蓝舌病毒BTV-HbC_3株与蓝舌病毒BTV-10型的比较研究
摘要
蓝舌病毒(Bluetongue Virus，BTV)是呼肠孤病毒科环状病毒属的成员，由昆虫传播，感染牛、羊等野生反刍动物。该病毒全世界己发现25个血清型，给畜牧业造成了严重的经济损失。BTV含10分子的双链RNA(dsRNA)作基因组，分别编码病毒7个结构多肽(VP1，VP2，VP3，VP4，VP5，VP6，VP7)和3个非结构蛋白多肽(NS1，NS2，NS3)。外衣壳由两种主要多肽VP2和VP5构成，内衣壳主要由VP3和VP7两种多肽构成，同时内衣壳包绕三种少量蛋白VP1、VP4和VP6以及基因组。VP7蛋白携带有群特异性抗原决定簇，在病毒装配中起重要作用，占衣壳蛋白组成的36％。由该病毒群特异性抗原决定簇产生的抗体多数是针对VP7的，且S7基因非常保守，是BTV新型疫苗的主要研究对象。VP2蛋白是蓝舌病毒的主要结构多肽，含有型特异性抗原决定簇，诱导机体产生中和抗体。
蓝舌病毒BTV-HbC_3株是我们实验室于1994年在襄樊某畜牧场所分离到的一株病毒。本研究通过对BTV-10型和BTV-HbC_3的增殖特征、免疫学特征、形态学特征以及分子生物学方面的比较进一步证实了BTV-HbC_3是一株蓝舌病毒，并且可能是一株新的基因型毒株。
1、蓝舌病毒BTV-HbC_3在Vero、MCF-7和Hep-3B细胞上的增殖特点以及与BTV-10的免疫学关系。发现BTV-HbC_3株和BTV-10株感染细胞后在48hr之内均出现CPE，表现为细胞收缩变圆、间隔变大等特征。通过琼脂糖双向扩散实验显示BTV-HbC_3和BTV-10型编码共同抗原。
2、根据已发表的BTV-10标准株S7基因序列，通过Primer Premier 5.0软件设计合成一对覆盖BTV-10 S7片段13.303bp之间区段的引物，经逆转录．聚合酶链反应(RT-PCR)扩增出蓝舌病毒BTV-HbC_3株和BTV-10型的S7基因5′非编码区(NonCoding Region，NCR)cDNA片段，以建立起dsRNA体外扩增系统。将扩增的BTV-HbC_3毒株的S7基因5′非编码区片段通过粘端连接克隆到pUCm-T载体中，用PCR技术和限制性内切酶分析鉴定，表明获得重组质粒pUCm-T-BTV-HbC_3-S7。在ABI PRISM377全自动序列分析仪上测序得到蓝舌病毒BTV-HbC_3株和BTV-10型S7
片段5‘一NCR区域分别长为277bp和290bp的产物。通过Blast软件将两株病毒核昔
酸序列在GenBank上比对发现，BTV一HbC3株57基因5‘非编码区与呼肠孤病毒一3
(Reovirus一3)型LZ片段3464bp一3739bp序列相同;然而就这一段序列比较而言，
BTV一HbC3株和BTV一10型之间并无同源。也许BTV一HbC3和BTV一10起源于两个基因
群，或是在双链RNA病毒中RNA多聚酶缺乏严格的校正活性以及在进化过程中与呼
肠孤病毒一3发生基因重配(reassortment)插入了一段呼肠孤病毒一3的序列使然。
3、MTT法确证了BTV一HbC3株和BTV一10型对乳腺癌细胞MCF一7有较强的抗增
殖作用;将两株病毒分别以1 MOI接种MCF一7细胞，通过流式细胞术检测凋亡率分
别为31.9%和29.1%。结果表明BTV一HbC3株和BTV一10型体外可以杀乳腺癌MCF一7
细胞。
结论:通过病毒与细胞相互作用的生物学特征、病毒分子生物学和免疫学特征等
进行系统研究，揭示出:BTV一HbC3与BTV一10能够感染肿瘤细胞，并且表现出基本相
同的细胞生物学特征和形态学特征;BTV一HbC3和BTV一10型编码共同抗原，由于VP7
是BTV的群特异性抗原，我们认为二者的共同抗原为VP7;利用BTV一10 57片段5
‘非编码区13一3O3bp区段设计的一对特异性引物能有效地扩增合成BTV一HbC3 277bp
大小特异性片段，所扩增片段大小与BTV一10并不相同;序列分析表明BTV一HbC3株
s7基因的5‘一NcR 277bp核昔酸与呼肠孤病毒一3 LZ基因3464一3739bP序列相同，替
换了BTV的57片段s‘一CR 13一303bp长为29obp的序列。这些结果表明BTV一HbC3
株是蓝舌病毒，并有可能是一种新的基因型蓝舌病毒。
Bluetongue virus (BTV) is the type species (one of twenty different species) of the genus Orbivirus, within the family Reoviridae. BTV can infect most ruminants and even some predatory carnivores. The virus can also infect and is transmitted by certain species of biting midges (Culicoides species) and is divided into 25 distinct serotypes in the world. BTV is a double-shelled virus with a genome consisting of 10 dsRNA segment. The virus has an icosahedral core that contains two major (VP7 and VP3) and