fish after challenge was recorded daily and expressed as the average cumulative percent mortality (CPM) for triplicate subgroups of 20 fish. Variation among subgroups is provided in Table 3. A) rVHSVmi series groups for experiments 1 and 2, B) rVHSVdk series groups for experiments 1 and 2

Fig. 5

Infectious virus titers in rainbow trout 7 days after challenge with low virulence rVHSVmi series viruses in experiments 1 and 2. Juvenile rainbow trout in low virulence rVHSVmi series groups in experiments 1 and 2 were sampled at 7 days post-challenge and tested for infection status by plaque assay. Nine fish were sampled for each experimental group, except the mock group in experiment 1 and the rVHSVmi-Gdk group in experiment 2, each of which had 6 fish. Titers of infectious virus are presented for individual fish in each group with a horizontal bar indicating the mean of virus-positive fish. Red dotted lines indicate the amount of virus in the inocula injected into each fish, so that virus titers above this line indicate ability of the virus to replicate in vivo. The detection limit for the plaque assay is 100 PFU, as indicated by the gray dotted line