间接ELISA检测犊牛轮状病毒抗体方法的建立及其疫苗的初步研制
|
摘要
本文在基础实验室条件下,通过普通高速离心机代替低温高速离心机、焦碳酸二乙酯(DEPC)处理水制胶、制胶板自制以及普通的电泳槽等改良措施,提取了含量较高的轮状病毒RNA,此法也适用于其他生物性病原的RNA抽提。虽然本法中有少量的RNA降解,但并不影响实验结果,这对于条件相对较薄弱的基层防疫、监督单位来说,仍具有较好的可行性和推广意义。
从山东暴发犊牛腹泻的地区采集腹泻犊牛的粪便, RT-PCR检测出了特异性目的条带,SDS-PAGE检测出轮状病毒特异性的11条带。证实是A组的轮状病毒。
以牛轮状病毒标准株NCDV作为抗原,以其制备的高免血清作为一抗,辣根过氧化物酶标记的羊抗牛IgG作为二抗,运用正交试验法结合方阵法建立了检测犊牛轮状病毒抗体的间接ELISA方法。本实验运用正交实验法结合方阵法确定间接ELISA最佳反应条件:包被抗原51.5 ng/孔,血清150倍稀释,酶标抗体2500稀释,对牛轮状病毒的检测灵敏度达36.78 ng。本次试验成功建立了检测牛轮状病毒抗体间接ELISA方法。
旨在研制出经济有效的轮状病毒疫苗,减少养牛业的经济损失。首先对种子批毒进行TCID50、病毒对小鼠毒力、特异性试验、免疫原性试验、纯粹性检查。制备的疫苗进行无菌检验和安全检验结果符合标准后,分别免疫犊牛和怀孕牛。本试验制备的种子毒符合标准,研制出犊牛轮状病毒灭活疫苗,分别免疫犊牛和怀孕牛,对照组和免疫组的犊牛IgG抗体水平差异显著;而对照组和免疫组的怀孕牛乳清IgG抗体效价差异不显著。分析免疫怀孕牛失败的原因可能是疫苗免疫量少、免疫次数少、饲喂环境影响等原因。本实验证明了疫苗免疫犊牛的可行性,为犊牛轮状病毒性腹泻的防治奠定基础。
Based on the basis of laboratory conditions, by an ordinary high-speed centrifuges instead of low-temperature high-speed centrifuges, Coke Diethyl (DEPC) water treatment system for plastic, rubber-made and the general improvement measures, such as electrophoresis tank, the extraction of higher rotavirus RNA, the law also applies to other sexually transmitted diseases of the original RNA extraction. Although this law in a small amount of RNA degradation, but does not affect the results, this relatively weak conditions for the grass-roots epidemic prevention and supervision units, still has good feasibility and significance of the promotion.
This study was collection from calf diarrhea outbreak in Shandong. RT-PCR detected the specific purpose of the zone, SDS-PAGE detected rotavirus in 11 specific zones. A group confirmed that the rotavirus.
Cattle NCDV as a standard strain of rotavirus antigen, its high for the preparation of an anti-serum as, horseradish peroxidase labeled goat anti-IgG as the anti-cattle, the use of orthogonal test with a phalanx of detecting calf Bovine rotavirus antibodies indirect ELISA method. The experimental use of orthogonal experiment with square indirect ELISA method to determine the best conditions: coating antigen 51.5 ng / hole, 150 times the serum dilution, dilution ELISA antibody 2500, the detection of bovine rotavirus sensitivity of 36.78 ng.
This experiment were aimed to develop the most-effective rotavirus vaccine for reduce economic losses of cattle. The first was approved virus TCID50, virulence of the virus in mice, specific tests, immunogenicity trial, and purity check. Preparation of the vaccine sterile test and safety test results were meet the standards, were immune calves and pregnant cows, respectively. The trial of the virus to accord with the standard, bovine rotavirus vaccine were immune calves and pregnant cows, respectively .The control group and the immune group IgG antibody levels were significantly of calves; the control group and immune group of Pregnant cow’s milk-IgG antibody titers were not significantly different. Analyze the failure of immune pregnant cow were vaccine may be less, the less time of immune, the feeding environmental impact and other reasons. The experimental has proved that feasibility of bovine rotavirus vaccine immune calves and lied the foundation of prevention of rotavirus diarrhea.vaccine immune calves and laid the foundation of prevention of rotavirus diarrhea.
从山东暴发犊牛腹泻的地区采集腹泻犊牛的粪便, RT-PCR检测出了特异性目的条带,SDS-PAGE检测出轮状病毒特异性的11条带。证实是A组的轮状病毒。
以牛轮状病毒标准株NCDV作为抗原,以其制备的高免血清作为一抗,辣根过氧化物酶标记的羊抗牛IgG作为二抗,运用正交试验法结合方阵法建立了检测犊牛轮状病毒抗体的间接ELISA方法。本实验运用正交实验法结合方阵法确定间接ELISA最佳反应条件:包被抗原51.5 ng/孔,血清150倍稀释,酶标抗体2500稀释,对牛轮状病毒的检测灵敏度达36.78 ng。本次试验成功建立了检测牛轮状病毒抗体间接ELISA方法。
旨在研制出经济有效的轮状病毒疫苗,减少养牛业的经济损失。首先对种子批毒进行TCID50、病毒对小鼠毒力、特异性试验、免疫原性试验、纯粹性检查。制备的疫苗进行无菌检验和安全检验结果符合标准后,分别免疫犊牛和怀孕牛。本试验制备的种子毒符合标准,研制出犊牛轮状病毒灭活疫苗,分别免疫犊牛和怀孕牛,对照组和免疫组的犊牛IgG抗体水平差异显著;而对照组和免疫组的怀孕牛乳清IgG抗体效价差异不显著。分析免疫怀孕牛失败的原因可能是疫苗免疫量少、免疫次数少、饲喂环境影响等原因。本实验证明了疫苗免疫犊牛的可行性,为犊牛轮状病毒性腹泻的防治奠定基础。
Based on the basis of laboratory conditions, by an ordinary high-speed centrifuges instead of low-temperature high-speed centrifuges, Coke Diethyl (DEPC) water treatment system for plastic, rubber-made and the general improvement measures, such as electrophoresis tank, the extraction of higher rotavirus RNA, the law also applies to other sexually transmitted diseases of the original RNA extraction. Although this law in a small amount of RNA degradation, but does not affect the results, this relatively weak conditions for the grass-roots epidemic prevention and supervision units, still has good feasibility and significance of the promotion.
This study was collection from calf diarrhea outbreak in Shandong. RT-PCR detected the specific purpose of the zone, SDS-PAGE detected rotavirus in 11 specific zones. A group confirmed that the rotavirus.
Cattle NCDV as a standard strain of rotavirus antigen, its high for the preparation of an anti-serum as, horseradish peroxidase labeled goat anti-IgG as the anti-cattle, the use of orthogonal test with a phalanx of detecting calf Bovine rotavirus antibodies indirect ELISA method. The experimental use of orthogonal experiment with square indirect ELISA method to determine the best conditions: coating antigen 51.5 ng / hole, 150 times the serum dilution, dilution ELISA antibody 2500, the detection of bovine rotavirus sensitivity of 36.78 ng.
This experiment were aimed to develop the most-effective rotavirus vaccine for reduce economic losses of cattle. The first was approved virus TCID50, virulence of the virus in mice, specific tests, immunogenicity trial, and purity check. Preparation of the vaccine sterile test and safety test results were meet the standards, were immune calves and pregnant cows, respectively. The trial of the virus to accord with the standard, bovine rotavirus vaccine were immune calves and pregnant cows, respectively .The control group and the immune group IgG antibody levels were significantly of calves; the control group and immune group of Pregnant cow’s milk-IgG antibody titers were not significantly different. Analyze the failure of immune pregnant cow were vaccine may be less, the less time of immune, the feeding environmental impact and other reasons. The experimental has proved that feasibility of bovine rotavirus vaccine immune calves and lied the foundation of prevention of rotavirus diarrhea.vaccine immune calves and laid the foundation of prevention of rotavirus diarrhea.