three minor (VP1,VP4 and VP6) structural proteins. VP7 is a dominant BTV serogroup-reactive antigen and is the major, constituent of capsomers on the surface of core particles, accounting for 36% of capside protein. And most amino acids of VP7 are conserved being the major research target as vaccine of BTV. The core is surrounding by an outer layer composed of two structural proteins. VP2 and VP5. In addition to seven structural proteins, BTV infected cells contain three virus-specific,non-structural proteins (
NS1,NS2 and NS3).
BTV-HbC3 strain is isolated by Dr. Dong Changyuan in Xiangfan City Hube Province in 1994. With the systemic analysis of our lab that the replicated characteristics of BTV-HbC3 between BTV-10 in different cells; the genome characteristics; the argrose diffusion test about immune across reaction between BTV-HbC3 and BTV-10; the characteristics of the sequence of RT-PCR of BTV-HbC3 S7 5' -NCR which proved that BTV-HbC3 strain is probably a new genome type of Bluetongue Virus.
1. With the replicated and morphologic characteristic of BTV-HbC3 infecting different cells such as Vero, MCF-7 and Hep-3B ,all the cells displayed a rounded, granular phenotype having the same CPE(characteristics of cytopathic effects) with that of BTV-10.Through agar gel immunodiffusion test, the cross-reactivities were detected between BTV-HbC3 and BTV-10 indicating that the common antigenic determinants on the VP7 proteins of BTV-HbC3 and BTV-10 shared some epitopes.
2. Using primers corresponding to the conserved sequence of BTV genomic dsRNA
segment previously published, S7 5' -NCR of BTV-HbC3 and BTV-10 were amplified by RT-PCR. And the PCR products is 277bp and 290bp respectively. Comparative sequence analyses of 5' -NCR show that 100% of the nucleotides are conserved between BTV-HbC3 and Reovirus-3 L2, while none of the bases are identical between BTV-HbC3 and BTV-10. In this study, partial sequence of S7 segment permitted us to investigate the role of genetic exchange(reassortment and recombination) in the evolutionary mechanisma deriving the evolution of the genus Orbivirus. Pehaps BTV-HbC3 and BTV-10 originated from two gene pools or some sequences of Reovirus-3 were inserted into BTV-HbC3 undergo genetic drift which contributed to diversification of individual gene segments of field strains of BTV.
