【作者】 李丹；

【导师】 宋德贵； 谢芝勋；

【作者基本信息】 广西师范大学 ， 生物学， 2017， 硕士

【摘要】 禽流感(Avian Influenza,AI)是由A型流感病毒属禽流感病毒(Avian Influenza virus,AIV)引起的一种禽类(家禽和野鸟)的传染病。根据其表面糖蛋白血凝素(HA)和神经氨酸酶(NA)的不同,将其分为不同的HA和NA亚型组合。其中H9亚型禽流感是目前对家禽养殖业危害最为严重的疾病之一,其在全球各个地方均有分布,而H9N2亚型AIV在我国鸡群中流行最为广泛。H9N2亚型AIV可以跨越种属屏障直接感染人类,且自2013年以来国内出现了 H9N2亚型AIV为H5N6、H10N8、H7N9和H6N1四种感染人的流感亚型提供内部基因的报道,因此H9N2亚型AIV还严重威胁人类公共健康。本研究为了了解2014年至2016年广西不同宿主H9亚型禽流感病毒的流行情况及其分离株内部基因遗传进化的特点,建立了关于H9亚型禽流感病毒的快速检测方法,并对分离到的部分病毒毒株进行了全基因测序及生物学特性的研究。其研究结果如下:(1)、近年来不同地区禽流感监测研究的结果表明H9与H6亚型低致病性AIV混合感染的情况在家禽养殖和出售的环节中是比较常见的。本研究针对H9与H6亚型AIV的HA基因设计了特异性引物,其片段大小分别为551bp与222bp。将两对引物进行组合,优化引物浓度与退火温度等条件建立可以同时检测H9与H6的二重RT-PCR的检测方法。该方法具有很高的特异性,其敏感性可达5×104拷贝/μL,而且可以同时在一管内检测H9与H6两种亚型的AIV,可为H9与H6亚型AIV的监测提供技术支撑。(2)、本研究针对广西2014-2016年从不同家禽和野禽中采集的棉拭子及组织样品进行H9亚型AIV的分离和鉴定,挑选毒株进行纯化。本研究共分离和纯化出来源于9种不同宿主的26株H9亚型AIV,HA和NA测序结果表明其均属于H9N2亚型AIV,裂解位点也符合低致病性AIV分子特征。不同宿主来源分离株HA基因与参考株在进化树中分别处于不同的进化分支上。26株代表性分离株与参考株遗传演化分析的结果表明大多分离株的8个基因片段有着程度不一的基因重组,共有7种不同的重组模式。(3)、本研究对测完全基因的26株毒株进行生物学特性的研究,研究结果表明其ELD50范围为10-4.5～10-8之间,EID50范围在10-6.2～10-8.5之间。受体亲和性测定结果表明26株毒株当中有20株毒株同时具有禽源SAα2,3与人源SAα2,6两种受体的亲和性特征,而另外6株则只有禽源S Aα2,3受体的亲和性特征。为了评价不同宿主来源H9N2亚型禽流感病毒对鸡的致病性强弱,我们根据近年广西不同宿主来源H9N2亚型AIV HA基因遗传进化分析的结果选取了处于不同的进化分支的病毒进行SPF鸡的致病性试验。试验的结果表明H9N2亚型禽流感病毒分离株可以感染SPF鸡,病毒在鸡体内不同脏器的组织分布也存在一定的差异。SPF鸡感染H9N2病毒后表现出一定的临床症状,感染鸡能够通过呼吸道和和泄殖腔向外排毒。剖检及组织病理观察的结果显示部分感染鸡出现气管充血等病变,肺组织等出现一定程度的病理损伤。此外,同居感染试验表明这两株病毒可以由感染组的鸡水平传播给同居组的鸡。综合以上所述,本研究中的不同宿主来源H9N2亚型禽流感病毒对SPF鸡具有一定的致病性,并存在很大的不同鸡群间水平传播的潜在威胁。因此,我们在家禽的流通环节及其饲养过程中尽量避免鸡群与其它野鸟的接触。更多还原

