猪繁殖与呼吸综合征病毒流行毒株NSP2基因遗传变异及GM-CSF对GP3/GP5的免疫增强作用研究

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英文题名：Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Isolates from China Based on NSP2 Gene and Imuno-Enhancing Effect of GM-CSF on the Viral GP3/GP5 Protein 作者：王兴龙 论文级别：博士 学科专业名称：预防兽医学 中文关键词：PRRSV ; NSP2 ; GM-CSF ; GP3-GP5 ; 重组腺病毒 英文关键词：PRRSV ; NSP2 ; GM-CSF ; GP3-GP5 ; Immunogenicity 学位年度：2010 导师：姜平 学科代码：090602 学位授予单位：南京农业大学 论文提交日期：2010-04-01

摘要

猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)属于动脉炎病毒科,基因组为单股正链RNA,具有快速变异特性。自1991年首次分离获得病毒以来,该病毒基因逐渐发生变化并伴随着给养猪业带来严重经济损失。2000年美国出现非典型PRRSV,免疫商品猪群和母猪群出现严重死亡。PRRS于1996年在我国首次爆发,并逐渐传播各省市。2006年在中国大陆地区多省份出现高致病性PRRS,主要临床表现为持续高热(41.0℃以上)、皮肤发红、发病率和死亡率高。病毒非结构蛋白NSP2上存在30个氨基酸缺失。近年来,PRRSV持续流行,造成了我国养猪业的巨大经济损失。为了深入阐明该病毒基因遗传变异规律和NSP2毒力作用,探索该病毒新的免疫防控方法,本研究从华东地区部分省份2004-2009年发病猪群分离获得38株PRRSV,并对其NSP2基因高变异区进行了克隆和序列分析；根据基因分析结果,从中选择4株PRRSV流行毒株进行仔猪人工感染发病试验,研究NSP2基因变异与病毒毒力间的关系；同时,设计构建共表达PRRSV GP3和GP5与猪粒细胞巨噬细胞集落细胞刺激因子(Granulocyte/macrophage colony stimulating factor, GM-CSF)融合蛋白重组腺病毒,并进行了小鼠和猪体免疫试验,具体研究内容和结果分为以下5个部分：
     1.我国猪繁殖与呼吸综合征病毒流行毒株分离与鉴定
     2004-2009年在临床流行病学研究基础上,从江苏、上海、山东、安徽、浙江和广西等省市收集临床发病猪场仔猪肺脏病料,经匀浆、冻融和过滤处理后接种Macr-145或猪肺泡巨噬细胞(PAM),通过CPE观察、RT-PCR检测和PRRS阳性抗体间接免疫荧光试验鉴定,分离获得38株PRRSV,其中,32株病毒直接采用Macr-145细胞分离获得,其余6株病毒则是先用PAM细胞分离,然后接种Marc-145细胞分离获得；分离病毒在Marc-145细胞上培养特性有一定差异,病毒毒价为103.75-106.33TCID50/ml,为PRRSV研究提供了重要物质基础。
     2.2004-2009年猪繁殖与呼吸综合征病毒流行毒株NSP2基因遗传变异分析
     根据PRRSV NSP2基因设计合成2对PCR引物,采用巢式RT-PCR方法对上述分离的38株PRRSV NSP2基因高变区进行克隆和测序分析,结果显示：不同毒株PCR扩增产物长度有所不同,共出现5种长度类型,分别为：1215、1275、1287、1373和1374bp。38株PRRSV核苷酸同源性为71.6%-99.6%,推测的氨基酸同源性为63.7%-98.3%。NSP2基因进化分析结果显示,38株分离株可以分为两个基因亚型,即NSP2基因亚型Ⅰ和NSP2基因亚型Ⅱ,其中,NSP2基因亚型Ⅰ内的毒株有28株,具有30个或更多个氨基酸的缺失,基因同源性为92.9%-99.2%;NSP2基因亚型Ⅱ内的毒株具有1个或不具有氨基酸的缺失,但基因差异较大,基因同源性为76.5%-99.6%。根据NSP2基因高变区基因缺失情况,38株PRRSV分离株分为5种类型,其中,30aa缺失型毒株比例最高(26/38),2种新的NSP2基因缺失类型的PRRSV毒株出现于2008年后。该研究结果表明,我国PRRSV流行毒株NSP2基因变异较大,并不断出现新的变异毒株,加强PRRSV流行毒株基因变异监测十分必要。
     3.猪繁殖与呼吸综合征病毒NSP2基因变异与毒力变化间的关系研究
