Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.
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.
Johnson
M C
MC
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA.
Simon
B E
BE
Kim
C H
CH
Leong
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005399:Fishes; D017353:Gene Deletion; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D012209:Rhabdoviridae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
D017931:DNA Primers; D018076:DNA, Complementary; C096112:NV protein, Rhabdovirus; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases