中国不同地区A、B组轮状病毒分离株VP7基因序列的比较分析
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摘要
目的：
研究从我国大庆地区婴幼儿腹泻临床粪便标本中分离的一株A组人轮状病毒阳性毒株(命名为DQ-1株)VP7基因的基因分型，对DQ-1株VP7基因序列和糖蛋白VP7的蛋白质结构进行分析，并与已测序同血清型标准株和国内外地方株VP7基因序列进行序列比对。
方法：
通过RT-PCR技术扩增人A组轮状病毒DQ-1株VP7全长基因，并将RT-PCR产物连接到pMD18-T载体，转化大肠杆菌JM109感受态细胞，在含有氨苄青霉素的LB琼脂培养基上培养，形成单菌落后挑取白色菌落进行检测，从中筛选出两个PCR阳性菌落，并提取质粒，双酶切鉴定，最后利用通用引物进行测序。利用DNAStar软件对DQ-1株VP7基因测序序列与其他A组轮状病毒株VP7基因序列进行同源性比较；用RNAstructure4.4软件绘制DQ-1株VP7基因的二级结构；用Predictprotein软件对DQ-1株糖蛋白VP7蛋白结构进行分析。
结果：
DQ-1株VP7基因与同型的我国已测序G1型地方株T73、R96-197株基因序列同源性均为97.6％，与G1型标准株Wa株核苷酸序列同源性为92.9％，与G2血清型代表株DS-1株基因序列同源性为73.8％，与G3血清型代表株SA11株核甘酸序列同源性为75.6％，与G4血清型代表株J-4621株(78.1％)同源性也不高；其VP7基因mRNA可折叠多达20个发卡结构。DQ-1株VP7蛋白是由354个氨基酸组成的多肽，含有2个潜在的糖基化位点和多个磷酰化位点；DQ-1株氨基酸序列与T73、R96-197株同源性都达97.5％，与Wa株达94.6％，而与其它型DS-1株、SA11株和J-4621株分别仅为74.4％、79.4％和75.2％。因此，我们推定DQ-1株属于G1血清型。
结论：
DQ-1株VP7基因序列与已测序我国G1型地方株VP7基因序列几乎没有差别，而与G1型标准株VP7基因序列存在细微差异，这为我国人A组轮状病毒疫苗的引进和研制提供了分子生物学依据。
目的：
克隆中国不同地区四个B组成人腹泻轮状病毒株结构蛋白VP7基因，通过测序得到其核苷酸序列和氨基酸序列，比较与其它B组轮状病毒毒株基因和氨基酸序列同源性，从而进一步分析我国不同地区流行的B组成人腹泻轮状病毒毒株之间的系统进化关系。
方法：
参照文献设计引物，对中国不同地区流行的4株B组轮状病毒株进行RT-PCR，并将RT-PCR产物克隆到PCR~(TM)Ⅱ载体，转化E．Coli感受态细胞，提取转化菌质粒经限制性内切酶HamHⅠ和BglⅡ双酶切分析，从中筛选阳性重组质粒进行测序，测序结果采用DNAStar6.0进行多重序列比对分析，并与B组轮状病毒代表株和其它已测序地方株的核苷酸及氨基酸序列进行同源性比较，绘制系统遗传进化树。
结果：
本研究获得的4株B组成人腹泻轮状病毒株核苷酸序列同源性为97.3％～98.8％，氨基酸序列同源性为95.2％～99.3％之间；与B组成人腹泻轮状病毒代表株ADRV核苷酸序列同源性为98.2％～100％，氨基酸序列同源性为97.4％～100％；与国外B组成人腹泻轮状病毒株印度、孟加拉国株核苷酸序列同源性为90.1％～92.5％，氨基酸序列同源性为92.3％～95.6％。
结论：
研究表明所有B组成人腹泻轮状病毒毒株均属于同一血清型，这为成人腹泻轮状病毒的流行病学监测以及将来研制成人腹泻轮状病毒疫苗提供了分子生物学依据，同时本研究可以从分子病毒学理论上证明“病毒是以居群的形式存在的”和“病毒的居群内部存在一个’差异谱’”的说法。
Objectives
To study strain DQ-1 VP7 gene variation of human group A rotavirus( GARV) from da-qing district, we analyzed and compared its gene sequence and protein structure with standard strain and field strains of China which had been sequenced.