     根据上述PRRSV NSP2基因序列分析结果,选择4个PRRSV毒株,包括YX0907、BB0907、SY0909和NT0801毒株,其中,YX0907、BB0907和SY0909毒株属于基因亚型Ⅰ,具有30个氨基酸缺失的特征,NT0801属于NSP2基因亚型Ⅱ,其NSP2上不存在缺失。GP5基因序列分析结果为：4株PRRSV毒株GP5基因同源性为98.0%-99.8%,其氨基酸同源性为97.5%-99.5%。选择23头45日龄PRRSV、猪圆环病毒2型(PCV2)和副猪嗜血杆菌(HPs)阴性健康商品仔猪,随机分为5组,第1-4组分别颈部肌肉注射上述4株PRRSV分离株(第3代),2×10432TCID50/1ml/头,第5组3头猪作为空白对照,隔离饲养21d,每天测量体温,观察临床症状；攻毒后第7d、14d和21d采血分离血清,测定PRRSV含量；对死亡猪和第21天处死猪进行病理解剖,观察肉眼病理变化和组织病理变化,并进行记分和比较,结果显示,BB0907毒株毒力最强,试验猪感染后体温明显升高,临床症状明显,可引起3/5死亡,病猪肺脏组织具有明显病理变化；YX0907毒株毒力较强,试验猪临床症状和病理变化明显,可引起2/5死亡；NT0801毒力较弱,部分猪感染后出现1-2d体温升高和轻度临床症状,但未引起死亡；SY0909毒株毒力最弱,感染猪出现1-3d体温升高,临床症状不明显,病理变化较轻；而对照组猪无临床异常表现和病理变化。该研究结果表明,PRRSV分离株毒力存在明显差异,4个毒株毒力高低为：BB0907> YX0907> NT0801> SY0909。具有NSP2相同基因缺失类型的3个PRRSV分离株毒力差异较大,证实PRRSV毒力与NSP2的30个氨基酸基因缺失没有直接联系。
     4.猪繁殖与呼吸综合征病毒GP3-GP5与猪GM-CSF融合基因重组腺病毒的构建与鉴定
     利用PCR扩增出高致病性PRRSV SY0608分离株的GP3和GP5基因及猪粒细胞巨噬细胞集落刺激因子(GM-CSF)基因,并按顺序将它们连入梭载体pShuttle-CMV,并在GM-CSF和GP3之间及GP3和GP5间分别插入FMDV 2A基因和"AAGCTT"连接子,重组穿梭载体经酶切和测序鉴定后用PmeⅠ线性化,然后电转化入基因工程菌株大肠杆菌BJ5183内,与腺病毒骨架载体pAdEasy-1重组,获得重组腺病毒质粒。将重组腺病毒质粒经PacⅠ酶切线性化,然后用脂质体法转染HEK-293A细胞,10d后获得重组腺病毒,命名为rAd-GF35。用RT-PCR方法检测,可以扩增获得长度约1.8kb的GM-CSF-GP3-GP5融合表达转录产物；用猪抗PRRSV阳性血清和小鼠免疫原核表达猪GM-CSF蛋白获得的血清进行间接免疫荧光试验(IFA),证明该重组腺病毒在293A细胞中可以表达GM-CSF和GP3-GP5目的蛋白；用Western blot检测,结果显示GM-CSF-GP3-GP5融合蛋白能够正确表达,且抗原性良好；猪骨髓细胞用感染rAd-GF35的PK-15细胞上清刺激,可以表现明显增殖并形成集落。该研究结果证明,本研究构建的重组腺病毒rAd-GF35可以有效表达GM-CSF-GP3-GP5重组蛋白,并具有GM-CSF生物活性。
     5.猪繁殖与呼吸综合征病毒GP3-GP5与猪GM-CSF融合基因重组腺病毒小鼠免疫特性研究取60只BALB/c小鼠,随机分为4组,每组15只。第1和第2组分别背部皮下注射PBS缓冲液和野生型腺病毒(wtAd)作为对照组,第3和第4组分别于背部皮下注射重组腺病毒rAd-GP35(表达GP3-GP5融合蛋白)和rAd-GF35(表达GM-CSF-GP3-GP5融合蛋白),接种剂量为1010TCID50/0.5ml/只,隔离饲养3周后用同样方法加强免疫一次。首次免疫后不同时间,随机抽取5只／组采血和取脾脏,分别检测PRRSV特异抗体、淋巴细胞增殖指数、IFN-y和IL-4,结果为：第3-4组小鼠在免疫后21d,均可以检测到PRRSV特异ELISA抗体产生,42d达到最高,抗体水平差异显著(P<0.05)；首次免疫后42d, rAd-GF35免疫组抗体最高可达1：128,平均效价为1:80,显著高于rAd-GP35免疫组(1:32)(P<0.05);同时,rAd-GF35免疫组小鼠淋巴细胞增殖指数(Stimulation Index,SI)平均值显著高于rAd-GP35免疫组(P<0.05); rAd-GF35免疫组小鼠脾细胞经PRRSV抗原体外刺激后,细胞培养上清中IFN-γ平均含量为175pg/ml, IL-4平均含量为87.3 pg/ml,而rAd-GP35免疫小鼠脾细胞培养上清中IFN-γ和IL-4的含量只有117.3 pg/ml和46.5pg/ml,差异显著(P<0.05)。该结果表明,重组腺病毒rAd-GF35诱导的体液免疫和细胞免疫明显高于重组腺病毒rAd-GP35, GM-CSF可以显著增强GP3/GP5蛋白在小鼠体内的免疫反应。
     6.猪繁殖与呼吸综合征病毒GP3-GP5与猪GM-CSF融合基因重组腺病毒仔猪免疫保护效力研究
