【作者】 帅江冰；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2008， 博士

【摘要】 猪圆环病毒(Porcine circovirus,PCV)属于圆环病毒科,圆环病毒属,无囊膜,直径约17nm,是最小的动物病毒之一,其因组为一单链环状DNA。PCV分为两个基因型,PCV1无致病性,广泛存在于猪群中。而PCV2被认为是断奶仔猪多系统衰竭综合征(PMWS)的主要致病因子,可引起消瘦、可视黏膜苍白、呼吸困难、间歇性腹泻及淋巴结肿胀等症状,对养猪业构成了严重威胁。PCVl和PCV2均包含ORF1、ORF2和ORF3三个主要的开放阅读框。其中ORF2编码病毒的衣壳蛋白,并且是PCV2的主要毒力因子和免疫原性蛋白。研究表明,植物单链环状病毒核衣壳蛋白核定位信号(NLS)的破坏或缺失直接导致ssDNA积聚减少,从而影响病毒的复制增殖。核衣壳蛋白有可能通过与复制相关蛋白的作用而参与调节病毒基因组的复制。对PCV2核衣壳蛋白的研究将有助于深入了解病毒复制、致病性,开发有效的病毒疫苗。而猪圆环病毒2型(PCV2)是引起猪多系统衰竭综合征的病原之一,严重影响养猪业的健康发展,目前还没有有效疫苗。本项目以PCV2 ORF2为基础,在对浙江省及周边地区猪场PCV2感染血清学和分子流行病学调查的基础上,比较深入地开展了PCV核衣壳蛋白核定位信号对PCV2复制的影响和重组病毒的免疫原性研究。由于该病毒在体外培养细胞中的增殖较慢,是影响疫苗研发的瓶颈问题,本研究针对PCV2复制问题开展的研究,具有重要的理论和实际意义。首先,分别应用PCR和ELISA方法对2003-2007年浙江省及其周边上海、江苏等地猪场采集的169份病猪组织和1779份血清进行了PCV2及其特异性抗体的检测。结果显示,淋巴结中PCV2的PCR阳性率为52.07%(88／169),猪群PCV2抗体阳性率为46.0%(819／1779)。其中断奶后仔猪(54.1%,223／412)和肥育猪血清(49.9%,423／848)PCV2抗体阳性率显著高于哺乳仔猪的阳性率(33.3%,173／519)。进一步扩增27株2004-2007年不同地区PCV2毒株的ORF2基因,进行测序并分析其分子流行特点。结果显示该地区PCV2毒株彼此间核苷酸的同源性较高(98%-100%),但与国内其它地区或国外毒株相比具有明显的氨基酸变异,特别是衣壳蛋白的两个主要抗原表位区内氨基酸位点变异率非常高,导致其亲水性发生明显改变。遗传进化分析发现该地区的所有PCV2毒株与部分国内毒株处于同一分支内,而与大部分国内或国外的PCV2毒株亲缘关系较远。以上结果说明PCV2感染在浙江省及周边省市呈广泛流行并呈上升趋势,而且其毒株具有独特的分子特征。PCV衣壳蛋白在胞浆内合成后通过其自身核定位信号的作用转运至细胞核内参与病毒的复制和组装。预测结果表明PCV1 ORF2蛋白的N端也含有多个可能的单一型或双分型核定位信号序列(NLS)。本研究将PCV1 ORF2及其突变体与eGFP融合,插入真核表达质粒pcDNA3,使其能融合表达两个不同基因。转染细胞后观察荧光,以分析和确证PCV1 ORF2蛋白中的核定位元件。试验结果表明,PCV1 ORF2蛋白C端190个氨基酸缺失后不影响融合蛋白的核定位,即蛋白均集中于细胞核内,与野生型ORF2-EGFP融合蛋白一致。而其N端43位氨基酸序列的缺失导致融合蛋白丧失了核定位功能而使蛋白散布于PK-15细胞的胞浆内。这说明PCV1 ORF2蛋白的核定位功能由其N端序列决定。进一步对N端NLS序列中的碱性氨基酸残基分别进行点突变,结果表明氨基酸短肽9RRRR12的突变和25RRPYLAHPAFRNRYRWRRK43中后6个碱性氨基酸的突变导致融合表达产物分布于整个细胞,丧失了完整的核定位功能,说明这两个序列为PCV1 ORF2蛋白的核定位元件,与PCV2 ORF2蛋白的NLS有较大差异。应用反向遗传学技术可以详细研究病毒的基因结构和编码蛋白的功能。我们将PCV2基因组克隆入pUC-18载体,并在其3’端亚克隆入一段重复序列,构建了mPCV2感染性克隆。其中的重复序列有助于DNA在细胞内通过同源重组自身环化并从质粒中释放。以同样策略构建了PCV1基因组为骨架、含有PCV2ORF2的杂合型PCV12感染性克隆以及PCV2 ORF2核定位信号序列(NLS)被PCV1 ORF2相应序列替换的PCV2-2NLS1和PCV12-2NSL1两种杂合型感染性克隆,以研究PCV核定位信号元件对于病毒复制的影响。结果表明4个感染性克隆转染PK-15细胞后均能产生感染性的病毒粒子。连续传代稳定后,mPCV2和PCV12病毒分别能达到105.5和105TCID50/ml,滴度与野毒株PCV2相似。而杂合病毒PCV2-2NLS1和PCV12-2NLS1的滴度则显著低于野毒PCV2(103.5TCID50/ml)。mPCV2和PCV12要比PCV2-2NLS1和PCV12-2NLS1在PK-15细胞具更强的复制能力：mPCV2和PCV12从感染后12h的102TCID50/ml达到96h时的104TCID50/ml左右,但PCV2-2NLS1和PCV12-2NLS1只达到103TCID50/ml左右。将各重组病毒以相同剂量接种8周龄的BALB/c小鼠,比较它们在体内的复制能力、致病性和免疫原性差异。虽然mPCV2组小鼠血清中病毒含量显著高于PCV12接种组,但它们诱导的血清抗体效价没有显著差异,表明它们在体内具有相似的免疫原性。而PCV2-2NLS1和PCV12-2NLS1组小鼠只有少数能从血清中检测到低水平的病毒量和PCV特异性抗体。致病性试验发现各病毒接种组均未出现明显的肺部病变。野毒株PCV2 (SH04)和(?)nPCV2组小鼠脾脏均出现了轻度的显微病变,与对照组相比差异显著。但三个杂合型病毒PCV12、PCV2-2NLS1和PCV12-2NLS1接种组小鼠脾脏的病变程度与对照组差异不显著。流式细胞检测结果显示SH04和mPCV2接种后,小鼠外周血CD4+和CD8+T淋巴细胞含量显著低于其余各组(p<0.05)。但PCV12接种组小鼠外周血T淋巴细胞亚群变化与对照组相比不明显。因此,PCV12在体内的致病性明显低于野毒株SH04和重组PCV2病毒,但与PCV2野毒株具有相似的免疫原性。然而,PCV2-2NLS1和PCV12-2NLS1在体内外都只表现较弱的复制增殖能力,表明将PCV1核衣壳蛋白NLS序列替换PCV2相应部位序列并没有提高PCV2病毒的复制能力。相反地,由于包含PCV1和PCV2 ORF2蛋白核定位信号的N端核衣壳序列的互换可能使杂合型核衣壳蛋白核定位功能减弱或丧失,甚至导致蛋白结构和功能的改变,从而影响病毒的复制和组装。综上所述,本论文对浙江省及其周边地区猪场中PCV2的流行进行了系统的调查,发现PCV2感染呈广泛流行并有明显上升趋势,且其毒株与国内外其它毒株相比在ORF2蛋白的两个主要抗原表位区具有较大差异。