辽宁省仔猪病毒性腹泻病的病原调查及PEDV的遗传变异分析
发布时间：2018-11-05 17:16
【摘要】：引起仔猪传染性腹泻病的病原主要有猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(RV),这三种病原的传染途径和发病过程相类似,具有发病急、传播速度快,哺乳仔猪感染率和死亡率都较高的特点,临床诊断和控制难度较大。近年来辽宁省哺乳仔猪传染性病毒性腹泻病发生较为严重,给养猪业造成了巨大的经济损失。为查明辽宁省仔猪病毒性腹泻病主要病原的种类,明确主要流行病原的分布情况,从而制定有针对性的防控措施,开展了辽宁省仔猪病毒性腹泻的病原调查及PEDV的遗传变异分析。1.仔猪病毒性腹泻的病原调查采用RT-PCR方法对2013年4月份至次年3月份辽宁省9个市采集的335份腹泻病料进行PEDV、TGEV、RV的检测,根据采集病料的时间和地点进行统计分析。结果显示阳性的病毒主要为PEDV和TGEV,其阳性率分别为38.2%和15.2%,二者在9个市均有分布。说明辽宁省仔猪病毒性腹泻的主要病原是PEDV,在辽宁省感染分布广泛,污染程度较高,并且全年持续存在。2.猪流行性腹泻LN-J毒株的分离鉴定临床调查发现已注射PEDV疫苗的猪场仍有本病发生,为查明辽宁地区PEDV流行毒株的来源及变异情况。采集辽宁某地区疑似PED腹泻仔猪的肠内容物,应用Vero细胞进行传代培养,分离到一株毒株,TCID50为10-4.30/0.1ml,通过电镜观察该病毒为130nm左右的球形颗粒;通过RT-PCR鉴定,该病毒为RNA病毒,而且核酸片段大小与所设计的PEDV引物相同,经过核酸序列分析证明为PEDV,暂命名为PEDV LN-J毒株。3.猪流行性腹泻LN-J毒株S基因的遗传变异分析由于PEDV S基因编码的表面纤突糖蛋白具有抗原表位,S基因的变异可能引起该病毒抗原性和毒力的变化,因此,选择S基因进行遗传变异分析。设计了2对扩增S全基因的引物S1和S2,进行RT-PCR扩增,成功获得了和目的片段大小相符的条带,并构建了pUM-S1和pUM-S2重组质粒,测序后通过生物软件进行分析、拼接,得到4161bp的全基因,利用DNAStar分析发现,PEDV LN-J毒株的S基因和其他许多地方毒株均有较高的同源性,构建系统进化树,发现该毒株与HB-2011-1、BJ-2001-2和CHGD-01亲缘关系较近。说明辽宁地区流行的PEDV属于输入性传播,但存在变异趋势。
[Abstract]:The main pathogens causing infectious diarrhea in piglets are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (RV),). The infection rate and mortality of suckling piglets are high, and the clinical diagnosis and control are difficult. In recent years, the infectious viral diarrhea of suckling piglets in Liaoning Province is more serious, which has caused huge economic losses to the pig industry. In order to find out the main pathogens of piglets viral diarrhea in Liaoning Province, and to determine the distribution of the main pathogens of the disease, and to formulate targeted prevention and control measures, The pathogen of viral diarrhea of piglets and the analysis of genetic variation of PEDV in Liaoning province were carried out. 1. RT-PCR method was used to detect 335 diarrhea samples collected from 9 cities in Liaoning Province from April 2013 to March of next year. The results were analyzed statistically according to the time and place of collection. The results showed that the positive rates of PEDV and TGEV, were 38.2% and 15.2% respectively. The results showed that the main pathogen of viral diarrhea of piglets in Liaoning Province was PEDV, which was widely distributed in Liaoning Province, and had a high degree of pollution. Isolation and Identification of Porcine epidemic diarrhea LN-J strain Clinical investigation showed that the disease still occurred in swine farm which had been injected with PEDV vaccine. In order to find out the source and variation of PEDV epidemic strain in Liaoning area. The intestinal contents of suspected PED diarrhea piglets in a certain area of Liaoning province were collected and subcultured with Vero cells. A virus strain with TCID50 of 10-4.30% 0.1 ml was isolated. The virus was observed to be a spherical particle about 130nm by electron microscope. The virus was identified as RNA virus by RT-PCR, and the nucleic acid fragment size was the same as that of the designed PEDV primer. The nucleic acid sequence analysis showed that the virus was tentatively named PEDV, as PEDV LN-J strain. Genetic variation Analysis of S Gene of Porcine epidemic diarrhea virus strain S the variation of S gene may cause the change of antigenicity and virulence of PEDV S gene because the surface fibrin encoded by PEDV S gene has antigen epitopes. S gene was selected for genetic variation analysis. Two pairs of primers S1 and S2 were designed to amplify the S gene, and the RT-PCR amplification was carried out, and the bands corresponding to the size of the target fragment were successfully obtained. The recombinant plasmids of pUM-S1 and pUM-S2 were constructed, and the recombinant plasmids were sequenced and analyzed by biological software. The whole gene of 4161bp was obtained. By DNAStar analysis, the S gene of PEDV LN-J strain was found to have high homology with many other local strains. A phylogenetic tree was constructed, and it was found that the S gene of PEDV LN-J strain was similar to that of HB-2011-1, strain. The relationship between BJ-2001-2 and CHGD-01 was close. The results showed that the prevalence of PEDV in Liaoning province belonged to imported transmission, but there was a trend of variation.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：2312751
