猪繁殖与呼吸综合征病毒基因1型毒株GZ11-G1的致病性及其分子基础

发布时间：2018-05-15 23:01

本文选题：猪繁殖与呼吸综合征病毒 + 基因1型　；参考：《中国农业大学》2016年博士论文

【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)是世界范围内对养猪业危害最严重的传染病之一,病原为猪繁殖与呼吸综合征病毒(PRRSV)。根据基因组与抗原性的差异PRRSV可分为2个基因型,即基因1型和基因2型。近年来,基因1型PRRSV在我国的存在和临床感染受到关注。因此,开展基因1型PRRSV的相关研究十分必要。本研究以基因1型PRRSV毒株GZ11-G1为对象,分析其对仔猪的致病性,构建感染性cDNA克隆,并采用定点突变技术分析与其致病性相关的分子基础,以期为深入开展基因1型PRRSV的致病机制研究奠定必要的技术和前期基础。将基因1型PRRSV毒株GZ11-G1及基因1型疫苗毒株分别接种6周龄SPF猪,观察并记录各实验组感染猪的体温变化、临床症状、病毒血症、体外排毒情况、血清抗体产生动态、组织病变及免疫组化结果。试验结果显示,基因1型PRRSV毒株GZ11-G1能够引起仔猪呼吸道症状并伴随一过性体温升高,体内增殖能力显著高于疫苗毒株,并且能够引起严重的弥漫性间质性肺炎,表明基因1型PRRSV毒株GZ11-G1对仔猪具有致病性。根据基因1型PRRSV毒株GZ11-G1与疫苗毒株的全基因组序列,分段进行RT-PCR扩增,通过同义突变引入酶切位点,连入低拷贝质粒pWSK-29T,获得全长cDNA质粒pWSK-GZ11-G1及pWSK-Amervac。经对全长cDNA质粒线化、体外转录和转染细胞,进行病毒拯救和鉴定。结果表明,构建的基因1型PRRSV毒株GZ11-G1及疫苗毒株的全长cDNA克隆具有感染性,可拯救出病毒,分别命名为Rv-GZ11和Rv-Amervac,拯救病毒具有与亲本病毒相似的体外增殖特性。对GZ11-G1、Amervac PRRS疫苗毒株在内的10株基因1型PRRSV相关区域(5’及3'UTR、ORF1b、ORF2-6)的序列及编码氨基酸序列进行了比对分析。结果表明,GZ11-G1在Nsp9-10区域及GP2分别存在4个和3个氨基酸突变位点,包括Nsp993位甘氨酸(G)、Nsp10281位脯氨酸(P)、304位缬氨酸(V)、401位赖氨酸(K)、GP25位组氨酸(H)、120位甘氨酸(G)和252位丝氨酸(S)。利用感染性克隆技术及定点突变技术拯救出6株突变病毒,分别命名为Rv-A-G-Nsp9-10、 Rv-A-G-GP2+Nsp9-10、Rv-A-G-GP2、Rv-G-A-Nsp9-10、Rv-G-A-GP2和Rv-G-A-GP2+Nsp9-10。体外增殖结果表明,突变GP2的3个氨基酸后,各突变病毒与相应的亲本毒株增殖水平均无显著差异,而突变Nsp9-10的4个氨基酸后,Rv-A-G-Nsp9-10、Rv-A-G-GP2+Nsp9-10早期的病毒滴度均显著提高,而Rv-G-A-Nsp9-10、Rv-G-A-GP2+Nsp9-10病毒滴度达到峰值的时间点分别滞后12h和24h,各时间点的病毒滴度也显著低于亲本拯救毒株(P0.05)。对突变病毒的致病性分析结果表明,与Rv-Amervac相比,Rv-A-G-Nsp9-10感染猪的临床症状、病毒血症和间质性肺炎更为严重,而Rv-G-A-Nsp9-10较亲本病毒的病毒血症持续时间缩短,感染猪的体温反应和肺脏的病理损伤减轻。结果表明,基因1型PRRSV毒株GZ11-G1的Nsp9的第93位,Nsp10的第261位、304位和401位氨基酸与其增殖能力及致病性增强有关。综上所述,研究结果表明基因1型PRRSV毒株GZ11-G1具有致病性,成功构建了基因1型PRRSV感染性克隆技术,揭示了基因1型PRRSV毒株GZ11-G1 Nsp9和Nsp10中与病毒增殖能力和致病性相关的氨基酸位点,为进一步研究基因1型PRRSV分子致病机制提供了技术平台,并为阐明基因1型PRRSV的致病机制提供了有价值的科学依据。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious infectious diseases in the world. The pathogen is porcine reproductive and respiratory syndrome virus (PRRSV). According to the difference between genome and antigenicity, PRRSV can be divided into 2 genotypes, that is, gene 1 and gene 2. Because of the existence and clinical infection of type 1 PRRSV in China, it is necessary to carry out the related research of gene 1 type PRRSV. This study is based on the gene 1 type PRRSV strain GZ11-G1, to analyze its pathogenicity to piglets, to construct infectious cDNA cloning, and to analyze the molecular basis related to its pathogenicity by site directed mutagenesis. In order to develop the pathogenic mechanism of gene 1 type PRRSV, the necessary technology and preliminary basis were laid. The gene 1 PRRSV strain GZ11-G1 and gene 1 vaccine strains were inoculated respectively for 6 weeks old SPF pigs, and the temperature changes, clinical symptoms, viremia, detoxification in vitro, the dynamic and tissue of serum antibodies were recorded and recorded in the experimental groups. The results of pathological changes and immunohistochemical staining showed that the gene 1 PRRSV strain GZ11-G1 could cause respiratory symptoms and a hyperthermia in piglets, and the ability to proliferate in vivo was significantly higher than that of the vaccine strain, and could cause severe diffuse interstitial pneumonia, indicating that the gene 1 PRRSV strain, GZ11-G1, has