Methods
Firstly we extract total RNA of DQ-1, the gene that encoded the structural protein VP7 gene of GARV was amplified from isolated strain DQ-1 by reverse transcription polymerase chain reaction(RT-PCR).Then the products of RT-PCR were ligated with plasmid pMD18-T and transformed into E.coli competent cells JM109. Analyzed by PCR detection ,it was showed that the VP7 gene was cloned into pMD18-T and the recombinant plasmids were constructed by PCR detection. The VP7 gene of GARV strain DQ-1 was then sequenced. The identity to other GARV was analyzed by software DNAStar,the secondary structure of DQ-1 VP7 mRNA was predicted by software RNA structure 4.4 ,and the protein of VP7 was analyzed by Predictprotein.
Results
The nucleotide sequence of DQ-1 VP7 gene showed high identities (more than 90 %) to that of other human GARV such as T73、R96-197 and Wa which belongs to G1 Genotype, while identities to that of G2 Genotype such as DS-1 and G3 Genotype such as SA11 and G4 Genotype such as J-4621 were lower than 80%.The secondary structure of DQ-1 VP7 mRNA was constructed by about 20 stem-loop structures.The polypeptide encoded by DQ-1 VP7 contains 354 amino acids,the protein contains two potential N-linked glycosylation sites and some phosphorylation sites. Compared with other GARV DQ-1 VP7 demonstrated 97.5% and 94.6% of amino acids similarity with T73 and Wa respectively,but only 74.4 %、79.4% and 75.2% with DS-1、SA11 and J-4621. So, we might draw the conclusion that strain DQ-1 belongs to G1 serotype.
Conclusions
The sequence analysis revealed that the VP7 gene of DQ-1 shared higher identities with field strains of G1 serotype ,but showed differences with standard strain ,these results might give a molecular biological evidence to the inducement and development of the GARV vaccine.
Objectives
To compare gene and amino acid sequence identity to other group B rotavirus,and further analyse the Phylogenetic relationship of group B ADRV rotavirus that spreads different areas of china, we cloned and sequenced the structural protein VP7 gene of four strains human group B ADRV rotavirus in different areas of china.
Methods
We designed primers accordinging to the article, and extracted the RNA from four strains,then,VP7 genes were amplified by reverse transcription polymerase chain reaction(RT-PCR). The products of RT-PCR were ligated with plasmid PCR~(TM)II and transformed into E.coli competent cells.Analyzed by PCR detection.it was showed that the VP7 gene was cloned into PCR~(TM)II and the recombinant plasmids were constructed. The VP7 genes of four strains were then sequenced. The identity to other group B rotavirus was analyzed by software DNAStar6.0, and thus the phylogenetic tree was established.
Results
The results show that the gene sequence identity between the four strains is 97.3%～98.8%, the ammo acid sequence identity is 95.2%～99.3%; the gene sequence identity to ADRV is 98.2%～100%, the amino acid sequence identity is 97.4%～100%; the gene sequence identity to foreign strains in India and Bangladesh is 90.1%～92.5%, the amino acid sequence identity is 92.3%～95.6%.
Conclusions
The sequence analysis revealed that all strains of human group B ADRV rotavirus belongs to the same serotype, these results can give a molecular biological evidence to the inducement of the group B ADRV rotavirus vaccine.They can also give a molecular virul evidence to the viewpoint that "Virus exists in the form of population"and "Inside the population of the Virus exists in a'variation spectrum'.