     取15头2周龄的PRRSV, PCV2和HPs阴性健康商品仔猪,随机分为3组,每组5头,隔离饲养。第1和第2组肌肉注射重组腺病毒rAd-GF35和rAd-GP35,第3组同样方法接种野生型腺病毒(wtAd)作为对照,接种剂量为5×10.0TCID50/1ml/头,间隔3周后用同样方法加强免疫一次,免疫后不同时间采血,测定ELISA抗体和猪外周血淋巴细胞(PBMC)增殖指数SI；首免后42d用PRRSV-SY0608强毒滴鼻,2×104.0TCID50/1ml/头,每个鼻孔1 ml,攻毒后分别观察临床症状、PRRSV病毒血症和病理变化,并进行综合记分和比较,结果为：免疫后21d,重组腺病毒rAd-GP35和rAd-GF35组ELISA抗体水平显著高于wtAd, rAd-GF35组中和抗体水平最高可达1:10,平均效价1:7.6,而rAd-GP35免疫组最高为1:8,平均效价<1:5;免疫后42d, rAd-GF35免疫组PBMC增殖指数SI平均值显著高于rAd-GP35免疫组(P< 0.05); rAd-GF35免疫组猪血清中IFN-γ与IL-4含量为276.2 pg/ml和312.7 pg/ml,显著高于rAd-GP35免疫猪血中的94.6 pg/ml和242.5pg/ml (P< 0.05).攻毒后,rAd-GF35免疫组临床症状明显减轻,综合评定数值为26.2,显著低于rAd-GF35免疫组的44和wtAd组的82.2(P<0.05)；攻毒后7d, rAd-GF35、rAd-GP35和wtAd组猪血清病毒含量分别为1.5×1030、3.3×1030和1.0×1040个TCID50,三个组间差异显著(P<0.05)。此外,对照组猪攻毒后出现明显病理变化,包括肺脏出血、炎性浸润和肺泡损伤等,其次为rAd-GP35,而rAd-GF35无明显病理变化。该研究结果证明,rAd-GF35免疫可以提供仔猪较好免疫保护作用。
     综上所述,我国流行PRRSV毒株发生了新的变异,多种NSP2基因缺失类型病毒株同时存在,丰富了PRRSV分子流行病学资料；人工感染发病试验进一步证实,NSP2基因缺失与病毒毒力间没有直接关系；用腺病毒表达的GM-CSF-GP3-GP5能够在小鼠和猪体内诱导PRRSV特异性体液和细胞免疫反应,证实GM-CSF能够显著增强GP3-GP5的免疫反应,提高对仔猪的攻毒保护,从而为PRRSV新型疫苗研究奠定了基础。
Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus, belonging to Arteriviridae. PRRSV was firstly isolated in the last century, and then it has kept varying and caused great economic losses in pig farms. In 2000, the atypical PRRSV appeared in America and the immunized herds still encountered with serious mortality. PRRSV firstly emergeed in China in 1996, and then rapidly spread out to all the provinces. In 2006, there was a harrowing outbreak of PRRSV in the Mid-East China, which was characterized by durative fever (>41.0℃) and high mortality in herds of each age. The pathogen was identified as a new variant of PRRSV, with a unique hallmark in NSP2 gene (with two discontinues deletion of 30aa). In order to reveal the variation rule of PRRSV genetics and the contribution of NSP2 to PRRSV virulence, thirty-eight PRRSV isolates were isolated from different farms in the southeast of China during 2004-2009, and the NSP2 of the isolates were sequenced partly and analyzed. Then 4 representative isolates were selected to challenge piglets and the relationship between the NSP2 variation and virulence change was analyzed. Meanwhile, the recombinant adenovirus co-expressing the porcine granulocyte/macrophage colony stimulating factor (GM-CSF) and PRRSV GP3/GP5 was constructed and the immunogenicity was detected in mice and piglets.
