环洞庭湖区新城疫病原监测及分子演化分析

发布时间：2018-07-26 11:35

【摘要】：新城疫(Newcastle disease, ND)的高致死性仍然是对世界各国养殖业的重大危害,随着发展中国家疫苗免疫为主的政策实施,疫苗得以广泛应用,新城疫的流行一定程度上得到控制,但其流行趋势却在发生改变：流行基因发生改变、宿主范围不但扩大、非典型性新城疫越来越普遍等。研究新城疫的分子流行病学,评估其流行特点和新城疫病毒的局部传播意义重大。为了解环洞庭湖地区新城疫的感染分布情况,本文通过对2013-2014年洞庭湖地区养禽场新城疫病原监测采样,采用鸡胚分离及血清学鉴定,分离到51株新城疫病毒,其中鸡源34株,鸭源2株,环境15株。选取不同时间、不同地点和宿主的16株代表毒株,采用R T-PCR方法扩增F基因和HN基因片段,并将扩增产物纯化后克隆于PMD18-T Easy Vector载体中进行测序,与Genbank中发表的参考序列进行遗传进化分析比对,结果表明1株NDV分离株属于Class Ⅱ基因Ⅱ型,F蛋白裂解位点的氨基酸序列为112GRQGRL117,属于疫苗类毒株；其余15株均为Class I基因3型,F蛋白裂解位点的氨基酸序列为I12ERQERL117, Class I各分离毒株之间F蛋白同源性在97.8%以上HN蛋白同源性在98.2%以上,与参考毒株吉林野鸟毒株J36/13的遗传关系最近。对洞庭湖周边地区活禽市场分离到的48株NDV(34株为鸡源病毒,4株鸭源病毒,1株鸽源病毒,9株分离自环境样本)测序及遗传进化分析,结果分为Class Ⅰ和Class Ⅱ两大类。其中Class Ⅰ毒株共41株,占样品分离率的3.66%,占总分离率的85.42%株,均为基因3型；Class Ⅱ毒株共7株,占样品分离率的0.62%,占总分离率的14.58%,包括基因Ⅰ型1株,基因Ⅱ型5株,基因Ⅵ型1株。据研究报道,Class Ⅰ这类弱毒株起初是来源于水禽,本文研究的Class Ⅰ毒株中的鸡源毒株占75.6%(31/41),鸭源毒株占2.4%(1/41),且均与野鸟毒株J36/13的同源性在97.4%以上,说明Class Ⅰ这类弱毒株存在水禽、家禽和野禽之间相互传播的可能。综合全文监测结果分析表明：环洞庭湖地区Class Ⅰ新城疫毒株在禽群中带毒率高,超过疫苗株和强毒株的带毒率；环境来源毒株多,存在一定的病毒污染扩散；主要流行毒株为Class Ⅰ基因3型,与野鸟毒株的遗传亲缘关系最近,分析可能来自同一毒株来源,所以,对洞庭湖区新城疫的病原监测及分子流行病学的研究工作还需高度重视。
[Abstract]:The high death rate of Newcastle disease (ND) is still a major harm to the breeding industry in the world. With the policy of vaccine immunization in developing countries, the vaccine is widely used and the epidemic of Newcastle disease is controlled to some extent, but the trend of the epidemic is changing: the change of the epidemic gene and the host range Not only expanded, and atypical Newcastle disease was becoming more and more common. The molecular epidemiology of Newcastle disease was studied, and its epidemic characteristics and local transmission of Newcastle disease virus were significant. In order to solve the infection distribution of Newcastle disease in the Dongting Lake area, this paper sampled the new town blight of the poultry farm in the Dongting Lake area for 2013-2014 years. 51 strains of Newcastle disease virus were isolated with chicken embryo isolation and serological identification, of which 34 strains of chicken, 2 ducks and 15 environment. 16 strains of F and HN genes were selected at different time, different locations and host. The F gene and HN gene fragments were amplified by R, and the amplified products were cloned in PMD18-T Easy Vector vector and were sequenced, and Gen The reference sequence published in bank was compared with the genetic evolution analysis. The results showed that 1 NDV isolates belonged to Class II type II, the amino acid sequence of the F protein cracking site was 112GRQGRL117, and the other 15 were Class I gene 3, the amino acid sequence of F protein cracking site was I12ERQERL117 and Class I was separated. The homology of F protein between 97.8% and more HN proteins was more than 98.2%, and the genetic relationship with the reference strain of Jilin wild bird virus strain J36/13. 48 strains of NDV (34 strains of chicken source virus, 4 duck source virus, 1 pigeon source virus, 9 isolated self environmental samples) and genetic evolution were isolated from the live bird market in the surrounding area of Dongting Lake. Analysis, the results are divided into Class I and Class II two categories, of which 41 strains of Class I, accounting for 3.66% of the sample separation rate, accounting for 85.42% of the total separation rate, are all gene 3, and 7 strains of Class II, accounting for 0.62% of the sample separation rate, accounting for 14.58% of the total separation rate, including gene type I 1, 5 gene type II and 1 gene type, according to the research report, The weak strain of Class I was originally from waterfowl. The chicken source in the Class I strain of this study accounted for 75.6% (31/41), the duck source was 2.4% (1/41), and the homology of the wild bird virus strain J36/13 was more than 97.4%. It showed that the weak strains of Class I existed in waterfowl, the possibility of mutual transmission between poultry and wild birds. Comprehensive full text monitoring The results showed that the virulent Class I strains of Newcastle disease in the Dongting Lake area were highly toxic in the fowl, exceeding the virulent rate of the vaccine strain and the strong virulent strain; there were many virulent strains of the environment source, and there was a certain virus contamination spread. The main epidemic strain was Class I gene 3, which was closely related to the relic relationship of the wild bird virus strain, and the analysis may come from the same poison. Therefore, the pathogen monitoring and molecular epidemiology of Newcastle disease in Dongting Lake area should be highly regarded.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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