| Since the isolation of the H9N2 subtype avian influenza virus(AIV)in turkey chickens in Wisconsin in the 1960s,the virus has spread worldwide.In order to control the spread of H9N2 avian influenza,the H9N2 influenza virus vaccination program has been widely implemented in mainland China.Although this immunization program effectively reduced the economic losses caused by the H9N2 flu virus to the flock,it did not prevent the spread of H9N2 avian flu in most parts of China.In order to study the evolution of H9N2 subtype avian influenza virus under vaccine antibody selection pressure,our laboratory had already carried out serial passage of H9N2 virus in chicken embryos,and also carried out related research on serial passage of H9N2 virus mixed from different SPF chickens.Changes in antigenicity and pathogenicity of the passaged virus were observed.In order to reduce the interference of the mixed virus on the research results,and to explore the variation of the virus after passage in each chicken individual,we chose the single-pass mode of one-to-one chicken under vaccine antibody selective pressure in this experiment.A/Chicken/Shanghai/F/98(H9N2,F/98)avian influenza virus was used as the research object,and the F/98 strain virus was continuously one-to-one passaged in SPF chickens with and without homologous vaccine antibody selection pressure.The antigenic drift,genetic evolution and virus virulence changes of the passaged viruses were analysised by HI test,chicken embryo infectivity RT-PCR and sequencing toprovide a new theoretical basis for the vaccine control of avian influenza.1.Serial passage of F/98 strain in chickens with and without vaccine antibody selective pressure,and analysis of antigenicity and chicken embryo infectivity of the passaged virusSix 21-day-old SPF chickens were divided into two groups:vaccine antibody selection pressure group(V)and no vaccine antibody selection pressure group(N),three chickens in each group.In V group,three independent passage series V1,V2 and V3 were established,and three chickens were vaccinated with oil emulsion vaccine of F/98 strain respectively.When the antibody titer HI of the immunized chicken was up to 24,and the infection experiment was performed.N groups established three independent passage series N1,N2 and N3,and directly carried out infection experiments.The first generation passage series of all V and N groups were infected with the virus allantoic fluid of F/98 strain collected in the same batch.Infection was carried out by intranasal,eye and throat injections at a dose of 106 EID50/0.2 mL,respectively.On the 3rd day after infection,the lung and trachea of each passage series of experimental chickens were separately sampled to prepare tissue culture medium in PBS,and stored at-70 0C.This was the first generation of tissue homogenate with and without vaccine antibody selective pressure.Then,six 21-day-old SPF chickens were taken and grouped according to the above method,and this method was used to carry out the passage series of the 9th generation.The 10-day-old chicken embryos were inoculated with tissue homogenates of the 5th and 9th generation lungs and trachea,respectively,and the virus allantoic fluid passaged in chickens with and without vaccine antibody selection pressure was collected.The HI titer of the passaged viruses against the positive polyclonal antiserum of the parental virus F/98 strain were measured.Compared with the HI titer of the parental virus F/98 strain,there was no significant difference in the HI titer of the passage virus isolated from the fifth generation of lung and trachea tissue with(V5L,V5T)or without(N5L,N5T)the pressure of vaccine selection.However,the HI titer of the passage virus isolated from the ninth generation of lung and trachea tissue with(V9L,V9T)or without(N9L,N9T)the pressure of vaccine selection showed significantly higer than that of the parental virus F/98 strain.The HI titer of the passage virus isolated from the ninth generation of lung tissue without vaccine selective pressure(N9L)and the passage virus isolated from the ninth generation of trachea without vaccine selective pressure(N9T)were 8 times lower than the HI titer of the parental virus F/98 strain.While the HI titer of the passage virus isolated from the ninth generation of lung tissue under the pressure of vaccine selection(V9L)and