引文
[1] Mebus CA, Underdahl NR, Rhodes MB, et al.Calf diarrhoea (scours) reproduces with a virus from a field outbreak. Bulletin: University of Nebraska Agricultural Experiment Station Resrarch, 1969,233.
[2] Stannard L M. Observation on the morphology of two rotaviruses. [J].Gen.Virol. 1977, (37):435~441
[3] Prasad BV,Wang G J,Cler JPM. et al.Three-demessional structure of rotavirus. [J]. MolBiol, 1988, 199:269~275
[4] Hsu GG, Bellamy AR,Yeager M. Projection structure of VP6,the rotavirus inner capsidprotein,and comparison with bluetongue VP7.[J].Mol Biol. 1997,272(3):362~368.
[5] Espul C,Cuello H,Navarta L M, et al. Characterization of antigenic types of circulating rotaviruses in Mendoza,Argentina based on typing of the external VP7 capsid protein.[J].Acta Gastroenterol Latinoam. 1993, 23(4):211~216.
[6]Gorziglia M,Larralde G,kapikian AA Z, et al. Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4 .[J].Proc Natl Acad Sci U S A. 1990,87(18):7155~7159.
[7] Johansen K, Svensson L. Neutralization of rotavirus and recognition of immunologically important epitopes on VP4 and VP7 by human IgA.[J].Arch Virol. 1997, 142(7):1491~1498
[8] Unicomb, L. E., G. Podder, et al. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995.[J]. Clin. Microbiol. 1999.37:1885~1891.
[9] Gouvea V, N. Santos. Rotavirus serotype G5: an emerging cause of epidemic childhood diarrhea.[J]. Vaccine 1999,17:1291~1292.
[10] Leite, J. ., A. A. Alfieri.Rotavirus G and P types circulating in Brazil: characterization by RT-PCR,probe hybridization, and sequence analysis.[J]. Arch. Virol. 1996,141:2365~2374.
[11] Santos, N., R. C. C. Lima, C. F. A. Pereira, et al. Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea.[J]. Clin. Microbiol. 1998, 36:2727~2729.
[12] Aijas, S., K. Gowda, H. V. Jagannath, et al.Epidemiology of symptomatic human rotaviruses in Bangladore and Mysore, India from 1988 to 1994 as determined by electropherotypes, subgroup and serotype analysis.[J]. Arch. Virol. 1996,141:715~726.
[13] Griffin, D. D, C. D. Kirkwood, U. D. Parashar, et al. The National Rotavirus Strain Surveillance System Collaborating Laboratories. Surveillance of rotavirus strains in the United States: identification ofunusual strains.[J]. Clin Microbiol. 2000, 38:2784~2787.
[14] Ramachandran M., J. R. Gentsch, U. D. Parashar, et al. TheNational Rotavirus Strain Surveillance System Collaborating Laboratories. Detection and characterization of novel rotavirus strains in the United States.[J]. Clin. Microbiol. 1998,36:3223~3229.
[15] Cunliffe, N. A., J. S. Gondwe, R. L. Broadhead, et al.Rotavirus Gand P types in children with acute diarrhea in Blantyre, Malawi, from 1997 to 1998: predominance of novel P[6]G8 strains.[J]. Med. Virol. 1999,57:308~312.
[16] Arista, S., E. Vizzi, D. Ferraro, et al. Distribution of VP7 and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea.[J]. Arch. Virol. 1997,142:2065~2071.