[Abstract]:The main pathogens causing infectious diarrhea in piglets are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (RV),). The infection rate and mortality of suckling piglets are high, and the clinical diagnosis and control are difficult. In recent years, the infectious viral diarrhea of suckling piglets in Liaoning Province is more serious, which has caused huge economic losses to the pig industry. In order to find out the main pathogens of piglets viral diarrhea in Liaoning Province, and to determine the distribution of the main pathogens of the disease, and to formulate targeted prevention and control measures, The pathogen of viral diarrhea of piglets and the analysis of genetic variation of PEDV in Liaoning province were carried out. 1. RT-PCR method was used to detect 335 diarrhea samples collected from 9 cities in Liaoning Province from April 2013 to March of next year. The results were analyzed statistically according to the time and place of collection. The results showed that the positive rates of PEDV and TGEV, were 38.2% and 15.2% respectively. The results showed that the main pathogen of viral diarrhea of piglets in Liaoning Province was PEDV, which was widely distributed in Liaoning Province, and had a high degree of pollution. Isolation and Identification of Porcine epidemic diarrhea LN-J strain Clinical investigation showed that the disease still occurred in swine farm which had been injected with PEDV vaccine. In order to find out the source and variation of PEDV epidemic strain in Liaoning area. The intestinal contents of suspected PED diarrhea piglets in a certain area of Liaoning province were collected and subcultured with Vero cells. A virus strain with TCID50 of 10-4.30% 0.1 ml was isolated. The virus was observed to be a spherical particle about 130nm by electron microscope. The virus was identified as RNA virus by RT-PCR, and the nucleic acid fragment size was the same as that of the designed PEDV primer. The nucleic acid sequence analysis showed that the virus was tentatively named PEDV, as PEDV LN-J strain. Genetic variation Analysis of S Gene of Porcine epidemic diarrhea virus strain S the variation of S gene may cause the change of antigenicity and virulence of PEDV S gene because the surface fibrin encoded by PEDV S gene has antigen epitopes. S gene was selected for genetic variation analysis. Two pairs of primers S1 and S2 were designed to amplify the S gene, and the RT-PCR amplification was carried out, and the bands corresponding to the size of the target fragment were successfully obtained. The recombinant plasmids of pUM-S1 and pUM-S2 were constructed, and the recombinant plasmids were sequenced and analyzed by biological software. The whole gene of 4161bp was obtained. By DNAStar analysis, the S gene of PEDV LN-J strain was found to have high homology with many other local strains. A phylogenetic tree was constructed, and it was found that the S gene of PEDV LN-J strain was similar to that of HB-2011-1, strain. The relationship between BJ-2001-2 and CHGD-01 was close. The results showed that the prevalence of PEDV in Liaoning province belonged to imported transmission, but there was a trend of variation.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
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