【作者】 陈庆新；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 博士

【摘要】 猪圆环病毒2型（Porcine circovims type 2，PCV2）属于圆环病毒科，基因组全长为1768nt或1767nt，除ORF1和ORF2两大阅读框外，还潜在9个ORFs。ORF1编码与病毒复制相关的Rep蛋白，ORF2编码PCV2免疫相关的蛋白Cap。PCV2是引起PMWS的主要病原，主要表现为免疫系统的损伤，使感染猪处于严重免疫抑制状态。本课题主要进行了PCV2的血清流行病学，分子流行病学和基因组各阅读框的致病性等方面的研究。应用无PCV污染的PK-15细胞分离病毒，从浙江省境内11个地市分离获得10株PCV2病毒。分离的病毒株经超速密度梯度离心纯化后，电镜下可观察到直径17-20nm的病毒粒子；分离病毒株的核苷酸测序结果显示，所用分离株的基因组长度均为1767bp。应用IFA技术，对浙江省内11个不同地区104个规模化猪场2000-2004年期间的4，307个血清样本进行PCV2的血清学流行病学调查，通过对不同地区、年份、年龄和品种猪的血清学数据分析，结果显示，所检测浙江省猪场均存在PCV2感染，猪群PCV2感染率为68.10％，种猪感染的血清阳性率为66.88％，断奶后猪感染的血清阳性率为82.10％。长白种猪感染的血清阳性率为44.83％、长白仔猪PCV2感染的血清阳性率为64.28％，明显高于大约克和杜洛克的种猪和仔猪。PCV2感染的血清阳性率高的种猪和仔猪群，其PRRSV和PRV的抗体阳性率低。通过对未免疫PRRSV疫苗的猪场进行血清学调查发现，PCV2感染率高的猪群，其PRRSV的感染率也随之升高。这些结果说明PCV2在猪群中的感染非常普遍，不同品种猪对PCV2的易感性不同，PCV2的感染降低了猪群对疫苗的免疫应答，PCV2的感染增加了PRRSV感染水平。以IFA和IPMA技术，对2004-2005年2，352份人血清与2005年奶牛、鸡、鸭、和山羊血清进行PCV2相关抗体的血清学检测，在689份人血清中检测到PCV2相关抗体，PCV2相关抗体阳性率为29.3％，没有在奶牛、鸡、鸭、和山羊中检测到PCV2相关抗体的存在。数据分析表明：2005年人群中PCV2的阳性率显著高于2004年（p＜0.01）；不同地区PCV2相关抗体阳性率差异不显著性（p＞0.05）；40至49年龄段人群抗体阳性率最高（OR=1.390[95％CI，1.011-1.9101，p=0.042），女性的阳性率高于男性；PCV2相关抗体阳性率与HBV感染和ALT水平的升高不存在相关性。根据PCV2-HZ0201株序列设计引物，扩增HZ0201的全基因组和各ORF突变全基因组，构建PCV2-HZ0201正常、ORF3和ORF4双突变（PCV2-MF3／4）、ORF5至11单ORF突变（PCV2-MF5至11）等基因组的重组质粒；将实验室保存的PCV2-MF3和PCV2-MF4与本研究中构建重组质粒中的PCV2全基因酶切，环化后转染PK-15细胞，转染细胞进行连续6次传代。PCV2正常的感染性克隆、PCV2-MF3、MF4、MF5、MF9、MF10和MF11在转染后和传代后均能够用PCR和IFA检测到PCV2病毒的存在，测序分析显示突变位点仍然存在；TCID₅₀测定结果显示与野毒株相比未见明显差异，而ORF3-ORF4、ORF6、ORF7和ORF8缺失后则不能检测到病毒抗原和核酸存在。这些结果表明，ORF3-ORF4、ORF6、ORF7和ORF8或其突变点可能与病毒的复制有关；而ORF5、ORF9、ORF10、和ORF11或其突变点与病毒的复制无关。将PCV2-HZ0201、正常感染性克隆（IC）及突变体（PCV2-MF3至5、PCV2-MF9-11），腹腔和鼻内接种8周龄BALB／c小鼠，研究突变后的PCV2对小鼠致病性变化。PCV2-HZ0201、IC、PCV2-MF3和PCV2-MF4组，在7-14天出现抗体，28天左右抗体水平最高，随后开始下降；PCV2-MF5、PCV2-MF9至11接种后14-21天出现抗体，35天左右抗体水平最高，随后开始下降。PCV2-HZ0201，IC，PCV2-MF4、PCV2-MF9和PCV2-MF10通过Caspase-3和Caspase-8途径诱导细胞凋亡，感染后21天脾脏凋亡细胞数显著上升，CD3⁺CD4⁺、CD4⁺CD8⁺和CD3⁺CD8⁺T淋巴细胞数量显著下降，病理切片可观察到淋巴细胞缺失和凋亡现象，并出现病毒血症；感染后42天有所恢复。PCV2-MF3和PCV2-MF5接种小鼠后未诱导脾脏内凋亡细胞水平升高，和CD3+CD4+、CD4+CD8+和CD3+CD8+T淋巴细胞数量显著降低，大部分小鼠切片未观察到明显病理变化和病毒血症。PCV2-MF11感染小鼠后21天未显著诱导脾脏细胞凋亡和降低CD3+CD4+和CD3+CD8+T淋巴细胞数量；但感染后42天显著降低CD3⁺CD4⁺和CD3⁺CD8⁺T淋巴细胞数量，并出现病毒血症和脾脏淋巴细胞缺失现象。以上结果表明，ORF3，ORF5和ORF11突变后部分的改变了PCV2的致病性，可能存在于PCV2基因组中，是构成致病力的一部分；而ORF9和ORF10突变后没有引起PCV2致病性发生明显变化，与PCV2的致病性无关，其存在的可能性很小。ORF4突变后造成了PCV2一定程度的降低，但不十分显著，或许其致病性的激活需要其他因素，如免疫刺激等，其存在性仍值得进一步探究。更多还原

【Abstract】 Porcine circovirus type 2 (PCV2), a member of the genus Circovirus of the Circoviridae family, is a small DNA virus with negative-sense circular genome (1767 or 1768bp in length). Two major open reading frames (ORFs) have been identified for PCV2, ORF1, named the rep gene, encodes a protein with a molecular weight of 35.7 kDa involved in virus replication, and ORF2, named the cap gene, encodes a immunogenic capsid