     1. Isolation and identification of porcine reproduction and respiratory syndrome virus
     Base on the clinical epidemiology of the pig disease, we collected the lung samples from pathogenic pig farm in Jiangsu, Shanghai, Shandong, Anhui, Zhejiang and Guangxi provinces. After homogenization, freeze-thawing and then filtrated with filter membrane, the supernatants were collected and inoculated into Macr-145 cells or PAM for virus isolation. Thirty-eight PRRSV isolates were isolated and identified by RT-PCR and IFA. Among them, thity-two were isolated directly with Marc-145 cells and 6 were recovered with first PAM and then passaged in Marc-145. The 38 PRRSV isolates had different characters for in vitro culture and graw up with different titers of 10-3.75-10-6.33TCID50/ml. These virus isolates could be used to study on the disese in the future.
     2. Genetic variation of porcine reproduction and respiratory syndrome virus based on NSP2 during the period of 2004-2009
     Base on NSP2 sequence of PRRSV, two pairs of primers were designed for Nest-PCR to amplify the high variation region of NSP2. Totally 5 fragments with different length,1215,1275,1287,1373 and 1374bp were obtained from the 38 isolates. The homology of the isolates base on the nucleotide sequence was 72.7%-99.6% and the derivation of amino sequence was 63.7%-98.3%. According to the polygenic analysis results, the 38 PRRSV isolates could be classified into two subgenomic groups:NSP2-subtypeⅠand NSP2-subtypeⅡ. Of the 38 isolates,28 were sorted into NSP2-subtypeⅠsharing with 1 and 29 amino acids deletion, and the homology of NSP2-subtypeⅠwas 92.9%-99.2%. The isolates in NSP2-subtypeⅡhad 1 or no amino acid deletion with 76.5%-99.6% genic homology. Moreover, five different deletion patterns were identified in NSP2 in these 38 isolates. The most popular isolates were the virus with 30aa deletion. Two new deletion patterns were identified in these 38 isolates, which isolated since 2008. It indicated that the NSP2 of the PRRSV isolates in China had high variety and it was need to monitor on the variation of NSP2 in the future.