[17] Bon, F., C. Fromantin, S. Aho, P. Pothier, et al. G and P genotyping of rotavirus strains circulating in France over a three-year period: detection of G9 and P[6] strains at low frequencies.[J]. Clin. Microbiol. 2000,38:1681~1683.
[18]. Clark, H. F., Y. Hoshino, L. M. Bell, et al.Rotavirus isolate W161 representing a presumptive new human serotype.[J]. Clin. Microbiol. 1987,25:1757~1762.
[19] Cubitt, W. D., A. D. Steele, M. Iturrize. Characterisation of rotaviruses from children treated at a London hospital during 1996: emergence of strains G9P2A[6] and G3P2A[6].[J]. Med. Virol. 2000,61:150~154.
[20] Fang, Z.-Y., H. Yang, J. Qi, et al.Diversity of rotavirus strains among children with acute diarrhea in China: 1998–2001 surveillance study.[J]. Clin. Microbiol. 2002,40:1875~1878.
[21] Iturriza-Gomara, M.. J. Green, D. W. G. Brown, et al.Molecular epidemiology of human group A rotavirus infections in the United Kingdom between 1995 and 1998.[J]. Clin.Microbiol. 2000,38: 4394~4401.
[22] Masendycz, P., N. Bogdanovic-Sakran, C. Kirkwood, et al. Report of the Australian rotavirus surveillance program, 2000/2001.[J]. Commun Dis. Intell. 2001, 25:144~146.
[23] Oka, T., T. Nakagomi, and O. Nakagomi. Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis. [J],Microbiol. Immunol. 2000, 44:957~961.
[24] Palombo, P. J. Masendycz, H. C. Bugg, et al. Emergence of serotype G9 human rotaviruses in Australia. [J] .Clin. Microbiol. 2000,38:1305~1306.
[25] Ramachandran, B. K. Das, A. Vij, et al.Unusual diversity of human rotavirus G and P genotypes in India. [J]. Clin. Microbiol. 1996, 34:436~439.
[26] Ramachandran, J. R. Gentsch, U. D. Parashar, et al.Detection and characterization of novel rotavirus strains in the United States. [J]. Clin. Microbiol. 1998, 36:3223~3229.
[27] Steele, A. D., L. Nimzing, I. Peenze, et al. Circulation of the novel G9 and G8 rotavirus strains in Nigeria in 1998/1999. [J]. Med. Virol. 2002, 67:608~612.
[28] Unicomb, G. Podder, J. R. Gentsch, et al. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. [J]. Clin. Microbiol. 1999, 37: 1885~ 1891 .
[29] Zhou, Y., J. Supawadee, C. Khamwan, et al. Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997. [J]. Med. Virol. 2001,65:619~628.
[30] Estes M K.Cohen J, et al.Rotavirus gene structure and function.[J].Microbiol Rev.1989,53(4):410~449.
[31] Racz M L,KroeffS S,Munford V, et al.Molecular characterization of porcine rotaviruses from the sounthern region og Brazil:characterization of an atypical genotype G [9] strain.[J] Clin Microbiol. 2000.38(6): 2443~2446.
[32] Chen D,Zeng CQY,Wentz.M J.Template-dependent in vitro replication of rotavirus RNA.[J ]. Virology. 1994,68: 7030~7039
[33] Desslberger U. Microbiology Immunol .1994.185:31~66.
[34] DuboisE,Le Guyader F, Haugarreau L.Molecular epidemiological survey of rotavirus in sewage by reversetranscriptase seminested PCR and restriction fragment length polymorphism assay.[J]. Appl Environ Microbiol.1997.63(5):1794~1800.
[35] Rao C D,Gowda K,Reddy B S. Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses[J].Virology. 2000,276(1):104~113
[36] Piec T L,Palombo E A. Sequence comparison of the VP7 of serotype G2 rotaviruses from diverse geographical locations[J].DNA Seq. 1998,9(5-6):369~373.
