Avian-derived NS gene segments alter pathogenicity of the A/Puerto Rico/8/34 virus

•Diverse avian-derived NS recombinant viruses in A/Puerto Rico/8/1934 (PR8) exhibited similar viral growth patterns in vitro and in vivo.•The NS recombinant viruses showed varied pathogenicity in infected mice.•Mice infected with the more highly-virulent recombinant viruses (PR8 and PR8 × 483NS) showed greater and more sustained cytokine production levels.•Avian-derived NS genes alter pathogenicity, histopathology, and cytokine production in mice.

While the effect of the influenza A virus non-structural protein (NS) on cytokine production during viral infection is well known, inconsistent results have been observed with some other influenza A virus backbone studied. In this study, in order to focus on the impact of the avian NS gene segments on viral virulence, the NS genes encoded by different strains of avian influenza A viruses were incorporated into an identical [A/Puerto Rico/8/1934(H1N1), PR8] virus background to generate various NS recombinant viruses. Thus, PR8NS, PR8 × [A/Hong Kong/483/97(H5N1) 483NS, PR8 × [A/Ck/Korea/150/03(H9N2) 150NS, and PR8 × [A/EM/Korea/W149/06(H5N1) W149NS were constructed utilizing reverse genetics. Here, we show the effects of each of these recombinant viruses upon viral pathogenesis and cytokine production during viral replication in vivo. In this regard, we found that infection of mice with the PR8 × 150NS recombinant virus resulted in the lowest pathogenicity (6.0 × 104 MLD50), yet elicited the highest levels of TNF-α production in bronchoalveolar lavage (BAL) fluid compared to infection with the other recombinant influenza viruses. In contrast, infection with the PR8 virus showed the highest pathogenicity (1.0 × 102 MLD50) as well as relatively high cytokine levels (IL-1α, IL-1β, IL-17, and eotaxin) in mouse BAL fluid. In addition, the PR8 and PR8 × 483NS viruses induced severe and extensive inflammation in infected lungs compared with that of PR8 × 150 NS recombinant virus-infected mice. These results clearly demonstrate that the NS genes of diverse influenza A strains can variable impact pathogenicity, histopathology, and cytokine production in mice even when expressed in an identical genetic background.