Evolution Analysis Of The Envelope Proteins Of H9N2 AIV From Broilers And Effect Of HA 145 Site Glycosylation On Viral Biological Characteristics

Avian influenza(AI)is a severe infectious disease in poultry caused by avian influenza virus(AIV).OIE has designated highly pathogenic avian influenza of animal disease in must report list.H9N2 virus is one of the low pathogenic avian influenza virues.When co-infected with other pathogens,it would result in high morbidity and mortality in poultry.H9N2 AIV can also infect mammals,which is harmful to the poultry industry and public health.Assessing whether H9N2 AIV will cause pandemic is the primary task of ensuring global public health security.With the increasing demand of broiler market in China,the poultry industry is gradually developing in the direction of scale and intensification.Therefore,the epidemiological investigation in farms is of great significance for the surveillance of epidemic diseases.To understand development of H9N2 AIV,we isolated and identified H9N2 AIV in a broiler company and analyzed the genetic evolution of the HA and NA genes.At the same time,the viral antigenicity and pathogenicity were explored through whole genome sequencing,reverse genetic system and mouse study.The main research is as follow.1.Evolution analysis of the envelope proteins of H9N2 AIV in a farm from 2012 to 2019This study conducted the isolation and identification of the H9N2 subtype AIV from chicken flocks in a large farm from 2012 to 2019,and the genetic evolution and key antigenic sites of HA and NA genes were analyzed.The results showed that the HA and NA genes of 25 H9N2 strains isolated from farms belonged to h9.4.2.5 branch in the phylogenetic tree.HA analysis showed that AA residues on the left edge of receptor-binding pocket were NGLMG,on the right edge of receptor-binding pocket were SKACS,STACS,SNTCS.Among the 25 strains in this study,19 isolates showed an A190T/V mutation,24 isolates showed a glycosylation site at position 313,23 isolates have deleted glycosylation site at position 218,2 isolates have a glycosylation site at position 145.NA analysis revealed that 2 strains had glycosylation site at position 70 or 143,and 2 strains had no glycosylation site at position 69 or 86.Deletion at positions 63-65 in the stalk of the NA protein was found in all strains.2.Pathogenesis study of H9N2 AIV isolates in miceSince the H9N2 AIV is easy to co-infect with other pathogens,the isolates were purified by plaque-assay.Through IFA and RT-PCR,three strains were obtained:WJ356-14,JX191-1,2019326-19,and performed whole genome sequencing of them.To further determine the pathogenicity of the virus to mammals,the pathogenesis was analyzed in mice.We found that the 190 receptor binding sites of virus showed three changes:A,V,and T.The NA protein of WJ356-14 possessed a potential glycosylation site NHS at position 143,and the PB2 protein was mutated from A to V at position 588 and E to V at position 627.The pathogenesis study demonstrated that the mice had no obvious clinical symptoms during the 14-day observation period,but JX191-1 virus could obviously cause slow down the weight gain of the mice and could cause more serious tissue lesions.Through the HI test,it was found that the HI antibody level of the mice infected with JX191-1 virus was higher than other groups.3.Effects of HA145 glycosylation site on viral biological characteristicsThe mutant virus was rescued by reverse genetic system with genes of the JX106 isolates.The virus growth kinetic,receptor affinity and pathogenesis in mice were determined to explore the effect of 145 glycosylation sites in HA on virus replication,receptor affinity and virulence.The study found that there was no significant difference between rgJX106-HA 145N and rgJX106-HA 145 S viruses on the infectivity to chicken embryos and the growth in A549 cells.While rgJX106-HA145S has better affinity with ? 2,6-sialic acid receptors.The pathogenesis study in mice demonstrated that viral titers of rgJX106-HA 145S in the lung showed significantly higher than rgJX106-HA145N at day 3 post infection.rgJX1 06-HA 145S can cause more weight significant loss in mice and induce more cross-reactive antibodies.