[37] Valenzuela S,Pizarro J,Sandino A M, et al. Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP:identification of the viral RNA polymerase[J].Gen Virol. 1991,65:3964~3967
[38] Liu M,Offit P A,Estes M K. Idetification of simian rotavirus SA11 genome segment 3 product[J].Virol.1988,163:26~32
[39] Hsu GG,Bellamy AR,Yeager M. Projection structure of VP6,the rotavirus inner capsid protein,and comparison with bluetongue VP7.[J].Mol Biol. 1997,272(3):362~368.
[40] Sandino A M,Jashes M,Faundex G, et al.Role of the inner protein capsid on invitro human rotavirus transcription.[J].Gen Virol. 1985,60:797~802
[41] Eequivel F R,Lopez S,Arias C. The internal rotavirus protein VP6 primes for an enhanced neutralizing antibody response.[J].Arch Virol. 2000,145(4):813~25.
[42] Rao C D,Gowda K,Reddy B S. Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses.[J].Virology. 2000,276(1):104~113
[43] Wang L,Huang J A,Nagesha H S. Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice.[J].Vaccine. 1999,17(20~21):2636~2645.
[44] Johnson M A,Misra R M,Lardelli M. Synthesis in Escherichia coli of the major glycoprotein of human rotavirus:analysis of the antigenic regions.[J].Gene. 1989,84(1):73~81
[45] Mendez E,Arias CF,Lopez S.Interactions between the two surface protein of rotavirus may alter the receptor-binding specificity of the virus.[J].Virol.1996,70(2):1218~1222.
[46] Graham KL, Halasz P, Tan Y, et al.Integrin-using rotaviruses bin alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognizalphaXbeta2 and alphaVbeta3 by using VP7 during cell entry.[J].J Virol.2003,77(18):9969~9978.
[47] Chemello ME, Aristimuno OC, Michelangeli F, et al.Requirement for vacuolarH+-ATPase activity and Ca2+ gradient during entry of rotaviruinto MA104 cells.[J].J Virol.2002,76(24):13083~13087.
[48] Ball JM,Tian P.Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.Science. 1996,272:101~104
[49] Sasaki S,Horie Y. Group C rotavirus NSP4 induces diarrhea in neonatal mice.[J].Arch Virol .2001,146:801~806
[50] Mori Y,Borgan MA, et al.Diarrhea inducing activity of avian rotavirus NSP4 glycoproteins,which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice.[J].Virol. 2002,76:5829~5834
[51] Horie Y,Nakagomi O, et al.Diarrhea inductionby rotavirus NSP4 in the homologous mouse model system.[J].Virology .1999,262:398~407
[52] Mirazimi A,Magnusson KE,Svens,son L.A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retentionin the endoplasmic reticulum.[J]. Gen Virol .2003,84:875~883
[53] Ruiz MC,Cohen J,Michelangeli F.Role of Ca2+ from the replication and pathogenesis of rotavirus and other viral infections.[J].Ceff Calcium .2000,28:137~149
[54] Tian P,Estes MK.The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+from the endoplasmic reficulum.[J] Virol .1995,69:5763~5772
[55] Dong V, Zeng CQ.The rotavirusenterotoxin NSP4 mobilizes intracellular calcium in humanintestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production.[J].Proc Natl Acad Sci USA .1997,94:3960~3965
[56] Tian P,Ball JM,Zeng CQ Estes MK.The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.[J]. Virol .1996,70:6973~6981
[57] Browne EP,Bellamy AR,Taylor JR.Membrane-destabilizingactiviW of rotavirus NSP4 is mediated by a membrane-proxi—mal amphipathic domain.[J]. Gen Virol .2000,81:1955~1959