研究从我国大庆地区婴幼儿腹泻临床粪便标本中分离的一株A组人轮状病毒阳性毒株(命名为DQ-1株)VP7基因的基因分型，对DQ-1株VP7基因序列和糖蛋白VP7的蛋白质结构进行分析，并与已测序同血清型标准株和国内外地方株VP7基因序列进行序列比对。
方法：
通过RT-PCR技术扩增人A组轮状病毒DQ-1株VP7全长基因，并将RT-PCR产物连接到pMD18-T载体，转化大肠杆菌JM109感受态细胞，在含有氨苄青霉素的LB琼脂培养基上培养，形成单菌落后挑取白色菌落进行检测，从中筛选出两个PCR阳性菌落，并提取质粒，双酶切鉴定，最后利用通用引物进行测序。利用DNAStar软件对DQ-1株VP7基因测序序列与其他A组轮状病毒株VP7基因序列进行同源性比较；用RNAstructure4.4软件绘制DQ-1株VP7基因的二级结构；用Predictprotein软件对DQ-1株糖蛋白VP7蛋白结构进行分析。
结果：
DQ-1株VP7基因与同型的我国已测序G1型地方株T73、R96-197株基因序列同源性均为97.6％，与G1型标准株Wa株核苷酸序列同源性为92.9％，与G2血清型代表株DS-1株基因序列同源性为73.8％，与G3血清型代表株SA11株核甘酸序列同源性为75.6％，与G4血清型代表株J-4621株(78.1％)同源性也不高；其VP7基因mRNA可折叠多达20个发卡结构。DQ-1株VP7蛋白是由354个氨基酸组成的多肽，含有2个潜在的糖基化位点和多个磷酰化位点；DQ-1株氨基酸序列与T73、R96-197株同源性都达97.5％，与Wa株达94.6％，而与其它型DS-1株、SA11株和J-4621株分别仅为74.4％、79.4％和75.2％。因此，我们推定DQ-1株属于G1血清型。
结论：
DQ-1株VP7基因序列与已测序我国G1型地方株VP7基因序列几乎没有差别，而与G1型标准株VP7基因序列存在细微差异，这为我国人A组轮状病毒疫苗的引进和研制提供了分子生物学依据。
目的：
克隆中国不同地区四个B组成人腹泻轮状病毒株结构蛋白VP7基因，通过测序得到其核苷酸序列和氨基酸序列，比较与其它B组轮状病毒毒株基因和氨基酸序列同源性，从而进一步分析我国不同地区流行的B组成人腹泻轮状病毒毒株之间的系统进化关系。
方法：
参照文献设计引物，对中国不同地区流行的4株B组轮状病毒株进行RT-PCR，并将RT-PCR产物克隆到PCR~(TM)Ⅱ载体，转化E．Coli感受态细胞，提取转化菌质粒经限制性内切酶HamHⅠ和BglⅡ双酶切分析，从中筛选阳性重组质粒进行测序，测序结果采用DNAStar6.0进行多重序列比对分析，并与B组轮状病毒代表株和其它已测序地方株的核苷酸及氨基酸序列进行同源性比较，绘制系统遗传进化树。
结果：
本研究获得的4株B组成人腹泻轮状病毒株核苷酸序列同源性为97.3％～98.8％，氨基酸序列同源性为95.2％～99.3％之间；与B组成人腹泻轮状病毒代表株ADRV核苷酸序列同源性为98.2％～100％，氨基酸序列同源性为97.4％～100％；与国外B组成人腹泻轮状病毒株印度、孟加拉国株核苷酸序列同源性为90.1％～92.5％，氨基酸序列同源性为92.3％～95.6％。
结论：
研究表明所有B组成人腹泻轮状病毒毒株均属于同一血清型，这为成人腹泻轮状病毒的流行病学监测以及将来研制成人腹泻轮状病毒疫苗提供了分子生物学依据，同时本研究可以从分子病毒学理论上证明“病毒是以居群的形式存在的”和“病毒的居群内部存在一个’差异谱’”的说法。
Objectives
To study strain DQ-1 VP7 gene variation of human group A rotavirus( GARV) from da-qing district, we analyzed and compared its gene sequence and protein structure with standard strain and field strains of China which had been sequenced.
Methods
Firstly we extract total RNA of DQ-1, the gene that encoded the structural protein VP7 gene of GARV was amplified from isolated strain DQ-1 by reverse transcription polymerase chain reaction(RT-PCR).Then the products of RT-PCR were ligated with plasmid pMD18-T and transformed into E.coli competent cells JM109. Analyzed by PCR detection ,it was showed that the VP7 gene was cloned into pMD18-T and the recombinant plasmids were constructed by PCR detection. The VP7 gene of GARV strain DQ-1 was then sequenced. The identity to other GARV was analyzed by software DNAStar,the secondary structure of DQ-1 VP7 mRNA was predicted by software RNA structure 4.4 ,and the protein of VP7 was analyzed by Predictprotein.
Results
The nucleotide sequence of DQ-1 VP7 gene showed high identities (more than 90 %) to that of other human GARV such as T73、R96-197 and Wa which belongs to G1 Genotype, while identities to that of G2 Genotype such as DS-1 and G3 Genotype such as SA11 and G4 Genotype such as J-4621 were lower than 80%.The secondary structure of DQ-1 VP7 mRNA was constructed by about 20 stem-loop structures.The polypeptide encoded by DQ-1 VP7 contains 354 amino acids,the protein contains two potential N-linked glycosylation sites and some phosphorylation sites. Compared with other GARV DQ-1 VP7 demonstrated 97.5% and 94.6% of amino acids similarity with T73 and Wa respectively,but only 74.4 %、79.4% and 75.2% with DS-1、SA11 and J-4621. So, we might draw the conclusion that strain DQ-1 belongs to G1 serotype.
