人微小病毒B19结构蛋白VP1/VP2区基因克隆及序列分析
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摘要
人微小病毒B19（human parvovirus B19，PVB19）是微小病毒科微
小病毒属中目前已知的唯一致人类疾病的病毒，也是动物病毒中体积最
小结构最简单的DNA病毒。现已证实该病毒同多种儿童及成人疾病有关，
且随着检测手段的改进及研究的深入，其疾病谱还在不断扩大。
本研究应用巢式聚合酶链反应（Nest-PCR）对473例临床标本进行
了检测，共发现17例阳性标本。其阳性疾病谱分别为过敏性紫癜1例，
急性淋巴细胞白血病2例，急性非淋巴细胞白血病1例，再生障碍性贫
血1例，肝癌术后1例，乙型病毒性肝炎4例，丙型病毒性肝炎1例，
非甲-戊型肝炎2例，查体（无症状）2例，皮疹1例，脑炎1例。
17例阳性标本中有2例的临床资料完整而清晰，因此，为初步了解
PVB19病毒广东分离株的基因变异情况，我们用加强聚合酶链反应
（re-PCR）扩增了这2例阳性标本（一例为活动性肝硬化伴再生障碍性贫
血患者，另一例为患急性淋巴细胞白血病的儿童）的PVB19病毒结构蛋白
VP1/VP2区（2909-3806 nt）长约898bp的基因片段。该目的片段经TA
克隆后进行核苷酸序列测定。测定序列与标准序列比较后发现：同一时
间，在广东地区分离的PVB19病毒株核苷酸变异率有很大差别，两株病
毒在该区段的核苷酸变异数分别为32和10，对应的氨基酸变异数分别
为6和1。通过与Genbank中已有的该区段核苷酸序列进行种系发生树
分析后发现PVB19病毒中国流行株属种系发生树分型的Ⅰ型且与欧美、
日本、韩国流行株在进化速度上有很大差异。另外还发现PVB19病毒株
种系发生树分型结果与感染PVB19后的临床表现关系不明确。
Human parvovirus B19 (PVB 19), the only parvovirus known to
be pathogenic in human , belongs to the genus Parvovirus in the family
Parvoviridae. It is the smallest animal DNA virus. PVB 19 infection often
results in a variety of clinical manifestations in adults and children . The
objective of this research was to preliminary study the variation of PVB19
in Guangdong Province. Firstly, we used Nest-PCR method to detect 496
serum samples obtained from 473 patients. 18 sampels obtained from 17
patients were positive for PVB 19 DNA. Of the 17 patients, two had clear
clinical record. one was a child with acute lymphocytic leukemia, the other
was a adult with chronic active hepatitis B associated with aplastic
anemia .Then a methodology combined with re-PCR amplifying, TA cloning
and nucleotide sequencing was used to analyses the 898bp fragment
(2909-3806 nt) of the two strains Compared with standard strain, the rate
of nucleotide variability of the two strains was very different, even the. two
strains were isolated in Guangdong Province in the same time. We also
compared the two strain抯 fragmentes with other associated strains抯
fragment, which was searched from Genbank . These data suggest that
PVB 19 isolates from China were significantly different from other isolates at
both the nucleotide and prediected amino acid levels, and that no particular
genotype of PVB19 was associated with the particular clinical outcome.
小病毒属中目前已知的唯一致人类疾病的病毒，也是动物病毒中体积最
小结构最简单的DNA病毒。现已证实该病毒同多种儿童及成人疾病有关，
且随着检测手段的改进及研究的深入，其疾病谱还在不断扩大。
本研究应用巢式聚合酶链反应（Nest-PCR）对473例临床标本进行
了检测，共发现17例阳性标本。其阳性疾病谱分别为过敏性紫癜1例，
急性淋巴细胞白血病2例，急性非淋巴细胞白血病1例，再生障碍性贫
血1例，肝癌术后1例，乙型病毒性肝炎4例，丙型病毒性肝炎1例，
非甲-戊型肝炎2例，查体（无症状）2例，皮疹1例，脑炎1例。
17例阳性标本中有2例的临床资料完整而清晰，因此，为初步了解
PVB19病毒广东分离株的基因变异情况，我们用加强聚合酶链反应
（re-PCR）扩增了这2例阳性标本（一例为活动性肝硬化伴再生障碍性贫
血患者，另一例为患急性淋巴细胞白血病的儿童）的PVB19病毒结构蛋白
VP1/VP2区（2909-3806 nt）长约898bp的基因片段。该目的片段经TA
克隆后进行核苷酸序列测定。测定序列与标准序列比较后发现：同一时
间，在广东地区分离的PVB19病毒株核苷酸变异率有很大差别，两株病
毒在该区段的核苷酸变异数分别为32和10，对应的氨基酸变异数分别
为6和1。通过与Genbank中已有的该区段核苷酸序列进行种系发生树
分析后发现PVB19病毒中国流行株属种系发生树分型的Ⅰ型且与欧美、
日本、韩国流行株在进化速度上有很大差异。另外还发现PVB19病毒株
种系发生树分型结果与感染PVB19后的临床表现关系不明确。
Human parvovirus B19 (PVB 19), the only parvovirus known to
be pathogenic in human , belongs to the genus Parvovirus in the family
Parvoviridae. It is the smallest animal DNA virus. PVB 19 infection often
results in a variety of clinical manifestations in adults and children . The
objective of this research was to preliminary study the variation of PVB19
in Guangdong Province. Firstly, we used Nest-PCR method to detect 496
serum samples obtained from 473 patients. 18 sampels obtained from 17
