| Since the late 1990 s,multiple genotype H9N2 and H5N1 influenza viruses have been circulating in China.The H9N2 AIV may become more efficient to transmit to other species,including humans,which was a threat to human.Clade 2.3.4.4 H5N6 subtype AIV is the current H5 subtype AIV in the most important epidemic virus.Moreover,humans can be infected with the clade 2.3.4.4 H5N6 subtype avian influenza virus.Sixteen H5N6 strains of clade 2.3.4.4 have caused human infections(World Health Organization,2016).The virus seriously threatens human health and the poultry industry.In this study,we determined the whole gene sequence of eight H5N6 strains which isolated from different species.Phylogenetic analyses and genotyping of all H5N6 AIV(include the sequences from GISAID's)were presented herein.Molecular evolution analysis of whole genome sequence found that the HA gene almost belong to clade 2.3.4.4 H5 subtype.And the NA gene with/without stalk region of deletion 11 amino acids at positions 58–68 can be divided into two clades,namely,Stalk Deletion or No Deletion.The analysis indicated that seven isolated and most of the H5N6 AIV belong to Stalk Deletion.The internal gene of H5N6 subtype AIV were found recombination with low pathogenic influenza virus.The PA,PB1 and PB2 genes derived from H5,H6(most closely related to H6 reported in the South Korean),H9 subtype virus and the NP,NS and M gene derived from to H5 and H9 subtype virus.In recent years,it is believed that H9N2 viruses continuously provided internal gene segments for H5,H10N8 and H7N9 subtype AIV.In addition,influenza virus polymerases have been proved that played an important role in viral pathogenicity,recombination,and widespread cross-host in previous studies.However,the function and mechanism PB2 protein function and mechanism were still not yet clearly.And our results revealed that PB2 amino acid 627(K)decided the virulence in mice in previous study,but possessing PB2 amino acid 627(E)were not highly pathogenic for mice.Therefore,we got recombined plasmids that express PB2 fused with Strep/Flag protein tag at carboxyl end and then we rescued recombined viruses by reverse genetic.In order to analyze how PB2 amino acid decides the virulence from the perspective of host,pull-down assay had been to capture host proteins DDX48 interacts with PB2 after HEK293 T cells were infected with recombined viruses.The interaction between AIV PB2 and cellular protein DDX48 had been determined by BiFC and Co-IP.Our results also showed that the interaction between AIV PB1 as well as NP and cellular protein DDX48 by Co-IP.Moreover,the binding region of DDX48 was identified at domain ?.The confocal microscopy analysis indicated that PB2 could co-localize with DDX48 in the cells and lead to the nuclear translocation of PB2 following the expressing plasmid transfection in HEK293 T cells.Subsequently,Additionally,we found that DDX48 played a crucial role in promoting the H9N2 AIV PB2(K627)replication and the synthesis of PB2 RNA in knock down or knock out DDX48 cells.Finally,the confocal microscopy analysis showed that the nuclear export of NP could be promoted.In conclusion,this study analyzed the whole gene sequence of H5N6 subtype AIV(our isolated strains and GISAID database).The results showed that the main prevalent H5N6 AIV belong to clade 2.3.4.4 and most of the NA genes with stalk deletion.Additionally,the internal gene of H5N6 subtype AIV were the result of dynamic reassortment with low pathogenic influenza virus.Moreover,we identified the host cellular protein DDX48 that interact with the PB2,PB1 and NP of AIV.And we also revealed that host cellular protein DDX48 is involved in the replication of H9N2 AIV PB2(K627)AIV and the nuclear export of NP.These results show that DDX48 is likely as drug target against influenza virus infection in applications and prospects. |
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