Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication

Abstract

Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

FIG. 1

Vector construction. (A) Locations of the six PCR fragments used to construct the full-length SHRV genomic clone and relevant restriction sites. Restriction site lines (vertical lines) which pass through the primer (arrow lines) indicate restriction sites that were designed into the primer and lines which pass through the representative genome line indicate sites naturally occurring in the genomic clone. (B) Schematic of the four plasmids used to produce recombinant SHRV in vTF7-3-preinfected EPC cells. Amp, ampicillin resistance gene; HDV, hepatitis delta virus; CMV, cytomegalovirus; Ori, origin.

FIG. 2

T7 luciferase reporter assays. (A) Luciferase activity from duplicate samples of cells transfected with pTM1-Luc and pcDNA3 and cells transfected with pTM1-Luc and pcDNA3-T7. Transfections were carried out in 6-well (10-cm²) tissue culture plates. (B) Luciferase activity from duplicate samples of cells that were mock infected and then transfected with pTM1-Luc and cells that were infected with vTF7-3 and then transfected with pTM1-Luc. Transfections were carried out in 98-well (1-cm²) tissue culture plates. Note that the transfection conditions in panels A and B were not identical and thus cannot be compared with one another directly.

FIG. 3

Schematic of the mutations introduced into pMJ-SHRV-NV⁻.

FIG. 4

Viral plaques of wild-type SHRV (A), pMJ-SHRV recombinant virus (B), and pMJ-SHRV-NV⁻ recombinant virus (C).

FIG. 5

Electron micrographs of virions of wild-type SHRV (A), pMJ-SHRV recombinant virus (B), and pMJ-SHRV-NV⁻ recombinant virus (C). Bars, 100 nm.