两株北京鸭源呼肠孤病毒生物学特性比较

发布时间：2018-03-20 08:51

本文选题：北京鸭脾坏死病　切入点：鸭呼肠孤病毒　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：北京鸭“脾坏死病”是由鸭呼肠孤病毒(Duck reovirus, DRV)引起的一种病毒性疾病,以往该病所引起的死亡率较低,但在2013年,河南某鸭场所饲养的北京鸭发生该病,导致两批雏鸭死亡60%左右。因此,本研究旨在对可能出现的DRV变异株进行分子检测、分离与鉴定,进而对病毒分离株的生物学特性进行研究。用DRV特异性RT-PCR进行检测,从7份病死鸭脾脏样品均可检出DRV的μB、σB和σC蛋白编码基因。经病毒分离、培养和空斑纯化,获得两株DRV克隆株(HN5d株和HN5c株)。对RT-PCR扩增产物进行测序和序列分析的结果显示,相对于以往所测DRV 091株,HN5d株的σC编码区存在54 bp的连续缺失。用两株病毒感染1日龄北京鸭,可导致20-40%的雏鸭死亡,并可复制出鸭“脾坏死病”的脾脏坏死病变,同时,还可引起肝脏出血和坏死病变。组织病理学检查结果进一步证实实验感染鸭的肝脏和脾脏病变。用免疫组化试验从实验感染鸭的肝脏和脾脏检出大量棕黄色阳性信号,用扩增DRV S1基因的RT-PCR从死亡鸭的各组织样品和存活鸭的肝脏和脾脏样品中均易检测到DRV核酸。结果表明,相对于以往的DRV 091株,HN5c株和HN5d株对雏鸭具有较高的致病性,属于新的变异株。HN5c株感染引起的死亡率略高于HN5d株,表现出更高的致病性,但二者所引起的死亡率之间并无显著性差异。在上述工作基础上,观察了两株病毒的分子特征及σC基因缺失对病毒生物学特性的影响。SDS-PAGE结果显示,两株病毒的基因组均含10个基因片段。基因组测序和序列分析结果显示,HN5c株和HN5d株与以往所测北京鸭源DRV 091株具有相近的遗传演化关系,均属水禽呼肠孤病毒的基因2型。基因组分析结果进一步证实HN5d株的σC蛋白存在18 aa的缺失。两株病毒在Vero细胞中均表现出良好的增殖性能,但相对而言,HN5d株感染可产生更高的滴度。交叉中和试验结果显示,HN5c株和HN5d株的抗原相关系数为56.2%。结果提示,HN5d株σC蛋白缺失的18 aa可能与病毒的复制能力有关,并影响到病毒的抗原性。用肽扫描法进行鉴定的结果表明,该18 aa序列至少含有1个3细胞线性表位,其缺失是导致两株病毒出现抗原性差异的重要原因。用荧光定量RT-PCR% 和 Western blotting评估了感染细胞内σC蛋白的表达量。结果显示,HN5d株σC蛋白编码基因的相对核酸表达量以及aC蛋白表达量均高于HN5c株。荧光／免疫荧光和免疫印迹结果显示,用HN5c株和HN5d株的aC基因真核表达质粒转染Vero细胞后,其σc蛋白均获得表达；在激光共聚焦荧光显微镜下观察,可见σc蛋白表达于细胞浆中。用Annexin V-FITC/PI细胞凋亡检测试剂盒对病毒感染和I真核质粒转染的Vero细胞进行了检测,结果表明,两株DRV及其σC蛋白均可诱导细胞凋亡。在病毒感染的细胞内,HN5d株诱导细胞凋亡的能力显著强于HN5c株(P0.05)；在真核质粒转染的细胞内,两株病毒的σC蛋白所诱导的细胞凋亡无显著差异(P0.05)。结果表明,HN5d株的σC蛋白的18 aa缺失对DRV及其σC蛋白所诱导的细胞凋亡程度无显著影响。综上所述,本研究从分子特征和生物学特性角度揭示了北京鸭源呼肠孤病毒的毒株多样性,并证实所分离的DRV及其σC蛋白均可诱导Vero细胞凋亡。本研究有助于理解北京鸭“脾坏死病”及其病原特征,亦为鸭呼肠孤病毒的致病机理提供了数据。
[Abstract]:Beijing duck spleen necrosis disease "by duck reovirus (Duck reovirus DRV) is a viral disease caused by the past, the disease caused by the low mortality, but in 2013, the occurrence of the disease in Beijing Henan duck duck rearing sites, resulting in two batches of about 60%. So the death of ducklings. The purpose of this study is to conduct molecular detection of possible DRV mutation, isolation and identification, and study the biological characteristics of the virus isolates were detected by DRV. The specificity of RT-PCR detection, B DRV from 7 dead duck spleen samples can be B and sigma Sigma C protein encoding gene by virus isolation. Culture, and plaque purification, to obtain two DRV clones (HN5d strain and HN5c strain). The display of the RT-PCR amplified products were sequencing and sequence analysis results, compared with the previous measured DRV 091 strains, 54 continuous deletion of BP encoding region of sigma C HN5d strain. By two strains of virus infection 1 days Beijing duck, 20-40% can lead to the death of ducklings, and can replicate the duck spleen necrosis of spleen necrosis lesions, at the same time, can also cause liver necrosis and hemorrhage lesions. The histopathological findings further confirmed experimentally infected duck liver and spleen disease. Immunohistochemical test detected a large number of brown positive signal from the experimental infection of duck liver and spleen, were easily detected DRV nucleic acid of each tissue sample from the dead ducks with amplification of DRV S1 gene of RT-PCR and survival of duck liver and spleen samples. The results show that, compared with the previous DRV 091 