同时,我们发现PCV1ORF2蛋白的核定位功能由其N端氨基酸短肽9RRRR12和25RRPYLAHPAFRNRYRWRRK43决定,与PCV2 ORF2蛋白的NLS有较大差异。重组病毒体内外特性研究表明PCV12对小鼠的致病性明显低于野毒株PCV2和mPCV2病毒,但它们却具有相似的复制能力和免疫原性。而PCV2-2NLS1和PCV12-2NLS1病毒在体内外复制能力都较弱,表明将PCV1核衣壳蛋白NLS序列替换PCV2相应部位序列并没有明显提高PCV2病毒的复制能力。提示PCV2-2NLS1和PCV12-2NLS1病毒核衣壳蛋白的核定位元件中可能存在关键碱性氨基酸位点对于病毒复制具有重要影响或致死性的,从而影响了病毒的复制和组装。进一步研究可对PCV1和PCV2核衣壳蛋白不同的核定位元件中的碱性氨基酸位点进行突变,从而在更大程度上保证Cap蛋白的完整性和兼容性,以研究核定位元件在病毒复制中的作用。而本研究中基于质粒的病毒感染性克隆构建方法的建立及成功运用将有助于我们对PCV病毒复制和蛋白功能的进一步研究。而小鼠感染模型也为进一步的研究提供了便利。更多还原

【Abstract】 Porcine circovirus (PCV), which belongs to the family Circoviridae, genus Circovirus, is one of the smallest animal viruses with unenveloped, single-stranded circular genome and a size of 17 nm in diameter. Two species of PCV, PCV1 and PCV2, have been characterized. PCV1 is considered to be non-pathogenic to pigs by experimental inoculation and was circulating widely in swine population in the world. PCV2 has been shown to be the causative agent of post-weaning multisystemic wasting syndrome (PMWS) of pigs. The affected pigs show progressive weight loss, respiratory and enlarged lymph nodes and characteristic microscopic lesions including granulomatous interstitial pneumonia, lymphadenopathy, hepatitis, nephritis and pancreatitis. PMWS has been a big threat to the swine industry worldwide. Three major open reading frames (ORF), ORF1, ORF2 and ORF3, are oriented in opposite directions in the genome of PCVs. ORF2 is highly variable between PCV1 and PCV2 (less than 60% homology) and encodes the only structural capsid (Cap) protein that contains the dominant immunological regions. It was reported that disruption or loss of nuclear localization of Cap might result in a reduced level of ssDNA in geminiviruses, which might result in low level of viral replication. Therefore, it is generally believed that PCV Cap is not merely involved in encapsidation and might contribute to replication control by way of interactions between Cap and Rep in the nucleoplasm. Mutagenic studies on PCV2 ORF2 could be helpful to understanding the mechenism of viral replication, pathogenesis and development of vaccines. The present study was aimed to investigate the status of PCV2 infection in swine population in southeastern China by ELISA and PCR based on PCV2 ORF2, the genetic diversity of PCV2 strains from southeastern China originating from PMWS cases, and the effects of nuclear localization signals (NLS) of PCV ORF2 protein on viral replication in vitro and in vivo using the recombinant PCV viruses by reverse genetic approaches.The prevalence of PCV2 infection in swine herds in southeastern China was investigated by ELISA and PCR. Seroprevalence of PCV2 in samples collected from 89 swine herds was significantly higher by ELISA in post-weaning (54.1%) and growing piglets (49.9%) than that of suckling pigs (33.3%) with an average rate of 46.0%(819/1779). Seventy-eight cases out of 159 diseased pigs from these herds were PCV2 