Conclusions
The sequence analysis revealed that the VP7 gene of DQ-1 shared higher identities with field strains of G1 serotype ,but showed differences with standard strain ,these results might give a molecular biological evidence to the inducement and development of the GARV vaccine.
Objectives
To compare gene and amino acid sequence identity to other group B rotavirus,and further analyse the Phylogenetic relationship of group B ADRV rotavirus that spreads different areas of china, we cloned and sequenced the structural protein VP7 gene of four strains human group B ADRV rotavirus in different areas of china.
Methods
We designed primers accordinging to the article, and extracted the RNA from four strains,then,VP7 genes were amplified by reverse transcription polymerase chain reaction(RT-PCR). The products of RT-PCR were ligated with plasmid PCR~(TM)II and transformed into E.coli competent cells.Analyzed by PCR detection.it was showed that the VP7 gene was cloned into PCR~(TM)II and the recombinant plasmids were constructed. The VP7 genes of four strains were then sequenced. The identity to other group B rotavirus was analyzed by software DNAStar6.0, and thus the phylogenetic tree was established.
Results
The results show that the gene sequence identity between the four strains is 97.3%～98.8%, the ammo acid sequence identity is 95.2%～99.3%; the gene sequence identity to ADRV is 98.2%～100%, the amino acid sequence identity is 97.4%～100%; the gene sequence identity to foreign strains in India and Bangladesh is 90.1%～92.5%, the amino acid sequence identity is 92.3%～95.6%.
Conclusions
The sequence analysis revealed that all strains of human group B ADRV rotavirus belongs to the same serotype, these results can give a molecular biological evidence to the inducement of the group B ADRV rotavirus vaccine.They can also give a molecular virul evidence to the viewpoint that "Virus exists in the form of population"and "Inside the population of the Virus exists in a'variation spectrum'.
引文
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[9] ihmed M U, Kobayashi N, Wakuda M, et aL. Genetic analysis of group B human rotaviruses detected in Bangladesh in 2000 and 2001[J].J Med Virol, 2004,72: 149-155.
[10] 杨红彦，纪绍忠，康志刚等．B组轮状病毒ADRV与RDRV主要基因的克隆及其相关性的初步研究[J]．中华微生物学和免疫学杂志，1999，19：364-368．
[1] 李维琳，唐力，王斌等．B组轮状病毒WH-1株NSP2基因序列和蛋白质结构分析[J]．中华微生物学和免疫学杂志，2003，23 (11)：853-857．
[12] 赵姜勤，陈广牧，Mackow ER，等．成人腹泻轮状病毒基因组的分子生物学研究．中华实验和临床病毒学杂志，1994，8：388-390．
[13] 金奇，曾力宇，Jing．H 等．世界范围内A组轮状病毒G1型毒株VP7基因及VP4基因高变区的分析[J]．中华实验和临床病毒学杂志，1997，11(1)：3-7．
[14] 徐倏，王健伟，温乐英等．G1 型轮状病毒中和抗原 VP7 基因变异特点[J]．中华实验和临床病毒学杂志．2001，15(3)：269-273．
[15] 陈湘，胡超文，黄华利等．怀化地区腹泻爆发流行的病原学及流行病学调查分析[J]．中华流行病学杂志，1987，11(腹泻病特刊)：34-36．
[16] Li Y Y, hi X, Zheng C X. Adult diarrhea rotavirus in Jilin, China[J]. J Uirusu, 1990 Jun;40 (1):9-12.
[17] 叶青，方肇寅，秦树民等．一起成人轮状病毒腹泻的接触型爆发流行[J]．中华流行病学杂志，1987，11(腹泻病特刊)：5-7．
[18] 纪邵忠，陈新生，杨凤蓉等．唐山赵各庄媒矿成人轮状病毒性腹泻暴发流行的病原学研究[J]．中华流行病学杂志，1988，9(腹泻病特刊)：10-12．
[19] Erich R M, Robin Wernereckert,Mary Ellen Fay, et al. Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV[J].J Virol ,1993,67:2730-2738.
[20] 唐少文，唐力，王斌等．人B组轮状病毒VP7基因的克隆和序列分析．中国病毒学杂志[J]，2003，18(5)：432-436．
[21] Chen G M, Hong T, Mackow E R. cDNA cloning of each genomic segment of the group B rotavirus ADRV: Molecular characterization of the eleventh gene segment [J]. Virology, 1990,175: 605-609.
[22] Yang J H, Kobayashi N, Wang Y H. Phylogenetic analysis of a human group B rotavirus WH-1 detected in China in 2002[J].J Med Virol, 2004 74 (4):662-667.
