牦牛口蹄疫病毒持续感染分离株主要功能蛋白基因的变异研究
摘要
在国内首次用全序列已知的原始毒株Akesu／58通过舌面穿刺感染牦牛，建立了FMDV持续感染动物模型，定期刮取食道／咽部(Oesophageal-Pharyngeal，O／P)黏液、采集血清，并跟踪检测了12个月，同时采用反转录—聚合酶链反应(RT-PCR)、分子克隆和核酸序列测定技术对其中阳性样品的VP1、L、3ABC基因进行了序列测定，通过计算机软件对序列进行比较分析，绘制系统发生树。结果表明，所感染的5头牦牛均持续带毒，只是由于个体差异，带毒的时间长短不一。在测定的持续感染毒株中，VP1、L、3ABC基因发生了不同程度的变异，其中VP1变异最大，并且细胞受体结合位点由原始毒株的SGD变为了RGD，这可能是发生了适应性突变。对于不同的个体，3ABC特别3A基因变化较大，它的变异有可能造成毒株对宿主适应性的改变。此外，将RT-PCR检测结果与血清中非结构蛋白抗体的检测结果相比较，发现非结构蛋白抗体水平较高的个体，持续带毒的可能性也越大，但两者之间没有线性化关系。
This is the first time in our country to construct animal model of FMDV persistent infection, five yaks were infected with the oringinal strain Akesu/58 that the full-length genome has known by puncturing in the tongue, then Oesophageal-Pharyngeal(O/P) fluid and serum were collected and detected, lasting 12 months. Towards the positive samples, by RT-PCR, molecular cloning, sequencing and biologic software, VP1, L and 3ABC genes sequences were achieved and analyzed. The results showed that the five infected yaks were in carrier state, but persistent time was various because of individual difference among them. VP1, L and 3 ABC genes of persistence strains had incoordinate variation and VP1 gene was the most evident. The central tripeptide of binding cell receptor in these strains was Arg-Gly-Asp(RGD) instead of Ser-Gly-Asp(SGD) in oringinal strain. This may be the result of adaptive mutation. As for different hosts, 3 ABC, especially 3 A gene, varied obviously and its variation contributed to the changes in the hosts of virus. In addition, the comparison of the result of RT-RCR and non-structural (NS) protein antibody in serum showed that the higher NS protein antibody was, the more possible persistent infection was, but there was not linear relationship between them.
This is the first time in our country to construct animal model of FMDV persistent infection, five yaks were infected with the oringinal strain Akesu/58 that the full-length genome has known by puncturing in the tongue, then Oesophageal-Pharyngeal(O/P) fluid and serum were collected and detected, lasting 12 months. Towards the positive samples, by RT-PCR, molecular cloning, sequencing and biologic software, VP1, L and 3ABC genes sequences were achieved and analyzed. The results showed that the five infected yaks were in carrier state, but persistent time was various because of individual difference among them. VP1, L and 3 ABC genes of persistence strains had incoordinate variation and VP1 gene was the most evident. The central tripeptide of binding cell receptor in these strains was Arg-Gly-Asp(RGD) instead of Ser-Gly-Asp(SGD) in oringinal strain. This may be the result of adaptive mutation. As for different hosts, 3 ABC, especially 3 A gene, varied obviously and its variation contributed to the changes in the hosts of virus. In addition, the comparison of the result of RT-RCR and non-structural (NS) protein antibody in serum showed that the higher NS protein antibody was, the more possible persistent infection was, but there was not linear relationship between them.
引文
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Immunal, 1975, 115: 1636-1641.
[12] Mason P W, Pieder E, Baxt B. RGD sequence of foot-and-mouth disease virus is essential for infecting cell via the natural receptor but can be bypassed by antibody dependent enhancement pathway [J]. Proc Natl Acad Sci USA, 1994, 91: 1932-1936.
[13] Lippert M, Beck E, Weiland F, et al. Point mutations within the G-H loop of foot-and-mouth disease virus 01K after virus attachment [J]. J Virol, 1997, 2: 1046-1051.
[14] 刘在新．口蹄疫病毒基因组及其编码蛋白一级结构研究[D]．兰州：中国农业科学院兰州兽医研究所，2002．
[15] Piccone M E, Rieder E, Mason PW, et al. The foot-and-mouth disease virus leader proteinase gene is not required for viral qeplication[J]. J Virol, 1995, 69: 5376-5382.
