江苏地区猪嵴病毒感染流行病学调查和全基因组遗传进化分析

发布时间：2018-04-28 23:18

本文选题：猪嵴病毒 + RT-PCR　；参考：《扬州大学》2015年硕士论文

【摘要】：猪嵴病毒(Porcine Kobuvirus, PKVs)是小RNA病毒科(Picornaviridae)嵴病毒属(kobuvirus)的成员,于2008年首次发现。在世界多个国家的猪群中均发现了该病毒,越来越多的证据表明该病毒与猪腹泻疫情有关。猪腹泻病影响和危害我国养猪生产和养猪业发展,自2010年以来,我国大部分省份爆发了以水样腹泻、脱水、呕吐为特征的哺乳仔猪病毒性腹泻疫情,发病率和死亡率高,给养猪业带来巨大损失,这其中病毒性腹泻疫情引起养猪业的普遍关注。在腹泻猪群中检测到了猪嵴病毒,并且猪嵴病毒可能会导致仔猪腹泻。猪嵴病毒目前尚未实现体外分离,对该病毒的研究大多是对其分子流行病学的调查研究。在PKVs的基因结构中,3D基因猪嵴病毒最保守的基因,其编码的蛋白在病毒增殖的动物机体内和感染的细胞培养液中发现的一种非结构性的病毒特异性蛋白,被称为病毒感染相关蛋白。VP1基因是嵴病毒基因组中最具可变性的区域,编码的蛋白是衣壳蛋白中暴露最多的免疫显性蛋白,同小RNA病毒科其他病毒一样VP1基因编码的蛋白可引起宿主机体产生免疫反应,是嵴病毒的抗原位点,是微RNA病毒分属的重要依据。本研究建立了针对PKVs 3D基因和VP1基因的RT-PCR检测方法,对3D基因和VP1基因进行基因特征分析并对江苏地区猪嵴病毒进行全基因组测序分析,旨在通过对江苏地区猪群中猪嵴病毒的分布流行情况进行分子流行病学调查,了解该病毒在猪群中的感染状况,丰富猪嵴病毒的流行病学数据,为防治猪腹泻疫情提供一定的参考。1猪嵴病毒3D基因RT-PCR检测方法的建立根据猪嵴病毒(Porcine kobuvirus, PKVs) 3D基因保守序列设计一对特异性引物,建立了检测猪棉拭样本中的猪嵴病毒的RT-PCR方法,该方法能特异性的检测猪嵴病毒,而对其它病原如猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪A群轮状病毒(Group A porcine rotavirus, GARV)、猪瘟病毒(Classical swine fever virus)、猪呼吸与繁殖障碍综合征病毒(Porcine Re productive and Respiratory Syndrome virus, PRRSV)、猪圆环病毒(porcine circovirus, PCV)、猪大肠杆菌则不能检出,用该方法能检测的浓度为0.1263pg,灵敏度高,可用于对江苏地区猪群中猪嵴病毒的检测。2猪腹泻病原混合感染检测及猪嵴病毒3D基因和VP1基因特征分析用建立的猪嵴病毒3D基因RT-PCR检测方法对2012年8月至2013年12月从江苏13个地区46个猪场采集的396份猪腹泻肛拭样本进行检测。结果显示猪场阳性率和病料阳性率分别为80.4%和45.7%,表明猪嵴病毒广泛存在于江苏地区猪群中。随机选取19份病料进行猪嵴病毒3D基因分析。19个猪嵴病毒3D基因的核苷酸和氨基酸相似性分别为88.0%～100%和69.4%～100%,而与其它嵴病毒的核苷酸和氨基酸相似性分别为69.6%～78.8%和27.8%～56.9%。进化树分析显示不同地区的猪嵴病毒3D基因处于同一分支,说明江苏地区猪嵴病毒没有一定的地理限制。185份病料经检测发现含有2种及两种以上其它病原,其中有68份PKVs病料检测阳性的样本有31份检测到其它病原,说明在江苏地区猪嵴病毒与其它病原发生混合感染。使用设计的针对猪嵴病毒VP1基因的特异性引物,对2014年8月至2015年2月从江苏地区采集的91份腹泻仔猪棉拭样本和126份临床健康仔猪棉拭样本进行检测,腹泻仔猪猪嵴病毒感染率64.8%(59/91)；临床健康仔猪感染猪嵴病毒感染率为19.8%(25/126)。随机选取28个VP1基因测序分析显示,28个VP1基因的氨基酸同源性为80.3%～100%,核苷酸同源性为79.6%-100%。进化树分析显示,28个VP1基因分为四个群,其中腹泻猪感染的猪嵴病毒VP1基因分布在四个不同的群；所有临床健康猪群感染的猪嵴病毒VP1基因分布在同一个群并显示较近的基因同源性。结果表明江苏地区腹泻猪群中流行的猪嵴病毒表现一定的基因多样性,而临床健康猪群中则不然。这种差异可能与猪嵴病毒的潜在致病性有关。3猪嵴病毒全基因组的测序与基因遗传进化分析根据GenBank公布的PKVs全基因组序列,设计12对针对PKVs全基因组的特异性引物,对从江苏地区检测的2株PKVs进行全基因测序分析。JS-01-CHN检测自腹泻仔猪,除去poly(A)基因组全长为8214nt,开放阅读框(ORF)基因全长为7470nt,5'-UTR长度为576nt,与参考毒株比较发现JS-01-CHN的VP1基因编码的氨基酸第237位插入一个氨基酸,在3'-UTR基因的108位插入一个胸腺嘧啶。JS-02a-CHN来自临床健康仔猪,除去poly (A)基因组全长为8121nt,在其2B编码区缺失30个氨基酸,这是首次发现感染健康PKVs的2B区的氨基酸缺失,这可能是PKVs进化过程中变异的结果。序列分析发现,两株PKVs全基因序列与参考序列编码区氨基酸和核苷酸同源性分别为94.3%-96.4%和87.8%-89.3%,而与其它嵴病毒则为56.2%-65.1%和59.0%-66.1%。5'-UTR和3'-UTR与其它PKVs核苷酸同源性为95.1%-97.0%和89.1%-97.0%。进化树分析显示JS-02a-CHN和JS-01-CHN处于同一分支并和PKVs较近。对PKVs全基因组进行重组分析发现,JS-01-CHN存在潜在的重组事件,这可能是全基因组不同基因片段拼接导致的结果,为了解PKVs的重组特点,需要对更多的全基因序列进行测序分析。
[Abstract]:Porcine Kobuvirus (PKVs) is a member of the small RNA virus family (Picornaviridae), a member of the cristovirus (kobuvirus) genus (kobuvirus). It was first discovered in 2008. The virus was found in pigs in many countries of the world. More and more evidence shows that the virus is related to the epidemic of porcine diarrhea. Since 2010, most provinces of our country have outbreak of viral diarrhea of suckling piglets characterized by water sample diarrhea, dehydration and vomiting. The incidence and mortality rate are high, which bring huge losses to the pig industry. In this case, the virus diarrhea epidemic causes the swine industry to pay attention to the swine industry. Porcine