     3. The relationship between the NSP2 variation and the virulence of porcine reproduction and respiratory syndrome virus
     Based on the NSP2 sequence of the 38 PRRSV iolates, four different isolates named as YX0907, BB0907, SY0909 and NT0801 were selected. Among them, YX0907, BB0907 and SY0909 isolates were ranged to NSP2-subtypeⅠwith 30aa deletion and NT0801 isolate to NSP2-subtypeⅡwithout any amino acids deletion. The homology of these 4 isolates base on GP5 nucleotide sequence was 98.0%-99.8% and the derivation of amino sequence was 97.5%-99.5%. Twenty-three 45-day old piglets free of PRRSV, PCV2 and HPs were selected and randomly separated into 5 groups. The first 4 groups were challenged individully with the 4 PRRSV isolates by injection of 2ml of 2×104.32TCID50/ml. Three pigs in the 5th group were served as control. After challeng, the rectal temperatures of the pigs were recorded every day. The sera were collected at 7,14 and 21 days post challenge (dpc) for detection of the viremia. The dead and sacrificed pigs at 21 dpc were autopsied and the gross lesions were valued. The results showed that BB0907 isolate had highest virulence. It caused apparent fever and clinical disease with great pathological changes. And 3 pigs out of 5 were dead after infection. Comparing with BB0907, YX0907 isolate had less virulent. The pigs in this group also showed apparent clinical syndrome and tissue gross lesion, and 2 pigs were dead after inoculation. But NT0801 isolate has relatively lower pathogenic ability and caused the pigs had 1-2 days fever with slight clinical signs. SY0909 isolates has lowest virulence. Only some pigs in this group only experienced 1-2 days fever without clinical sign. Meanwhile, the pigs in the control group were quite healthy throughout the trail. These results revealed that the level of the virulence of these four isolates is BB0907> YX0907> NT0801> SY0909, indicating that the isolates with 30aa deletion have different pathogenic ability and the 30aa deletion has no relationship with the virulence of isolate.
     4. Construction and identification of the recombinant adenovirus co-expressing pig GM-CSF and GP3 and GP5 of porcine reproduction and respiratory syndrome virus
     PRRSV GP3 and GP5 were amplified from high pathogenic isolate SY0608 and pig GM-CSF was amplified from stimulated PBMC by RT-PCR, respectively. And then they were inserted into the different sites of the adenovirus shuttle vector one by one. Between GM-CSF and GP3, the gene of 2A protein from FMDV was used as a linker, and "AAGCTT" as a linker between GP3 and GP5. The recombinant plasmid was identified by restriction enzyme digestion and then sequenced. The linearized positive shuttle plasmid was electro-transformated into BJ5183 and recombined with the adenovirus backbone vector, and then recombined plasmid linearized with PacⅠ. After that, the recombinant adenovirus, named as pAd-GF35, were transfected into 293A cells. At 10 days post incubation, the recombinant adenovirus came up with CPE. Then, the expressions of these target proteins were identified by IFA and western blot, the bioactivity of the GM-CSF was confirmed by MTT assay and colony formation assay. It indicated that a recombinant adenovirus, rAd-GF35, co-expressing the GM-CSF-GP3-GP5 fusion protein was successfully constructed and it could be used to deteced the immunogenicity in the future.
