H9N2亚型禽流感病毒感染水貂的研究
发布时间：2018-10-31 16:42
【摘要】：H9N2-型禽流感病毒在我国广泛流行和存在,不仅给养禽业带来巨大危害,且能够跨越种属屏障获得了感染人等哺乳动物的能力,还因其易发生变异和重组,可作为内部基因组供体,将其他亚型的禽流感病毒“引渡”到人群中,对人类的健康造成了极大的威胁。自1956年我国从国外引种开始饲养毛皮动物以来,养貂规模不断扩大,但养貂场养殖模式不规范、管理水平参差不齐,长期饲喂禽类副产品及貂舍暴露于外界易接触飞鸟粪便、羽屑等状况,使水貂感染流感病毒的风险日益加大,致使我们期望全面掌握我国养殖水貂中感染流感病毒的情况及其危害,研究水貂是否可以作为“病毒携带者”或“中间宿主”在流感病毒传播过程中起到推波助澜的作用。所以本文通过水貂群中流感病毒血清学调查、流感病毒的分离鉴定、对分离毒株的生物学特性研究和全基因测序分析、H9N2亚型禽源禽流感分离株对水貂致病性的研究及水貂呼吸道组织中流感受体的分布分析,系统研究了H9N2亚型禽流感病毒对水貂的感染情况。首先,为评估水貂感染H9N2禽流感的风险,在山东省不同地区采集血清样品560份,开展了禽流感H9N2亚型抗体的血清学调查。HI试验显示,被检血清中对A/Chicken/Hebei/4/2008 (H9N2)和A/chicken/Shanghai/10/01 (H9N2)抗原的阳性反应百分比分别为45.4%和47.5%。无论在成年貂还是幼貂中,H9N2禽流感的抗体阳性普遍存在,阳性率高达40%以上。说明山东省不同养殖区域貂群均不同程度地感染禽流感H9亚型病毒,具有重大的感染风险。血清学调查提示养殖水貂中存在感染H9N2禽流感病毒的风险,所以本研究试图从水貂中分离得到H9N2病毒株,并进行全面的生物学特性研究。2014年从一只有咳嗽、发热、流鼻涕症状的发病水貂分离得到一株流感病毒,经鉴定为H9N2亚型流感病毒,命名为A/Mink/Shandong/wm/2014(H9N2).生物学特性分析表明,接种SPF鸡后,该分离株对SPF鸡致病性较低,未表现明显临床症状,属低致病性流感病毒。但接种小鼠后可引起小鼠显著的临床变化,肺部明显水肿、淤血和出血；病毒可在在小鼠体内复制并引起小鼠发病,说明该株病毒对小鼠的致病性较强。对该分离株8个基因片段进行分子遗传进化分析表明,HA基因属于Y280-like分支,M基因属于G1-like分支,NA、NP、NS、PB1、PB2、PA基因均属于F98-like分支,说明该毒株是一株H9N2大陆流行谱系的低致病性禽流感病毒。与近两年在山东及其周边地区流行的H0N2禽流感分离株同源性较高,其中M、NS、NP、PA、PB1基因与多株H7N9毒株同源性较高,说明这些基因片段与H7N9分离株有相同的来源。HA基因裂解位点氨基酸序列为PSRSSR↓GL,符合低致病性禽流感的特征。HA基因含有8个潜在糖基化位点,与经典毒株Y280相比,在313位和551位出现两个潜在的糖基化位点。HA基因受体结合相关位点发生了N191H与E198T的突变；Q234L发生突变,具有了与哺乳动物唾液酸α,2-6受体结合的特性。NA基因有7个潜在的糖基化位点,在其茎部缺失3个氨基酸,造成61位潜在糖基化位点的缺失；三处红细胞受体结合位点在368位点(K→N)、400(S→N)及401(D→E)三处氨基酸发生突变,而且368位点的突变与同源性较高的近几年H9N2的流行株突变相同。HA与NA基因这些位点的突变与缺失是否与其宿主范围扩大和对哺乳动物致病性增强有关,有待于进一步研究。M2基因出现了S31N变异,说明该病毒可能对离子通道抑制剂类药物不敏感。为研究禽源H9N2亚型禽流感病毒对水貂的致病性,本试验通过鼻腔接种Ck/HB/4/08分离株感染幼貂,观察其临床症状、病理变化、各脏器感染情况、排毒情况及抗体变化。结果显示,攻毒后水貂未出现明显的临床症状；但肺脏淤血,局部轻微实变。感染后第3、7d的肺脏中均可检测到病毒。攻毒后3-11d内均可在鼻拭子中检测到病毒。攻毒后第7d抗体水平开始升高,持续到第15d。表明禽源H9N2亚型禽流感病毒可以直接感染水貂,且对水貂造成了一定的致病性,临床症状不显著,病毒可在其体内有效复制,并可向外排毒。为了检测唾液酸受体在水貂呼吸道组织中的类型和分布,本研究利用免疫组化方法对水貂呼吸道组织进行了禽型SAa-2,3Gal受体和人型SAa-2,6Gal受体两种受体的分析。分析显示,两种唾液酸受体在水貂的呼吸道组织中均有表达,但以SAa-2,6Gal受体为主,尤其在上呼吸道以SAa2,6Gal受体表达较多,说明水貂呼吸道含有人型与禽型两种受体,提示水貂可能作为一种甲型流感病毒“基因混合器”或“中间宿主”。总之,本研究提供了临床和实验室两方面的研究资料,提示H9N2亚型流感病毒在养殖水貂中普遍存在,病毒可以感染水貂,引起水貂发病,病毒可在水貂体内复制并向外排毒。禽源流感病毒传播到了水貂中,使水貂成为病毒的侵入者和携带者,提醒我们务必加强水貂群中流感病毒的监测,对防控禽流感以及人类流感大流行均具有的重要意义。
[Abstract]:The H9N2-type avian influenza virus is widely popular and exists in China, not only brings great harm to the poultry industry, but also has the ability to cross the species barrier to obtain mammals such as infectious people, and also can be used as an internal genome donor because of its easy mutation and recombination. Other subtypes of avian influenza virus" Extradition "In the crowd, there is a great threat to human health. Since China introduced the fur animals from abroad in 1956, the scale of breeding mink has been expanding, but the breeding mode of mink farms is not standardized, the management level is uneven, and the long-term feeding of poultry by-products and mink houses are exposed to the conditions such as excrement and feather of birds, which are easy to contact with the outside. The risk of influenza virus infection is increasing, so that we expect to master the situation and harm of influenza virus infection in China's aquaculture industry, and study whether the virus can be used as" Viral Carriers" or" Intermediate Host "plays an important role in the propagation of influenza virus. Therefore, through serologic investigation of influenza virus, isolation and identification of influenza virus, the study on the biological characteristics of isolated strains and the analysis of whole gene sequencing, H9N2 subtype avian influenza isolate (H9N2) was used to study the pathogenic avian influenza virus (H9N2) and the distribution of influenza receptor in respiratory tract tissues. The infection of H9N2 subtype avian influenza virus (H9N2) on the pathogenic bacteria was studied. First, in order to assess the risk of H9N2 avian influenza infection, 560 samples of serum samples were collected in different regions of Shandong Province, and the serological investigation of H9N2 subtype antibody of avian influenza virus was carried out. HI test showed that the positive percentage of A/ Chicken/ Hebeei/ 4/ 2008 (H9N2) and A/ chenken/ Shanhai/ 