patients were positive for PVB 19 DNA. Of the 17 patients, two had clear
clinical record. one was a child with acute lymphocytic leukemia, the other
was a adult with chronic active hepatitis B associated with aplastic
anemia .Then a methodology combined with re-PCR amplifying, TA cloning
and nucleotide sequencing was used to analyses the 898bp fragment
(2909-3806 nt) of the two strains Compared with standard strain, the rate
of nucleotide variability of the two strains was very different, even the. two
strains were isolated in Guangdong Province in the same time. We also
compared the two strain抯 fragmentes with other associated strains抯
fragment, which was searched from Genbank . These data suggest that
PVB 19 isolates from China were significantly different from other isolates at
both the nucleotide and prediected amino acid levels, and that no particular
genotype of PVB19 was associated with the particular clinical outcome.
引文
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3． PattisonJR, Jones SE, Hodgson J et al. Parvovirus infections and hypoplastic crisis in sickle-cell anaemia. Lancet 1981:1:664-665
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5． White DG, Woolf AD, Mortimer PP et al. Human parvovirus arthropathy. Lancet 1985:1:419-422
6． Pattison JR Diseases caused by human parvovirus B19． Arch. Dis. Child 1988:63:1426-1427
7． Yoto Y, Kudoh T, Haseyama K et al. Huamn parvovirus B19 infection associated with acute hepatitis. Lancet 1996:347:868-869
8． Chia JKS, Jackson B Myopericarditis due to parvovirus B19 in the adult. Clin. Infect Dis. 1996:23:200-201
9． Torok TJ, Wang QY, Gary GW et al. Prenatal diagnosis of intrauterine infection with parvovirus B19 by the polymerase chain reaction technique. Clin. Infect Dis. 1992;14:149-155
10． Brown KE, Young NS, Liu JM Molecular, cellular and clinical aspects of parvovirus B19 infection. Critical Reviews in Oncology and Hematology 1994:16(1) :1-31
11． Kerr JR, Curran MD, Moore JE et al. Persistent parvovirus B19 infection. Lancet 1995:345:1118
12． Shade RO, Blundell MC, Cotmore SF et al. Nucleotide sequence and genome organization of human parvovirus B19 isolated from
the serum of a child during aplastic crisis. J.Virol. 1986;58:921-936
13． Blundell MC, Beard C, Astell CR In vitro identification of a B19 parvovirus promoter. Virology 1987;157:534-538
14． Hick KE, Beard S, Cohen BJ et al. A simple and sensitive DNA hybridisation assay used for the diagnosis of human parvovirus B19 infection. J. Clin. Microbiol. 1995:33:2473-2475
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31．张国成 许东亮 聚合酶链反应诊断小儿微小病毒B19感染 《临床儿科杂志》1997；15(3)：213-214
32．张国成 许东亮 王绮云等 国内人细小病毒B19特异基因靶区扩增产物的核苷酸序列分析 《中华微生物学和免疫学杂志》1995；15(1)：13
33．蔡定邦 郭亮 陈享等 聚合酶链反应法检测人细小病毒B19DNA及相关片段克隆鉴定 《中华实验和临床病毒学杂志》1996；12(3)：268
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2． leads from the MMWR, Risks associated with human parvovirus B19 infection. JAMA 1989:261:1406-1408
3． PattisonJR, Jones SE, Hodgson J et al. Parvovirus infections and hypoplastic crisis in sickle-cell anaemia. Lancet 1981:1:664-665
4． Anderson MJ, Khousam MN, Maxwell DJ et al. Human parvovirus B19 and hydrops fetal is. Lancet 1988;1 : 535
5． White DG, Woolf AD, Mortimer PP et al. Human parvovirus arthropathy. Lancet 1985:1:419-422
6． Pattison JR Diseases caused by human parvovirus B19． Arch. Dis. Child 1988:63:1426-1427
