A Single Mutation N166D In Hemagglutinin Alters Antigenicity And Pathogenesis Of H9N2 Avian Influenza Virus

H9N2 avian influenza virus is endemic in Asia,the Middle East and North Africa.As low pathogenic influenza virus,H9N2 mainly causes symptoms such as difficulty breathing and decreased egg production.When co-infected with other pathogens,H9N2 can cause a significantly economic loss to poultry industry due to high morbidity and mortality.H9N2 can infect not only poultry,but also mammal including human,swine,cat and dog.Moreover,H9N2 can offer internal genes to high pathogenic influenza viruses such as H5N1,H5Nx,H7N9 and H10N8,which are beneficial to generate novel influenza viruses with potential pandemic by reassortment.These indicate that H9N2 not only affects the development of poultry industry,but also poses a potential threat to public health safety.Although the widespread use of H9N2 vaccine can effectively control the epidemic,in recent years,H9N2 still can be frequently isolated from the vaccinated chicken flocks in China.The constant emergence of H9N2 immune escape mutants indicates that the antigenicity of H9N2 epidemic strains is drifting.Therefore,to identify the antigenic sites in HA and evaluate their impacts on the virus is critical for H9N2 surveillance and vaccine design.Some antigenic sites in H9 have been identified,the discovery of new antigenic amino acids may facilitate the understanding of their roles in the antigenicity and pathogenesis of H9N2 influenza virus.In this study,we identified several novel B cell epitopes in HA of H9N2,and found that a single mutation N166D in HA could alter the antigenicity and pathogenesis of H9N2.1.Selection and identification of escape mutants derived from H9N2 AIVTo select and identify escape mutants,four identified anti-HA monoclonal antibodies by our research group?3F2,2G10,7F4,5C7?were used to perform an embryo neutralization test with H9N2 isolate XZ299.Following two selection of MAbs,four immune escape mutants were found,named as m3F2,m2G10-1,m2G10-2 and m5C7 respectively according to the corresponding antibodies.By sequencing the HAs of the mutants,seven critical antigenic sites were identified.These mutated sites are N166D,N167K,A168L,D207N,N218D and L234M,respectively,and Q133L and N218D were first reported.All these seven sites were located in the surface of the head of HA,and some of them is located near the receptor binding region.The m3F2 mutant only had a single mutation N166D,suggesting the N166D mutation may significantly alter the antigenicity and pathogenesis of H9N2.The acquisition of these immune escape mutants and the identification of the critical amino acid mutations not only enriches the B cell epitopes of H9N2 HA protein,but also lays the foundation for further analysis of the molecular antigen variation of H9N2 epidemic strains and the development of efficient vaccines.2.Construction and rescuing of recombinant H9 virus expressing HA_N166D mutationFor further study of the effect of mutation N166D of escape mutant m3F2 on the antigenicity and pathogenesis of H9N2,the cDNA were generated from viral RNA of H9N2 isolate XZ299.Then the HA gene of XZ299 was amplified by PCR,and were cloned into the linear influenza vector pDP2002 by one-step recombination method to generate the rescuing plasmid HA₁66N.A mutant HA₁66D rescuing plasmid was constructed by Over-lap PCR technology by using HA₁66N as a template.For viral rescuing,the HA plasmid HA 166D or HA₁66N and seven plasmid?NA,NP,PB1,PB2,PA,MP,NS?derived from PR8 were co-transfected into MDCK and 293T co-cultured cells.The rescued viruses were amplified by chicken embryos and verified by sequencing,designated as rgPR8-H9 166N and rgPR8-H9 166D,with virus titers of 1.58×107 TCID50 and 2.32×106 TCID50,respectively.The HI assay further showed that the rescued virus rgPR8-H9 166D similiar to the escape mutant m3F2,was not effectively recognized by monoclonal antibody 3F2.The rescued rgPR8-H9 166N and rgPR8-H9 166D provided materials for further research on the effect of N166D mutation on the pathogenicity of H9N2.3.Pathogenicity of recombinant H9N2 expressing HA_N166D mutationTo investigate the impact of N166D mutation on the pathogenesis of H9N2,the viral growth kinetics and animal infection experiments were performed for four viruses rgPR8-H9166N,rgPR8-H9 166D,XZ299 and m3F2.Viral growth kinetics demonstrated that N166D mutation in H9 did not affect the viral replication of H9N2 in MDCK cells.The infection study in mice showed that the mice infected with rgPR8-H9 166N had a significantly body weight loss and higher viral shedding in the lungs in comparison with rgPR8-H9 166D.The infection study in SPF chickens demonstrates that both viruses XZ299 and m3F2 caused a similar virus shedding in the larynx and cloaca.Notably,the HI assay for sera from infected chickens showed that N166D mutation resulted in the low HI antibody response.All these data suggested that the N166D mutation not only affects the antigenecity of H9N2,but also attenuated the viral pathogenesis.Therefore,to monitor the mutation N166D of HA in the clinical H9N2 isolates in China is of great significance for preventing immune escape and updating efficient vaccine.