[23] 陈锦生，邓涤夷，陈霞，等．福建省首次检出新的成人轮状病毒[J]．中国人兽共患病杂志，1988，4：22-24．
[24] Wang C A，Yang R J，Chen G M et al．Molecular epJdemiological study of ADRV from 12 outbreaks[J].Mei Sheng Wu Xue Bao. t985 .Jun, 25(2):153-157.
[25] 病毒的进化：病毒存在的基本方式[M]．医学分子病毒学．北京：科学出版社，2001：2-3．
[26] 萨姆布鲁克主编．分子克隆实验指南．北京：科学出版社，第二版．1996．366-371．
[27] 蒋盛军、纪绍忠、唐青等．新成人腹泻轮状病毒J19株VP2、VP3的编码基因序列分析[J]．病毒学报，2006，22(3)：172-175．
[28] 蒋盛军、纪绍忠、唐青等．成人腹泻轮状病毒J19株依赖RNA的RNA聚合酶基因序列分析[J]．中华微生物学和免疫学杂志，2006，26(8)：739～742．
[29] 蒋盛军 纪绍忠 唐青等．新成人腹泻轮状病毒J19株NSPl、NSP2和NSP3基因序列分析[J]．中华实验和临床病毒学杂志，2006，29(11)：994-997．
[1] Glass R I, Parashar U D, Brasee J S. Rotavirus vaccines: current prospects and future challenges [R]. Lancet. 2006 Jul 22;368 (9532) "323-332.
[2] 王蓓，方肇寅，高倩等．中国轮状病毒腹泻的经济负担浅析，第二届国际轮状病毒疫苗研讨会，2005，36-40．
[3] Prasad B V, Estes M K. Molecular basis of rotavirus replication:struc ture-function correlationsM. Led, New York NYOxford University. J Nature, 1990,343 (6257):476-479.
[4] Jayasinghe S M, Palombe EA. Genetic homogeneity of human serotype G1 rotaviruses isolated during a single epidemic season implications for vaccine strategies[J], icta Virol, 1999,43:53-55.
[5] 白植生，陈冬梅，申颈．口服轮状病毒活疫苗LLR—85株的选育及鉴定[J]．中国生物制品学杂志．1994，7(2)49～52．
[6] 陈冬梅．口服轮状病毒活疫苗疫苗资料汇编[M]．兰州生物制品研究所．2003。1—4．
[7] 刘东磊，杨洁，李安等．轮状病毒疫苗免疫效果及人体反应观察[J]．中国计划免疫杂志2002，8(3)：138～140．
[8] 《中国生物制品规程》2000年版增补本[S]．2002，8-10．
[9] SoaresW, eiser K, Goldberg E, Tam im i G, etal. Rotavirus vaccine for preventing diarrhoea[R]. Coch rane Database Syst Rev, 2004, (1) : CD002848.
[10] 杨国峰，周鹏，王建伟．轮状病毒研究进展及其转基因植物疫苗的开发前景[R]．生物技术通报，2001，1：16-19．
[11] 金奇主编医学分子病毒学[M]．北京：科学出版社，2001．
[12] Wlliams C J. New rotavirus vaccines protect against diarrhoea in children[R], Euro Surveillance. 2006 Jan 19;11(1):E0601195.
[13] 方肇寅．轮状病毒疫苗和肠套叠发病关系研究进展[M]．国外医学病毒学分册，2003，10 (1)：5-7．
[14] Centers for Disease Control and Prevention (CDC).Postmarketing monitoring of intussusception after RotaTeq vaccination--United States, February 1, 2006-February 15, 2007[J].MMWR Morb Mortal Wkly Rep, 2007 Mar 16;56(10):218-222.
[15] Buttery JP, Kirkwood C. Rotavirus vaccinesindeveloped countries[J]. Curr Opin Infect Dis. 2007 Jun;20(3):253-258.
[16] Himshi U. Rotavirus infection in Asia[J]. Pediatrics International. 2000, 42: 392～394.
[17] Rodriguez ZM, Goveia M G, Stek J E, et al. Concomitant use ofan oral live pentavalent human-bovine reassortant rotavirus vaccine with licensed parenteral pediatric vaccines in the United States[J]. Pediatr Infect Dis J, 2007 Mar;26(3):221-227.
[18] Denis F, Alain S, Ploy M C. New routes of administration: epidermal, transcutaneous mucosal ways of vaccination[J].Med Sci (Paris),2007 Apt, 23(4) :379-85.
[19] Chiappini E, Galli L, de Martino M. New rotavirus vaccines: renewed optimism and reason for caution[J].J Pediatrics, 2007 May, 150 (5):86-87.
