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Journal of Virology, March 2000, p. 2343-2350, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Production of Recombinant Snakehead Rhabdovirus: the NV Protein Is Not Required for Viral Replicationdagger

Marc C. Johnson,1 Benjamin E. Simon,1 Carol H. Kim,2 and Jo-Ann C. Leong1,*

Department of Microbiology, Oregon State University, Corvallis, Oregon,1 and Department of Microbiology, University of Maine, Orono, Maine2

Received 22 July 1999/Accepted 1 December 1999

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°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.

* Corresponding author. Mailing address: Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, OR 97331. Phone: (541) 737-1834. Fax: (541) 737-1841. E-mail: leongj{at}orst.edu.

dagger Technical paper 11623 of the Oregon State University Agricultural Experiment Station.

Journal of Virology, March 2000, p. 2343-2350, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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