猪圆环病毒2型：流行毒株基因组序列分析及其对Toll样受体传导通路的影响
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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,其基因组为一单链环状DNA。是最小的动物病毒之一。根据PCV的致病性、抗原性及核苷酸序列,PCV可分为PCV1和PCV2两个型,PCV1对猪为非致病性的,但是PCV2与猪的很多疾病综合征有关,统称为猪圆环病毒病(Porcine circovirus disease, PCVD),以断奶仔猪多系统衰竭综合症(Post-weaning Multisystemic Wasting Syndrome, PMWS)最为常见,主要感染7-15周龄仔猪,临床症状包括消瘦、呼吸困难、腹泻、可视粘膜苍白、也会伴发皮炎,剖检可见淋巴结肿胀,肾脏损伤,具有高死亡率。
本研究的主要目的是：(1)在全基因组水平分析浙江地区猪圆环病毒2型的流行毒株的分子特征；(2)应用双报告基因以及荧光定量检测法,建立研究TLRs通路的技术体系；(3)初步探索猪圆环病毒2型基因组中影响Toll样受体传导通路的核酸片段。
1.本研究应用PCR方法对2008-2009年99份疑似病猪脾脏、肺脏、淋巴结混合组织样本进行PCV2检测,并以组织DNA为模板进行PCV2的全基因组扩增,克隆后进行测序。结果显示,2008与2009年浙江地区PCV2的平均阳性率47.5%,与往年相比基本持平。随机选取18份阳性病料进行PCV2全基因组测序分析,18个毒株均属于Group1,其中5个毒株(27.8%)属于1A分支,3个毒株(16.7%)属于1B分支,9个毒株(50%)属于1C分支。可见Group1依然是浙江地区PCV2的主导基因型,随着PCV2的流行,1C分支从无到有,并逐渐占据主导地位,但该分支的致病性特征还有待进一步研究。
2.为了建立与先天免疫相关的TLR通路研究平台,本研究利用TLRs的配体[Poly(I:C)、ODN1826]和胞内TLRs通路抑制剂[氯喹(Chloroquine,Chq)],运用双报告基因的相对荧光定量检测方法,通过分析NF-κB以及相关TLRsmRNA表达量的变化,建立研究TLRs通路的技术体系。50μg/mL Poly(I:C)与50μM ODN1826刺激RAW264.7后,细胞内NF-κB表达量分别比未刺激细胞增高238%与113%,差异极显著(P＜0.01)；相应TLR配体TLR3与TLR9的mRNA表达量是未刺激细胞的12.9与1.5倍,前者差异极显著(P＜0.01)。因此,利用双报告基因以及相对荧光定量检测方法研究TLRs通路是可行的。利用此方法初步研究了PCV2基因组中影响TLRs通路的核酸片段,结果表明ssDNA比dsDNA对NF-κB这一TLR通路上的关键分子影响更大。
综上所述,本研究初步探明了浙江地区PCV2流行毒株的分子特征,建立了研究先天免疫相关的TLR通路技术平台,探讨了PCV2基因组对TLR通路的影响。为深入研究PCV2氨基酸变异与病毒致病性关系、PCV2感染与免疫特性奠定了良好基础。
Porcine circovirus (PCV) is a member of the family Circoviridae, a recently established virus family composed of small, non-enveloped viruses, with a circular, single-stranded DNA genome, it is one of the smallest animal viruses. According to the pathogenicity, antigenicity and nucleotide sequence of PCV, PCV can be divided into two types, PCV1 and PCV2. 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 recently associated with a number of disease syndromes, which have been collectively named porcine circovirus diseases (PCVD). Post-weaning multisystemic wasting syndrome (PMWS) is the most common. PMWS is distributed worldwide, and occurred in 7-15 weeks old piglets. Its clinical symptoms include weight loss, respiratory problems, diarrhea, visible mucous membranes pale, dermatitis, Lymph node swelling and kidney damage are often seen upon necropsy. Its mortality rate is high, particularly when co-infected with other pathogens.
The main objectives of this study were (1) to characterize the genomic structure of PCV2 isolates prevalent in Zhejiang; (2) to establish a research platform to study TLRs pathway by using dual reporter gene assay and relative quantitative RT-PCR; and (3) to probe the effect of nucleotide fragments of PCV2 on the pathway of Toll-like receptors.
1. To gain insights into the molecular epidemiological characteristics of PCV2 isolates prevalent in Zhejiang, PCV2-specific PCR method was used for mixed tissue samples, including spleen, lung and lymph nodes of diseased pigs during 2008-2009. Eighteen PCV2 isolates were then randomly selected for sequencing and sequence analysis. Out of 99 samples,47 were PCV2 positive (47.5%), and PCV2-positive rates in 2008 and 2009 remained at similar level to those during 2004-2006. Among 18 PCV2 isolates sequenced,5 belonged to subgroup 1A (27.8%),3 to subgroup 1B (16.7%), and 9 to subgroup 1C (50%). Group 1 remains to be the predominant PCV2 genotype in Zhejiang. Remarkably, subgroup 1C isolates has emerged in the region in recent years, and account for the majority at present, suggesting that this genotype probably plays a predominant role in the prevalence of PCV2. However, the pathogenic features of subgroup 1C isolates require further study.
