【作者】 张琳；

【导师】 孙淑红；

【作者基本信息】 山东农业大学 ， 兽医学， 2022， 硕士

【摘要】 我国于1992年首次发现H9N2亚型禽流感病毒(Avian influenza virus,AIV),此后的20多年该病毒持续存在,并且逐渐成为我国流行率最高的禽流感亚型。H9N2亚型禽流感虽然为低致病性禽流感,但感染后会引起禽鸟产蛋下降以及呼吸道症状,并且经常容易发生继发感染,加重发病率和死淘率,对养禽业造成巨大的经济损失。为了解2019～2020年我国部分地区H9N2亚型AIV的抗原变异情况,本研究对本实验室自2019～2020年分离的我国部分地区的1152株H9N2亚型AIV进行分子流行病学调查,研究毒株的进化变异情况;利用血清学试验技术对挑选毒株进行抗原性分析后挑选了疫苗候选株,并对疫苗候选株的免疫效力做出评价。1.H9N2亚型禽流感病毒分子流行病学调查对2019～2020年我国部分地区分离到的H9N2亚型AIV测序并对其遗传进化特点进行分析,结果显示,所有病毒均属于h9.4.2亚系,分离株在系统发育树上分布比较集中,均同属于h9.4.2.5进化分支,但已经形成多个单独的进化分支。除3株病毒外,其余毒株的HA蛋白都有Q226L的突变,有潜在感染人类的风险。NA蛋白根据是否发生颈部氨基酸的缺失,明显分为两个类型,由于颈部缺失,会导致NA蛋白的61位氨基酸的潜在糖基化位点缺失,颈部缺失的毒株与Y280-Like毒株的潜在糖基化位点类型一致,颈部没有缺失的毒株与A/Duck/Hong Kong/Y439/97(H9N2)毒株的潜在糖基化位点类型一致。在分析的1152株病毒中,有8株病毒存在第61位潜在糖基化位点。对11株代表毒株的内部基因测序分析后,3株病毒与其他8株病毒的遗传距离较远,属于单独的分支,且与其他亚型(H10N3、H7N9、H10N8)相似度更高,H9N2的内部基因可作为其他亚型内部基因的供体,如H7N9,可引起严重的呼吸道疾病。2.H9N2亚型禽流感病毒抗原性分析通过氨基酸同源性比对及遗传进化分析,挑选9株病毒对其抗原性进行分析。9个毒株免疫鸡群后,抗体水平普遍较高(抗体效价≥8log2),具有良好的免疫原性;中和试验结果显示,毒株H2049和J1443两个毒株只对自身和互相产生较高的保护力,而对其他毒株保护力较低,并且H2049、J1443毒株与其他毒株间的R值都较小,表示抗原之间抗原性差异大。综合交叉血凝抑制试验和中和试验的结果,证明存在两个抗原群。我们根据交叉血凝抑制试验和中和试验的结果,选择抗原性好,遗传稳定的一株毒毒株Y2432作为疫苗候选株免疫鸡群,21 d后用两个抗原群的病毒去攻击鸡群,鸡群均未排毒,证明该疫苗可以完全保护鸡群。制备的候选灭活疫苗免疫SPF鸡连续监测20周,观察抗体消长规律,14d即可产生高水平抗体,最高平均抗体可达到14.4 log2,且抗体可维持140 d以上。利用反向遗传技术成功拯救一株病毒Re-Y2432,比较亲本毒与重组毒的异同,结果显示重组毒与亲本毒HI相差不超过1 log2,差距较小。MDT结果表明,重组病毒对鸡胚致死力低于野毒。重组病毒的构建为后续分子定点研究搭建平台,也为新型H9N2亚型禽流感疫苗提供思路。更多还原

【Abstract】 The H9N2 subtype avian influenza virus(AIV)was first discovered in China in 1992,and the virus persisted for more than 20 years,and gradually became the most prevalent avian influenza subtype in China.Although H9N2 subtype avian influenza is a low pathogenic avian influenza,after infection,it will cause a decline in egg production and respiratory symptoms in avian,and secondary infections often occur,aggravating the morbidity and mortality,and causing huge economic losses to the poultry industry.In order to understand the antigenic variation of H9N2 subtype AIV in China from 2019 to 2020,this study conducted a molecular epidemiological investigation on 1152 H9N2 subtype AIV isolates in some areas of China from2019 to 2020 in our laboratory to study the evolutionary variation of the virus strain.using serological test technology to analyze the antigenicity of the selected strains,select vaccine candidate strains,and evaluate the immune efficacy of the vaccine candidate strains.1.Molecular epidemiological investigation of H9N2 subtype AIVThe H9N2 subtype AIV isolated in some parts of China from 2019 to 2020 was sequenced and its genetic evolution characteristics were analyzed.The results showed that all viruses belonged to the h9.4.2 subline,and the isolates were relatively concentrated on the phylogenetic tree.All belong to the h9.4.2.5 clade,but multiple separate clade have been formed.Except for3 strains,the HA proteins of the other strains have the Q226 L mutation,which has the potential to infect humans.The NA protein is obviously divided into two types according to whether the deletion of the neck amino acid occurs.Due to the deletion of the neck,the potential glycosylation site of the 61 amino acid of the NA protein is deleted.The neck deletion strain is similar to the Y280-Like.The potential glycosylation site types of the strains were consistent,and the strains without neck deletion were consistent with the potential glycosylation site types of the A/Duck/Hong Kong/Y439/97(H9N2)strain.Among the 110 viruses analyzed,8 viruses had the potential glycosylation site at position 61.After the internal gene sequencing analysis of 11 representative strains,the genetic distance between 3 viruses and the other 8 viruses is farther,they belong to a separate branch,and they are more similar to other subtypes(H10N3,H7N9,H10N8),and H9N2 The internal gene can serve as a donor for other subtype internal genes,such as H7N9,which can cause severe respiratory disease.2.Analysis of antigenicity of H9N2 subtype AIVThrough amino acid homology comparison and genetic evolution analysis,9 strains of viruses were selected to analyze their antigenicity.After 9 strains immunized chickens,the antibody level was high(antibody titer ≥ 8log2)and had good immunogenicity;the results of neutralization test showed that the two strains H2049 and J1443 only produced relatively high levels of antibodies against themselves and each other.High protective power,but low protection against other strains,and the R values between H2049,J1443 strains and other strains are all small,indicating that antigenic differences between antigens are significant.Combining the results of the cross-hemagglutination inhibition assay and the neutralization assay,it was demonstrated that there were two antigen groups.Based on the results of the crosshemagglutination inhibition test and neutralization test,we selected a Y2432 strain with good antigenicity and genetic stability as the vaccine candidate strain to immunize the chickens.After21 days,the chickens were challenged with viruses from the two antigen groups.,the flocks did not detoxify,proving that the vaccine can completely protect the flocks.The prepared candidate inactivated vaccine immunized SPF chickens were continuously monitored for 20 weeks,and the regularity of antibody fluctuation was observed.High-level antibodies could be produced within 14 days,and the highest average antibody could reach 14.4 log2,and the antibody could be maintained for more than 140 days.A virus was successfully rescued by reverse genetic technology,and the similarities and differences between the parental virus and the recombinant virus were compared.The results showed that the HI difference between the recombinant virus and the parental virus did not exceed 1 log2,and the gap was small.MDT results showed that the lethality of recombinant virus to chicken embryos was lower than that of wild virus.The construction of the recombinant virus provides a platform for the follow-up molecular targeted research,and also provides ideas for the new H9N2 subtype avian influenza vaccine.更多还原