Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Abstract

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 mutantswere 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.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

“…Effects of NS1 mutations on pH1N1 virus growth and induction of IFN-␤ in cell cultures. IAV NS1 has previously been shown to play a significant role in viral pathogenesis in vivo (15,44,45). Generally, mutations decreasing the ability of NS1 to counteract host innate immune responses have been shown to reduce the virulence of IAV in vivo (46,47).…”

Novel Influenza Virus NS1 Antagonists Block Replication and Restore Innate Immune Function

“…NS1 is a nonstructural protein encoded by segment 8 of influenza virus A. Genetic analyses of NS1 have shown that viral replication, spread, and pathogenesis are very dependent on the function of this protein (3,4,6,10,11,13,17,18,22,27,29,30,36,63,66,74). This satisfies an important criterion for an anti-influenza virus target, since drugs that inhibit the action of the target must be able to slow virus production and/or pathogenesis as a consequence.…”

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents

“…The EHF sequels develop in the presence of a cytokine storm and immunoparalisis due to infection of macrophages and dendrite cells and a block of synthesis and functional activity of interfer ons [23][24][25][26]. The Ebola virus came into focus of relevant studies [22,23] soon after a discovery of virus proteins acting as interferon antagonists [29]. As a result, VP35 was identified as a protein that to blocks interferon pro duction in early infection [22,23].…”

“…Large scale generation of immune complexes in circulation is also considered to be a factor that provokes blood clotting [28,[43][44][45]. The disease outcome is usually determined on days 6-11, when either the acute phase crisis resolves or a transition to the terminal stage occurs [14,29,[31][32][33][34][35][36][37][38][39]. This circumstance shows that the therapy window is limited and that a medical decision is to be made within a short while; it is therefore of immense impor tance to detect the turning period [25,[40][41][42]46].…”