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Amino Acid Mutations Identification And Inducting Specific Neutralizing Antibody Of H9N2 AIV With Antigenic Variation Under Antibody Pressue In Chicken

Posted on:2020-05-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:R ZhuFull Text:PDF
GTID:1360330602962560Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
Although a vaccination program for the H9N2 AIV has been widely implemented in China since 1998,the H9N2 AIV is still the most prevalent subtype influenza virus in our country.The reason for this is,that the H9N2 subtype avian influenza virus(H9N2 AIV)is a kind of virus with only single-stranded negative-strand RNA,and its RNA polymerase and reverse transcriptase has no strict self-correcting mechanism,which leads to its nucleic acid replication can't be completed with 100%correct.Eventually,the mutated nucleotides can be accumulated to cause viral antigenic variation of the virus to achieve immune escape.Through our previous experiment,we have successfully isolated the antigenic variants that were serially passaged in SPF chicken with and without antibody selection pressure,and found that antigenic variation could be driven by the antibody selection pressure.This study aims to understand the roles of the antibody selection pressure on antigenic variation and inducting specific neutralizing antibody of H9N2 subtype avian influenza virus in chicken.By reverse genetics technology,serological experiments,qPCR and animal experiments,identification and inducting specific neutralizing antibody of the amino acids on hemagonium(HA)causing antigenic variation of H9N2 AIV were conducted systematically.The results addressed as follows:1.Identification of the amino acid mutations on HA associated with antigenic variation of H9N2 virus passaged in SPF eggIn the previous experiment,four amino acid mutations(K131R,S145N,G181E,and A198V)in HA gene of antigenic variant serially passaged in SPF eggs with and without antibody selection pressure.In this study,the parental virus F/98(alanine at residue on the position 198 of HA,A198)was used as the backbone,and a total of 9 recombinant viruses of HA gene were rescued by reverse genetics technology respectively,including the single mutant virus,rF/HAK131R,rF/HAs145N,rF/HAG181E and rF/HAA198v,and multi-mutants virus,rF/HA348,rF/HA349,rF/HA389,rF/HA489 and rF/HA3489.The antigenicity analysis between these 9 HA recombinant viruses and the parental virus F/98 was performed by HI assay,and the results showed that,comparing with the F/98 virus,the HI titer of rF/HAK131R and rF/HAG181E was the same as that of the F/98 virus,the HI titer of rF/HAs145N and rF/HA348 decreased by 1.67-fold,and the HI titer of the rest 5 recombinant viruses(rF/HAA198V,rF/HA349,rF/HA389,rF/HA489 and rF/HA3489)decreased by 5-fold(4 times difference in HI titers can be determined as antigen drift,?HI titers?4-fold),indicating that the antigenic variation of the recombinant viruses occurred obviously.2.Identification of the amino acid mutations on HA associated with antigenic variation of H9N2 virus passaged in SPF chickenAccording to the HA sequencing results of 390 antigenic variants,which were serially passaged in SPF chicken with and without antibody selection pressure in our previous experiment,the parental virus F/98 was used as the backbone,and a total of 13 HA single-mutant viruses were rescued by reverse genetics technology in this study,including rF/HAK131R,rF/HAQ133H,rF/HAQ164L,rF/HAA168T,rF/HAA198V,rF/HAM224K,rF/HAQ234L,rF/HAY264H,rF/HAG270R,rF/HAG274R,rF/HAK278E,rF/HAI386V and rF/HAK399N.The antigenicity analysis between the above viruses and the parental virus F/98 was performed by HI assay,and the results showed that,comparing with the F/98 virus,most of the HA single-mutation couldn't change the HI titer of the virus,but three HA mutations(Q164L,A168T and M224K)caused a 1.67-fold decrease in HI titer,the Q234L mutation caused a 1.2-fold increase in HI titer,and the A198V mutation caused a 5-fold decrease in HI titer(?HI titers>4-fold)?indicating the antigenic variation occurred obviously.The results of both the micro-neutralization experiment(the viruses rF/HAA198V(V198)and F/98(A198))and the cross-HI assay(the viruses rF/HAA198V(V198),F/98(A198),GD/SS/94(A198)and JS/YZ618/12(T198))sufficiently proved that,the A198V mutation can escape from the polyclonal antibody,and the 198th amino acid on HA supposes to be one of the antigenic sites of H9N2 AIV.3.The effects of the antibody selection pressure on viral receptor binding