the passage virus isolated from the ninth generation of trachea under the pressure of vaccine selection(V9T)were 16 times lower than the HI titer of the parental virus F/98 strain.These resulted indicated that after 5 passages.the antigenicity of the passaged virus was substantially unchanged compared to the antigenicity of the parental virus F/98 strain.However,after 9 passages,the antigenicity of the passaged virus was significantly different from the antigenicity of the parental virus F/98.The adaptability of the 9th passage of the virus with and without vaccine antibody selective pressure not only included antigenic changes,but also showed changes in chicken embryo infectivity and lethality.The EID50 of V9L and V9T viruses was 1.77 times higher than that of the parental virus F/98 strain,and there was no significant difference.However,the EID50 of N9L and N9T viruses was 25.4 times and 17.7 times higher than the EID50 of the parental virus F/98,respectively.This indicated that the chicken embryo infectivity of the passaged viruses was significantly increased.In addition,the V9L and V9T viruses lost the ability to kill chicken embryos compared to the F/98 strain.However,the ELD50 of the N9L and N9T viruses was only increased by 0.77 times,which was not significantly different from the parental virus.In summary,the passaged virus without vaccine selective pressure had a significantly higher infectivity to chicken embryos than the parental virus,while the passaged virus with vaccine selective pressure had little difference in the infectious effect on chicken embryo than the parental virus,but they lost the ability to kill the chicken embryo.2.Genetic analysis of the passaging F/98 strain virus with and without vaccine antibody selection in the 9th generationCompared with the parental virus F/98 strain,the superior passage virus of the ninth generation showed some the amino acid change of each gene fragment in addition to the antigenicity of the virus.Compared with the parental virus,the V9L virus has a total of 13 amino acid mutations,including 5 PB2 genes(T296K,A322E,R327K,H366Q,Y369S),2 PB1 genes(T251V,A669G),and 2 HA genes(K131R,A198V),1 NP gene(A100R),1 NA gene(C385T),2 M genes(P154A,A246Q).Compared with the parental virus,the V9T virus had a total of 11 amino acid mutations,including 4 PB2 genes(A322E,R327K,H366Q,Y369S),1 PA gene(S148H),2 HA genes(A 198V,Q234L),One NP gene(G45T),one NA gene(C385T),and two M genes(P154A,A246Q).Compared with the parental virus,the N9L virus had a total of 20 amino acid mutations,including 6 PB2 genes(T138A,T296K,I298Q,R327K,Y369S,E526R),1 PB1 gene(T348A),and 4 PA genes(S148H,L336M,K544M,S640K),two HA genes(N167G,N285S),three NP genes(G52A,D65L,L187G),two M genes(H23I,T331A),and two NS genes(S81L,H85S).Compared with the parental virus,the N9T virus had a total of 18 amino acid mutations,including 5 PB2 genes(T296K,I298Q,R327K,Y369S,E526R),1 PB1 gene(T348A),and 2 PA genes(S148H,L336M),HA gene 3(A198V,Q234L,N285S),NP gene 4(G52A,S65L,L187G,G419V),M gene 1(H23I),NS gene 2(S81L,H85S).In addition,the NA protein of the V9L and V9T viruses were deleted 10 amino acids at positions 67-76(?67-76).These results show that vaccine selection pressure causes amino acid deletion of 67-76 of the NA gene.The number of amino acid mutations in the passaged virus with vaccine selective pressure was significantly less than the number of amino acid mutations in the passage virus without vaccine selection pressure.The number of amino acid mutations in the passaged virus isolated from lung tissue was slightly more than the number of amino acid mutations in the passaged virus isolated from the trachea under the same passage conditions.In summary,after 9 generations of continuous passage with and without vaccine antibody selection pressure,the antigenic variation occurred in the F/98 virus.The selection pressure of the vaccine caused the F/98 strain to lose the ability to kill the chicken embryo,and the NA protein deleted the 67-76 amino acid,resulting in a significantly fewer genomic mutations in the F/98 strain than without vaccine antibody selection pressure.These results indicate that vaccine antibody selection pressure played an important role in the evolution of H9N2 avian influenza virus and changes in some biological properties. |
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