[20] 洪涛，陈广牧，方肇寅等．成人腹泻轮状病毒感染．医学病毒学基础及实验技术．北京：科学出版社，1990，636-639．
[2] Bishop R F, Davidson G P, Holmes I Het al. Virus particles in epithElial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis[J].Lancet, 1973,2:1281-1283.
[3] 黄祯祥主编．医学病毒学基础与实验技术〔M〕．北京：科学出版社，1990．
[4] Noel J S, Beards G M , Cubitt W D. Epidemiological survey of human rotavirus serotypes and electropherotypes in young children admitted to two children' s hospitals in Northeast London from 1984 to 1990 [J]. J Olin Microbiol, 1991,29: 2213-2219.
[5] 萨姆布鲁克主编．分子克隆实验指南．北京：科学出版社，第二版．1996．366-371．
[6] 何金生．人轮状病毒重要抗原在重组腺病毒中表达与免疫效果研究．博士学位论文．北京：中国预防医学科学院病毒学研究所，2001．
[7] 方肇寅，晋圣瑾，秦树民，等．PCR 方法用于我国 A 组轮状病毒的分型研究[J]．病毒学报，1994，10：316-321．
[8] 陈均，纪绍忠，谈幸之等．轮状病毒1、2血清型VP7蛋白基因的扩增及其克隆[J]．中华实验和临床病毒学杂志，1994，8 (4)：312-314．
[9] 李国华，钱渊，熊朝晖等．北京地区G1～G4 型人轮状病毒地方株VP7编码基因的序列分析[J]．病毒学报，1998，14(2)：126-132．
[10] 陈东梅，钱渊，张又等．A组轮状病毒地方株VP7基因序列的比较分析 〔J〕．中华实验和临床病毒学杂志，2003，17 (2)：165-168．
[11] Dunn S J ,Ward R L ,McNeal M M,et al. Identificationof new neutrallization epitopeon VP7 of human serotype 2 rotavirus and evidence for electropherotype differences caused by single nucleotide substitutions[J].Virology, 1993,197:397-404.
[12] Gouvea V, Glass R I, Woods P, et al. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens[J]. J Clin Microbiol, 1990, 28: 276-282.
[13] 赵姜勤等．成人腹泻轮状病毒基因组的分子生物学研究〔J〕．中华实验和临床病毒学杂志，1994，4(8)：94-97．
[14] Green K, Sears J F. Prediction of human rotavirus serotype by nucleotide sequence analysis of the VP7 protein gene[J]. J Virol, 1988, 62:1819-1823.
[15] Jayasinghe S M, Palombe E A. Genetic homogeneity of human serotype Gl rotaviruses isolated during a single epidemic season implications for vaccine strategies[J]. Acta Virol ,1999 ,43:53-55.
[16] Green K Y, Hoshino Y, Ikegami N. Sequence analysis of the gene encoding the serotype specific glycoprotein (VPT) of two new human rotavirus serotypes[J]. Virology, 1989,168:429-433.
[17] 金奇，曾力宇，Jing．H等．世界范围内A组轮状病毒Gl型毒株VP7基因及VP4基因高变区的分析[J]．中华实验和临床病毒学杂志，1997，11(1)：3—7．
[18] 徐倏，温乐英，王健伟等．中国部分城市 A 组轮状病毒感染状况调查及 VP7血清型分析[J]．中华实验和临床病毒学杂志，2001，15(1)：77-80．．
[19] 徐倏，王健伟，温乐英等．G1型轮状病毒中和抗原VP7基因变异特点[J]．中华实验和临床病毒学杂志，2001，15(3)：269-273．
[20] 唐少文，王斌，李燕等。婴幼儿腹泻A组轮状病毒VP7基因型别的研究[J]．华中医学杂志，2004，28 (2)：73-74。
[21] Coulson B S, Kirkwood C. Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus Gonotype[J].J Virol, 1991,65:5968-5974.
[22] 方肇寅，齐锦，杨辉等．我国 1998～1999 年流行的婴幼儿腹泻轮状病毒的分型研究[J]．病毒学报，2001，17(1)：17-23．
[23] Jayasinghe S M, Palombe E A. Genetic homogeneity of human serotype Gl rotaviruses isolated during a single epidemic season implications for vaccine strategies[J]. Acta Virol, 1999,43:53-55.
