犬瘟热病毒对毛皮动物致病性及变异株致病性增强机制的初步研究

发布时间：2018-09-18 18:44

【摘要】：犬瘟热病毒(Canine distemper virus,CDV)感染引起的犬瘟热(Canine distemper,CD)是一种多种属动物共患传染病,不同动物对CDV易感性和致死率不同。因毛皮动物(貉、狐和水貂)是CDV自然易感宿主,该病一直是我国毛皮动物养殖业的“头号杀手”。H蛋白为病毒主要保护性抗原蛋白,除在介导病毒毒力、SLAM/Nectin-4受体识别和辅助F蛋白诱导细胞膜融合中发挥重要作用外,编码基因的高变异性也使其成为CDV基因分型及遗传变异研究的主要靶基因。为建立用于病毒动物致病性研究及疫苗免疫效力评价的CDV毛皮动物致病模型,本研究用我国CDV主要流行基因型(Asia-1型)LN(10)1株实验感染3组狐狸、貉和水貂(每组5只)。通过设置评价毒力和免疫抑制指标的赋分系统,建立了CDV感染毛皮动物的致病模型。试验结果表明CDV对3种动物致病性(毒力和免疫抑制)差异显著(P0.05):感染貉出现明显发热、结膜炎等典型CD症状和病理变化并于15 dpi内100%(5/5)死亡,获得高致病分数(2.6和2.9);狐狸表现出相对温和的CD症状并于21 dpi内40%(2/5)死亡,获得中等致病分数(2.15和2.25);而水貂仅显示温和CD症状并于21 dpi内全部存活,获得低致病分数(0.9和1.95)。此外,死亡和存活动物IFN-γ和IL-4在mRNA转录水平上分别表现下调和上调;发病动物TNF-ɑ转录水平上调,表明CDV对动物致病性差异与细胞因子变化具有相关性。为阐明近年来我国毛皮动物CD频繁暴发的原因,本研究对2011-2013年来自我国山东、河北、辽宁和黑龙江省16份水貂、狐狸和貉源CDV-H基因序列分析表明:与2012年之前的CDV流行株不同,10株来自山东省的CDV-H蛋白与SLAM受体的结合区(RBS)发生了I542N和Y549H突变,在系统进化树上处于Asia-1基因型独立遗传分支,我们将其命名为CDV变异株(HI542N/Y549H)。三维结构分析表明Y549H突变改变了H蛋白与水貂SLAM(mSLAM)的分子间作用力,I542N突变导致H蛋白542-544位产生了一个潜在N-连接糖基化位点。通过对CDV-H基因群体进化分析显示变异株2006年起源于我国毛皮动物,Y549H突变在H基因进化过程中受到选择压力影响。该突变株暴发可能预示着CDV在宿主或免疫压力下对毛皮动物适应性增强。为评价CDV变异株对毛皮动物致病性和疫苗免疫保护效果,基于我们建立的CDV动物致病模型,本研究对CDV变异株SD(14)7和经典株LN10(1)1进行动物感染试验。实验结果表明CDV变异株对毛皮动物(尤其水貂)毒力(发热、死亡率和病理变化等)和诱导免疫抑制(淋巴细胞减少症、抑制中和抗体和拮抗细胞因子表达等)水平有所增强,但当前疫苗能提供免疫动物对该变异株的攻毒保护。我们通过对2株CDV-H基因542和549位氨基酸人工突变和回复突变、CDV-H蛋白与mSLAM免疫共沉淀及细胞融合试验,在H蛋白表达、受体亲和力和细胞感染水平来探究氨基酸突变对病毒生物学特性的影响。研究结果表明:(1)I542N突变未使H蛋白在该位点被糖基化修饰;(2)I542N和Y549H突变降低了H蛋白表达水平但增强了H蛋白与mSLAM相互作用。(3)I542N和Y549H突变未增强CDV-H/F蛋白对表达mSLAM细胞的融合能力。综上研究表明,近年来我国毛皮动物CD频繁暴发可能是由于强毒力CDV变异株的出现,初步揭示了CDV变异株对毛皮动物(尤其水貂)致病性增强的分子机制。
[Abstract]:Canine distemper (CD) caused by Canine distemper virus (CDV) infection is a common infectious disease of many animals. Different animals have different susceptibility and mortality to CDV. Because fur animals (mink, Fox and mink) are the natural susceptible hosts of CDV, the disease has been the "top killer" of fur animal breeding industry in China. Proteins are the main protective antigen proteins of viruses, which play an important role in mediating virulence, SLAM/Nectin-4 receptor recognition and assistant F protein-induced cell membrane fusion. The high variability of the coding genes also makes them the main target genes for CDV genotyping and genetic variation research. In this study, three groups of foxes, minks and minks (5 in each group) were experimentally infected with LN (10), a major epidemic genotype of CDV (Asia-1), in China. The pathogenic model of CDV infection in fur animals was established by setting up a scoring system for evaluating virulence and immunosuppressive index. Pathogenicity (virulence and immunosuppression) of the infected raccoons were significantly different (P 0.05). Typical CD symptoms and pathological changes such as conjunctivitis occurred in infected raccoons, and 100% (5/5) died within 15 