7． Yoto Y, Kudoh T, Haseyama K et al. Huamn parvovirus B19 infection associated with acute hepatitis. Lancet 1996:347:868-869
8． Chia JKS, Jackson B Myopericarditis due to parvovirus B19 in the adult. Clin. Infect Dis. 1996:23:200-201
9． Torok TJ, Wang QY, Gary GW et al. Prenatal diagnosis of intrauterine infection with parvovirus B19 by the polymerase chain reaction technique. Clin. Infect Dis. 1992;14:149-155
10． Brown KE, Young NS, Liu JM Molecular, cellular and clinical aspects of parvovirus B19 infection. Critical Reviews in Oncology and Hematology 1994:16(1) :1-31
11． Kerr JR, Curran MD, Moore JE et al. Persistent parvovirus B19 infection. Lancet 1995:345:1118
12． Shade RO, Blundell MC, Cotmore SF et al. Nucleotide sequence and genome organization of human parvovirus B19 isolated from
the serum of a child during aplastic crisis. J.Virol. 1986;58:921-936
13． Blundell MC, Beard C, Astell CR In vitro identification of a B19 parvovirus promoter. Virology 1987;157:534-538
14． Hick KE, Beard S, Cohen BJ et al. A simple and sensitive DNA hybridisation assay used for the diagnosis of human parvovirus B19 infection. J. Clin. Microbiol. 1995:33:2473-2475
15． Gallinell G, Venturoli S B19genome sequence and structure analysis. Clinical and Experimental Medicine 1999;23(7) ;9-22
16． Agbandje M, Kaajigaga S, Mckenna R et al. The structure of human parvovirus B19at 8 A resolution. Virology 1994:203(1) 106-115
17． Kawase M, Momneda M, Young NS et al. Most of the VP1 unique region of B19parvovirus is on the capsid surface. Virology 1995;21(2) :359-366
18． Brown KE, Hibbs JR, Gallinella G et al. Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen). N.Engl.Med. 1994;330(17) :1192-96
19． Brown KE, Anderson SM, Young NS Erythrocyte P antigen: cellular receptor for B19 parvovirus. Science 1992;262:114-116
20． Mori BJ, Beattie P, Melton DW et al. Structure and mapping of the DNA of human parvovirus B19． J. Gen. Virol. 1987:68:2797-2806
21． Kerr JR, Curran MD, Moore JE et al. Genetic diversity in the non-structural gene of parvovirus B19 detected by single-stranded conformational polymorphism assay (SSCP) and partial nucleotide sequencing. J. Virol. Methods 1995;53(2-3) :213-222
22． Gray A, Guillou L, Zufferey J et al. Persistence of parvovirus
B19 DNA in testiS of patients with testicular germ cell tumours. J.Gen.Virol.1998;79(3)573-579
23．钟酶 曹虹，温淑鹃等 正常孕妇，新生儿及异常胎儿血清中人微小病毒B19感染的研究。《中华妇产科杂志》1997；32(4)：205-207
24．钱新宏 焦富勇 细小病毒B19与儿童疾病 《中华儿科杂志》1997；35(2)107-109
25．严延生 李世清 王惠榕等 福建省B19微小病毒感染的病原学证据《中国人兽共患病杂志》1997；13(1)27-29
26．吴晓玲 付德庆 郭莉萍等 自然流产妇女HPVB19感染情况调查《贵州医药》1997；21(2)：125-126
27．唐清 12省市人群中微小病毒B19-IgG抗体水平调查 《中国公共卫生学报》1997；16(4)：203-205
28．姜静雯 王荣平 陈星琪 西安地区新生儿人细小病毒B19感染的初步观察 《陕西医学杂志》1997；26(3)：167-168
29．曹虹，卓礼梅，郭辉玉等 原位杂交检测人微小病毒B19方法的建立 《中华实验和临床病毒学杂志》1996；10(1)：9
30．杨洪江 张桦 林书祥 用PCR检测临床标本中人类细小病毒B19 DNA 《病毒学报》1995；11(4)：371-380
31．张国成 许东亮 聚合酶链反应诊断小儿微小病毒B19感染 《临床儿科杂志》1997；15(3)：213-214
32．张国成 许东亮 王绮云等 国内人细小病毒B19特异基因靶区扩增产物的核苷酸序列分析 《中华微生物学和免疫学杂志》1995；15(1)：13
33．蔡定邦 郭亮 陈享等 聚合酶链反应法检测人细小病毒B19DNA及相关片段克隆鉴定 《中华实验和临床病毒学杂志》1996；12(3)：268
34．曹虹 姚海军 郭辉玉 应用PCR-SSCP方法初步分析人微小病毒B19基因变异 《第一军医大学学报》 1996；16(1)：20-21
35. Salimans MMM,Holsappel S, Vande Rijke FM et al. Rapid detection of human parvovirus B19 DNA by dot-hybridization and the polymerase chain reaction. J. Virol. Methods.1989; 23(1): 19-28
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