2. In order to set up a platform for research on TLR-mediated innate immune pathways, we used TLRs ligand (Poly (I:C) and ODN1826) as well as intracellular TLRs pathway inhibitor Chloroquine (Chq) as model molecules to detect NF-κB and TLRs mRNA expression levels by means of dual reporter gene assay and the relative quantitative RT-PCR. With 50μg/mL Poly (I:C) and 5μM ODN1826 stimulation, the cellular expression of NF-κB in RAW264.7 was significantly higher than unstimulated cells (238% and 113%, P＜0.01); the corresponding TLR ligands of TLR3 and TLR9 mRNA expression were also significantly higher than unstimulate cells (12.9(P<0.01) and 1.5). Therefore, the combination of dual reporter gene assay with quantitative RT-PCR may be used to examine the TLRs pathway by PCV2. We found that single strand DNA fragment of PCV2 was more potent than double strand DNA in inducing NF-κB expression.
In summary, the present study revealed the molecular characteristics of PCV2 isoaltes prevalent in recent years in Zhejiang. The dual reporter assay in combination with quantitative RT-PCR could be used to examine the effect of PCV2 nucelotide fragments on expression of molecules along the TLR pathways. These works has laid the foundation for studying the relationship between amino acid mutations and PCV2 pathogenicity as well as between PCV2 infection and host immune responses.
本研究的主要目的是：(1)在全基因组水平分析浙江地区猪圆环病毒2型的流行毒株的分子特征；(2)应用双报告基因以及荧光定量检测法,建立研究TLRs通路的技术体系；(3)初步探索猪圆环病毒2型基因组中影响Toll样受体传导通路的核酸片段。
1.本研究应用PCR方法对2008-2009年99份疑似病猪脾脏、肺脏、淋巴结混合组织样本进行PCV2检测,并以组织DNA为模板进行PCV2的全基因组扩增,克隆后进行测序。结果显示,2008与2009年浙江地区PCV2的平均阳性率47.5%,与往年相比基本持平。随机选取18份阳性病料进行PCV2全基因组测序分析,18个毒株均属于Group1,其中5个毒株(27.8%)属于1A分支,3个毒株(16.7%)属于1B分支,9个毒株(50%)属于1C分支。可见Group1依然是浙江地区PCV2的主导基因型,随着PCV2的流行,1C分支从无到有,并逐渐占据主导地位,但该分支的致病性特征还有待进一步研究。
2.为了建立与先天免疫相关的TLR通路研究平台,本研究利用TLRs的配体[Poly(I:C)、ODN1826]和胞内TLRs通路抑制剂[氯喹(Chloroquine,Chq)],运用双报告基因的相对荧光定量检测方法,通过分析NF-κB以及相关TLRsmRNA表达量的变化,建立研究TLRs通路的技术体系。50μg/mL Poly(I:C)与50μM ODN1826刺激RAW264.7后,细胞内NF-κB表达量分别比未刺激细胞增高238%与113%,差异极显著(P＜0.01)；相应TLR配体TLR3与TLR9的mRNA表达量是未刺激细胞的12.9与1.5倍,前者差异极显著(P＜0.01)。因此,利用双报告基因以及相对荧光定量检测方法研究TLRs通路是可行的。利用此方法初步研究了PCV2基因组中影响TLRs通路的核酸片段,结果表明ssDNA比dsDNA对NF-κB这一TLR通路上的关键分子影响更大。
综上所述,本研究初步探明了浙江地区PCV2流行毒株的分子特征,建立了研究先天免疫相关的TLR通路技术平台,探讨了PCV2基因组对TLR通路的影响。为深入研究PCV2氨基酸变异与病毒致病性关系、PCV2感染与免疫特性奠定了良好基础。
Porcine circovirus (PCV) is a member of the family Circoviridae, a recently established virus family composed of small, non-enveloped viruses, with a circular, single-stranded DNA genome, it is one of the smallest animal viruses. According to the pathogenicity, antigenicity and nucleotide sequence of PCV, PCV can be divided into two types, PCV1 and PCV2. 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 recently associated with a number of disease syndromes, which have been collectively named porcine circovirus diseases (PCVD). Post-weaning multisystemic wasting syndrome (PMWS) is the most common. PMWS is distributed worldwide, and occurred in 7-15 weeks old piglets. Its clinical symptoms include weight loss, respiratory problems, diarrhea, visible mucous membranes pale, dermatitis, Lymph node swelling and kidney damage are often seen upon necropsy. Its mortality rate is high, particularly when co-infected with other pathogens.
The main objectives of this study were (1) to characterize the genomic structure of PCV2 isolates prevalent in Zhejiang; (2) to establish a research platform to study TLRs pathway by using dual reporter gene assay and relative quantitative RT-PCR; and (3) to probe the effect of nucleotide fragments of PCV2 on the pathway of Toll-like receptors.
