H4亚型禽流感病毒的分离鉴定与遗传进化及其对SPF鸡致病性的研究

发布时间：2018-05-02 08:24

本文选题：H4亚型禽流感病毒 + 分离鉴定　；参考：《广西大学》2017年硕士论文

【摘要】：H4亚型禽流感病毒(avian influenza virus,AIV)是一种比较常见的低致病性AIV(low pathogenic,LPAIV),尤其在我国南方地区,H4亚型AIV在流感病毒监测中的分离率较高,且常与H1、H3、H5、H6和H9等多种亚型的AIV混合感染,较易发生病毒的重排和变异。因此,了解H4亚型AIV的遗传进化关系及其致病性对我国禽流感的防控具有重要意义。本研究对2015-2016年广西活禽市场H4亚型AIV进行分离与鉴定及其生物学研究,并对分离株进行病毒全基因组序列分析。结果共分离出6株H4亚型AIV,均为LPAIV,同时具备SAα2,3和SAα2,6受体的亲和特性,但其结合SAα2,3受体的能力更高。遗传进化分析表明6株广西H4亚型AIV分离株的8个基因均属于欧亚谱系,不同基因组之间的来源比较复杂,其中HA基因的核苷酸序列主要与蒙古A/duck/Mongolia/OIE-7438/2011(H4N6)分离株具有较高的同源性。H4亚型AIV分离株主要与N2和N8亚型组合,其中H4N2亚型的NA基因与蒙古的A/duck/Mongolia/154/20 1 5(H 1N2)和浙江的 A/duck/Zhejiang/727042/2014(H6N2)分离株的NA基因核苷酸序列具有较高的同源性;H4N8亚型的NA基因与越南A/duck/Vietnam/OIE-2577/2011(H3N8)分离株NA基因核苷酸序列具有较高同源性,表明广西H4亚型AIV分离株极有可能与上述不同国家的分离株发生重组,推测其可能是由于候鸟迁徙等原因导致的病毒重组。为了建立快速H4亚型AIV检测方法,根据GenBank中H4亚型AIV的HA基因保守序列,设计1对特异性引物,通过优化反应条件,建立了 H4亚型AIVSYBRGreenI实时荧光定量检测方法。并以β-actin作为内参基因对各组织样品中细胞的起始数量进行校正,为准确确定不同组织样品中H4亚型AIV的拷贝数提供依据。为了掌握H4亚型AIV分离株对鸡群的致病情况,筛选了 6株H4亚型AIV分离株。对4周龄SPF鸡以106 EID50/只进行滴鼻感染,结果6株H4亚型AIV分离株均不需要提前适应即可在SPF鸡体内进行有效地复制,且6株H4亚型AIV分离株均可向外界排毒。其中水禽源毒株 A/duck/Guangxi/101DI8/2011(H4N6)、A/duck/Guangxi/030E93/2015(H4N2)和A/goose/Guangxi/220G30/2016(H4N2)均具有水平传播的能力。整个试验过程中病毒未引起鸡群的全身性感染或者死亡,感染3 d后剖检部分感染鸡可见肺脏和肾脏有轻微的充血,脾脏有轻微的肿大,其余器官无明显的解剖学病变;病理切片观察可见气管、肺脏、肝脏、心脏和肾脏出现淋巴细胞浸润,肺脏和肾脏出现充血等组织学病变。同时用鸡胚对气管、胸腺、脑、肝脏、脾脏、心脏、肺脏、胰腺、肾脏、法氏囊和盲肠扁桃体11个不同组织进行病毒滴定,结果显示在气管和肺脏中病毒滴度较高,呈现出较强的组织嗜性。H4亚型AIV属于LPAIV,目前也尚未引起家禽发病及流行的情况,但当家禽群中存在H4亚型AIV和其它亚型AIV混合感染时,不同亚型AIV的基因极易发生交换及重排产生新亚型AIV。因此加_7H4亚型AIV的监测,对AIV的防控有重要意义。
[Abstract]:H4 subtype avian influenza virus (Ave) is a common low pathogenic AIV(low pathogenetic AIV, especially in southern China, where the isolation rate of H4 subtype AIV in influenza virus surveillance is high, and it is often co-infected with H1H3, H5, H6, H9 and other subtypes of AIV. It is easy to rearrange and mutate the virus. Therefore, it is important to understand the genetic and evolutionary relationship of H4 subtype AIV and its pathogenicity for the prevention and control of avian influenza in China. In this study, the AIV of H4 subtype in Guangxi live poultry market from 2015 to 2016 was isolated and identified, and the whole genome sequence of the virus was analyzed. Results A total of 6 H4 subtype AIVs were isolated, all of them were LPAIV.The affinity of SA 伪 2C 3 and SA 伪 2O 6 receptors was also found, but the ability of binding SA 伪 2O 3 receptor was higher. Genetic evolution analysis showed that the 8 genes of 6 AIV isolates of H4 subtype in Guangxi belonged to Eurasian lineage, and the origin of different genomes was complex. The nucleotide sequence of HA gene was highly homologous to the Mongolian A / Duck / Mongolia- / OIE-7438 / 2011H4N6) isolate. H4 subtype AIV was mainly combined with N2 and N8 subtypes. The na gene of the H4N2 subtype was