strains of pathogenic HN5c strain and HN5d strain of duck is high, belong to the new the mutant strain.HN5c infection induced mortality was slightly higher than that of HN5d strain showed a higher pathogenicity, but not between the two caused by the mortality significantly. On the basis of the above work, the molecular observation of two strains The characteristics of C gene deletion and sigma effect on virus biological characteristics of.SDS-PAGE showed that two strains of the virus genome contains 10 genes. The genome sequencing and sequence analysis showed that HN5c and HN5d strains with the previous measured Beijing duck DRV 091 strains with similar genetic evolutionary relationship as Waterfowl reovirus genotype 2. Genome analysis results further confirmed the lack of HN5d strain C protein 18 sigma AA. Two strains of virus in Vero cells showed a proliferation of good performance, but relatively speaking, HN5d strains can produce higher titers. Cross and test results showed that HN5c strain and HN5d strain the correlation coefficient of 56.2%. antigen showed that HN5d strain C protein deletion of 18 sigma AA may be associated with virus replication, and affect the antigenicity identification by peptide scanning method. The results show that the aa sequence containing at least 18 1 3 cell linear epitopes, its deficiency is an important cause of the emergence of antigenic differences between two strains of the virus. Using fluorescence quantitative RT-PCR% and Western blotting were evaluated expression of sigma C protein in infected cells. The results showed that the amount and the amount of protein expression of aC was higher than that of HN5c strain HN5d strain. C relative nucleic acid protein encoding gene expression. Fluorescence and immunofluorescence and Western blot results showed that HN5c strain and HN5d strain aC gene eukaryotic expression plasmid was transfected into Vero cells, the expression of sigma C protein were obtained; in the observation of laser confocal fluorescence microscope, a visible expression of C protein in the cytoplasm. The virus infection and I plasmid the grain of Vero cells transfected with V-FITC/PI cell apoptosis by Annexin assay kit were detected, the results show that the two strains of DRV and sigma C protein could induce apoptosis in virus infected cells, HN5d strain can induce apoptosis The force was stronger than that of HN5c strain (P0.05); in eukaryotic plasmid transfected cells, no significant difference in apoptosis induced by two strains of the virus. C protein (P0.05). The results showed that no significant influence of HN5d cell apoptosis. C protein 18 AA deletion induced by DRV and C. Protein. In summary, this study from the perspective of molecular characteristics and biological characteristics reveal that the Beijing duck reovirus strains diversity, and confirmed by the separation of DRV and sigma C proteins can induce apoptosis in Vero cells. This study is helpful to understand the characteristics of the original "Beijing duck spleen necrosis disease and disease, provide the data for the pathogenic mechanism of duck reovirus.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.32

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