1. To gain insights into the molecular epidemiological characteristics of PCV2 isolates prevalent in Zhejiang, PCV2-specific PCR method was used for mixed tissue samples, including spleen, lung and lymph nodes of diseased pigs during 2008-2009. Eighteen PCV2 isolates were then randomly selected for sequencing and sequence analysis. Out of 99 samples,47 were PCV2 positive (47.5%), and PCV2-positive rates in 2008 and 2009 remained at similar level to those during 2004-2006. Among 18 PCV2 isolates sequenced,5 belonged to subgroup 1A (27.8%),3 to subgroup 1B (16.7%), and 9 to subgroup 1C (50%). Group 1 remains to be the predominant PCV2 genotype in Zhejiang. Remarkably, subgroup 1C isolates has emerged in the region in recent years, and account for the majority at present, suggesting that this genotype probably plays a predominant role in the prevalence of PCV2. However, the pathogenic features of subgroup 1C isolates require further study.
2. In order to set up a platform for research on TLR-mediated innate immune pathways, we used TLRs ligand (Poly (I:C) and ODN1826) as well as intracellular TLRs pathway inhibitor Chloroquine (Chq) as model molecules to detect NF-κB and TLRs mRNA expression levels by means of dual reporter gene assay and the relative quantitative RT-PCR. With 50μg/mL Poly (I:C) and 5μM ODN1826 stimulation, the cellular expression of NF-κB in RAW264.7 was significantly higher than unstimulated cells (238% and 113%, P＜0.01); the corresponding TLR ligands of TLR3 and TLR9 mRNA expression were also significantly higher than unstimulate cells (12.9(P<0.01) and 1.5). Therefore, the combination of dual reporter gene assay with quantitative RT-PCR may be used to examine the TLRs pathway by PCV2. We found that single strand DNA fragment of PCV2 was more potent than double strand DNA in inducing NF-κB expression.
In summary, the present study revealed the molecular characteristics of PCV2 isoaltes prevalent in recent years in Zhejiang. The dual reporter assay in combination with quantitative RT-PCR could be used to examine the effect of PCV2 nucelotide fragments on expression of molecules along the TLR pathways. These works has laid the foundation for studying the relationship between amino acid mutations and PCV2 pathogenicity as well as between PCV2 infection and host immune responses.
引文
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[1]Hirai T, Nunoya T, Ihara T, et al. Dual infection with PCV-2 and porcine epidemic diarrhoea virus in neonatal piglets[J]. The Veterinary Record.2001.148(15):482-484.
[2]Madec F, Rose N, Grasland B, et al. Post-weaning multisystemic wasting syndrome and other PCV2-related problems in pigs:a 12-year experience[J]. Transboundary and Emerging Diseases.2008.55(7):273-283.
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[4]Gillespie J, Opriessnig T, Meng XJ, et al. Porcine circovirus type 2 and porcine circovirus-associated disease[J]. Journal of Veterinary Internal Medicine.2009.23(6):1151-1163.
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[6]de Boisseson C, Beven V, Bigarre L, et al. Molecular characterization of Porcine circovirus type 2 isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs[J]. The Journal of General Virology.2004.85(2):293-304.
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[14]Larochelle R, Magar R, D'Allaire S. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions [J]. Virus Research.2002.90(1-2):101-112.
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[23]Grenfell BT, Pybus OG, Gog JR, et al. Unifying the epidemiological and evolutionary dynamics of pathogens[J]. Science.2004.303(5656):327-332.
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[1]Hirai T, Nunoya T, Ihara T, et al. Dual infection with PCV-2 and porcine epidemic diarrhoea virus in neonatal piglets[J]. The Veterinary Record.2001.148(15):482-484.
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[16]Pallares FJ, Halbur PG, Opriessnig T, et al. Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS)[J]. Journal of Veterinary Diagnostic Investigation.2002.14(6):515-519.
[17]Drolet R, Larochelle R, Morin M, et al. Detection rates of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia[J]. Veterinary Pathology.2003.40(2):143-148.
[18]Grau-Roma L, Crisci E, Sibila M, et al. A proposal on porcine circovirus type 2 (PCV2)
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[20]Shi KC, Guo X, Ge XN, et al. Cytokine mRNA expression profiles in peripheral blood mononuclear cells from piglets experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2[J]. Veterinary Microbiology. 140(1-2):155-160.
[21]Li WL, Wang XW, Ma T, et al. Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009:genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China[J]. Virus Genes.2010.40:244-251.
[22]Cheung AK. Homologous recombination within the capsid gene of porcine circovirus type 2 subgroup viruses via natural co-infection[J]. Archives of Virology.2009.154(3):531-534.
[23]Grenfell BT, Pybus OG, Gog JR, et al. Unifying the epidemiological and evolutionary dynamics of pathogens[J]. Science.2004.303(5656):327-332.
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