Characterization Of Stability Of H9N2 Subtype Avian Influenza Virus Hemagglutinin And Its Influence On The Immunogenicity Of Candidate Vaccine

The H9N2 subtype avian influenza virus first emerged in a chicken farm in China in 1992.The virus has gradually spread widely among po μLtry and become endemic in China.Vaccination is the most efficient strategy for preventing H9N2 subtype avian influenza virus infection.The surface glycoprotein hemagglutinin(HA)is the main component of influenza vaccine and the main protective antigen of influenza virus,and mediates virus entry into host cells and fusion of virus envelope with cell membrane during virus infection.In recent years,the isolation rate of the H9N2 virus has increased in China,and it can even be isolated in the hot summer,which is different from the previous seasonal distribution characteristics of the obvious high isolation rate in winter and spring.Thermostability and acid stability of HA are one of the important biological characteristics of influenza virus,which have an important influence on the biological characteristics of the virus,such as pathogenicity,immunogenicity,replication ability,etc.,but the current research in this area is relatively less.More efforts were made to modify the HA protein to change the stability and immunogenicity of influenza vaccine strains.In this study,H9N2 isolates from different isolation year were selected to analyze the thermostability and acid stability,and the key gene region and amino acid substitution that affected the stability of the virus and their impact on the immunogenicity of the vaccine.1.Characterization of stability of H9N2 subtype avian influenza virus hemagglutininDuring the prvious epidemiological surveillence,we found that H9N2 subtype avian influenza virus isolates have no obvious seasonality compared with the early isolates.In recent years the virus can be isolated in hot season,indicating that the viruses had high thermostability.We selected seventeen strains based on the isolation time and genetic distribution of H9N2 viruses and analyzed the thermostability and acid stability of the virus heamagglutinin.Among the seventeen H9N2 strains,six strains were more heat-resistant,nine strains were moderately heat-resistant and two were sensitive to heat.Five of heat-resistant strains were isolated after 2012,while both of heat-sensitive strains were isolated before 2012.The acid stability of the strains has no obvious time distribution characteristics.However,some strains were both resistant to lower pH and heat,such as AH463,FJ1802 and C1,while 5F2 and SQ68 were sensitive to lower pH and heat.Therefore,we selected AH463 and 5F2 strains as the model viruses for HA stability analysis.We constructed the plasmids with the HA and NA gene inserted into pHW2000 vector,respectively.The recombinant virus,whose HA and/or NA gene derived from AH463 or 5F2,and six internal genes provided by the H9N2 strain AH320 constructed in the previous studies.The res μLts showed that the recombinant virus with the HA from AH463 had higher thermostability,while the virus with HA from 5F2 was sensitive to heat.Therefore,the thermostabilty of the recombinant virus was consistent with that of HA gene donor virus,which indicated the close relation between HA and thermostabilty.This resμLt co μLd also be seen for acid stability.Then,we constructed the chimeric plasmid pHA-AH320HA1+5F2HA2 and pHA-5F2HA1+AH320HA2,which exchanged HA1 and HA2 between AH320 and 5F2,and rescued the recombinant virus from the chimeric HA plasmid and the other seven genes(PB2,PB1,PA,NA,NP,M,NS)from AH320.The res μLts showed that the thermostability and acid stability of the recombinant virus was consistent with the parent virus providing HA1,indicating that the main gene region affecting the stability of the virus was HA1.Furthermore,recombinant virus rHA-5F2HA1+AH463HA2 and rHA-AH463HA1+5F2HA2 were obtained from the HA chimeric plasmid exchaning the HA1 and HA2 between 5F2 and AH463 HA gene,and the remaining seven fragment(PB2,PB1,PA,NA,NP,M,NS)plasmids from AH320.The res μLts of the stability experiment further proved that the critical gene region that affected the thermostability and acid stability of the virus was located in HA1.2.The amino acid mutation that determins the stability of the virus and the immunogenicity of H9N2 vaccine candidateIn order to further explore the amino acid sites that affect HA stability,we downloaded all HA sequences of H9N2 virus from NCBI isolated after 2010,and used treesub software to draw evolutionary trees to analyze amino acid substitutions.Meanwhile,we analyzed the amino acid substitution in HA between the heat-and acid-resistant strains and heat-and acid-sensitive strains in the previous chapter.Then,six mutations in HA1 were finally selected(from heat-and acid-resistant to heat-and acid-sensitive):L79S,A138V,Q156R,T163I,K246R,D248N(H3 numbering).The mutation was introduced into HA plasmid of the vaccine candidate strain AH463,respectively,and the point mutation virus was rescued after co-transfection with the AH463 NA plasmid and 6 internal gene plasmids provided by AH320.It showed that the mutant virus rAH320-AH463-A138V with a mutation A138V in HA had a decrease in the thermostability and acid stability compared with AH463,while the mutants while other single mutation did not cause significant changes in the thermostability and acid stability.It indicated that the position138 in HA is a key amino acid site that affects the stability of the virus HA.The A138V mutation conferred the decreased ability to replicate in CEF and MDCK.The res μLts of immunogenicity experiments showed that rAH320-AH463-A138V had the significant decreased immunogenicity compared with the vaccine candidate strain AH463 and had a 16-180-fold lower HI titre than that of wt AH463 1-3 weeks post immunization.Therefore,changes in the HA stability had an important impact on viral immunogenicity,and replication ability in vitro,but the underlying mechanism needed to be further explored.In summary,in this study we analyzed the thermostability and acid stability of H9N2 subtype avian influenza virus isolated in different years.The difference in stability was mainly affected by HA1.The A138V mutation is the key amino acid position that affected the stability of the virus.At the same time,A138 V mutation affected the level of replication of the virus in vitro,and the immunogenicity of the vaccine candidate.