protein with a molecular weight of 27.8 kDa. There are still nine potential ORFs besides ORF1 and ORF2 in the genome. PCV2 is the primary cause of postweaning multisystemic wasting syndrome (PMWS) in the pig. PCV2, resulting in immunosuppression due to lymphocyte depletion. The objects of study were to undertake systemic serological and epidemiological investigations of PCV2 infections in pig herds, human and other spicies, and to investigate the pathogenicity of potential ORFs (except ORF1 and 2) in genome of PCV2.Using PCV-free PK-15 cell line, ten isolates of porcine circovirus type 2 were isolated from 11 districts in China Zhejiang province. The isolated virus is a 17nm in diameter and icosahedral virion by transmission electron microscope. The complete genome of ten isolates is composed of 1767 nucleotides.A total of 4307 serum samples were collected from 104 pig farms in Zhejiang province from 2000 to 2004 years. Serum samples were tested for the presence of antibodies against porcine circovirus 2 (PCV2) using indirect immunefluorescent sassay (IFA). Antibodies against PCV2 were detected in all 104 pig farms. Overall seroprevalences were 68.10% for all, 66.88% for sows, 82.10% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalences of Landrace sows was higher than Yorkshire and Duroc sows. In the same pig herd, the higher seroprevalences of antibodies against PCV2 were; the lower seroprevalences of antibodies against PRRSV and PrV were. On the contrary, there were positive relation between rate of PCV2 infection and PRRSV infection in the pig farms without PRRSV vaccine. These results revealed that the infection of PCV2 was prevalent in the pig herds, the susceptibility of the various pig species was diverse to PCV2, the PCV2 infection reduced the immune responses to vaccine, and PCV2 infection enhanced PRRSV infection.No antibody against PCV2 were detected in 227 duck serum samples, 204 chicken serum samples, 493 cattle serum samples and 38 goat serum samples in Zhejiang province. By screening 2,352 human serum samples, using IFA and IMPA, the antibody against PCV2-like virus was detected in 689 human serum samples collected from 2004 to 2005 years. The seroprevalence of PCV2 was 29.3%. Statistical analysis showed that PCV2 seroprevalence in 2005 increased significantly, compared with that in 2004 (p<0.01). Logistic regression analysis indicated that positive ratios of PCV2 antibody in women was higher than that in men, and the percent of anti-PCV2 like virus antibody in 40 to 49 years old humans was higher than that in other human populations. The seroprevalence of PCV2 like virus in human populations was not associated with elevated ALT level and HBV infection significantly.The genomes of PCV2-HZ0201 or with