J Virol, August 1998, p. 6437-6441, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Institute of Applied Microbiology,
Received 18 February 1998/Accepted 30 April 1998
We established a reverse genetics system for the nonstructural (NS)
gene segment of influenza A virus. This system is based on the use of
the temperature-sensitive (ts) reassortant virus 25A-1. The
25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene,
a plasmid-derived NS gene from influenza A/PR/8/34 virus was
ribonucleoprotein transfected into cells that were previously infected
with the 25A-1 virus. Two subsequent passages of the transfection
supernatant at 40°C selected viruses containing the transfected NS
gene derived from A/PR/8/34 virus. The high efficiency of the selection
process permitted the rescue of transfectant viruses with large
deletions of the C-terminal part of the NS1 protein. Viable
transfectant viruses containing the N-terminal 124, 80, or 38 amino
acids of the NS1 protein were obtained. Whereas all deletion mutants
grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length
of the deletions. In Vero cells expression levels of viral proteins of
the deletion mutants were similar to those of the wild type. In
contrast, in MDCK cells the level of the M1 protein was significantly
reduced for the deletion mutants.
*
Corresponding author. Mailing address: Institute of
Applied Microbiology, University of Agriculture, Muthgasse 18b,
A-1190 Vienna, Austria. Phone: 43-1-36006-6593. Fax: 43-1-3697615. E-mail: A.Egorov{at}iam.boku.ac.at.
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