【Abstract】 Avian Influenza(AI)is a highly contagious disease of poultry and birds caused by influenza A viruses,a member of Influenza virus A genis.According to its surface glycoprotein haemagglutinin(HA)and neuraminidase(NA),the virus is divided into different HA and NA subtype combination.The H9 subtype of avian influenza is one of the most serious diseases to the poultry,it was scattered in various locations around the world,and H9N2 subtype AIV which is the most widely popular in chicken flocks in China.H9N2 subtype AIV also can cross the species barrier to infect humans directly,and that appeared H9N2 subtype AIV provide internal genes for H5N6,H10N8,H7N9 and H6N1 four different human infection with avian influenza in China since 2013,therefore H9N2 subtype AIV also has become a serious threat to human public health.In order to understand the prevalence of different host H9 subtype avian influenza viruses in Guangxi from 2014 to 2016 and genetic evolution of the internal characteristics of the isolates,we established rapid detection method for H9 subtype avian influenza virus,and some of the isolates were performed whole-genome sequencing and biological characterization.The results were as follows:(1)In recent years,different parts of the bird flu monitoring results show that low pathogenic AIV subtype H9 and H6 mixed infection situation in poultry production and sale link is relatively common.In this study H6 and H9 subtypes of AIV HA gene specific primers were designed to amplify 551 bp and 222 bp amplicon respectively.Amplification conditions were optimized including primer concentrations and annealing temperature for simultaneousdetection of H9 and H6 by double RT-PCR detection method.The detection limit of triplex PCR was 5×104 copies/μL.It suggests that this duplex RT-PCR assay is a specific,sensitive,stable and repeatable method for the detection of H9 and H6 subtype of AIV,can provide technical support for the monitoring of H9 and H6 subtype AIV.Using the newly developed method samples origonatong from nine different host 26 strains of H9 subtype AIV was detected and sequencing of their HA and NA gene showed that they all belong to H9N2 subtype AIV,cracking site also conforms to the low pathogenic AIV molecular features.Source separation of different host plants HA gene and reference strains respectively in the evolution of the different branches in the evolutionary tree.Genetic evolution analysis result of 26 strains representative isolates and reference strains of showed that most of the isolates eight gene segments with varying degrees of genetic recombination,a total of seven different restructuring mode.(2)In this study biological characterization of the isolates were also done along with their whole-genome sequencing.The results show that ELD5O range between 10-4.5 and 10-8,EID50 range between 10-6.2 and 10-8.5.Receptor affinity determination results showed that 17 strains of 26 strains have poultry at the same time SAα 2,3 and anthropogenic SAα 2,6 characteristics of two kinds of receptor affinity,and the other 9 cases with poultry SAa 2,3 receptor affinity characteristics.In order to evaluate different sources of host H9N2 subtype of avian influenza virus pathogenicity we used SPF and infected them with the isolateschicken,we according to different sources of host in recent years,Guangxi H9N2 subtype AIV HA selection genetic evolution analysis of the results in different branches of the evolution of the virus of SPF chicken pathogenic experiment was carried out.Test results show that isolates H9N2 subtype avian influenza viruses can infect SPF chicken,virus in chicken body there exist certain differences in tissue distribution in different organs.SPF chickens infected H9N2 virus showed certain clinical symptoms,can through the respiratory tract infection chicken and cloacal poison outward.Autopsy and histopathological observation results showed that the part of the airway infection chicken hyperemia lesions,lung tissue also showed certain degree of pathological damage,etc.Cohabitation infection test,moreover,that the two strains of the virus can be spread by infected group level of chicken to cohabitation group.Inoculation of H9N2 subtype avian influenza virus in SPF chicken demonstrated pathological lesions.And these viruses poses a big potential threat for chickens.We recommend to avoid contact of chickens with other species of birds.更多还原