positive by PCR. To provide new insights into the extent of genetic heterogeneity of PCV2 isolates in southeastern China, the ORF2 genes of 27 isolates from the area from January 2004 to March 2007 were sequenced and aligned. While closely related to each other with identity of 98.0%-100%, these isolates displayed lower homologies to those from other regions of China or to some foreign isolates. Alignment of deduced amino acid sequences of capsid protein identified two major hypervariable regions (positions 53-91 and 185-215) in isolates obtained in this study, which were within or close to the putative epitope domains. The substitutions consequently resulted in higher hydrophilicity of the epitope region (positions 47-85). Phylogenetic analysis revealed two clusters of 48 isolates including those from Genbank:the larger cluster I consisting of two subgroups and cluster II containing most of foreign isolates owing to the residue substitutions in epitope domains (amino acid positions 80,86,88 and 91). While the subgroup Ib contained all the isolates with ORF2 of 705 bp in length, the 27 isolates we sequenced were clustered exclusively in subgroup la together with some other Chinese strains. We conclude that PCV2 isolates prevailing in southeastern China were genetically different from those of other countries.Since PCV2 ORF2 protein was synthesized in cytoplasm and imported into nucleus via its own nucleus localization signal (NLS) to participate in viral replication and assembly, it was predicted that PCV1 ORF2 protein contains several potential monopartite or bipartite nuclear localization signals in its N-terminus. Contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (eGFP). The C-terminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed throughout cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap, which was different from that of PCV2 Cap.We constructd a DNA clone that contains a PCV2 genome and partial duplication in pUC-18. Also, a chimeric PCV12 DNA clone was consucted, in which PCV1 ORF2 was replaced by PCV2 ORP2 in PCV1 genome. Other two chemeric DNA clones were constucted based on PCV2 and PCV12 DNA clones, in which the nuclear localization signal sequence of PCV2 ORF2 was replaced by that of PCV1 ORF2, resulting in PCV2-2NLS1 and PCV12-2NLS1 infectious clones. The transfection of PK-15 cells with each of the four DNA clones led to the expression of a functional virus genome, indicating that they are all infectious. To confirm the viability of the progeny viruses after initial transfection and to compare the in vitro replication levels, the four recombinant viruses were subjected to 14 passages in PK-15 cells and the virus titers reached 105.5 and 105 50% tissue culture infective doses (TCID50/ml) for progeny virues PCV2 (mPCV2) and PCV12. However, PCV2-2NLS1 and PCV12-2NLS1 had lower titers of only 103.5 TCID50/ml. To further determine the growth characteristics of the four