[58] Zhang M,Zeng CQ,et al.Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence.[J]. Virol .1998,72:3666~3672
[59] Zhang M,Zeng CQ,Moms AP,Estes MK.A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.[J] Virol .2000,74:11663~11670
[60] Huang H,Schroeder F,Zeng C, et al.Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.[J].Biochemistry. 2001,40:4169~4180
[61] Jourdan N,Brunet JP.Rotavirus infection re-duces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization ofmicrovillar cytoskeleton.[J] Virol. 1998,72:7228~7236
[62] Halaihel N,Lievin V, et al.Direct inhibitory effect of rotavirus NSP4(114~135)peptide on the Na(+)-D-glucose symporter of rabbit intestinalbrush border membrane.[J] Virol .2000,74:9464~9470
[63] Halaihel N,Lievin V, et al.Rotavirus infection impairs intestinal brush-border membrane Na(+)-solutecotransport activities in young rabbits.[J].Am Physiol Gastrointest Liver Physiol. 2000, 279: G587~G596
[64]Ashby MC,Tepikin AV.ER calcium and the functions ofintracellular organelles.[J].Semin Cell Dev Biol .2001,12:1 1~17
[65] COrbett EF,Michalak M.Calcium, a signaling molecule in theendoplasmic reticulum.[J].Trends Biochem Sci. 2000,25:307~311
[66] Brunet JP,Cotte-Laffitte J, et al.Rotavirus infection induces an inherem intracellular calcium concentration in human intestinal epi-thelial cells:role in microvillar actin alteration.[J] Virol .2000,74:2323~2332
[67] Ma n,Tran D.Mechanism of extracellular calcium reffulation of intestinal epithelial tight iunction perrneability:role of cytoskeletal involvement.[J].Microsc Res tech. 2000,51:156~168
[68] Obeft G,Peiffer I,Servin AL.Rotavirus-induced structuraland functional alterations in tight iunctions of polarized intestinal Caco-2 cell monolayers.[J]. Virof .2000,74:4645~4651
[69] Dickman KG,Hempson SJ, et al.Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells.[J].Am Physiol Gastrointest Liver Physiol. 2000,279:G757~G766
[70] Bowman GD,Nodelman IM.Crystal structure of the oligomerization domain of NSP4 from rotavirusreveals a core metalbinding site.[J ].Mol Biol. 2000,304:861~871
[71] Angel J,Tang B.Studies of therole for NSP4 in the pathogenesis of homologous murinerotavirus diarrhea.[J].Infic Dis .1998,177:455~458
[72] Horie Y,Masamune O,Nakagomi 0.rnuee major alleles ofrotavirus NSP4 proteins identified by sequence analysis.[J]. GeH Virol. 1997,78:2341~2346
[73] Mori Y,Borgan MA.Sequential analysis of nonstructural protein NSP4s derived from Group A avian rotaviruses.[J].Virus Res .2002,89:145~151
[74] Mohan KV,KDlkarni S.Ahuman vaccine strain of lamb rotavirus(Chinese)NSP4 gene:complete nucleotide sequence and phylogenetic analyses.[J].Vi-rus GeHEs .2003,26:1R5~192
[75] Ciadet M,Liprandi F,Conner ME,Estes MK.Species specificity and interspecies relatedness of NSP4 genetic groups bycomparative NSP4 sequence analyses of animal rotaviruses.[J].Arch Virol. 2000,145: 371~383
[76] Lee CN,Wang YL,Kao CL, et al.NSP4 gene analysis of rotaviruses recovered from infected childrenwith and without diarrhea.[J] .Clin Microbiof .2000,38:4471~4477
[78] Kirkwood CD,Gentsch JR,Glass RI.Sequence analvsis of theNSP4 gene from human rotavirus strains isolated in the United States.[J].Virus Genes. 1999,19:113~122