[16] 孙乃恩，孙东旭，朱德煦．分子遗传学[M]．南京：南京大学出版社，1996，178-180．
[17] Cao X M, Bergmann I E, Beck E. Comparison of the 5' and 3' untranslated genomic regions of virulent and attenuated foot-and-mouth disease viruses strains O1 Campos and C3 Resende[J].J ournal of general virology, 1991, 72: 2821-2825.
[18] Falk MSobrino F, Beck E. Vpg gene amplication correlates with infective particle formation in foot-and-mouth disease virus [J]. J Virol, 1992, 66: 2251-2260.
[19] 江鹏斐，赵启祖，谢庆阁译．区分口蹄疫感染动物和注苗动物．家畜口蹄疫学术讨论会论文集[C]．兰州：中国农业科学院兰州兽医研究所，2000，394-398．
[2] Geysem H M, Meloem R H, Barteling S J, et al. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid [J]. Proc Nati Acad Sci, 1984, 81: 3998-4002.
[3] Medina M, Domingo E, Brangwyn J K, et al. The Two Species of the Foot-and-Mouth Disease Virus Leader Protein, Expressed Individually, Exhibit Same Activitie [J]. Virology, 1993, 194: 355-359.
[4] Piccone M E, Zellner M, Komosinski TF, et al. Identification of the Activesite residues of the L Proteinase of Foot-and-Mouth Disease Virus[J]. J Virol, 1995, 69: 4950-4956.
[5] Piccone M E, Rieder E, Mason P W, et al. The foot-and-mouth disease virus leader proteinase gene is not required for viral replication [J]. J Virol, 1995, 69: 5376-5382.
[6] 张显升．口蹄疫病毒China／99基因组全序列及结构与功能分析[D]．兰州：中国农业科学院兰州兽医研究所，2002．
[7] Sobrino F, Saizm M, Jimenez-Clarero M A, et al. Foot-and-mouth disease virus: a long known virus, but a current threat [J]. Vet Res, 2001, 3: 21-30.
[8] Vosloo W, Kirkbride E, Bengis R G, et al. Genome Variation in the SAT types of foot-and-mouth disease viruses prevalent in buffalo (Syncerus caffer) in the Kruger National Park and other regions of southern Africa, 1986-93[J]. Epidemiol Infect, 1995, 114: 203-218.
[9] Dawe P S, Sorensen K, Ferris N P, et al. Experimental transmission of foot-and-mouth disease virus from carrier African buffalo(syncerus caffer) to cattle in Zimbabwe. Vet Res, 1994, 134(9): 211-215.
[10] Pfaff E, Mussgay M, Bohm H O, et al. Antibodies against a preselected peptide recognize and neutralize foot-and-mouth disease virus[J]. EMBO J, 1982, 1(7): 869-874.
[11] Bachrach H D, Moore D M, Mekercher P D, et al. Immune and antibody responses to an isolation capsid protein of foot-and-mouth disease virus [J]. J
Immunal, 1975, 115: 1636-1641.
[12] Mason P W, Pieder E, Baxt B. RGD sequence of foot-and-mouth disease virus is essential for infecting cell via the natural receptor but can be bypassed by antibody dependent enhancement pathway [J]. Proc Natl Acad Sci USA, 1994, 91: 1932-1936.
[13] Lippert M, Beck E, Weiland F, et al. Point mutations within the G-H loop of foot-and-mouth disease virus 01K after virus attachment [J]. J Virol, 1997, 2: 1046-1051.
[14] 刘在新．口蹄疫病毒基因组及其编码蛋白一级结构研究[D]．兰州：中国农业科学院兰州兽医研究所，2002．
[15] Piccone M E, Rieder E, Mason PW, et al. The foot-and-mouth disease virus leader proteinase gene is not required for viral qeplication[J]. J Virol, 1995, 69: 5376-5382.
[16] 孙乃恩，孙东旭，朱德煦．分子遗传学[M]．南京：南京大学出版社，1996，178-180．
[17] Cao X M, Bergmann I E, Beck E. Comparison of the 5' and 3' untranslated genomic regions of virulent and attenuated foot-and-mouth disease viruses strains O1 Campos and C3 Resende[J].J ournal of general virology, 1991, 72: 2821-2825.
[18] Falk MSobrino F, Beck E. Vpg gene amplication correlates with infective particle formation in foot-and-mouth disease virus [J]. J Virol, 1992, 66: 2251-2260.
[19] 江鹏斐，赵启祖，谢庆阁译．区分口蹄疫感染动物和注苗动物．家畜口蹄疫学术讨论会论文集[C]．兰州：中国农业科学院兰州兽医研究所，2000，394-398．