坦布苏病毒毒株间毒力差异的分子基础

发布时间：2018-04-04 09:24

本文选题：坦布苏病毒　切入点：雏鸭　出处：《中国农业大学》2017年博士论文

【摘要】：坦布苏病毒(Tembusu virus,TMUV)感染是危害养鸭业的新发传染病,对于我国养鸭业具有重要的经济意义。迄今为止,TMUV的分子致病机制尚不清楚。本研究以毒力存在差异的TMUV分离株为材料,对决定毒株间毒力差异的关键基因和位点进行分析和鉴定,以期为阐明TMUV的分子致病机制提供数据。2014年10月,两个3～4周龄麻鸭群发生了一种以瘫痪为特征的疾病。经RT-PCR检测、病毒分离、间接免疫荧光检测和动物试验,确定致病病原为TMUV。将第3代鸭胚分离株(GL株)经脑内途径接种2日龄北京鸭,可复制出与自然病例相似的症状,在接种后3～6d雏鸭死亡率达100%。结果表明,该TMUV分离株对北京鸭雏鸭具有高致病性。为观察TMUV不同毒株的毒力差异,以2日龄北京鸭为动物模型,用PS株、Y株和GL株经脑内途径进行感染试验。结果显示,感染Y株和GL株后,雏鸭表现出严重的临床症状和组织病理变化,且死亡率分别为80%和70%。而PS株未导致雏鸭死亡,仅影响雏鸭增重,所引起的组织病理学变化也较轻微。由此可见,Y株和GL株对雏鸭的毒力显著高于PS株。基因组测序和序列比较结果显示,在非编码区,PS株分别与Y株和GL株存在3个和4个碱基差异;在聚蛋白氨基酸水平,PS株分别与Y株和GL株存在16个和19个氨基酸差异,结果提示,这些差异位点可能是决定毒株间毒力差异的分子基础。为鉴定TMUV毒力相关基因和位点,构建了PS株的反向遗传操作技术平台。用RT-PCR对PS株基因组进行了分段扩增,获得5个(称为A、B、C、D和E)基因片段,并由此构建重组质粒pBR322-AB和pBR322-BCDE。以这两个质粒为模板进行PCR扩增,并利用融合PCR方法获得PS株的全长cDNA。经体外转录,用纯化后的RNA转染BHK-21细胞,成功拯救出病毒。比较结果显示,拯救病毒在BHK-21细胞上的生长特性以及对小鼠的致病性均与亲本病毒相似,除遗传标记外,拯救病毒与亲本病毒序列一致。在上述工作基础上,以PS株基因组为骨架,分别构建了含Y株不同基因区(5'UTR、3'UTR、E 和 NS1-3'UTR)的嵌合毒株(rPSY5'UTR、rPSY3UTR、rPSYE和 rPSYNS1-3'UTR)。致病性试验结果显示,嵌合病毒rPSYE可使70%的雏鸭死亡,与Y株的毒力相近,由此可见,用Y株的E基因替换PS株的E基因,可显著提高PS株对雏鸭的致病性,提示E基因是决定TMUV PS株和Y株毒力差异的关键基因。运用定点突变技术,将PS株E蛋白304位精氨酸(R)突变为Y株E蛋白对应位置上的蛋氨酸(M),构建了突变病毒 rPSYER304M。致病性试验结果显示,用 rPSYER304M感染2日龄北京鸭,可引起60%的死亡率,与Y株所致死亡率相似,而亲本拯救毒株rPS感染雏鸭的死亡率仅为10%。结果表明,E蛋白304位氨基酸(R/M)是决定TMUV PS株和Y株毒力差异的关键位点。
[Abstract]:Tembusu virus Tembusu virus (TMUV) infection is a new infectious disease that endangers the duck industry and has important economic significance for the duck industry in China.Up to now, the molecular pathogenesis of TMUV is still unclear.In this study, TMUV isolates with different virulence were used as materials to analyze and identify the key genes and loci that determine virulence differences among strains, in order to provide data for elucidating the molecular pathogenicity of TMUV.A disease characterized by paralysis was found in two 3-and 4-week-old ducks.By RT-PCR detection, virus isolation, indirect immunofluorescence detection and animal test, the pathogenic agent was identified as TMU V.The third generation of duck embryo isolate strain GL) was inoculated into the brain of 2 day old Beijing duck. The symptoms similar to those of natural cases could be replicated. The death rate of ducklings reached 100 at 3 ~ 6 days after inoculation.The results showed that the TMUV isolate had high pathogenicity to Peking duck ducklings.In order to observe the virulence difference of different strains of TMUV, two day old Beijing ducks were used as animal models. The infection test was carried out by using PS strain Y strain and GL strain through brain pathway.The results showed that after infection with Y strain and GL strain, ducklings showed severe clinical symptoms and histopathological changes, and the mortality was 80% and 70%, respectively.PS strain did not cause the death of ducklings, but only affected the weight gain of ducklings, and the histopathological changes were slight.Therefore, the virulence of Y strain and GL strain to ducklings was significantly higher than that of PS strain.The results of genomic sequencing and sequence comparison showed that there were 3 and 4 base differences between PS strain and Y strain and GL strain in non-coding region, 16 and 19 amino acid differences between PS strain and Y strain and GL strain at polyprotein amino acid level, respectively.The results suggest that these differential sites may be the molecular basis for the virulence difference among strains.In order to identify the virulence related genes and loci of TMUV, a platform for reverse genetic manipulation of PS strain was constructed.The genome of PS strain was amplified by RT-PCR, and five fragments were obtained, and the recombinant plasmids pBR322-AB and pBR322-BCDE were constructed.The two plasmids were used as templates for PCR amplification, and the full-length cDNA of PS strain was obtained by fusion PCR method.After transcription in vitro and transfection of BHK-21 cells with purified RNA, the virus was successfully saved.The results showed that the growth characteristics and pathogenicity of the rescue virus on BHK-21 cells were similar to those of the parent virus, except for genetic markers, the sequence of the rescue virus was consistent with that of the parent virus.On the basis of the above work, the chimeric strains rPSY5UTR3UTRE and NS1-3UTRE were constructed with the genome of PS strain as the skeleton, and the chimeric strains rPSY5UTR3UTRYE and rPSYNS1-3UTRYE were constructed respectively.The results of pathogenicity test showed that the chimeric virus rPSYE could kill 70% of ducklings and the virulence of Y strain was similar to that of Y strain. Thus, the pathogenicity of PS strain could be significantly improved by replacing the E gene of PS strain with Y strain.It is suggested that E gene is the key gene to determine the virulence difference between TMUV PS strain and Y strain.Using site-directed mutagenesis technique, the mutant virus rPSYER304M was constructed by mutating E protein 304 arginine R) into methionine MN on the corresponding position of Y strain E protein.The results of pathogenicity test showed that the death rate of Beijing duck infected with rPSYER304M for 2 days was 60%, similar to that caused by Y strain, while the death rate of parent saving strain rPS infected ducklings was only 10%.The results showed that the amino acid R / M at position 304 of E protein was the key site to determine the virulence difference between TMUV PS strain and Y strain.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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