dpi, resulting in high pathogenic fraction (2.6 and 2.9); foxes showed relatively mild CD symptoms and died within 21 DPI in 40% (2/5) with moderate pathogenic fraction (2.15 and 2.25). In addition, IFN-gamma and IL-4 were down-regulated and up-regulated at the mRNA transcription level in dead and surviving animals, respectively. TNF-_transcription level was up-regulated in the pathogenic animals, indicating that there was a correlation between CDV pathogenicity and cytokine changes. In this study, the CDV-H gene sequences of 16 minks, foxes and minks from Shandong, Hebei, Liaoning and Heilongjiang provinces in China from 2011 to 2013 were analyzed. Compared with the prevalent strains of CDV before 2012, 10 strains of CDV-H protein from Shandong Province and the binding region (RBS) of SLAM receptor occurred in I542N. Y549H and Y549H mutations, which are independent of the genotype of Asia-1 in phylogenetic trees, are named CDV variants (HI542N/Y549H). Three-dimensional structural analysis shows that Y549H mutation alters the intermolecular interaction between H protein and mink SLAM (mSLAM), and I542N mutation results in a potential N-linked glycosylation site at 542-544 of H protein. Population evolution analysis of CDV-H gene revealed that the mutant strain originated from fur animals in China in 2006. The Y549H mutation was affected by selection pressure during the evolution of H gene. The outbreak of the mutant strain may indicate that the CDV is more adaptable to fur animals under host or immune pressure. Based on the animal pathogenic model of CDV established by us, the animal infections of CDV variants SD (14) 7 and classical strain LN10 (1) 1 were tested. The results showed that the CDV variants were toxic (fever, mortality and pathological changes, etc.) to fur animals (especially minks) and induced immunosuppression (lymphopenia, suppression of neutralizing antibodies). The level of CDV-H gene 542 and 549 amino acid mutation, CDV-H protein and mSLAM immunoprecipitation and cell fusion test, protein expression in H, receptor affinity and cell infection were carried out. The results showed that: (1) I542N mutation did not cause H protein to be glycosylated at this site; (2) I542N and Y549H mutations decreased H protein expression but enhanced the interaction between H protein and mSLAM. (3) I542N and Y549H mutations did not enhance the effect of CDV-H/F protein on mSLAM-expressing cells. In conclusion, the frequent outbreaks of CDs in fur animals in China in recent years may be due to the emergence of highly virulent CDV mutants, which preliminarily reveals the molecular mechanism of increased pathogenicity of CDV mutants to fur animals, especially minks.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.92

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