[79] Jaup EA,Timar Peregrin A,Jodal M, et al.Nervouscontrol of alkaline secrefion in the duodenum as studied bythe use of cholera toxin in the anaesthetized rat.[J].Acta Physiol Scand. 1998,162:165~174
[80] Lundgnm O,Peregrin AT,Person K, et al.Role of the enteric nervous systern in the fluid andelectrolytesecretion of rotavirus diarrhea,Science 2000,287:491~495
[81] Ltmdgren 0,Jodal M.The entericnervous system and choleratoxin induced secretion.[J].Comp Biochem Physiol A Phvsiol 1997,118:319~327
[82] Kagnoff MF,Eckmann L.Epithelial cells as sensors for micro-bial infection.[J].Clin Invest 1997,100:6~10
[83] Sansonetti P.Host-pathogen interactions:the seduction ofmolecular cross talk.Gut. 2002,50(Suppl 3):IIl2~IIl8
[84] Neish AS.The gut microflora and intestinal epithelial cells:a continuing dialogue.[J].Microbes Infect 2002,4:309~317
[85] Philpott DJ,Girardin SE,Sansonetti PJ.Innate immune re.sponses of epithelial cells following infection with bacterialpathogens.CUTT Opin ImmunoI. 2001,13:410~416
[86] Rollo EE,KDmar KP,Reich NC, et al.The epithelial cell response to rotavirus infection.[J] Immunol 1999,163:4442~4452
[87] Casola A,Garofalo RP,Crawford SE, et al.Intedeukin-8 gene regulation in intesti-nal epithelial cells infected with rotavirus:role of viral-irI.duced IkappaB kinase activation.[J].ViroloKg. 2002,298:8~19
[88] Kim J,Sanders SP,Siekierski Es, et al.Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection.[J] Immunol 2000,165:3384~3392
[89] Sugawara T,Yamabe H,Osawa H, et al.way inhibitor production by human proximal tubular epithe-lial cells in culture.[J].Thromb Res .2003,110:141~147
[90] KirKDp Al,Brunsden AM,Grundy D.Receptors and trans.mission in the brain-gut axis:potential for novel therapies.[J].Am J Physiol Gastrointest LiverPhysiol. 2001,280:G787~G794
[91] Booth CE,KirKDp AJ,Hicks GA, et al.Somatostatin sst(2) receptor-mediated inhibition of mesen.teric afferent nerves of the iejunum in the anesthetized rat.[J]. GastroenteroloKg .2001,121:358~369
[92] KirKDp AJ,Booth CE,Chessell IP, et al.Excitatory effect of P2xreceptor activation on mesenteric after-ent nerves in the anaesthetised rat.[J] PhlJsiol .1999,520:551~563
[93] AzimT,Zaki MH,Podder G, et al.Rotavirus-specific subclass antibody ancytokine responses in Bangladeshi children with rotavirus diarrhoea.[J].Med Virol,2003,69(2):286~295.
[94] CuadrasMA,Feigelstock DA,An S, et al.Gene expression pattern in Caco-cells following rotavirus infection.[J].J Virol,2002,76(9):4467~4482.
[95] Colomina J,GilMT,Codoner P, et al.Viral proteinsVP2,VP6 and NSP2 arstrongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection.[J]. Med Virol.1998,56(1):58~65.
[96] Iosef C,Chang KO,Azevedo MS, et al.Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pimodel of human rotavirus disease.[J]. Med Virol,2002.68(1):119~128.
[97] Blutt SE,Warfield KL,Lewis DE, et al.Early response to rotavirus infectio involves massive B cell activation.[J].J Immunol.2002,168(11):5716~5721.
[98] KDshnir N,Bos NA,Zuercher AW, et al.B2 but not B1 cells can contribute to CD4+T-cell-mediated clearance of rotavirus in SCID mice.[J].J Virol. 2001,75(12):5482~5490.