     5. Immunogenicity of recombinant adenovirus co-expressing GM-CSF and GP3-GP5 of porcine reproduction and respiratory syndrome virus in mice
     Sixty BALB/c mice were randomly separated into 4 groups with15 in each. The mice in the first two groups were ranged as control, empty control with PBS and scramble control with wtAd, respectively. Group3 were immunized with rAd-GP35 (expressing GP3-GP5 fusion protein) and group 4 with rAd-GF35 (expressing the GM-CSF-GP3-GP5 fusion protein). After 3 weeks, the all groups were boosted vaccined except those mice were slaughtered. And PRRSV-specific antibody, lymphocyte proliferation index, IFN-γand IL-4 were detected to evaluate the immunization at different time after vaccination. The results showed that the antibody against PRRSV became detectable in group3 and group4 at 21 days post immunization (dpi), and reached to the peak at 42dpi. At 42dpi, the neutralizing antibody (NA) titers in some mice from rAd-GF35 group could reach to 1:128 and the average level was 1:80. It was significantly higher than that in rAd-GP35 group with the level of 1:32. The lymphocytes proliferation index in rAd-GF35 group was significant higher than that of the rAd-GP35 group. The secretion of IFN-γand IL-4 of the lymphocytes from the mice immunized with rAd-GF35 is 175pg/ml and 87.3pg/ml and which are significant higher than that with rAd-GP35 (117.3 pg/ml and 46.5pg/ml). These results indicated that the recombinant adenovirus rAd-GF35 could induce distinctly higher humoral and cell immune response than rAd-GP35. GM-CSF could enhance the immune response induced by GP3/GP5.
     6. Protective efficacy of recombinant adenovirus co-expressing GM-CSF and GP3-GP5 of porcine reproduction and respiratory syndrome virus
     Fifiteen 2-week old commericall pigs free of PRRSV, PCV2 and HPS were randomly separated into 3 groups with 5 in each. The first two groups were immunized with the recombinant adenovirus rAd-GP35 and rAd-GF35 (5×10.0TCID50/ml), respectively, and the last one as control, inoculated with wtAd. After 3 weeks, the all groups were boosted vaccined. PRRSV specific antibody, lymphocyte proliferation index, IFN-γand IL-4 were detected at different time after vaccination. And at 42 days post immunized (dpi), all the animals were challenged with high pathogenic PRRSV isolate SY0608 (2×104.0 TCID50/ml). And then the rectal temperature, virema, gross tissue lesion and microscope lesion were used to judgment the protective efficacy. The results showed that at 21dpi, rAd-GP35 and rAd-GF35 immunized pigs developed significant higher level of PRRSV-specific antibodies compared with the control. The highest NA titer in rAd-GF35group was 1:10 and the average level was 1:8. But the average NA titer in group rAd-GP35 was less than 1:4. The lymphocytes proliferation index of rAd-GF35 group was significantly higher than rAd-GP35 group. The contents of IFN-γand IL-4 in sera were 276.2pg/ml and 312.7pg/ml in rAd-GF35 immunized pigs, which are significantly higher than that immunized with rAd-GP35 (117.3 pg/ml and 46.5pg/ml). The pigs immunized with rAd-GF35 showed less clinical signs with integrated score 26.2, while the score in rAd-GP35 group is 44 and that in wtAd is 82.2. The pigs in rAd-GF35 group developed lower level of viremia (1.5×103.0 TCID50), which was significant with other two groups (3.3×103.0 and 104.0TCID50). Meanwhile, the piglets in control groups experienced the most serious tissue lesion. All results indicated rAd-GF35 could provide partly a protection against PRRSVchalleng in piglets.
     In summary, based on the NSP2 sequences of PRRSV isoaltes, it proved that there were a lot of PRRSV variants with various kinds of NSP2 deletion in China, which enriched our current epidemiologic knowledge of PRRSV. The results of challenge experiment revealed that there was no relationship between the 30 aa deletion of NSP2 gene and the virulence of PRRSV. The recombinant adenoviruses co-expressing porcine GM-CSF and PRRSV GP3/GP5 could induce PRRSV-specific immune response in mice and pigs. GM-CSF could enhance the immune response induced by GP3-GP5 and increased the protective effect to PRRSV challenge. This study provides new strategies for developing new efficient PRRSV vaccine.

引文

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