10/ 01 (H9N2) antigen in the tested serum was 44.5% and 47.5%, respectively. The positive rate of H9N2 avian influenza is more than 40% in adult mink or mink. It is indicated that the mink groups of different breeding regions in Shandong province are infected with H9 subtype avian influenza virus in different degrees, and have significant risk of infection. Serological investigations suggest that there is a risk of infection of H9N2 avian influenza virus in aquaculture, so this study attempts to isolate H9N2 virus strains from the population and conduct a comprehensive biological characterization study. In 2014, from a cough and fever, An influenza virus was isolated from the onset of runny nose symptom, identified as H9N2 subtype influenza virus, named A/ Mink/ Shanong/ wm/ 2014 (H9N2). The biological characteristic analysis indicated that after inoculation of SPF chicken, the isolate had low pathogenicity to SPF chicken, and did not show obvious clinical symptoms, belonging to the low pathogenic influenza virus. But after inoculation, the mice can cause significant clinical changes, obvious edema, congestion and bleeding in the lungs; the virus can be replicated in the mice and cause the onset of the mice, which indicates that the strain has strong pathogenicity to the mice. The molecular genetic evolutionary analysis of 8 gene fragments of the isolate showed that the HA gene belongs to Y280-like branch, and M gene belongs to G1-like branch, and NA, NP, NS, PB1, PB2 and PA genes belong to the F98-like branch, which indicates that the strain is a low pathogenic avian influenza virus of the epidemic genealogy of the H9N2 continent. Compared with H0N2 avian influenza isolates prevalent in Shandong and its surrounding areas in the past two years, the homology of M, NS, NP, PA, PB1 gene and multi-strain N9 strain was higher. The amino acid sequence of HA gene cleavage site was PSRSSR-GGL, which was consistent with the characteristics of low pathogenic avian influenza. The HA gene contains 8 potential glycosylation sites, two potential glycosylation sites at 313 and 551 sites compared to classical strain Y280. The mutation of N191H and E198T occurred in the binding sites of HA gene receptor, and the mutation of Q234L had the characteristics of binding to mammalian sialic acid receptor, 2-6 receptor. There are 7 potential glycosylation sites in NA gene, deletion of 3 amino acids in its nucleus, deletion of 61 potential glycosylation sites, mutation of three red cell receptor binding sites at 368 sites (K/ N), 400 (S/ N), and 401 (D/ E) 3 amino acids, Moreover, the mutation of 368 sites was the same as that of H9N2 in recent years. The mutation and deletion of these sites of HA and NA genes are related to the expansion of their host range and the enhancement of pathogenicity in mammals, which need to be further studied. The M2 gene showed S31N mutation suggesting that the virus could not be sensitive to ion channel inhibitor drugs. In order to study the pathogenicity of avian influenza virus (H9N2) subtype avian influenza virus (H9N2), the clinical symptoms, pathological changes, organ infection, toxin expelling and antibody change were observed by inoculating Ck/ HB/ 4/ 08 isolate in the nasal cavity. The results showed that there were no obvious clinical symptoms after attack. The virus was detectable in the lungs of days 3, 7 after infection. The virus can be detected in the nasal swab within 3-11d after the attack. The level of antibody 7d after the challenge began to rise and continued to day 15d. The avian influenza virus (H9N2) subtype avian influenza virus (H9N2) can be directly infected with dengue virus, which causes some pathogenicity and clinical symptoms. The virus can be effectively replicated in the body and can detoxify the virus. In order to detect the type and distribution of sialic acid receptors in respiratory tract tissues, this study conducted an analysis of the two receptors of avian type SA-2, 31R receptor and human SAa-2, 61R receptor by immunohistochemistry. The results show that the two sialic acid receptors are expressed in the