[24] (参考) 蒋胜军．Ⅰ：利用一种改良的方法扩增和克隆新成人腹泻轮状病毒J19株的大片段基因：Ⅱ：新成人腹泻轮状病毒 J19 株的全基因组序列分析．博士后研究工作报告．北京：中国疾病预防控制中心，2006．
[25] (参考) 马卫胜．2003—2005年山东省肾综合征出血热宿主变化及流行病学特征的研究．硕士学位论文．济南：山东大学，2006．
[1] 陈均，纪绍忠，谈幸之等．轮状病毒1、2血清型VP7蛋白基因的扩增及其克隆[J]．中华实验和临床病毒学杂志，1994，8(4)：312-314．
[2] 方肇寅，晋圣瑾，秦树民，等．PCR方法用于我国 A 组轮状病毒的分型研究[J]．病毒学报，1994，1O：316-321．
[3] Hung T, Chen G M, Wang C A, etal. Rotavirus-like agent in adult nonbacterial diarrhea in China [J]. Lancet, Ⅱ(8385): 1983, 2: 1078-1079.
[4] Hung T, Chen G M, Wang C, et al. Waterborne outbreak of rotavirus diarrhea in adults in China caused by a novel rotavirus [J]. Lancet, 1984,1: 1139-1142.
[5] Chen G M, Hung T, Bridget J C, et al. Chinese adult rotavirus is a group B rotavirus [J]. Lancet, 1985, 2:1123-1124.
[6] 洪涛，陈广牧，方肇寅等．成人腹泻轮状病毒感染[M]．医学病毒学基础及实验技术．北京：科学出版社，1990，636-639．
[7] krishnan T, Sen A, Choudhurg JS, et al . Emergence of adult diarrhea rotavirus in Calcutta[J]. India. Lancet, 1999,353:380-381.
[8] Kobayshi N, Naik T N, Kushhara Y, et al. Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India [J]. J Med Virology, 2001, 64: 583-588.
[9] ihmed M U, Kobayashi N, Wakuda M, et aL. Genetic analysis of group B human rotaviruses detected in Bangladesh in 2000 and 2001[J].J Med Virol, 2004,72: 149-155.
[10] 杨红彦，纪绍忠，康志刚等．B组轮状病毒ADRV与RDRV主要基因的克隆及其相关性的初步研究[J]．中华微生物学和免疫学杂志，1999，19：364-368．
[1] 李维琳，唐力，王斌等．B组轮状病毒WH-1株NSP2基因序列和蛋白质结构分析[J]．中华微生物学和免疫学杂志，2003，23 (11)：853-857．
[12] 赵姜勤，陈广牧，Mackow ER，等．成人腹泻轮状病毒基因组的分子生物学研究．中华实验和临床病毒学杂志，1994，8：388-390．
[13] 金奇，曾力宇，Jing．H 等．世界范围内A组轮状病毒G1型毒株VP7基因及VP4基因高变区的分析[J]．中华实验和临床病毒学杂志，1997，11(1)：3-7．
[14] 徐倏，王健伟，温乐英等．G1 型轮状病毒中和抗原 VP7 基因变异特点[J]．中华实验和临床病毒学杂志．2001，15(3)：269-273．
[15] 陈湘，胡超文，黄华利等．怀化地区腹泻爆发流行的病原学及流行病学调查分析[J]．中华流行病学杂志，1987，11(腹泻病特刊)：34-36．
[16] Li Y Y, hi X, Zheng C X. Adult diarrhea rotavirus in Jilin, China[J]. J Uirusu, 1990 Jun;40 (1):9-12.
[17] 叶青，方肇寅，秦树民等．一起成人轮状病毒腹泻的接触型爆发流行[J]．中华流行病学杂志，1987，11(腹泻病特刊)：5-7．
[18] 纪邵忠，陈新生，杨凤蓉等．唐山赵各庄媒矿成人轮状病毒性腹泻暴发流行的病原学研究[J]．中华流行病学杂志，1988，9(腹泻病特刊)：10-12．
[19] Erich R M, Robin Wernereckert,Mary Ellen Fay, et al. Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV[J].J Virol ,1993,67:2730-2738.
[20] 唐少文，唐力，王斌等．人B组轮状病毒VP7基因的克隆和序列分析．中国病毒学杂志[J]，2003，18(5)：432-436．
[21] Chen G M, Hong T, Mackow E R. cDNA cloning of each genomic segment of the group B rotavirus ADRV: Molecular characterization of the eleventh gene segment [J]. Virology, 1990,175: 605-609.
[22] Yang J H, Kobayashi N, Wang Y H. Phylogenetic analysis of a human group B rotavirus WH-1 detected in China in 2002[J].J Med Virol, 2004 74 (4):662-667.
