| Over the last decades, the Avian Influenza Virus(AIV) especially the H9N2 subtype AIV was widely circulating in China and resulted huge economic losses to poultry industries.H9N2 subtype AIV can infect mammalian species without adaptation, including mice, guinea pigs, ferrets, rhesus monkeys and other companion animals like domestic cats and dogs. More importantly, several human cases caused by H9N2 subtype AIV infection have been reported in China. The results of serological surveys also show that 2.3%-13.7% of poultry industry workers have detectable antibodies against H9N2 virus. All these indicated that H9N2 AIV has obtained the capacity to cross species barriers and then infect mammals even human.H9N2 subtype AIV has a potential threat to cause influenza pandemic. At the same time,H9N2 AIV became gene donors to provider gene segments to other subtype AIVs, especially for the H5N1 and H7N9 AIVs. Therefore, it is important to strengthen the cross-species infection and pathogenesis studies of H9N2 subtype AIV which is significant for poultry industry development and human health.In this study, to explore receptor binding ability and pathogenesis of H9N2 subtype AIV,two mainly studies were carried as follows:Firstly, receptor binding abilities of H9N2 viruses circulating in China during the last decades was analyzed. The HA gene of H9N2 viruses which were collected on Genbank since1999 to 2014 were analyzed. HA-226 site which associated with receptor binding ability were compared and the host distribution of viruses were statistically analyzed. The results show that the HA-226 site has presented an obvious diversity which including HA-226L(81.85%),HA-226Q(17.57%), HA-226M(4.81%) and HA-226F(0.11%); the results of host distribution show that a majority of H9N2 subtype viruses(97.92%) were from birds species,then pigs(1.81%) and humans(0.27%). Further, receptor binding capacity of three H9N2 subtype AIVs including HA-226 Q, HA-226 M or HA-226 F were detected by chicken erythrocytes method and solid-phase ELISA, respectively. We found that the virus processing HA-226L(CK/JN/3)has the ability to bind human-like receptor(SAα2,6Gal), the virusprocessing HA-226Q(CK/JL/6) has the ability to bind avian-like receptor(SAα2,3Gal),while the virus processing HA226F(CK/JN/2) has the ability to bind dual receptors. These data indicated that most of H9N2 viruses circulating in China could bind SAα2,6Gal receptor. The results suggested that most H9N2 viruses of China have obtained the ability to recognize human-like receptor and to transmit among mammal hosts.On the other hand, impacts of internal genes on pathogenicity of H9N2 subtype AIV were analyzed. We have found three amino acid substitutions(PB2-E627 K, HA-N313 D and HA-N496S) in mouse-adapted virus compared with parental virus A/Chicken/JN/Li-2/2010(H9N2) in previously study. Here, we introduced the three PB2-E627 K, HA-N313 D and HA-N496 S to parental virus by Site-directed mutagenesis, and virus strains with alone mutation or different combine mutations were rescued by reverse genetics technique respectively. The virulence of the rescued viruses were evaluated with mouse model. The results showed that, compared with the parental virus, PB2-E627 K mutation significantly increased virus replication ability in MDCK cells and lungs of mice, while virus only with HA-313D/496 S mutation was similar to the parental strain; in terms of pathogenicity in mice,the virus with PB2-E627 K was lethal and its MLD50 increased at least 316-fold, while virus only with HA-313D/496 S mutation was not lethal in mice like parental strain. Histology assay showed that lesions of mice lungs were observed around small blood vessels and bronchioles,including slight perivascular edema, lymphocytic infiltration around bronchioles, and slight hemorrhage near the bronchiole in the group that virus with PB2-E627 K mutation, no obvious pathological changes were observed in the group that virus with HA-313D/496 S mutation or parental virus group. The results of polymerase activity assay showed that viral polymerase activity of the adapted virus with PB2-E627 K was significantly enhanced at least 16-fold. No significant changes were observed in thermo stability and acid stability of the adapted virus.These results suggested that PB2-627 K is the key amino acid residue to enhance the virulence of the adapted virus and affected the viral pathogenicity to hosts by enhanced polymerase activity.The above results of receptor binding assay and pathogenicity analysis indicated that H9N2 subtype AIV has obtained the ability to recognize receptors on upper respiratory tract of humans, and could gain high pathogenicity to mammals by a single amino acid residuemutation. Our studies provide foundations to further explore the molecular mechanisms of pathogenic and transmission of H9N2 AIV in mammals. |
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