3. Flow cytometry (FCM) and MTT analysis was used to detect the CPE of MCF-7 which was infected by BTV-HbC3 and BTV-10 strain with 1MOI. And the results demonstrated that the apoptosis rate is 31.9% and 29.1% respectively. The CPE of cells infected by these two BTV strain indicated that BTV can induce tumour cells qpoptosis, and
it is the foundation which we use them as oncolytic virus theraping cancer.
Conclusions: With the replicated and morphologic characteristic of BTV-HbC3 and BTV-10 infecting different cells, agar gel immunodifrusion test between BTV-HbC3 and BTV-10, the analysis of the sequence results of BTV-HbC3 and BTV-10 S7 5' -NCR showed that BTV-HbC3 is the prototype virus of the Orbivirus genus, probably a new genome type of Bluetongue Virus.
蓝舌病毒BTV-HbC_3株是我们实验室于1994年在襄樊某畜牧场所分离到的一株病毒。本研究通过对BTV-10型和BTV-HbC_3的增殖特征、免疫学特征、形态学特征以及分子生物学方面的比较进一步证实了BTV-HbC_3是一株蓝舌病毒，并且可能是一株新的基因型毒株。
1、蓝舌病毒BTV-HbC_3在Vero、MCF-7和Hep-3B细胞上的增殖特点以及与BTV-10的免疫学关系。发现BTV-HbC_3株和BTV-10株感染细胞后在48hr之内均出现CPE，表现为细胞收缩变圆、间隔变大等特征。通过琼脂糖双向扩散实验显示BTV-HbC_3和BTV-10型编码共同抗原。
2、根据已发表的BTV-10标准株S7基因序列，通过Primer Premier 5.0软件设计合成一对覆盖BTV-10 S7片段13.303bp之间区段的引物，经逆转录．聚合酶链反应(RT-PCR)扩增出蓝舌病毒BTV-HbC_3株和BTV-10型的S7基因5′非编码区(NonCoding Region，NCR)cDNA片段，以建立起dsRNA体外扩增系统。将扩增的BTV-HbC_3毒株的S7基因5′非编码区片段通过粘端连接克隆到pUCm-T载体中，用PCR技术和限制性内切酶分析鉴定，表明获得重组质粒pUCm-T-BTV-HbC_3-S7。在ABI PRISM377全自动序列分析仪上测序得到蓝舌病毒BTV-HbC_3株和BTV-10型S7
片段5‘一NCR区域分别长为277bp和290bp的产物。通过Blast软件将两株病毒核昔
酸序列在GenBank上比对发现，BTV一HbC3株57基因5‘非编码区与呼肠孤病毒一3
(Reovirus一3)型LZ片段3464bp一3739bp序列相同;然而就这一段序列比较而言，
BTV一HbC3株和BTV一10型之间并无同源。也许BTV一HbC3和BTV一10起源于两个基因
群，或是在双链RNA病毒中RNA多聚酶缺乏严格的校正活性以及在进化过程中与呼
肠孤病毒一3发生基因重配(reassortment)插入了一段呼肠孤病毒一3的序列使然。
3、MTT法确证了BTV一HbC3株和BTV一10型对乳腺癌细胞MCF一7有较强的抗增
殖作用;将两株病毒分别以1 MOI接种MCF一7细胞，通过流式细胞术检测凋亡率分
别为31.9%和29.1%。结果表明BTV一HbC3株和BTV一10型体外可以杀乳腺癌MCF一7
细胞。
结论:通过病毒与细胞相互作用的生物学特征、病毒分子生物学和免疫学特征等
进行系统研究，揭示出:BTV一HbC3与BTV一10能够感染肿瘤细胞，并且表现出基本相
同的细胞生物学特征和形态学特征;BTV一HbC3和BTV一10型编码共同抗原，由于VP7
是BTV的群特异性抗原，我们认为二者的共同抗原为VP7;利用BTV一10 57片段5
‘非编码区13一3O3bp区段设计的一对特异性引物能有效地扩增合成BTV一HbC3 277bp
大小特异性片段，所扩增片段大小与BTV一10并不相同;序列分析表明BTV一HbC3株
s7基因的5‘一NcR 277bp核昔酸与呼肠孤病毒一3 LZ基因3464一3739bP序列相同，替
换了BTV的57片段s‘一CR 13一303bp长为29obp的序列。这些结果表明BTV一HbC3
株是蓝舌病毒，并有可能是一种新的基因型蓝舌病毒。
Bluetongue virus (BTV) is the type species (one of twenty different species) of the genus Orbivirus, within the family Reoviridae. BTV can infect most ruminants and even some predatory carnivores. The virus can also infect and is transmitted by certain species of biting midges (Culicoides species) and is divided into 25 distinct serotypes in the world. BTV is a double-shelled virus with a genome consisting of 10 dsRNA segment. The virus has an icosahedral core that contains two major (VP7 and VP3) and three minor (VP1,VP4 and VP6) structural proteins. VP7 is a dominant BTV serogroup-reactive antigen and is the major, constituent of capsomers on the surface of core particles, accounting for 36% of capside protein. And most amino acids of VP7 are conserved being the major research target as vaccine of BTV. The core is surrounding by an outer layer composed of two structural proteins. VP2 and VP5. In addition to seven structural proteins, BTV infected cells contain three virus-specific,non-structural proteins (