[23] 陈锦生，邓涤夷，陈霞，等．福建省首次检出新的成人轮状病毒[J]．中国人兽共患病杂志，1988，4：22-24．
[24] Wang C A，Yang R J，Chen G M et al．Molecular epJdemiological study of ADRV from 12 outbreaks[J].Mei Sheng Wu Xue Bao. t985 .Jun, 25(2):153-157.
[25] 病毒的进化：病毒存在的基本方式[M]．医学分子病毒学．北京：科学出版社，2001：2-3．
[26] 萨姆布鲁克主编．分子克隆实验指南．北京：科学出版社，第二版．1996．366-371．
[27] 蒋盛军、纪绍忠、唐青等．新成人腹泻轮状病毒J19株VP2、VP3的编码基因序列分析[J]．病毒学报，2006，22(3)：172-175．
[28] 蒋盛军、纪绍忠、唐青等．成人腹泻轮状病毒J19株依赖RNA的RNA聚合酶基因序列分析[J]．中华微生物学和免疫学杂志，2006，26(8)：739～742．
[29] 蒋盛军 纪绍忠 唐青等．新成人腹泻轮状病毒J19株NSPl、NSP2和NSP3基因序列分析[J]．中华实验和临床病毒学杂志，2006，29(11)：994-997．
[1] Glass R I, Parashar U D, Brasee J S. Rotavirus vaccines: current prospects and future challenges [R]. Lancet. 2006 Jul 22;368 (9532) "323-332.
[2] 王蓓，方肇寅，高倩等．中国轮状病毒腹泻的经济负担浅析，第二届国际轮状病毒疫苗研讨会，2005，36-40．
[3] Prasad B V, Estes M K. Molecular basis of rotavirus replication:struc ture-function correlationsM. Led, New York NYOxford University. J Nature, 1990,343 (6257):476-479.
[4] Jayasinghe S M, Palombe EA. Genetic homogeneity of human serotype G1 rotaviruses isolated during a single epidemic season implications for vaccine strategies[J], icta Virol, 1999,43:53-55.
[5] 白植生，陈冬梅，申颈．口服轮状病毒活疫苗LLR—85株的选育及鉴定[J]．中国生物制品学杂志．1994，7(2)49～52．
[6] 陈冬梅．口服轮状病毒活疫苗疫苗资料汇编[M]．兰州生物制品研究所．2003。1—4．
[7] 刘东磊，杨洁，李安等．轮状病毒疫苗免疫效果及人体反应观察[J]．中国计划免疫杂志2002，8(3)：138～140．
[8] 《中国生物制品规程》2000年版增补本[S]．2002，8-10．
[9] SoaresW, eiser K, Goldberg E, Tam im i G, etal. Rotavirus vaccine for preventing diarrhoea[R]. Coch rane Database Syst Rev, 2004, (1) : CD002848.
[10] 杨国峰，周鹏，王建伟．轮状病毒研究进展及其转基因植物疫苗的开发前景[R]．生物技术通报，2001，1：16-19．
[11] 金奇主编医学分子病毒学[M]．北京：科学出版社，2001．
[12] Wlliams C J. New rotavirus vaccines protect against diarrhoea in children[R], Euro Surveillance. 2006 Jan 19;11(1):E0601195.
[13] 方肇寅．轮状病毒疫苗和肠套叠发病关系研究进展[M]．国外医学病毒学分册，2003，10 (1)：5-7．
[14] Centers for Disease Control and Prevention (CDC).Postmarketing monitoring of intussusception after RotaTeq vaccination--United States, February 1, 2006-February 15, 2007[J].MMWR Morb Mortal Wkly Rep, 2007 Mar 16;56(10):218-222.
[15] Buttery JP, Kirkwood C. Rotavirus vaccinesindeveloped countries[J]. Curr Opin Infect Dis. 2007 Jun;20(3):253-258.
[16] Himshi U. Rotavirus infection in Asia[J]. Pediatrics International. 2000, 42: 392～394.
[17] Rodriguez ZM, Goveia M G, Stek J E, et al. Concomitant use ofan oral live pentavalent human-bovine reassortant rotavirus vaccine with licensed parenteral pediatric vaccines in the United States[J]. Pediatr Infect Dis J, 2007 Mar;26(3):221-227.
[18] Denis F, Alain S, Ploy M C. New routes of administration: epidermal, transcutaneous mucosal ways of vaccination[J].Med Sci (Paris),2007 Apt, 23(4) :379-85.
[19] Chiappini E, Galli L, de Martino M. New rotavirus vaccines: renewed optimism and reason for caution[J].J Pediatrics, 2007 May, 150 (5):86-87.
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