crista virus may cause diarrhea in piglets. The swine crista virus has not yet been isolated in vitro. Most of the studies on the virus are investigated for its molecular epidemiology. In the gene structure of PKVs, the most conservative gene of the 3D gene porcine crista virus is encoded in the animal body and infected cell culture fluid of the virus. A non structural virus specific protein known as the virus infection related protein.VP1 gene is the most mutable region in the crista virus genome, the encoded protein is the most exposed immune dominant protein in the capsid protein, and the VP1 gene encoded by the protein of the other viruses of the small RNA family can cause the host organism to produce The immunoreaction, an antigen site of crest virus, is an important basis for the classification of RNA virus. In this study, a RT-PCR detection method for the PKVs 3D gene and VP1 gene was established. The genomic characteristics of the 3D and VP1 genes were analyzed and the whole genome sequencing of the swine crista virus in Jiangsu area was analyzed. The aim is to pass the pig cristae in the pigs in the Jiangsu region. The epidemic situation of the virus distribution is investigated by molecular epidemiology, to understand the infection status of the virus in the pigs, to enrich the epidemiological data of porcine crista virus, and to provide a reference for the prevention and control of porcine diarrhea in the epidemic situation of porcine crest virus (.1) 3D gene RT-PCR detection method based on the conservative sequence of the Porcine kobuvirus (PKVs) 3D gene. A pair of specific primers was designed to establish a RT-PCR method for detecting porcine crest virus in pig's cotton swab samples. This method can specifically detect porcine crista virus, while other pathogens such as Porcine epidemic diarrhea virus (PEDV), swine infectious gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), and A group of pigs Group A porcine rotavirus (GARV), swine fever virus (Classical swine fever virus), porcine respiratory and reproductive disorder syndrome virus (Porcine Re productive), porcine Escherichia coli, can not be detected. The method can detect the concentration of 0.126 3pg, with high sensitivity, it can be used to detect the swine crista virus in the pigs in Jiangsu area and to detect the mixed infection of.2 porcine diarrhea pathogen and the 3D gene and VP1 gene of porcine crista virus (crest virus). The 3D gene of swine crista virus (crest virus 3D) gene RT-PCR was used to collect 396 porcine anal swabs from 46 pig farms from August 2012 to December 2013. The results showed that the positive rate and positive rate of pig farm were 80.4% and 45.7% respectively, indicating that the swine crista virus was widely found in the pigs in Jiangsu area. The nucleotide and amino acid similarity of the 3D gene of the.19 crest virus 3D gene was divided into 88% to 100% and 69.4% to 100% in the random selection of the swine crista virus 3D gene. The nucleotide and amino acid similarity of crest virus was 69.6% to 78.8% and 27.8% ~ 56.9%. phylogenetic tree analysis showed that the 3D gene of swine crista virus in different regions was in the same branch. It