Variation Of H9N2 Avian Influenza Virus To ?-2,6 Receptor Binding And The Effects Of Virus HA Protein Glycosylation To Pathogenicity

Avian influenza virus is an infectious disease caused by avian influenza virus type A, based on the pathogenicity it can be divided into LPAIV and HPAIV. Influenza virus has 8 segments, in which hemagglutinin and neuraminidase are the major glycoprotein?Influenza viruses infect host initials with HA binding to host cells receptor,HA mainly binding to two forms sialic acids namely:?-2,3 sialic acids glactose and ?-2,6 sialic acids glactose.Different hosts can secret different sialic acids,to some extent,this determines the range of hosts. Until now, we find that H9N2 avian influenza virus can infect poultry, it can also infect mammal including Mice, guinea pigs, ferrets, primates. Serological survey found that C hina Livestock employees H9N2 influenza virus serum antibody-positive rate can reach 2.3%-13.7%, suggesting that the H9N2 avian influenza virus has been cross species barrier to infect mammals and even human, which has caused a new influenza pandemic potential threat. AIV H9N2 subtype influenza viruses can be the internal genes donors, such as the 1997 Hong Kong H5N1 subtype of influenza virus, genes may be derived from the A / Quail / Hong Kong / G1 / 97(H9N2)?H7N9 AIV in C hina,among the internal genes,6 of 8 from the H9N2 subtype AIV. Therefore, monitoring the H9N2 subtype AIV receptor affinity and studying the mechanisms to mammalian pathogenic have great Public health significance.By analysis 12 isolates between 2001-2013 H9N2 subtype avian influenza virus HA phylogenetic we found that although 12 AIV both belonging to the Y-280 branch, but they can be further divided into four small branch, this indicates that H9N2 subtype avian influenza virus also mutates, although slowly. Solid phase binding assays results showed that: different H9N2 subtypes AIV receptor affinity to ?-2,6 sialic acid are different, and the overall trend is: With the approaching era of separation, virus receptor affinity to ?-2,6 sialic acid gradually increased, which in 2013 isolate(a/C hicken/Shandong/903) receptor affinity to ?-2,6 sialic acid is the strongest while the 2006 isolate(a/Chicken/Shandong/274) is weakest. Followed by CK / SD / 903 and CK / SD / 274 of these two strains HA protein glycosylation sites comparison: the hemagglutinin protein of CK / SD / 903 has two glycosylation sites,while CK/SD/274 lack two N-glycosylation in the same sites.In order to verify that glycosylation sites have impact on the virus receptor affinity, we constructed CK / SD / 903 8 plasmids reverse genetics system, and on the 200 and 295 amino acid CK / SD / 903 of the hemagglutinin protein is mutated to obtain two mutant viruses. Solid-phase binding assay results indicate that HA N200Q(200 N to Q), and HA N295Q(295 N to Q) can weaken the virus receptor affinity to ?-2,6 sialic acid receptor.In order to further verify these glycosylation sites affect virus pathogenicity to mammals, 106EID50 wild virus and mutant virus(CK / SD / 903(HA N200Q), CK / SD / 903 and CK / SD / 903)were challenged 6-week-old mice.The results showed that: HAN200Q(200 N to Q), though the mutant virus have no significant effect on body weight of mice, but can improve the relative expression levels of the virus in mice kidneys and other organs(viral genes are wild srains 936.32 fold); HA N295Q(295 N to Q) mutations can increase viral replication in the liver and other organs of mice(viral gene expression relative to the amount of 56 times the wild poisonous).In this study, we systematicaly study the 12 strains H9N2 subtype avian influenza virus HA gene isolates between 2001-2013 and viral receptor affinity to ?-2,6 sialic acid,the results showed: as the time approaching, virus ?-2,6 sialic acid receptor affinity gradually strong. More over we use reverse genetics technology to verify the 903 strain of HA protein 200 and 295 glycosylation virus affinity to ?-2,6 sialic acid receptor changes, and the two glycosylation sites can affect the virus tissue tropism in mice. This provides an important theoretical and experimental basis for elucidating the molecular genetic mechanisms of H9N2 subtype of influenza virus among cross-species transmission, and can provide early warning for the H9N2 subtype of influenza A virus infection in a mammal or human, this study has important guiding significance and veterinary public health significance.