The Influences Of G1-like PB2 And M Genes In The Survival Advantage Of S Genotype H9N2 Influenza A Virus And Their Mechanism

The H9N2 AIVs prevalent in China belong to Eurasia lineage and are divided into multiple groups represented by A/chicken/Beijing/1/1994(BJ/94-like),A/duck/Hong Kong/Y280/1997(Y280-like),A/quail/HongKong/G1/1997(G1-like),A/duck/HongKong/Y439/1997(Y439-like),and A/chicken/Shanghai/F/1998(F/98-like).The genotype S(G57),emerging in 2007 with the substitution of G1-like PB2 gene for F98-like ones,has become the overwhelming predominant genotype for the past 10 years since 2010 and frequently donate their whole internal gene segments to other emerging influenza A viruses,such as the novel H7N9,H5N6 and H10N8 viruses.Hao et al also demonstrated that G1-like PB2 gene played a more important role in the pathogenicity of H5Nx and H7N9 influenza A viruses.However,whether G1-like PB2/M play roles in the survival advantage of genotype S H9N2 virus and whether effect their competitive advantage as an internal gene donor for the new recombinant virus are unclear.In this study,we first compared the different effects in the survival advantage of the reassortant H9N2 viruses between F/98-like M/PB2 and G1-like M/PB2 genes.Second,we constructed reassortant viruses with exchanged packaging signals between G1-like and F98-like PB2 genes based on the nucleotides differences between G1-like and F/98-like PB2 genes.And to investigate the contribution of Gl-like PB2 packaging signals to the fitness of genotype S H9N2 viruses,we evaluated the effects of package signal sequence on the protein expression and RNA synthesis level of PB2 gene as well as on the replication capacity and competitive advantage of H9N2 virus.On the other hand,to explore the possible advantages of S genotype H9N2 virus as an internal gene donor for other novel recombinant influenza viruses,we compared the competitive incorporation between F/98-like M/PB2 genes from SH14 virus and G1-like M/PB2 gene from different virus strains.1.Compare the competitive advantages between G1-like PB2 and F/98-like PB2 genes by nine plasmids cotransfection and coinfection experimentsIn this study we assessed the RNA synthesis and protein expression of Gl-like and F98-like PB2,Briefly,the G1-like PB2AH320 or F/98-like PB2SH14 plasmid was transfected into 293T cells together with three plasmids for the expression of PB1,PA and NP of AH320 virus.Forty-eight hours post-transfection,the RNA and protein levels of PB2AH320 and PB2SH14 were analyzed by quantitative real-time RT-PCR and western-blot experiments.The results showed that the protein expression level of PB2AH320 was significantly higher than that PB2SH14,whereas the mRNA,vRNA and cRNA synthesis levels of PB2AH14 were similar to that of PB2AH320.On the other hand,the reassortant wild type AH320 virus and the reassortant H9N2 virus AH320-PB2AH14 that harboring F/98-like PB2 gene from SH14 virus was generated in AH320 virus background.We tested the hemagglutination(HA)and TCID50 titer of WT-AH320 and AH320-PB2SH14 upon obtaining the reassortant viruses.The two virus stocks had a similar amount of viral particle titer,but more infective virions were produced by WT-AH320 virus.Moreover,we purified the above viruses by plaque experiment,and found that the replication of the plaque purified viruses in MDCK cell improved significantly.Besides,we performed coinfection with the two viruses at 1MOI,and NH4C1 was added to restrict the infection to one cycle.The results demonstrated that the copies of PB2AH320 and PB2SH14 were similar in cells and supernatant when the virus infected cells respectively.However,when the two viruses coinfected cells,the copies of PB2AH320 gene in the supernatant were significantly greater than that of PB2SH14 gene.2.Packaging signals of G1-like PB2 gene are involved in the prevalence of genotype S H9N2 subtype AIVIn this study,we revealed close amino acid sequences similarity and far nucleotide genetic distance between F/98-like PB2 and G1-like PB2 genes by phylogenetic analysis.The discovery implied that the variations in RNA level may also play important roles in virus adaptability Packaging signals are specific structures of each segment that contribute to mediating the packaging of the vRNA into virions.To investigate whether the packaging signals of PB2 gene were involved in the adaptability of H9N2 viruses,we constructed reassortant viruses with chimeric PB2 segments by exchanging the packaging signals of G1-like PB2 and F98-like PB2 genes.Besides,we constructed a segment PB2mutSH with S44A and T676V mutation in PB2SH14,and a segment PB2mutAH with A44S and V676T mutation in PB2AH320 to exclude the effect of the two amino acid differences(at position 44 and 676)that resulted from the nucleotides variation between PB2SH14 and PB2AH320 genes.Subsequently,we assessed the RNA synthesis levels and protein expressions of the chimerical segments by transfection experiments.The RNA synthesis and protein expression of most mutant segments based on PB2SH14 increased significantly.And most packaging signals substitutions in PB2AH320 reduced its RNA synthesis.Remarkably,replacing the packaging signals at 5'end of PB2SH14 or PB2AH320 with the sequence of each other had little influence on their RNA synthesis.In addition,the replacement of packaging signals in PB2SH14 by