NS1,NS2 and NS3).
BTV-HbC3 strain is isolated by Dr. Dong Changyuan in Xiangfan City Hube Province in 1994. With the systemic analysis of our lab that the replicated characteristics of BTV-HbC3 between BTV-10 in different cells; the genome characteristics; the argrose diffusion test about immune across reaction between BTV-HbC3 and BTV-10; the characteristics of the sequence of RT-PCR of BTV-HbC3 S7 5' -NCR which proved that BTV-HbC3 strain is probably a new genome type of Bluetongue Virus.
1. With the replicated and morphologic characteristic of BTV-HbC3 infecting different cells such as Vero, MCF-7 and Hep-3B ,all the cells displayed a rounded, granular phenotype having the same CPE(characteristics of cytopathic effects) with that of BTV-10.Through agar gel immunodiffusion test, the cross-reactivities were detected between BTV-HbC3 and BTV-10 indicating that the common antigenic determinants on the VP7 proteins of BTV-HbC3 and BTV-10 shared some epitopes.
2. Using primers corresponding to the conserved sequence of BTV genomic dsRNA
segment previously published, S7 5' -NCR of BTV-HbC3 and BTV-10 were amplified by RT-PCR. And the PCR products is 277bp and 290bp respectively. Comparative sequence analyses of 5' -NCR show that 100% of the nucleotides are conserved between BTV-HbC3 and Reovirus-3 L2, while none of the bases are identical between BTV-HbC3 and BTV-10. In this study, partial sequence of S7 segment permitted us to investigate the role of genetic exchange(reassortment and recombination) in the evolutionary mechanisma deriving the evolution of the genus Orbivirus. Pehaps BTV-HbC3 and BTV-10 originated from two gene pools or some sequences of Reovirus-3 were inserted into BTV-HbC3 undergo genetic drift which contributed to diversification of individual gene segments of field strains of BTV.
3. Flow cytometry (FCM) and MTT analysis was used to detect the CPE of MCF-7 which was infected by BTV-HbC3 and BTV-10 strain with 1MOI. And the results demonstrated that the apoptosis rate is 31.9% and 29.1% respectively. The CPE of cells infected by these two BTV strain indicated that BTV can induce tumour cells qpoptosis, and
it is the foundation which we use them as oncolytic virus theraping cancer.
Conclusions: With the replicated and morphologic characteristic of BTV-HbC3 and BTV-10 infecting different cells, agar gel immunodifrusion test between BTV-HbC3 and BTV-10, the analysis of the sequence results of BTV-HbC3 and BTV-10 S7 5' -NCR showed that BTV-HbC3 is the prototype virus of the Orbivirus genus, probably a new genome type of Bluetongue Virus.