avidityReceptor binding avidity is an important factor for the antigen drift of influenza viruses.In this study,the receptor binding avidity of the 20th generation viruses passaged in SPF chicken with and without vaccine selection were all analyzed by RDE assay,including the parental virus F/98(A198),the 20th generation pF/NL,isolated and purified from the lungs,and pF/NB,isolated and purified from the trachea,without antibody selection pressure,the 20th generation pF/VL isolated and purified from the lungs,and pF/VB,isolated and purified from the trachea,with antibody selection pressure,and the HA single-mutation virus rF/HAK131R,rF/HAA168T,rF/HAA198V,rF/HAM224K and 1F/HAQ234L,whose HA mutations were from 20th antigenic variation generations.The results show that,the receptor binding avidity of the parental virus F/98(A198)was 6.25,and that of the viruses pF/NL and pF/NB passaged without antibody selection pressure was 50 and 167 respectively.Meanwhile,the receptor binding avidity of the viruses pF/VL and pF/VB with antibody selection pressure were both over 200,which showed that the receptor binding avidity from virus passaged with antibody selection pressure was significantly higher than those from virus without antibody selection pressure.What's more,the receptor binding avidity of rF/HAM224K was 12.5,which was double times of F/98;and the receptor binding avidity of rF/HAA198V and rF/HAQ234L were also both over 200,which were 32 times of F/98,indicating that the receptor binding avidity was significantly increased by A198V or Q234L HA mutant.4.Comparison of specific neutralizing antibody in chickens induced by antigen variants passaged in chicken with and without antibody selection pressureExperimental infection on SPF chickens of the antigenic variants pF/NL and pF/VL isolated in the lung,was set up between different groups,one with antibody selection pressure and one without antibody selection pressure.The AID mRNA changes in lung,spleen and bursa of fabricius and serological kinetics in different groups were analyzed by qPCR technology,HI assay and ELISA assay.The results showed that,during the infection of SPF chickens without antibody selection pressure,the pF/VL virus with antibody selection pressure induced higher expression of AID in the lung tissue,while induced lower expression of AID in the spleen tissue.Furthermore,the pF/VL virus with vaccine selection could induce lower levels of serum-specific antibodies in the chicken during the infection of SPF chickens without antibody selection pressure.During the infection of SPF chickens with antibody selection pressure,the pF/VL virus with antibody selection pressure induced higher expression of AID in the lung tissue,while induced lower expression of AID in the bursa of fabricius,inducing higher levels of serum-specific antibodies with high specific antiviral activity.The results also suggested that,during the infection of SPF chickens without antibody selection pressure,the AID mRNA was up-regulated in lung and spleen,and down-regulated in the bursa of fabricius,while during the infection of SPF chickens with antibody selection pressure,the AID mRNA was down-regulated in lung and spleen,and up-regulated in the bursa of fabricius.5.The role of the A198V HA mutation of H9N2 AIV in inducing host innate immune response and specific neutralizing antibodyTo clarify the molecular mechanism of the A198V HA mutation during virus escaping from the polyclonal antibody,the binding of polyclonal antibodies based on the A198 V HA mutation had been tested by ELISA assay.The results showed that,the A198V or V198A mutation had no significant effect on the binding of virus and polyclonal antibody.The main reason for the A198 V HA mutation escaping from polyclonal antibody is the extremely significant increase in the receptor binding avidity of the rF/HAA198v virus.In addition,the A198V HA mutation doesn't affect the viral infection,and the replication of the virus rF/HAAi98V was decreased during the early infection stage,while the replication of the virus rF/HAA198V was increased during the mid-to-late infection stage.To study the effect of the A198V HA mutation on innate immune-related genes expression in chicken,the mRNA levels of the innate immune-related genes were detected in HD 11 cells and lungs,respectively.The results showed that,the A198V HA mutation could significantly increase the innate immune response of HD11 cells at late stage,especially in TLR3,TLR21 and IFN-?reaction;it could also significantly increase the innate immune response of SPF