that of G1-like increased the replication of H9N2 virus.Otherwise,substituting the 3' end packaging signal of PB2AH320 with that of F/98-like decreased the replication of H9N2 virus significantly.Replacing the two ends packaging signals of PB2SH14 with that of PB2AH320 sequences(P-AH320-PB2asa virus),or replacing the 3'-terminal packaging signals of PB2AH320 with that of PB2SH14 sequences(P-AH320-PB2As)increased this ratio in both the coinfected cells and supernatants.5-week-old SPF chickens were inoculated 106 EID50 of the reassortant viruses in a volume of 0.1 ml,respectively.We found that replacing the packging signals of PB2SH14 with that of PB2AH320 or performed S44A and T676V mutation in PB2SH14 increased the number of chickens whose lungs and brain are positive for H9N2 viruses.In addition,compared with AH320-PB2SH14,the virus shedding of reassortant viruses lasted up to 7 dpi.And the reassortant viruses had more chances to shed viruses.Substituted the 3'end packaging signal of PB2AH320 with that of PB2SH14,or performed A44S and V676T mutation in PB2AH320 lead the virus shedding lasted for 5 days.3.Competitive incorporation between Gl-like PB2/M and F/98-like PB2/M genes in background of H7N9 virusIn the first chapter,it was found that G1-like PB2 are more competitive than F/98-like PB2 when the two H9N2 viruses were co-infected cells.And Hao et al.recently reported that G1-like PB2 and M genes of H9N2 virus enhance the fitness of H5Nx and H7N9 avian influenza viruses in chickens and mice.However,whether the G1-like M and PB2 genes are preferentially incorporated into H7N9 progeny virions during virus reassortment remains unclear;whether the M and PB2 genes from different G1-like subtypes can be differentially incorporated new virion progeny is unknown.In this study,we employed a novel reassortment approach in background of a H7N9 virus and found that G1-like M/PB2 genes showed preference in progeny virions over F/98-like M/PB2 genes.Importantly,the preference varied among G1-like M/PB2 genes that derived from different virus strains.When competing with F/98-like M/PB2 genes during reassortment,both the M and PB2 genes from an H7N9 virus GD15 showed an advantage,whereas only the PB2 gene from H9N2 virus CZ73 and the M gene from H9N2 virus AH320 displayed the advantage.Our results highlighted the preference for H9N2-derived Gl-like M and PB2 genes into H7N9 progeny virions,which provides insights into the mechanism underlying the genesis of emerging influenza viruses.4.The influence of G1-like M2 protein on the prevalence of genotype H9N2 viruses.Pu et al.found that five highly represented signature amino acid residues(37A,95K,224N,and 242N in the M1 protein and 21G in the M2 protein)encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity.In this study we found that compared with B J/94-like M gene,the signature amino acid 13N,16E,18N and 21G in G 1-like M2 protein ectodomain(ecto),the 31S in G1-like M2 protein transmembrane domain(TM),and 68M,82N,97K in G1-like M2 cytoplasmic tail(cyto)also deserve attention.To further clarify whether the above changes in the amino acids of M2 protein are related to the epidemic of genotype S H9N2 influenza virus,based on F/98-like MSH14 plasmid in Chapter 3,amino acids in the above regions were mutated to G1-like respectively to construct mutant M plasmid.On the one hand,293T cells were co-transfected with the mutant M plasmid and PB2,PB1,PA and NP of WT-AH320 virus;on the other hand,the recombinant virus with M gene mutation was rescued to infect MDCK cells.And the RNA synthesis and protein expression of M genes in the transfected and infected cells were detected by qPCR and Western-blot.Our data demonstrated that mutating the amino acids in F/98-like M2 protein to the amino acids mentioned above improved the protein expression and RNA synthesis level of Ml and M2 genes.We further measured the stability of the M mutant H9N2 virus at different temperatures.The results showed that the HA titers of all the viruses were thermally stable at 56?.When placed the mutant viruses at 42? for 24h or at room temperature for 72h,the HA titer of all mutant viruses decreased within 2 titiers,and the difference of TCID50 among the mutant viruses was not significantly at room temperature.However,after treatment at 42? for 24h,the TCID50 of WT-AH320 and AH320-MTM viruses decreased significantly less than that of other viruses,indicating that 3 1N in M2 is conducive to improve the stability of H9N2 virus at 42? which may be of positive to the prevalence of H9N2 virus.To sum up,this study,we revealed close amino acid sequences similarity and far nucleotide genetic distance between F/98-like PB2 and G1-like PB2 genes by phylogenetic analysis.Our results demonstrated that G1-like PB2 packaging signals contributed to increasing the production of infectious virus particles of H9N2 viruses.Furthermore,the packaging signals of G1-like PB2 gene were involved in PB2 protein expression,RNA synthesis,and their competitive advantage during coinfections,and are conducive to extend the virus shedding time in chicken.Besides,we found that 3 1N in G1-like M2 is conducive to improve the stability of H9N2 virus at 42?.Thus,we suggested that the packaging signals of G1-like PB2 and 3 1N of G1-like M2 protein plays important roles in the epidemic of genotype S H9N2 influenza A virus.