Studies On Genetic Compatibility Of H5N1 Aiv And H1N1/2009 And Mechanisms Of Oxidative Stress And Mitochondrial Dynamic Disorder Induced By H5N1

The genetic feature of influenza virus determines that reassortment is the most important way to drive influenza evolution,interspecies transmission,and ongoing prevalence.H5N1 highly pathogenic avian influenza virus has been considered to have the potential to cause pandemic,especially when the outbreak of H1N1 pandemic virus in 2009,which enhance public's worry about emerge of new virus with great hazard due to reassortment.This study is trying to uncover the characteristics and tendency of reassortment between H5N1 and H1N1/2009 by studying in vivo co-infection of H5N1 and H1N1/2009 in mouse.Oxidative stress is featured by the pro-oxidant-antioxidant balance in favor of the former,and abundant ROS accumulation.ROS plays significant roles in regulating cell bioprocesses,such as inflammatory response and apoptosis.NADPH oxidase is a major source of ROS production,and thus contributes greatly to oxidative stress.Antioxidant system can function to combat or attenuate excessive ROS production,such as superoxide dismutase 1(SOD1).However,the effects of NADPH oxidase and SOD1 on H5N1 replication and it's pathogenesis are unknown.Based on this,we investigates the roles of NOX4 and SOD1 in H5N1 replication and pathogenesis,and try to reveal the implication of ROS in H5N1 infection.Normally,mitochondria is balanced in fusion and fission,which is tightly regulated.When response to physiology disorder or virus invasion,mitochondrial dynamic would be disrupted to turn to enhance fusion/elongation or fission/fragment.mitochondrial dynamic disorder not only affects energy production,but also play key roles in regulation of immune response and apoptosis.Our study suggests that H5N1 infection can disrupt mitochondrial dynamic and enhance mitochondrial fusion.Main contents in this study:1 Study on genetic compatibility of H5N1 and H1N1/2009We co-infected mice with H5N1 and H1N1/2009,48hpi,a series of viruses were recovered from lungs of infected mice through plaque-purification,and sequenced 624 purified viruses.Sequences analysis indicated that 20 reassortant viruses with different genetic compositions were obtained,among which,virus consists with PB2 derived H1N1/2009 and other genes from HM/06(L1)account for about 53.5%,suggesting this type recombination possesses highest reassortment selectivity probability.And that all the reassortments contained HA from HM/06,suggesting the important role of H5N1-derived HA in reasortment.Besides,most reassortments possess H1N1-derived PB2,indicating that PB2 plays key role in host adaption.2 Replication and pathogenicity of reassortantsOur results demonstrated that all reassortants could replicate well in A549 cells;however,different virus exhibited varied pathogencity in mouse.L1 was most pathogenic,suggesting this virus has great potential hazard.Our study reveals that HM/06 and H1N1/2009 have great genetic compatibility,and highlighs the importance to monitor virus with genetic constitution like L1 that contains PB2 from H1N1/2009 and others from H5N1.3 NOX4 increases H5N1 replicationOur results demonstrated H5N1 infection could induce intensity oxidative stress,and NOX4 was increased upon virus infection.We fund that NOX4 could promote H5N1-induced ROS production,and increase H5N1 virus replication.However,knockdown of NOX4 could attenuate H5N1-induced ROS production and virus proliferation.4 SOD1 overexpression inhibits H5N1 virus replicationWe fund that H5N1 infection could down-regulate SOD1 expression in A549 cells.Further results demonstrated SOD1 overexpression could inhibits H5N1 replication in A549 cells and mouse primary lung epithelial cells?ROS production?and virus-induced apoptosis and pro-inflammatory response.5 H5N1 infection disrupts mitochondrial dynamic and enhance mitochondrial fusionWe fund that H5N1 infection caused significant damage to mitochondria and promoted mitochondrial hyperfusion.Furthermore,H5N1 infection could down-regulate Drpl and MFF expression,two important genes participating in mitochondrial fission regulation.Drpl and MFF knockdown could promote virus-induced apoptosis.We proposed that H5N1 can promote virus apoptosis by enhancing mitochondrial fusion.6 Different effects of Drpl and MFF on H5N1 replicationOur data demonstrated Drpl can increase H5N1 replication,while MFF can inhibit H5N1 replication.Drpl knockdown can strengthen anti-viral immune response,and thus inhibit H5N1 replication;MFF may positively regulate anti-viral immune response to inhibit H5N1 replication.