a pathogenicity to piglets. The whole genome sequence of the gene 1 PRRSV strain GZ11-G1 and the vaccine strain was amplified by RT-PCR, and the whole length cDNA plasmid pWSK-GZ11-G1 and pWSK-Amervac. were obtained by introducing the synonymous mutation into the enzyme cut site and joining the low copy plasmid pWSK-29T. The whole length cDNA plasmid was linearized, and the transfected cells were transferred and transfected in vitro. The results of the virus rescue and identification were carried out. The results showed that the full-length cDNA clone of the gene 1 PRRSV strain GZ11-G1 and the vaccine strain was infectious, and could save the virus, named Rv-GZ11 and Rv-Amervac respectively. The rescue virus was similar to the parent virus in vitro proliferation characteristics. 10 gene 1 PRRSV related regions (5 and 3'UTR) for GZ11-G1 and Amervac PRRS vaccine strains (5 'and 3'UTR). The sequence and encoded amino acid sequences of ORF1b, ORF2-6) were compared and analyzed. The results showed that GZ11-G1 had 4 and 3 amino acid mutation sites in Nsp9-10 region and GP2, including Nsp993 site glycine (G), Nsp10281 proline (P), 304 - position valine (V), 401 - bit lysine (K), GP25 position histidine, 120 glycine and 252 silk ammonia Acid (S). Using infectious cloning technology and site directed mutation technology to save 6 mutant viruses, named Rv-A-G-Nsp9-10, Rv-A-G-GP2+Nsp9-10, Rv-A-G-GP2, Rv-G-A-Nsp9-10, Rv-G-A-GP2 and Rv-G-A-GP2+Nsp9-10. in vitro proliferation results showed that after 3 amino acids mutation of GP2, the proliferation level of each mutant virus and the corresponding parent strain were both. There was no significant difference, but after 4 amino acids mutation of Nsp9-10, the virus titer of early Rv-A-G-Nsp9-10 and Rv-A-G-GP2+Nsp9-10 increased significantly, while Rv-G-A-Nsp9-10, Rv-G-A-GP2+Nsp9-10 virus titer at the peak time lag 12h and 24h respectively, and the virus titer at each time point was significantly lower than that of the parent rescue strain (P0.05). The results of pathogenicity analysis showed that compared with Rv-Amervac, the clinical symptoms of Rv-A-G-Nsp9-10 infected pigs, viremia and interstitial pneumonia were more serious, while the duration of Rv-G-A-Nsp9-10 was shorter than that of the parent virus, and the temperature response of the infected pigs and the pathological damage of the lungs were reduced. The results showed that the N of the gene 1 PRRSV strain of GZ11-G1 was N. The ninety-third, 261st, 304 and 401 amino acids of SP9 are related to their proliferative ability and pathogenicity. To sum up, the results show that the gene 1 PRRSV strain GZ11-G1 has pathogenicity. The gene 1 PRRSV infection cloning technology has been successfully constructed, and the gene 1 PRRSV strain GZ11-G1 Nsp9 and Nsp10 and the virus proliferative energy are revealed. The amino acid sites associated with virulence and virulence provide a technical platform for further research on the pathogenesis of gene 1 PRRSV molecules, and provide a valuable scientific basis for elucidating the pathogenesis of gene 1 type PRRSV.

【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】：S852.651

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