respiratory tract tissues of the urinary tract, but are mainly expressed by SAa-2 and 61R receptors, especially in upper respiratory tract with SAa2 and 61R receptors, suggesting that the respiratory tract contains both human and avian type receptors, suggesting that it may be a type of influenza A virus. gene mixer or" Intermediate Host ". In summary, this study provides clinical and laboratory research data suggesting that the H9N2 subtype influenza virus is ubiquitous in aquaculture, and the virus can be infected with dengue virus, causing the virus to develop in vivo, and the virus can be replicated and detoxified in vivo. The spread of avian influenza virus into the virus causes the virus to become the invading and carrier of the virus. It is important to remind us to strengthen the monitoring of influenza virus in the avian influenza virus group, and have important significance for preventing and controlling avian influenza and the pandemic of human influenza.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.92
，
本文编号：2302909
[Abstract]:The H9N2-type avian influenza virus is widely popular and exists in China, not only brings great harm to the poultry industry, but also has the ability to cross the species barrier to obtain mammals such as infectious people, and also can be used as an internal genome donor because of its easy mutation and recombination. Other subtypes of avian influenza virus" Extradition "In the crowd, there is a great threat to human health. Since China introduced the fur animals from abroad in 1956, the scale of breeding mink has been expanding, but the breeding mode of mink farms is not standardized, the management level is uneven, and the long-term feeding of poultry by-products and mink houses are exposed to the conditions such as excrement and feather of birds, which are easy to contact with the outside. The risk of influenza virus infection is increasing, so that we expect to master the situation and harm of influenza virus infection in China's aquaculture industry, and study whether the virus can be used as" Viral Carriers" or" Intermediate Host "plays an important role in the propagation of influenza virus. Therefore, through serologic investigation of influenza virus, isolation and identification of influenza virus, the study on the biological characteristics of isolated strains and the analysis of whole gene sequencing, H9N2 subtype avian influenza isolate (H9N2) was used to study the pathogenic avian influenza virus (H9N2) and the distribution of influenza receptor in respiratory tract tissues. The infection of H9N2 subtype avian influenza virus (H9N2) on the pathogenic bacteria was studied. First, in order to assess the risk of H9N2 avian influenza infection, 560 samples of serum samples were collected in different regions of Shandong Province, and the serological investigation of H9N2 subtype antibody of avian influenza virus was carried out. HI test showed that the positive percentage of A/ Chicken/ Hebeei/ 4/ 2008 (H9N2) and A/ chenken/ Shanhai/ 10/ 01 (H9N2) antigen in the tested serum was 44.5% and 47.5%, respectively. The positive rate of H9N2 avian influenza is more than 40% in adult mink or mink. It is indicated that the mink groups of different breeding regions in Shandong province are infected with H9 subtype avian influenza virus in different degrees, and have significant risk of infection. Serological investigations suggest that there is a risk of infection of H9N2 avian influenza virus in aquaculture, so this study attempts to isolate H9N2 virus strains from the population and conduct a comprehensive biological characterization study. In 2014, from a cough and fever, An influenza virus was isolated from the onset of runny nose symptom, identified as H9N2 subtype influenza virus, named A/ Mink/ Shanong/ wm/ 2014 (H9N2). The biological characteristic analysis indicated that after inoculation of SPF chicken, the isolate had low pathogenicity to SPF chicken, and did not show obvious clinical symptoms, belonging to the low pathogenic influenza virus. But after inoculation, the mice can cause significant clinical