LAUSR

BACKGROUND The level of virulence of H5N1 highly pathogenic avian influenza (HPAI) virus was higher than those of the other virus subtypes. It has been suggested that the nonstructural (NS) gene might be a factor contributing to H5N1 HPAI virulence. OBJECTIVES To determine the efficiency of the NS genomic segment of H5N1 HPAI virus on governing viral infectivity and cytokine induction in monocytic cells compared to other virus strain/subtypes. METHODS By reverse genetics, five reassortant influenza viruses carrying the NS genomic segment derived from seasonal influenza A(H1N1), 2009 pandemic A(H1N1), A(H3N2) or H5N1 HPAI virus in the backbone of A/Puerto Rico/8/34 H1N1 (PR8) virus were constructed together with the reassorted PR8 virus control, i.e., rgH1N1sea-NS, rgH1N1pdm-NS, rgH3N2-NS, rgH5N1-NS and rgPR8 viruses, respectively. These reverse genetics-derived viruses (rg-viruses) were used to infect monocytic cells for 24 hours prior to determining intracellular influenza nucleoprotein (NP) levels and cytokine induction by flow cytometry. RESULTS U937 cells were significantly more susceptible to rgPR8 control virus than THP-1 cells; thus, U937 cells were chosen for further study. The number of U937-infected cells (NP+ cells) and the numbers of infected cells that expressed IFN-α (NP+IFN-α+ cell) obtained with rgH5N1-NS virus infection were significantly higher than the others, except for cells infected with the rgH1N1pdm-NS virus. Nevertheless, the numbers of U937 cells that expressed NP+IL-1β+ were comparable upon infection with any of the rg-viruses; almost none expressed TNF-α. CONCLUSIONS The H5N1 NS genomic segment distinctly up-regulated the viral infectivity and induction of IFN-α compared to the rgPR8, rgH1N1sea-NS and rgH3N2-NS viruses.