毛皮動物犬瘟熱的流行情況及一株犬瘟熱病毒的生物學(xué)特性研究
發(fā)布時間：2018-10-20 15:23
【摘要】：犬瘟熱(Canine distemper,CD)是由犬瘟熱病毒(Canine distemper virus,CDV)引起的一種以發(fā)熱、胃腸炎、過度角質(zhì)化、神經(jīng)癥狀等為特征的高度接觸性傳染病。2013年8月-2014年8月,山東省部分地區(qū)毛皮動物養(yǎng)殖場暴發(fā)了CD,給養(yǎng)殖戶帶來了巨大的經(jīng)濟損失。為了解山東省部分地區(qū)毛皮動物犬瘟熱流行情況,揭示CDV的流行和遺傳變異規(guī)律,為預(yù)防和控制CD提供更全面的理論依據(jù),本研究進行了以下幾方面的工作:1.對山東省諸城、青島、文登、榮成、臨沂、臨朐、煙臺、聊城、泰安、萊蕪等10個城市的25個規(guī)�；游镳B(yǎng)殖場送檢的665份臨床病例進行統(tǒng)計并分析CD在季節(jié)分布上的差異、與其他細菌混合感染情況及諸城市100份出欄水貂血清犬瘟熱抗體(CDV-Ab)情況。結(jié)果顯示,犬瘟熱陽性檢出率為22.6%(150/665)。其中,犬瘟熱單獨感染陽性率為46.7%(70/150),犬瘟熱與細菌性疾病混合感染占陽性病例的53.3%(80/150),尤其是與綠膿桿菌混合感染率高達31.3%(25/80);對諸城市出欄水貂血清犬瘟熱抗體檢測結(jié)果發(fā)現(xiàn)犬瘟熱抗體平均陽性率為35%,且免疫次數(shù)的多少與犬瘟熱抗體陽性率呈正相關(guān)。2.對篩選出的6株CDV陽性樣本的血凝素基因(HA)進行了全基因序列測定及遺傳變異分析。序列分析結(jié)果表明,6個山東分離株HA基因的閱讀框全長均為1824bp,編碼607個氨基酸(aa),未發(fā)現(xiàn)堿基的插入和缺失。分離株間核苷酸(98.7%-99.5%)及氨基酸(97.9%-99.2%)同源性較高,而與CDV疫苗株間核苷酸(89.8%-90.6%)和氨基酸(88.2%-90.4%)同源性均較低。6個山東CDV分離株與分離自遼寧省的水貂株LN(13)2和SD(12)1以及貉源株SD(13)1共同歸屬于亞洲-1(Asia-1)型,而與CDV疫苗株遺傳距離較遠。其中,SD(13)9株含有10個潛在N-連接糖基化位點(N-X-S/T),其他5個山東分離株均有9個且456-458位N-X-S/T缺失,而疫苗株含有4個或7個,6個山東分離株均在542-544位出現(xiàn)一個新的N-X-S/T。6株山東CDV分離株HA蛋白530位氨基酸位點均為甘氨酸(G),549位氨基酸位點出現(xiàn)酪氨酸(Y)到組氨酸(H)的替換。3.通過細胞傳代分離得到一株適應(yīng)VERO細胞的CDV毒株ZC株,并對其進行了全基因序列測定與分析。序列分析結(jié)果表明,CDV-ZC株全基因組共包含15690nt,GenBank登錄號為KJ994343,該基因組推導(dǎo)的某些氨基酸位點發(fā)生了突變,CDV-ZC株與其他毒株的核苷酸同源性為91.6%-97.8%,與CDV PS株同源性高達97.8%;與Onderstepoort株同源性最低(91.6%)�；贖A基因構(gòu)建的系統(tǒng)發(fā)生樹與全基因分析結(jié)果一致,基于全基因序列繪制的系統(tǒng)發(fā)生樹表明,所有CDV分離株分為疫苗株和野毒株2支,CDV-ZC株和PS株位于同一支且親緣關(guān)系最近,歸屬于Asia-1型。HA基因的限制性片段多態(tài)性分析(RFLP)表明CDV-ZC株為野毒株。微量中和實驗結(jié)果表明,CDV-ZC株與疫苗株存在抗原表位差異。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.92
本文編號：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.92
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