changes, obvious edema, congestion and bleeding in the lungs; the virus can be replicated in the mice and cause the onset of the mice, which indicates that the strain has strong pathogenicity to the mice. The molecular genetic evolutionary analysis of 8 gene fragments of the isolate showed that the HA gene belongs to Y280-like branch, and M gene belongs to G1-like branch, and NA, NP, NS, PB1, PB2 and PA genes belong to the F98-like branch, which indicates that the strain is a low pathogenic avian influenza virus of the epidemic genealogy of the H9N2 continent. Compared with H0N2 avian influenza isolates prevalent in Shandong and its surrounding areas in the past two years, the homology of M, NS, NP, PA, PB1 gene and multi-strain N9 strain was higher. The amino acid sequence of HA gene cleavage site was PSRSSR-GGL, which was consistent with the characteristics of low pathogenic avian influenza. The HA gene contains 8 potential glycosylation sites, two potential glycosylation sites at 313 and 551 sites compared to classical strain Y280. The mutation of N191H and E198T occurred in the binding sites of HA gene receptor, and the mutation of Q234L had the characteristics of binding to mammalian sialic acid receptor, 2-6 receptor. There are 7 potential glycosylation sites in NA gene, deletion of 3 amino acids in its nucleus, deletion of 61 potential glycosylation sites, mutation of three red cell receptor binding sites at 368 sites (K/ N), 400 (S/ N), and 401 (D/ E) 3 amino acids, Moreover, the mutation of 368 sites was the same as that of H9N2 in recent years. The mutation and deletion of these sites of HA and NA genes are related to the expansion of their host range and the enhancement of pathogenicity in mammals, which need to be further studied. The M2 gene showed S31N mutation suggesting that the virus could not be sensitive to ion channel inhibitor drugs. In order to study the pathogenicity of avian influenza virus (H9N2) subtype avian influenza virus (H9N2), the clinical symptoms, pathological changes, organ infection, toxin expelling and antibody change were observed by inoculating Ck/ HB/ 4/ 08 isolate in the nasal cavity. The results showed that there were no obvious clinical symptoms after attack. The virus was detectable in the lungs of days 3, 7 after infection. The virus can be detected in the nasal swab within 3-11d after the attack. The level of antibody 7d after the challenge began to rise and continued to day 15d. The avian influenza virus (H9N2) subtype avian influenza virus (H9N2) can be directly infected with dengue virus, which causes some pathogenicity and clinical symptoms. The virus can be effectively replicated in the body and can detoxify the virus. In order to detect the type and distribution of sialic acid receptors in respiratory tract tissues, this study conducted an analysis of the two receptors of avian type SA-2, 31R receptor and human SAa-2, 61R receptor by immunohistochemistry. The results show that the two sialic acid receptors are expressed in the respiratory tract tissues of the urinary tract, but are mainly expressed by SAa-2 and 61R receptors, especially in upper respiratory tract with SAa2 and 61R receptors, suggesting that the respiratory tract contains both human and avian type receptors, suggesting that it may be a type of influenza A virus. gene mixer or" Intermediate Host ". In summary, this study provides clinical and laboratory research data suggesting that the H9N2 subtype influenza virus is ubiquitous in aquaculture, and the virus can be infected with dengue virus, causing the virus to develop in vivo, and the virus can be replicated and detoxified in vivo. The spread of avian influenza virus into the virus causes the virus to become the invading and carrier of the virus. It is important to remind us to strengthen the monitoring of influenza virus in the avian influenza virus group, and have important significance for preventing and controlling avian influenza and the pandemic of human influenza.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.92
，
本文编号：2302909
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