引文
[1] 董长垣．现代分子病毒学[M]．武汉：武汉大学出版社，1996．184-185．
[2] Loudon PT, Roy R Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins[J]. Virology, 1991, 180(2):798-802.
[3] Monastyrsjaya K, Staeuber N, et al. Effect of domain-switching and site-directed mutagens is on the properties and functions of the VP7 proteins of two orbivirus[J]. Virology, 1997, 237(2):217-227.
[4] 鄂征．组织培养和分子细胞学技术[M]．北京：科学出版社，1996，78-98
[5] Timothy F, Kowalik, Li J K. K. Bluetongue Virus Evolution. Sequence Analysis of the Genomic S7 Segment and Major Core Protein VP7[J]. Virology, 1991, 181:749-755.
[6] Wilson WC, Ma HC, Venter EH, et al. Phylogenetic relationships of bluetongue viruses based on gene S7[J]. Virus Res, 2000, 67(2):141-151.
[7] Bonneau KR, Zhang NZ, Wilson WC, et al. Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype[J]. Arch Virol, 2000;145(6): 1163-1171.
[8] Norman KL, Lee PW. Reovirus as a novel oncolytic agent[J]. J Clin Invest. 2000, 105(8): 1035-1038.
[9] Joensuu H, Pylkkaenen L, Toikkanen S. Bcl-2 protein expression and long-term survival in breast cancer[J]. Am J Pathol, 1994, 145:1191-1194
[10] Hiramatus N, Hayashi N, Katayama K, et al. Immunohistochemical detection of Fas antigen in liver tissue of patient with chronmic hepatitis C. Hepatology, 1994, 19:1354-1359.
[11] Willims GT, Smith CA. Molecular regulation of apoptosis: genetic control on cell death. Cell, 1993, 74:777-782
[12] Chirillo P, Natoli G, Falco M, et al. P53 status, pX induced hepatocytes transformation. Hepatology, 1995, 22pt. 2: 157A-163.
[2] Loudon PT, Roy R Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like proteins[J]. Virology, 1991, 180(2):798-802.
[3] Monastyrsjaya K, Staeuber N, et al. Effect of domain-switching and site-directed mutagens is on the properties and functions of the VP7 proteins of two orbivirus[J]. Virology, 1997, 237(2):217-227.
[4] 鄂征．组织培养和分子细胞学技术[M]．北京：科学出版社，1996，78-98
[5] Timothy F, Kowalik, Li J K. K. Bluetongue Virus Evolution. Sequence Analysis of the Genomic S7 Segment and Major Core Protein VP7[J]. Virology, 1991, 181:749-755.
[6] Wilson WC, Ma HC, Venter EH, et al. Phylogenetic relationships of bluetongue viruses based on gene S7[J]. Virus Res, 2000, 67(2):141-151.
[7] Bonneau KR, Zhang NZ, Wilson WC, et al. Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype[J]. Arch Virol, 2000;145(6): 1163-1171.
[8] Norman KL, Lee PW. Reovirus as a novel oncolytic agent[J]. J Clin Invest. 2000, 105(8): 1035-1038.
[9] Joensuu H, Pylkkaenen L, Toikkanen S. Bcl-2 protein expression and long-term survival in breast cancer[J]. Am J Pathol, 1994, 145:1191-1194
[10] Hiramatus N, Hayashi N, Katayama K, et al. Immunohistochemical detection of Fas antigen in liver tissue of patient with chronmic hepatitis C. Hepatology, 1994, 19:1354-1359.
[11] Willims GT, Smith CA. Molecular regulation of apoptosis: genetic control on cell death. Cell, 1993, 74:777-782
[12] Chirillo P, Natoli G, Falco M, et al. P53 status, pX induced hepatocytes transformation. Hepatology, 1995, 22pt. 2: 157A-163.