viruses, a one-step growth curve was performed simultaneously for each virus. By 96 h postinfection, mPCV2 and PCV12 had a titer of 104.5 and 104 TCID50/ml respectively, while PCV2-2NLS1 and PCV12-2NLS1 titers were only 103 TCID50/ml, indicating that mPCV2 and PCV12 showed higher replication levels in vitro.To evaluate the immunogenicity and pathogenicity of the recombinant viruses, ninety-six 8 weeks-old BALB/c mice were randomly assigned into six groups,16 per group, and inoculated with the viruses or MEM as control. The results showed that they had similar level of antibody to PCV2 ORF2 although the mice inoculated with mPCV2 developed more viremic than those inoculated with PCV12. However, only a few mice inoculated PCV2-2NLS1 or PCV12-2NLS1 had low level of viremia and low PCV antibody response. Almost none of the mice in the study had lung lesions. Nevertheless, mild to moderate microscopic lesions in spleens of mice inoculated with wild type PCV2 and mPCV2 were observed, which were significantly different from those of mice in other groups (p<0.05). However, there were no statistical differences of spleen lesions between mice inoculated with three chimeric viruses and those in control group. Consistently, mice inoculated with wild type PCV2 and mPCV2 showed significant downshifts of the CD4+ and CD8+ T-cell subsets of peripheral blood lymphocytes compared with the control mice (p<0.05), while the proportions of the CD4+ and CD8+ T-cells in PCV 12 inoculated mice were similar to that of control mice (p>0.05). Thus, PCV 12 could serve as a vaccine candidate as it showed high replication level both in vitro and in vivo, and induced specific antibody response similar to that of wild PCV2 but was somehow attenuated in vivo. However, PCV2-2NLS1 and PCV12-2NLS1 showed only low level of replication in vitro or in vivo, indicating that replacement of N-terimus of PCV2 ORF2 containing NLS of PCV1 may result in the disruption of the structure or function of the Cap, and lead to low level of viral replication.In conclusion, PCV2 infections were spreading widely in southeastern China by an epidemiology study and the PCV2 isolates in this area were significantly different from those of other regions especially on its epitopes in ORF2 protein. And we first reported that the nuclear localization of PCV1 ORF2 protein was directed by two motifs (9RRRR12 and 25RRPYLAHPAFRNRYRWRRK43) in its N-terminus. The progeny virus of PCV12 displayed a low pathogenesis in mice copared with that of PCV2 SH04 and mPCV2, however, they all showed similar replicating characteristic and immunogenicity. The construction and application of infectious clones of PCV based on pUC-18 plasmid and infection of viruses in mice are useful for further study which may focus on the mutation of the key amino acids of PCV2 NLS rather than the whole N-terminal sequence, which can ensure the integrality and compatibility of ORF2 protein.更多还原