H9N2亞型禽流感病毒感染水貂的研究
發(fā)布時間：2018-10-31 16:42
【摘要】：H9N2-型禽流感病毒在我國廣泛流行和存在,不僅給養(yǎng)禽業(yè)帶來巨大危害,且能夠跨越種屬屏障獲得了感染人等哺乳動物的能力,還因其易發(fā)生變異和重組,可作為內(nèi)部基因組供體,將其他亞型的禽流感病毒“引渡”到人群中,對人類的健康造成了極大的威脅。自1956年我國從國外引種開始飼養(yǎng)毛皮動物以來,養(yǎng)貂規(guī)模不斷擴(kuò)大,但養(yǎng)貂場養(yǎng)殖模式不規(guī)范、管理水平參差不齊,長期飼喂禽類副產(chǎn)品及貂舍暴露于外界易接觸飛鳥糞便、羽屑等狀況,使水貂感染流感病毒的風(fēng)險日益加大,致使我們期望全面掌握我國養(yǎng)殖水貂中感染流感病毒的情況及其危害,研究水貂是否可以作為“病毒攜帶者”或“中間宿主”在流感病毒傳播過程中起到推波助瀾的作用。所以本文通過水貂群中流感病毒血清學(xué)調(diào)查、流感病毒的分離鑒定、對分離毒株的生物學(xué)特性研究和全基因測序分析、H9N2亞型禽源禽流感分離株對水貂致病性的研究及水貂呼吸道組織中流感受體的分布分析,系統(tǒng)研究了H9N2亞型禽流感病毒對水貂的感染情況。首先,為評估水貂感染H9N2禽流感的風(fēng)險,在山東省不同地區(qū)采集血清樣品560份,開展了禽流感H9N2亞型抗體的血清學(xué)調(diào)查。HI試驗(yàn)顯示,被檢血清中對A/Chicken/Hebei/4/2008 (H9N2)和A/chicken/Shanghai/10/01 (H9N2)抗原的陽性反應(yīng)百分比分別為45.4%和47.5%。無論在成年貂還是幼貂中,H9N2禽流感的抗體陽性普遍存在,陽性率高達(dá)40%以上。說明山東省不同養(yǎng)殖區(qū)域貂群均不同程度地感染禽流感H9亞型病毒,具有重大的感染風(fēng)險。血清學(xué)調(diào)查提示養(yǎng)殖水貂中存在感染H9N2禽流感病毒的風(fēng)險,所以本研究試圖從水貂中分離得到H9N2病毒株,并進(jìn)行全面的生物學(xué)特性研究。2014年從一只有咳嗽、發(fā)熱、流鼻涕癥狀的發(fā)病水貂分離得到一株流感病毒,經(jīng)鑒定為H9N2亞型流感病毒,命名為A/Mink/Shandong/wm/2014(H9N2).生物學(xué)特性分析表明,接種SPF雞后,該分離株對SPF雞致病性較低,未表現(xiàn)明顯臨床癥狀,屬低致病性流感病毒。但接種小鼠后可引起小鼠顯著的臨床變化,肺部明顯水腫、淤血和出血；病毒可在在小鼠體內(nèi)復(fù)制并引起小鼠發(fā)病,說明該株病毒對小鼠的致病性較強(qiáng)。對該分離株8個基因片段進(jìn)行分子遺傳進(jìn)化分析表明,HA基因?qū)儆赮280-like分支,M基因?qū)儆贕1-like分支,NA、NP、NS、PB1、PB2、PA基因均屬于F98-like分支,說明該毒株是一株H9N2大陸流行譜系的低致病性禽流感病毒。與近兩年在山東及其周邊地區(qū)流行的H0N2禽流感分離株同源性較高,其中M、NS、NP、PA、PB1基因與多株H7N9毒株同源性較高,說明這些基因片段與H7N9分離株有相同的來源。HA基因裂解位點(diǎn)氨基酸序列為PSRSSR↓GL,符合低致病性禽流感的特征。HA基因含有8個潛在糖基化位點(diǎn),與經(jīng)典毒株Y280相比,在313位和551位出現(xiàn)兩個潛在的糖基化位點(diǎn)。HA基因受體結(jié)合相關(guān)位點(diǎn)發(fā)生了N191H與E198T的突變；Q234L發(fā)生突變,具有了與哺乳動物唾液酸α,2-6受體結(jié)合的特性。NA基因有7個潛在的糖基化位點(diǎn),在其莖部缺失3個氨基酸,造成61位潛在糖基化位點(diǎn)的缺失；三處紅細(xì)胞受體結(jié)合位點(diǎn)在368位點(diǎn)(K→N)、400(S→N)及401(D→E)三處氨基酸發(fā)生突變,而且368位點(diǎn)的突變與同源性較高的近幾年H9N2的流行株突變相同。HA與NA基因這些位點(diǎn)的突變與缺失是否與其宿主范圍擴(kuò)大和對哺乳動物致病性增強(qiáng)有關(guān),有待于進(jìn)一步研究。M2基因出現(xiàn)了S31N變異,說明該病毒可能對離子通道抑制劑類藥物不敏感。為研究禽源H9N2亞型禽流感病毒對水貂的致病性,本試驗(yàn)通過鼻腔接種Ck/HB/4/08分離株感染幼貂,觀察其臨床癥狀、病理變化、各臟器感染情況、排毒情況及抗體變化。結(jié)果顯示,攻毒后水貂未出現(xiàn)明顯的臨床癥狀；但肺臟淤血,局部輕微實(shí)變。感染后第3、7d的肺臟中均可檢測到病毒。攻毒后3-11d內(nèi)均可在鼻拭子中檢測到病毒。攻毒后第7d抗體水平開始升高,持續(xù)到第15d。表明禽源H9N2亞型禽流感病毒可以直接感染水貂,且對水貂造成了一定的致病性,臨床癥狀不顯著,病毒可在其體內(nèi)有效復(fù)制,并可向外排毒。為了檢測唾液酸受體在水貂呼吸道組織中的類型和分布,本研究利用免疫組化方法對水貂呼吸道組織進(jìn)行了禽型SAa-2,3Gal受體和人型SAa-2,6Gal受體兩種受體的分析。分析顯示,兩種唾液酸受體在水貂的呼吸道組織中均有表達(dá),但以SAa-2,6Gal受體為主,尤其在上呼吸道以SAa2,6Gal受體表達(dá)較多,說明水貂呼吸道含有人型與禽型兩種受體,提示水貂可能作為一種甲型流感病毒“基因混合器”或“中間宿主”�？傊�,本研究提供了臨床和實(shí)驗(yàn)室兩方面的研究資料,提示H9N2亞型流感病毒在養(yǎng)殖水貂中普遍存在,病毒可以感染水貂,引起水貂發(fā)病,病毒可在水貂體內(nèi)復(fù)制并向外排毒。禽源流感病毒傳播到了水貂中,使水貂成為病毒的侵入者和攜帶者,提醒我們務(wù)必加強(qiáng)水貂群中流感病毒的監(jiān)測,對防控禽流感以及人類流感大流行均具有的重要意義。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.92
，
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.92
，
本文編號：2302909
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