Prem Prasad Lamichhane1, Kobporn Boonnak2, Don Changsom1,3, Pirom Noisumdaeng4, Kantima Sangsiriwut5, Sa-Nga Pattanakitsakul6, Pilaipan Puthavathana1,3. 1. Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University. 2. Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University. 3. Center of Research and Innovation, Faculty of Medical Technology, Mahidol University. 4. Faculty of Public Health, Thammasat University. 5. Department of Preventive Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University. 6. Divison of Molecular Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University.
Abstract
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.
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.