highly homologous to the na gene of the Mongolian A/duck/Mongolia/154/20 15 (H1N2) and Zhejiang A / Duck / Zhejiangr 727042 / 2014 H6N2) isolates, and the na gene of the H4N8 subtype was highly homologous to the na gene sequence of the Vietnam A / Duckr / Vietnam OIE-2577 / 2011H3N8 (A / Duckr / Vietnam OIE-2577 / 2011H3N8) isolate. It was suggested that the AIV isolates of H4 subtype in Guangxi were likely to recombine with the isolates from different countries mentioned above, which might be caused by the migration of migratory birds. In order to establish a rapid detection method for H4 subtype AIV, a pair of specific primers were designed according to the conserved HA gene sequence of H4 subtype AIV in GenBank. A real-time fluorescence quantitative detection method for H4 subtype AIVSYBRGreenI was established by optimizing the reaction conditions. 尾 -actin was used as an internal reference gene to correct the initial number of cells in each tissue sample, which provided the basis for determining accurately the copy number of H4 subtype AIV in different tissue samples. In order to understand the pathogenicity of H4 subtype AIV isolates to chickens, 6 H4 subtype AIV isolates were screened. SPF chickens aged 4 weeks were infected by nasal drip only with 106 EID50/. The results showed that 6 AIV isolates of H4 subtype could be replicated effectively in SPF chickens without prior adaptation, and 6 AIV isolates of H4 subtype could detoxify the environment. Among them, the waterfowl strain A / Duck / Guangxi / 101 / DI8 / 2011 / H4N6 / A / P / 030E93 / 2005 / H4N2) and A / R / G / Guangdong / 220G / 30 / 2016 / H4N _ 2 have the ability to transmit horizontally. During the whole experiment, the virus did not cause systemic infection or death in chickens. After 3 days of infection, some infected chickens showed slight congestion of lung and kidney, slight enlargement of spleen, and no obvious anatomical changes in other organs. Histological changes such as infiltration of lymphocytes in trachea, lung, liver, heart and kidney, congestion in lung and kidney were observed in pathological sections. The titer of virus in trachea, thymus, brain, liver, spleen, heart, lung, pancreas, kidney, bursa of Fabricius and cecal tonsil was also titrated with chicken embryo. H4 subtype AIV belongs to LPAIV.H4 subtype, which has not caused the disease and epidemic of poultry, but when there is mixed infection of H4 subtype AIV and other subtype AIV in poultry population, The genes of different subtypes of AIV are easy to be exchanged and rearranged to produce new subtypes of AIV. Therefore, it is important for the prevention and control of AIV to add the monitoring of subtype 7 H 4 AIV.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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