Effect And Mechanism Of H9N2 Internal Gene Cassette On Pathogenicity Of H5 Subtype Avian Influenza Reassortants

Genotype S H9N2 avian influenza viruses(AIVs)have become the predominant one and frequently donate all their internal genes as a whole cassette to facilitate the generation of novel reassortants such as H5N6,H10N8 and H7N9 AIVs.Currently,the co-endemicity of both H5 and H9 subtype AIV in China has made their natural reassortment quite easily.Furthermore,it is worth noting that the novel reassorted H5 viruses harboring the whole set of internal genes from circulating H9N2 have already emerged in poultry and human.In order to evaluate the biohazard of these H5 variants,it is highly needed to investigate the potential effect of the internal gene cassette of current dominant genotype S H9N2 virus on the pathogenicity of H5 reassortants.Genotype S H9N2 virus was generated by replacing the F/98-like M and PB2 genes with G1-like M and PB2 genes on the genetic backbone of genotype H,respectively.However,what are the biological effects and the potential mechanism that the different lineages of H9N2 PB2 and M genes have on the pathogenicity of H5 reassortants?In this study,we investigated the molecular basis and host mechanism of the different geotype H9N2 internal gene cassette on the pathogenicity of H5 subtype reassortants.We found the H5 reassortants contained genotype S H9N2 subtypes internal gene cassette(H5/S)all showed attenuated pathogenicity in mice and chickens.However,the reassortant H5 viruses with genotype H internal gene cassette(H5/H)displayed attenuated virulence than those of the H5/S viruses in chicken and mouse models.Furthermore,H5 influenza viruses possessing H9N2 virus G1-like M and PB2 but not F/98-like M and PB2 contributed to their increasing fitness in mice and chickens.We then further determined the functional significance of M or PB2 gene in conferring increased polymerase activities and improved nucleus transportation efficiency by G1-like PB2,and increased spherical virions and facilitated efficient translocation of RNP to cytoplasm by Gl-like M.We also investigated the host factors that account for their virulence discrepancy.We found that the H5/S reassortants induced much stronger response that related with stimulus-related functions and more innate immune response-related pathways than H5/H,which may be assoiated with the high virulence of H5/S in chickens.1.Effect of internal gene cassette from S genotype H9N2 Avian Influenza Virus on the pathogenicity of H5 reassortantsH9N2 avian influenza virus(AIV)of the genotype S frequently donates internal genes to facilitate the generation of novel reassortants such as H7N9,H10N8,H5N2 and H5N6 AIVs,posing an enormous threat to both human health and poultry industry.However,the pathogenicity and transmission of reassortant H5/S viruses carrying internal gene cassette of genotype S H9N2-origin in chickens and mice remain unknown.In this study,four H5 reassortants carrying the HA and NA genes from different clades of H5 viruses and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics.We found that all the four H5/S reassortant viruses showed attenuated virulence in both chickens and mice,and lead to increased mean death times compared to the corresponding parental viruses.Consistently,the polymerase activity and replication ability in mammalian and avian cells,and the cytokine responses in the lungs of chickens and mice were also decreased when compared to their respective parental viruses.Moreover,these H5/S reassortants transmitted from birds to birds by direct contact but not by an airborne route,and H5/S virus shedding from the trachea lasted at least 7 days.Therefore,our data indicated that the internal genes as a whole cassette from genotype S H9N2 viruses play important roles in reducing the pathogenicity of the H5 reassorants in chickens and mice with lengthened virus shedding time,and might contribute to the circulation of virus in avian or mammalian hosts.2.Effect and mechanism of different lineages of H9N2 PB2 and M genes on pathogenicity of H5 subtype avian influenza reassortants in poultry models Genotype S H9N2 avian influenza viruses(AIVs),which have been predominant in China since 2010,frequently donate their internal gene cassette to facilitate the generation of novel reassortants such as H5N6,H10N8 and H7N9,etc.Genotype S was originated from the replacement of F/98-like M and PB2 genes of the genotype H internal gene cassette with those from G1-like lineage.However,whether this gene substitution will influence the viral fitness of emerging influenza viruses in animals remains unclear.In this study,we found that reassortant H5 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence than those with the genotype H internal gene cassette in cell cultures and in chicken models.We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared to the F/98-like counterpart,while the G1-like M gene facilitated the generation of more spherical virions and more effective translocation of RNP to cytoplasm.Our findings suggestted that the genotype S H9N2 internal gene cassette,which possesses G1-like M and PB2 genes,are superior to that of genotype H in optimizing viral fitness,thus may partially explain why the former has been frequently involved in the genesis of emerging influenza viruses that caused clinical infections in humans.3.Effect and mechanism of different lineages of H9N2 PB2 and M genes on pathogenicity of H5 subtype avian influenza reassortants in mammalian modelsGenotype S H9N2 avian influenza viruses(AIVs)frequently donate their internal genes to facilitate the generation of novel reassortants such as H5N6,H10N8 and H7N9 AIVs.A key feature of the S genotype is the substitution of the earlier F/98-like M and PB2 genes with the G1-like M and PB2 genes.However,which internal gene combination of H9N2 confers the optimal viral adaptability in mammals remains unclear.In this study,we found that the H5 viruses with G1-like M or PB2 gene,but not F/98-like M or PB2 gene,manifested higher virulence and replication in mice.Consistently,H5 viruses carry the G1-like M or PB2 gene,in place of F/98-like M or PB2 gene,showed higher viral replication and more viral protein production in mammalian cells.Moreover,compared with M gene,the PB2 gene had a greater contributioa We further determined the functional significance of PB2 gene in H5 viruses in conferring the increased polymerase activity and enhanced nuclear accumulation,and the G1-like M in increasing spherical virions release and enhancing the translocation of RNP to cytoplasm from mammalian cells.Therefore,our results suggest that the G1-like M and PB2 genes of H9N2 virus confer survival advantage to H5 reassortants and contribute to their increased fitness,and might increase the infection ability of the novel reassortant viruses in mammals.4.The differential proteomics of the H5/S and H5/H recombinants in chicken lungs using TMT-based quantitative proteomicsWe previously reported the two reassorted H5 viruses harboring the gene cassette of internal genes from S or H genotype H9N2 differ greatly in their virulence in chickens.H5/S is highly lethal to chickens,whereas H5/H is low lethal to chickens.In this study,to investigate the host factors that account for their virulence discrepancy,we compared the pathology and host proteome of the H5/S-or H5/H-infected chicken lungs.Severe lung injury was observed from H5/S-infected chickens.However,only mild lesions were observed in H5/H-infected chickens.Using the quantitative TMT coupled LC-MS/MS method,we first found that more significantly differentially expressed(DE)proteins were stimulated by H5/S when compared with H5/H.Furthermore,biofunction analysis of the DE proteins suggested that H5/S induced much stronger response of stimulus-related functions than H5/H,including inflammatory response,cell killing and response to cytokines,etc.Consistently,KEGG pathway analysis also demonstrated that H5/S activated more the innate immune response-related pathways than did H5/H,which results in a wide range of biological activities in response to viral infection,including NOD-like receptor signaling pathway,MAPK signaling pathway,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway and influenza A virus infection.Additionally,the H5/S virus induced the targeted DE proteins,RELA,actively involved in these important regulatory pathways in response to viral infection.Therefore,our findings suggest that the increased lung injury-associated host response induced by H5/S may contribute to the lung pathology and the subsequent high viral virulence in chickens.