毛皮动物犬瘟热的流行情况及一株犬瘟热病毒的生物学特性研究
发布时间：2018-10-20 15:23
【摘要】：犬瘟热(Canine distemper,CD)是由犬瘟热病毒(Canine distemper virus,CDV)引起的一种以发热、胃肠炎、过度角质化、神经症状等为特征的高度接触性传染病。2013年8月-2014年8月,山东省部分地区毛皮动物养殖场暴发了CD,给养殖户带来了巨大的经济损失。为了解山东省部分地区毛皮动物犬瘟热流行情况,揭示CDV的流行和遗传变异规律,为预防和控制CD提供更全面的理论依据,本研究进行了以下几方面的工作:1.对山东省诸城、青岛、文登、荣成、临沂、临朐、烟台、聊城、泰安、莱芜等10个城市的25个规模化毛皮动物养殖场送检的665份临床病例进行统计并分析CD在季节分布上的差异、与其他细菌混合感染情况及诸城市100份出栏水貂血清犬瘟热抗体(CDV-Ab)情况。结果显示,犬瘟热阳性检出率为22.6%(150/665)。其中,犬瘟热单独感染阳性率为46.7%(70/150),犬瘟热与细菌性疾病混合感染占阳性病例的53.3%(80/150),尤其是与绿脓杆菌混合感染率高达31.3%(25/80);对诸城市出栏水貂血清犬瘟热抗体检测结果发现犬瘟热抗体平均阳性率为35%,且免疫次数的多少与犬瘟热抗体阳性率呈正相关。2.对筛选出的6株CDV阳性样本的血凝素基因(HA)进行了全基因序列测定及遗传变异分析。序列分析结果表明,6个山东分离株HA基因的阅读框全长均为1824bp,编码607个氨基酸(aa),未发现碱基的插入和缺失。分离株间核苷酸(98.7%-99.5%)及氨基酸(97.9%-99.2%)同源性较高,而与CDV疫苗株间核苷酸(89.8%-90.6%)和氨基酸(88.2%-90.4%)同源性均较低。6个山东CDV分离株与分离自辽宁省的水貂株LN(13)2和SD(12)1以及貉源株SD(13)1共同归属于亚洲-1(Asia-1)型,而与CDV疫苗株遗传距离较远。其中,SD(13)9株含有10个潜在N-连接糖基化位点(N-X-S/T),其他5个山东分离株均有9个且456-458位N-X-S/T缺失,而疫苗株含有4个或7个,6个山东分离株均在542-544位出现一个新的N-X-S/T。6株山东CDV分离株HA蛋白530位氨基酸位点均为甘氨酸(G),549位氨基酸位点出现酪氨酸(Y)到组氨酸(H)的替换。3.通过细胞传代分离得到一株适应VERO细胞的CDV毒株ZC株,并对其进行了全基因序列测定与分析。序列分析结果表明,CDV-ZC株全基因组共包含15690nt,GenBank登录号为KJ994343,该基因组推导的某些氨基酸位点发生了突变,CDV-ZC株与其他毒株的核苷酸同源性为91.6%-97.8%,与CDV PS株同源性高达97.8%;与Onderstepoort株同源性最低(91.6%)。基于HA基因构建的系统发生树与全基因分析结果一致,基于全基因序列绘制的系统发生树表明,所有CDV分离株分为疫苗株和野毒株2支,CDV-ZC株和PS株位于同一支且亲缘关系最近,归属于Asia-1型。HA基因的限制性片段多态性分析(RFLP)表明CDV-ZC株为野毒株。微量中和实验结果表明,CDV-ZC株与疫苗株存在抗原表位差异。
[Abstract]:Canine distemper (Canine distemper,CD) is a highly contagious disease caused by canine distemper virus (Canine distemper virus,CDV) characterized by fever, gastroenteritis, hyperkeratosis, neurological symptoms, etc. The outbreak of CD, in fur animal farms in some areas of Shandong Province has brought huge economic losses to farmers. In order to understand the prevalence of canine distemper in fur animals in Shandong province, reveal the prevalence and genetic variation of CDV, and provide a more comprehensive theoretical basis for the prevention and control of CD, this study has carried out the following work: 1. For Zhucheng, Qingdao, Wendeng, Rongcheng, Linyi, Linqu, Yantai, Liaocheng, Tai'an, A total of 665 clinical cases were collected from 25 large-scale fur animal farms in 10 cities, including Laiwu, and the differences in seasonal distribution of CD were analyzed. Mixed with other bacteria and 100 mink serums in Zhucheng city, canine distemper antibody (CDV-Ab). The positive rate of canine distemper was 22.6% (150 / 665). Of which, The positive rate of canine distemper infection alone was 46.7% (70 / 150). The mixed infection rate of canine distemper and bacterial disease was 53.3% (80 / 150), especially with Pseudomonas aeruginosa as high as 31.3% (25 / 80). The average positive rate of plague antibody was 35%, and the number of times of immunization was positively correlated with the positive rate of canine distemper antibody. 2. 2. The total gene sequence and genetic variation of hemagglutinin gene (HA) in 6 CDV positive samples were analyzed. The results of sequence analysis showed that the reading frame of HA gene of 6 Shandong isolates was 1824 BP, encoding 607 amino acid (aa), no insertion or deletion of the base was found. The homology of nucleotides (98.7- 99.5%) and amino acids (97.9- 99.2%) between isolates was higher. The nucleotides (89.8-90.6%) and amino acids (88.2- 90.4%) of CDV vaccine strains were lower. The six Shandong CDV isolates, mink strains LN (13) 2 and SD (12) 1 isolated from Liaoning Province and raccoon dog source strain SD (13) 1 belonged to Asia-1 (Asia-1) type, but had a long genetic distance with CDV vaccine strain. Among them, 9 strains of, SD (13 contained 10 potential N-linked glycosylation sites (N-X-S/T), 9 of the other 5 Shandong isolates had N-X-S/T deletion at 456-458 positions. However, there were 4 or 7 vaccine strains, and a new amino acid locus of HA protein of Shandong CDV isolate was found at 542-544. The amino acid locus of HA protein of Shandong CDV isolate was the amino acid site of glycine (G), 549 and the substitution of tyrosine (Y) to histidine (H). A CDV strain ZC adapted to VERO cells was isolated by cell passage, and the whole gene sequence was determined and analyzed. The results of sequence analysis showed that the whole genome of the CDV-ZC strain contained 15690ntTX GenBank accession number. Some amino acid sites derived from the genome were mutated. The nucleotide homology of CDV-ZC strain and other strains was 91.6-97.8, which was 97.8% with CDV PS strain, and the lowest homology with Onderstepoort strain (91.6%). The phylogenetic tree constructed based on HA gene was consistent with that of the whole gene analysis. The phylogenetic tree based on the whole gene sequence showed that all CDV isolates were divided into two strains: vaccine strain and wild strain. CDV-ZC strain and PS strain were located in the same branch and had the closest genetic relationship. The restriction fragment polymorphism (RFLP) analysis of HA gene belonging to Asia-1 type showed that CDV-ZC strain was a wild strain. The results of microneutralization test showed that there were antigenic epitope differences between CDV-ZC strain and vaccine strain.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
