Adaptive Mutations In Receptor Binding Domain And Adjacent Region Of HA In Collaboration With PB2 627K Contribute To Increasing Pathogenicity Of H9N2 Subtype AIV In Mice

H9N2 avian influenza viruses(AIV)which are currently endemic in poultry in China,not only cause considerable economic loss in the poultry industry,but also can induce cross-species transmission that poses a continual threat to human health.The high antibody positive rate among workers in the live poultry market indicates that the H9N2 virus infection in humans is very common,while its low pathogenicity in humans results in fewer reported cases.AIV pathogenicity in mice is a polygenic trait.Among them,HA,which affects the receptor-binding preference,and the PB2,which affects the ability of AIV to replicate in mammalian cells,play important roles in virulence.Therefore,exploring key amino acid sites in HA and PB2 of H9N2 AIVs that affect the pathogenicity to mammals is of significant importance for the prevention and control of potential H9N2 pandemics in the future.In this study,an H9N2 SDKD1/15 strain which naturally harbors mammalian-adaptive molecular markers in the HA and PB2 proteins,but with low pathogenicity for mice was isolated from LPMs in Eastern China during routine epidemiological surveillance.We identified some novel key sites in HA protein associated with increased pathogenicity in mice,when we performed a gain-of-function experiment by using the strain passaged in mice and sequence analysis of natural viruses.In addition,we found that these novel sites in HA act synergistically with 627K in PB2 to increase pathogenicity in mice.1.Biological characteristics of an H9N2 strain naturally harbored mammalian-adaptive molecular factorsDuring epidemiological surveillance of AIVs in Eastern China,we isolated an avian-origin H9N2 strain,named A/chicken/Eastern China/SDKD1/2015(SDKD1/15).SDKD1/15 had the Gln-to-Leu mutation at position 226 and the Gly-to-Ser mutation at position 228 of the HA protein(H3 numbering).We also identified Glu-to-Lys mutation at position 627 of the PB2 protein,which is critical for AIV to support viral replication in mammalian cells.SDKS 1/15 showed dual receptor-binding affinity and exhibited more efficient replication in mammalian cells than in avian cells,showing good adaptability to mammals.Notably,despite having the mammalian-adaptive molecular markers in HA(226L,228S)and PB2(627K),median lethal dose in mice(MLD50)of SDKD1/15 was>107-5 EID50(50%egg infectious dose),suggesting that SDKD1/15 had low pathogenicity in mice.When mice were inoculated with a dose of 106.0 EID50,SDKD1/15 just replicated in limited organs and the duration of infection was short,suggesting of poor adaptability in mice.Furthermore,SDKD1/15 could spread from chicken to chicken and from chicken to guinea pig via the airborne route,demonstrating the potential for the pandemic in poultry and cross-species transmission.Therefore,this study shows that although the SDKD1/15 strain is adapted to mammals to a certain extent,other mutations are required for working synergistically to increase the pathogenicity in mammals.2.Adaptation of H9N2 virus SDKD1/15 in mice by serial passagesTo find other key virulence markers of the H9N2 virus in mammals,we conducted a gain-of-function experiment using the SDKD1/15 viral strain in mice by serial passages.Expectedly,infected mice showed clinical symptoms and died at sixth passage(SDKD1-P6).Comparison of the full coding sequences between the wild-type(SDKD1-WT)and SDKD1-P6 revealed two amino acid substitutions(T187P and M227L)and three consecutive amino acid deletions(?222-224)in the HA protein.We investigated the contribution of each amino acid substitution in H9N2 via reverse transcription.HA with ?222-224 alone and with the combined T187P+M227L mutation were associated with pathogenicity increase in mice.The H9N2 virus with these HA mutations showed increased affinity to human-type SA?-2,6-Gal receptors and significantly enhanced viral attachment to mammalian cells and mouse lung tissues.These changes resulted in enlarged plaque sizes on Madin-Darby canine kidney(MDCK)cells,enhanced early-stage viral replication in mammalian cells,and increased viral replication and virulence in mice.Additionally,HA with the T187P+M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact.AIV pathogenicity in mice is a polygenic trait.We also found that these HA mutations combined with the PB2-627K mutation able to increased the virulence in mice.More importantly,these HA and PB2 changes in combination contributed not only to the pathogenicity increase of the SDKD1-WT virus but also to that of other H9N2 strains in mice,indicating that that this characteristic is not a strain specific one.In summary,our study have identified novel key markers in the receptor binding domain and adjacent region of HA in collaboration with PB2 627K contribute to increased pathogenicity in mice and provided valuable information for pandemic preparedness against H9N2 subtype AIVs.3.Mutation at position 198 in the receptor binding domain of HA protein affecting the pathogenicity of H9N2 subtype AIV in miceAIVs acquire and select mutations during evolution process,however,accumulation of multiple mutations is necessary for gain-of-function with viral cross-species transmission and enhanced virulence.Many novel variations leading to pandemic potential cannot be recognized in time,causing public health problems.We observed two H9N2 field isolates with PB2-627K but with different HA mutations differed significantly in pathogenicity in mice.We therefore employed two strategies to mutate amino acid 198 of the HA protein,N198T and T198N simultaneously.The results revealed that 198N significantly decreased the replication and pathogenicity of H9N2 viruses in mice while 198T in vice versa.Amino acid 198 was located in the receptor binding domain of 190-helix in HA head region,and N198T mutation resulted in the deletion of glycosylation sites in HA,which would alter receptor binding properties.Based on this,we found that 198T promoted virus affinity to lung tissue from mice,while 198N mutation reduced that affinity,demonstrating direct association with pathogenicity in mice.Consistent with chapter one research,PB2-627K was also necessary for 198T mutation to enhance virulence in mice.In addition,amino acid 198 mutation had no effect on antigenicity and thermo stability of H9N2 viruses.Importantly,among all H9N2 isolations,198T accounted for 93.82%,indicating a potential threat to public health of H9N2.Hence,these findings have identified a novel key marker 198T in the receptor binding domain of HA in collaboration with PB2 627K contribute to increased pathogenicity in mice and deepen our understanding of pathogenesis for pandemic preparedness against H9N2 subtype AIVs.4.Host factors for enhanced pathogenicity in mice infected with H9N2 subtype AIV lacking 222-224 amino acids in the receptor binding domain of HAAn infection outcome of the disease depends on both pathogen and host defense elements.We sequenced the transcriptomes and proteomes of lungs of rSDKD1-?222-224 and rSDKDl-WT infected mice,respectively.Integrative analysis revealed good correlation between transcriptomic and proteomic data.rSDKD1-?222-224 induced a significantly larger number of differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)in host.Further biological function analysis demonstrated DEGs and DEPs induced by rSDKD1-?222-224 were mainly enriched in immunity-related biological processes,significantly higher than those by rSDKD1-WT.KEGG pathway analysis was consistent with GO term.During rSDKD1-?222-224 infection process,DEGs and DEPs were involved in inflammation-and immunity-related pathways.On the contrary,only a limited number of DEGs and DEPs in rSDKD1-WT infected mice contributed to RIG-I-like receptor pathway.Meanwhile,the number of DEGs and DEPs showing up-regulation was far higher than the number of down-regulation.These results indicated rSDKD1-?222-224 triggered broader and more vigorous immune responses and overwhelming immune responses resulted in severe pathological changes in lungs,thereby enhancing pathogenicity.To explore the underlying pathogenic mechanisms of the H9N2 virus with ?222-224 in the receptor binding domain of HA at the host level could help us understand the antiviral defense mechanisms.