| H9N2 Avian influenza virus is low pathogenic,and was first isolated from turkeys in the United States in 1966.In the early 1990s,the virus was first isolated from chickens in China and in the following decades H9N2 viruses has become one of the most wide spread avian influenza subtype in poultry in China.Production performance reduced in poultry infected with H9N2 subtype avian influenza virus.It is easy for AIV to mixed-infect with other pathogens aggravating morbidity and mortality,and causing economic losses to poultry industry.It was reported that H9N2 virus can be transmitted from poultry to human,and provide internal genes to other subtype influenza virus,which will have pandemic potential and can be a major threat to public health.In order to prevent and control H9N2 subtype avian influenza,China has used immunization plan of H9N2 vaccine for most poultry flocks since 1998,but the virus is still spreaded through most provinces,and becomes endemic in poultry.The reason may be that the RNA polymerase of influenza virus lacks the correction mechanism and the virus undergoes antigen drift under the continuous immune pressure.The renewal speed of new vaccine is also limited,so the vaccine can not provide effective protection for the current epidemic strains.In this study,we selected 57 strains of H9N2 subtype avian influenza virus isolated by our laboratory from 2008 to 2019 and analyzed the antigenicity of these strains by serological methods.At the same time,we selected the 3 viruses as vaccine candidate strains for immune-protection test based on the antigenicity analysis results to evaluate the efficacy of the vaccine candidates.1.Antigenicity analysis of H9N2 subtype avian influenza virusesIn order to analyze the antigenic characteristics of the H9N2 subtype avian influenza,We selected 57 strains(at least 2 strains per year)of the H9N2 subtype avian influenza viruses isolated during 2008 to 2019 according to the isolation time,location and genetic evolution of HA gene.We prepared antisera against 29 strains from 57 strains of the H9N2 viruses in chicken,and carried out the cross hemagglutination inhibition assays using antiserum to 57 viruses.Cross HI titer of the antiserum against the early vaccine strains F98 and WJ57 had a difference of a 2-32-fold,especially compared with the strains of 2017-2019.Based on the data of cross hemagglutination inhibition assay,we draw the antigenic map,which showed that the 57 strains clustered in two major antigen group(?,?).The viruses in antigen group ? involved 35(35/57)strains of virus in the 2008-2019.A new antigenic group,named as antigenic group ?,was emerged in 2014 including the viruses(18/57)in 2014-2019 because of antigenic evolution.The antigen distance of the viruses in antigen group of ? gradually enlarged with high discrete degree compared with antigen group ?.Amino acid substitutions,reported to related with antigenic drift,occurred in HA protein of H9N2 viruses of antigen group?:G72E,D135G,T182R,Q146H,G149D/K,I217L/M/Q,etc.,compared with the antigen group ?.This may result into new antigenic group when antigen distance is larger.The neutralization assays showed that the neutralization titer of the same antigenic group was higher than that of different antigenic groups,which was consistent with the results of cross-hemagglutination inhibition.The neutralization titer of the antiserum from the virus in antigenic group ? is higher to antigenic group I(PD50:447-14125)and group ?(PD50:447-3548)than that of antisera of group ?.Three strains were selected from two antigenic groups as vaccine candidate:AH463(antigen group ?)and FJ1802 and 292HI(antigen group ?),which showed good cross-reaction with most strains in both of cross hemagglutination inhibition and neutralization tests.The three viruses could be used as vaccine candidate strains of H9N2 subtype avian influenza in next chapter.2.Evaluation of immune efficacy of vaccine candidate of H9N2 subtype avian influenzaAccording to the results of antigenicity analysis in the previous chapter,we selected four H9N2 strains:WJ57(current vaccine strain),AH463,292HI and FJ1802 as immunogen to assess the ffficacy of vaccine candidates.H9N2 strains WJ57 and AH463 belong to antigen group ?,and FJ1802 belong to 292HI belong to antigen group?.The four viruses were inactivated to produce the inactivated vaccines for immunization in SPF chicken.After immunization with these four inactivated vaccines,antibodies in serum of SPF chickens were detected with an average HI antibody titer?8log2,reaching the level of protection in chicken.The immunized chickens in each group was challenged with H9N2 strains AH463 and 292HI respectively.The chickens were monitored every day,and no clinical symptoms were observed.Viral shedding was detected in each group at the 3dpc(day post challenge),5dpc and 7dpc.The viral shedding in the throat had a high rate and the continued for a longer period.The viral shedding rate at 7dpc was lower than the rate at 3dpc and 5dpc in the challenge group.Moreover,the virus could also be detected in the throat and cloaca of chickens in the direct or indirect contact groups till to 7dpc.It indicated that immune escape occurred in the immunized chicken.Second-generation sequencing of the immune escapee strain showed that amino acid substitutions at 17 positions in HA,among which postions 88,172 and 196 located at major antigenic sites in HA and readily caused antigenic drift.The implied mechanism of effect of amino acid substitution in HA protein on antigenicity and immune recognition of H9N2 virus remains to need further research. |
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