showed that the swine crista virus in Jiangsu region had no geographical limitation and found that there were 2 species and more than two other pathogens, including 68 PKVs. 31 samples of the positive samples were detected, indicating that the swine crista virus was mixed with other pathogens in Jiangsu. Using specific primers designed for the porcine crest virus VP1 gene, 91 cotton swabs and 126 healthy piglets were collected from the Jiangsu region from August 2014 to February 2015. The infection rate of swine cristae virus was 64.8% (59/91), and the infection rate of swine crista virus infection was 19.8% (25/126) in healthy piglets. The sequence analysis of 28 VP1 genes showed that the amino acid homology of 28 VP1 genes was 80.3% to 100%. The nucleotide homology was 79.6%-100%. evolution tree analysis, 28 VP1 genes were divided into two genes. Four groups, among which the swine crista virus VP1 gene infected by diarrhea pigs was distributed in four different groups, and the VP1 gene of swine crista virus infected by all clinical healthy pigs was distributed in the same group and showed the homologous gene. The results showed that the swine cristae virus in the diarrhea pigs in Jiangsu region showed a certain genetic diversity, and the clinical health was healthy. This difference may be related to the potential pathogenicity of porcine crista virus, which is related to the sequencing of the whole genome of.3 swine crista virus and gene genetic evolution analysis based on the PKVs genome sequence published by GenBank, and designed 12 pairs of specific primers for the whole genome of PKVs, and complete the whole gene sequencing of 2 strains of PKVs detected from Jiangsu region. The total length of the poly (A) genome was 8214nt, the full length of the open reading frame (ORF) gene was 7470nt, the 5'-UTR length was 576nt, and the 237th bits of the JS-01-CHN's VP1 gene encoded amino acids were inserted into one amino acid, and a thymine.JS-02a-CHN from the 3'-UTR gene was inserted in the 108 bit of the 3'-UTR gene to insert a thymine.JS-02a-CHN from.JS-01-CHN. In clinical healthy piglets, the total length of the poly (A) genome is 8121nt, and 30 amino acids are missing in its 2B coding region. This is the first discovery of the amino acid deletion in the 2B region infected with healthy PKVs. This may be the result of the variation in the evolutionary process of PKVs. The sequence analysis found that the sequence of the two PKVs whole bases was homologous with the amino acids and nucleotides in the reference sequence coding region. The homology of 56.2%-65.1%, 59.0%-66.1%.5'-UTR and 3'-UTR with other PKVs nucleotides for 95.1%-97.0% and 89.1%-97.0%. evolution tree analysis showed that JS-02a-CHN and JS-01-CHN were in the same branch and were similar to PKVs. The recombinant analysis of the whole genome of PKVs was found to be 94.3%-96.4% and 87.8%-89.3%. There is a potential recombination event, which may be the result of the splicing of different genome segments in the whole genome. In order to solve the recombinant characteristics of PKVs, more whole gene sequences need to be sequenced.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

【相似文献】