mutated ORFs (ORF3/4, ORF5 to 11) were amplified by the special primers and cloned into pMD18T vector. The circular DNA of PCV2 normal infectious clones (IC), PCV2-MF3/4 (ORF3 and ORF4 mutation), PCV2-MF3-4 (ORF3-4 single mutation, kept in our laboratory) and PCV2-MF5 to 11 (ORF5-11 single mutation) were generated by ligating the genome with normal or mutational sequence from recombinant plasmids. These circular DNA clones were transfected into PK-15 cells, respectively. Transfected cells were passaged 6 generations. Transfected and passaged cells were detected by IFA and PCR. The results indicated that IC, PCV2-MF3, PCV2-MF4, PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11 could produce progeny; whereas PCV2-MF3/4, PCV2-MF6, PCV2-MF7 and PCV2-MF8 could not produce PCV2 virions. The TCID50 of rescued virus was the same with wild PCV2. Sequenceing showed the mutated site in PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11 still existed in the progeny virus. The results suggested that ORFs5, 9, 10 and 11 were not necessary for virus replication, whereas ORF3-ORF4, ORFs 6, 7 and 8 were essential in virus repllication.To evaluate the roles of ORFs 3, 4, 5, 10 and 11 in viral pathogenicity in vivo, 8-week-old BABL/c mice inoculated intraperitoneally and intranasally with PCV2-HZ0201 virions, infectious clones (IC) and mutants (PCV2-MF3, PCV2-MF4, PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11). Mice inoculated with HZ0201, IC, PCV2-MF3 and PCV2-MF4 had seroconversion to PCV2 at 14 days postinoculation (dpi) and reached a peak at 28 dpi. Mice inoculated with PCV2-MF5, MF9, MF10, and MF11 had seroconversion to PCV2 at 21dpi and reached a peak at 35 dpi. At 21 dpi, mice infected with the PCV2-HZ0201, normal infectious clones, PCV2-MF9 and PCV2-MF10 showed significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets, and significant up-regulation of apoptotic cells in spleen compared to negative control mice (p<0.05). The apoptosis was induced by Caspase-3 and Caspase-8 pathway in mice infected with the PCV2-HZ0201, normal infectious clones, PCV2-MF9 and PCV2-MF10. At 42 dpi, the above-mentioned lesions discovered. There were not significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets in spleen, and apoptotic cell up-regulation in mouse spleen inoculated with PCV2-MF3 and PCV3-MF5 at 21 and 42 dpi (p>0.05). There were significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets, and significant up-regulation of apoptotic cells in mouse spleen infected with PCV2-MF11 at 42 dpi but not 21 dpi (p>0.05). These data suggest that ORF3, ORF5 and ORF11 play an important role in viral pathogenesis, ORF9, ORF10 are non-necessary to viral pathogenesis, and ORF4 should be identified in further study.更多还原