本文選題：豬繁殖與呼吸綜合征病毒 + 基因1型�。� 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome, PRRS)是世界范圍內(nèi)對(duì)養(yǎng)豬業(yè)危害最嚴(yán)重的傳染病之一,病原為豬繁殖與呼吸綜合征病毒(PRRSV)。根據(jù)基因組與抗原性的差異PRRSV可分為2個(gè)基因型,即基因1型和基因2型。近年來(lái),基因1型PRRSV在我國(guó)的存在和臨床感染受到關(guān)注。因此,開(kāi)展基因1型PRRSV的相關(guān)研究十分必要。本研究以基因1型PRRSV毒株GZ11-G1為對(duì)象,分析其對(duì)仔豬的致病性,構(gòu)建感染性cDNA克隆,并采用定點(diǎn)突變技術(shù)分析與其致病性相關(guān)的分子基礎(chǔ),以期為深入開(kāi)展基因1型PRRSV的致病機(jī)制研究奠定必要的技術(shù)和前期基礎(chǔ)。將基因1型PRRSV毒株GZ11-G1及基因1型疫苗毒株分別接種6周齡SPF豬,觀察并記錄各實(shí)驗(yàn)組感染豬的體溫變化、臨床癥狀、病毒血癥、體外排毒情況、血清抗體產(chǎn)生動(dòng)態(tài)、組織病變及免疫組化結(jié)果。試驗(yàn)結(jié)果顯示,基因1型PRRSV毒株GZ11-G1能夠引起仔豬呼吸道癥狀并伴隨一過(guò)性體溫升高,體內(nèi)增殖能力顯著高于疫苗毒株,并且能夠引起嚴(yán)重的彌漫性間質(zhì)性肺炎,表明基因1型PRRSV毒株GZ11-G1對(duì)仔豬具有致病性。根據(jù)基因1型PRRSV毒株GZ11-G1與疫苗毒株的全基因組序列,分段進(jìn)行RT-PCR擴(kuò)增,通過(guò)同義突變引入酶切位點(diǎn),連入低拷貝質(zhì)粒pWSK-29T,獲得全長(zhǎng)cDNA質(zhì)粒pWSK-GZ11-G1及pWSK-Amervac。經(jīng)對(duì)全長(zhǎng)cDNA質(zhì)粒線化、體外轉(zhuǎn)錄和轉(zhuǎn)染細(xì)胞,進(jìn)行病毒拯救和鑒定。結(jié)果表明,構(gòu)建的基因1型PRRSV毒株GZ11-G1及疫苗毒株的全長(zhǎng)cDNA克隆具有感染性,可拯救出病毒,分別命名為Rv-GZ11和Rv-Amervac,拯救病毒具有與親本病毒相似的體外增殖特性。對(duì)GZ11-G1、Amervac PRRS疫苗毒株在內(nèi)的10株基因1型PRRSV相關(guān)區(qū)域(5’及3'UTR、ORF1b、ORF2-6)的序列及編碼氨基酸序列進(jìn)行了比對(duì)分析。結(jié)果表明,GZ11-G1在Nsp9-10區(qū)域及GP2分別存在4個(gè)和3個(gè)氨基酸突變位點(diǎn),包括Nsp993位甘氨酸(G)、Nsp10281位脯氨酸(P)、304位纈氨酸(V)、401位賴氨酸(K)、GP25位組氨酸(H)、120位甘氨酸(G)和252位絲氨酸(S)。利用感染性克隆技術(shù)及定點(diǎn)突變技術(shù)拯救出6株突變病毒,分別命名為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。體外增殖結(jié)果表明,突變GP2的3個(gè)氨基酸后,各突變病毒與相應(yīng)的親本毒株增殖水平均無(wú)顯著差異,而突變Nsp9-10的4個(gè)氨基酸后,Rv-A-G-Nsp9-10、Rv-A-G-GP2+Nsp9-10早期的病毒滴度均顯著提高,而Rv-G-A-Nsp9-10、Rv-G-A-GP2+Nsp9-10病毒滴度達(dá)到峰值的時(shí)間點(diǎn)分別滯后12h和24h,各時(shí)間點(diǎn)的病毒滴度也顯著低于親本拯救毒株(P0.05)。對(duì)突變病毒的致病性分析結(jié)果表明,與Rv-Amervac相比,Rv-A-G-Nsp9-10感染豬的臨床癥狀、病毒血癥和間質(zhì)性肺炎更為嚴(yán)重,而Rv-G-A-Nsp9-10較親本病毒的病毒血癥持續(xù)時(shí)間縮短,感染豬的體溫反應(yīng)和肺臟的病理?yè)p傷減輕。結(jié)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位氨基酸與其增殖能力及致病性增強(qiáng)有關(guān)。綜上所述,研究結(jié)果表明基因1型PRRSV毒株GZ11-G1具有致病性,成功構(gòu)建了基因1型PRRSV感染性克隆技術(shù),揭示了基因1型PRRSV毒株GZ11-G1 Nsp9和Nsp10中與病毒增殖能力和致病性相關(guān)的氨基酸位點(diǎn),為進(jìn)一步研究基因1型PRRSV分子致病機(jī)制提供了技術(shù)平臺(tái),并為闡明基因1型PRRSV的致病機(jī)制提供了有價(jià)值的科學(xué)依據(jù)。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.651

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1 王星晨;豬繁殖與呼吸綜合征病毒基因1型毒株GZ11-G1的致病性及其分子基礎(chǔ)[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

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本文編號(hào)：1894313