本文编号：2283561
[Abstract]:Canine distemper (Canine distemper,CD) is a highly contagious disease caused by canine distemper virus (Canine distemper virus,CDV) characterized by fever, gastroenteritis, hyperkeratosis, neurological symptoms, etc. The outbreak of CD, in fur animal farms in some areas of Shandong Province has brought huge economic losses to farmers. In order to understand the prevalence of canine distemper in fur animals in Shandong province, reveal the prevalence and genetic variation of CDV, and provide a more comprehensive theoretical basis for the prevention and control of CD, this study has carried out the following work: 1. For Zhucheng, Qingdao, Wendeng, Rongcheng, Linyi, Linqu, Yantai, Liaocheng, Tai'an, A total of 665 clinical cases were collected from 25 large-scale fur animal farms in 10 cities, including Laiwu, and the differences in seasonal distribution of CD were analyzed. Mixed with other bacteria and 100 mink serums in Zhucheng city, canine distemper antibody (CDV-Ab). The positive rate of canine distemper was 22.6% (150 / 665). Of which, The positive rate of canine distemper infection alone was 46.7% (70 / 150). The mixed infection rate of canine distemper and bacterial disease was 53.3% (80 / 150), especially with Pseudomonas aeruginosa as high as 31.3% (25 / 80). The average positive rate of plague antibody was 35%, and the number of times of immunization was positively correlated with the positive rate of canine distemper antibody. 2. 2. The total gene sequence and genetic variation of hemagglutinin gene (HA) in 6 CDV positive samples were analyzed. The results of sequence analysis showed that the reading frame of HA gene of 6 Shandong isolates was 1824 BP, encoding 607 amino acid (aa), no insertion or deletion of the base was found. The homology of nucleotides (98.7- 99.5%) and amino acids (97.9- 99.2%) between isolates was higher. The nucleotides (89.8-90.6%) and amino acids (88.2- 90.4%) of CDV vaccine strains were lower. The six Shandong CDV isolates, mink strains LN (13) 2 and SD (12) 1 isolated from Liaoning Province and raccoon dog source strain SD (13) 1 belonged to Asia-1 (Asia-1) type, but had a long genetic distance with CDV vaccine strain. Among them, 9 strains of, SD (13 contained 10 potential N-linked glycosylation sites (N-X-S/T), 9 of the other 5 Shandong isolates had N-X-S/T deletion at 456-458 positions. However, there were 4 or 7 vaccine strains, and a new amino acid locus of HA protein of Shandong CDV isolate was found at 542-544. The amino acid locus of HA protein of Shandong CDV isolate was the amino acid site of glycine (G), 549 and the substitution of tyrosine (Y) to histidine (H). A CDV strain ZC adapted to VERO cells was isolated by cell passage, and the whole gene sequence was determined and analyzed. The results of sequence analysis showed that the whole genome of the CDV-ZC strain contained 15690ntTX GenBank accession number. Some amino acid sites derived from the genome were mutated. The nucleotide homology of CDV-ZC strain and other strains was 91.6-97.8, which was 97.8% with CDV PS strain, and the lowest homology with Onderstepoort strain (91.6%). The phylogenetic tree constructed based on HA gene was consistent with that of the whole gene analysis. The phylogenetic tree based on the whole gene sequence showed that all CDV isolates were divided into two strains: vaccine strain and wild strain. CDV-ZC strain and PS strain were located in the same branch and had the closest genetic relationship. The restriction fragment polymorphism (RFLP) analysis of HA gene belonging to Asia-1 type showed that CDV-ZC strain was a wild strain. The results of microneutralization test showed that there were antigenic epitope differences between CDV-ZC strain and vaccine strain.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
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