chickens' lung tissues at the mid-to-late stage,especially in TLR4,TLR3,TLR7,MDA5 and IFN-?.In addition,experimental infection on SPF chickens of the viruses F/98 and rF/HAAi98V,was set up between different groups,one with antibody selection pressure and one without antibody selection pressure.The AID mRNA changes in lung,spleen and bursa of fabricius and serological kinetics in different groups were analyzed by qPCR technology,HI assay and ELISA assay.The results showed that,during the infection of SPF chickens without antibody selection pressure,the virus rF/HAAi98v could induce higher expression of AID in the lung tissue and spleen in chicken,and produce the antibodies with higher antiviral activity,but these antibodies had lower IgG level and the level of specific antibodies for effective neutralization against virus was a little;during the infection of SPF chickens with antibody selection pressure,the AID mRNA lever of the rF/HAAi98v virus inducing in the lung tissue,spleen and bursa of fabricius on days 2 and 4 post-infection were lower,but could produce antibodies with higher IgG level and more specific antibodies for effective neutralization against virus.And the AID mRNA lever of the rF/HAAi98v virus inducing in the lung tissue and spleen on days 7 post-infection were lower.6.The role of the NA gene with mutation deletion of 67-76 amino acids of H9N2 AIV in inducing host innate immune response and specific neutralizing antibodyIn the previous experiment,in which the parental H9N2 virus F/98 was serially passaged in SPF chicken with and without antibody selection pressure,compared with F/98 strain,the NA with mutation deletion of 67-76 amino acids(NA?67-76)occurred in the viruses passaged with antibody selection pressure,which had never occurred in the wild strains and the viruses passaged without antibody selection pressure.In this study,the parental virus F/98 was used as the backbone,and the recombinant virus rF/NA?67-76 possessing NA with mutation deletion of 67-76 amino acids was rescued by reverse genetics technology,and the antigenicity and biological characteristics were explored by a set of experiments.The results addressed as follows:The virus rF/NA?67-76 could impro ve both NA activity and receptor binding avidity.Moreover,the replication of the virus rF/NA?67-76 were decreased during the early infection stage,while the replication of the virus rF/NA?67-76 were increased during the mid-to-late infection stage.The results also showed that,the NA?67-76 determined the tissue tropism of the virus,the virus was easily to replicate in trachea tissue efficiently.To study the effect of the virus rF/NA?67-76 on innate immune-related genes expression in chicken,the mRNA levels of the innate immune-related genes were detected in HD 11 cells and lungs in chicken,respectively.The results showed that,the virus rF/NA?67-76 could significantly increase the innate immune response of HD 11 cells at late stage,especially in TLR4,TLR7,TLR21 and NLRP3 reaction;it could also significantly increase the innate immune response of SPF chickens' lung tissues at the mid-to-late stage,especially in TLR2,TLR3,TLR7,TLR21 and MDA5.Experimental infection on SPF chickens of the viruses F/98 and NA?67-76,was set up between different groups,one with antibody selection pressure and one without antibody selection pressure.The AID mRNA changes in lung,spleen and bursa of fabricius and serological kinetics in different groups were analyzed by qPCR technology,HI assay and ELISA assay.The results showed that,during the infection of SPF chickens without antibody selection pressure,the virus rF/NA?67-76 could induce higher expression of AID in the lung tissue and spleen of chicken,and produce the antibodies with higher antiviral activity,but these antibodies had lower IgG level and the level of specific antibodies for effective neutralization against virus was a little;during the infection of SPF chickens with antibody selection pressure,the AID mRNA lever of the rF/NA?67-76 virus inducing in the lung tissue on days 2 post-infection were lower,the AID mRNA lever of the rF/NA?67-76 virus inducing in the spleen and bursa of fabricius on days 7 post-infection were higher,and the AID mRNA lever of the rF/NA?67-76 virus inducing in the bursa of fabricius on days 14 post-infection was lower,inducing less specific neutralization antibodies in serum.
Keywords/Search Tags:H9N2 avian influenza virus, antibody selection pressure, antigenic sites, immune escape, specific neutralizing antibody
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