[99] Flores J.Boggeman E,Purcall R A, et al.A dot hybridization assay for detection of rotavirus[J].Lancet. 1983,1:555~558
[100] K.O.Chang. Detection of Group B Rotaviruses in Fecal Samples from Diarrheic Calves and Adult Cows and Characterization of Their VP7 Genes.[J]. JOURNAL OF CLINICAL MICROBIOLOGY Aug. 1997, 2107~2110
[101] Manuel G.Claros. RNA isolation from plant tissues: a practical experience for biological undergraduates.[J]. Biochemical Education 27,1999,110~113
[102] Fedorcsak I, Ehrenberg L.Effects of diethyl pyrocarbonate and methyl methanesulfonate on nucleic acids and nucleases.[J]. Acta Chem Scand 1966,20: 107~112
[103] Maria Marone. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.[J]. Biol.Proced ,Online 2001,3(1):19~25
[104] Gouvea. V, R. I. Glass, P. Woods, et al.Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.[J].J Clin Microbiol, 1990, 28:276~282.
[105] Acres SD, Babiuk LA.Studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay.[J]. J Am Vet Med Assoc. 1978, 1:173(5 Pt 2):555~9
[106] A H Hussein, E Cornaglia, M S Saber, et al.Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec .[J]. Can J Vet Res. 1995 July; 59(3): 235–237.
[107] Niedrig.M., Sonnenberg.K, Steinhagen.K, et al.Comparison of ELISA and immunoassays formeasurement of IgG and IgM antibody to West Nile virus in human sera against virus neutralization. [J].Virol, 2007, 103~105.
[108] Thurber ET, Bass EP, Beckenhauer WH. Field trial evaluation of a reo-coronavirus calf diarrhea vaccine. [J].Can J Comp Med.1977,41:131~136.
[109] Waltner-Toews D, Martin SW, Meek AH, et al. A field trial to evaluate the efficacy of a combined rotavirus-coronavirus/Escherichia coli vaccine in dairy cattle.[J].Can Comp Med. 1985,49:1~9
[110] Collins JK, Riegel CA, Olson JD, et al.Shedding of enteric coronavirus in adult cattle. [J].Am J Vet Res. 1987,48:361~365
[111]Myers H, Snodgrass DR. Colostral and milk antibody titers in cows vaccinated with a modified live-rotavirus –coronavirus vaccine.[J]. Am Vet Med Assoc. 1982,181:289~298.
[112] Estes M K,Cohen J. Rotavirus gene structure and function.[J].Microbiol Rev. 1989,53(4):410~449
[113] 张礼壁等,ELISA方法用于诊断病毒病的进展.病毒学报.[J].1988,4(1)91~95.
[114] 殷震,刘景华.动物病毒学(第二版).[M].北京:科学出版社,1997.562~571.
[115] 蔡宝祥.家畜传染病(第四版).[M].北京:中国农业出版社,2001,401~410.
[116] 何孔旺,林继煌,江杰元,等.牛轮状病毒性腹泻的研究.[J].中国兽医学报,1998,18(2):224~227.
[145] 庞其芳,丘福禧,愈富荣,等.婴幼儿秋季急性胃肠炎病原的轮状病毒的研究.[J].中华医学杂志,1979,59:589~591.
[117] 于恩庶等主编,中国人畜共患病学(第二版).[M],福建科学技术出版社.1996.672~683
[118] 宣长和,任凤兰,孙富先.猪病学(第一版).[M],北京,中国农业出版社,1996.69~71.
[119] 庞昕,柴家前,赵宏坤.猪轮状病毒感染研究进展.[J].吉林农业大学学报. 1999,21(增刊),77~79
[120] 斯特劳B E,阿莱尔S D,蒙加林W L主编2000,猪病学.[M].第八版.中国农业大学出版社,263~276
[121] 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
[123] 李明锐,李春玲,李浩等.牦犊牛流行性腹泻病院的调查,中国兽医科技,199,2:12~13.
[124] 黄文林主编 M 分子病毒学(供研究生用).[M].人民卫生出版社. 2002,84~85、321~333
[125] 温乐英.轮状病毒蛋白的研究进展[J].国外医学病毒学分册. 1996,3(3):65~67
[126] 陈军华,刘作义.轮状病毒感染发病机制研究进展.[J].国外医学儿科学分册 2004.9.31(5)237~239.
[127] 王大燕 , 王 健 伟 , 于 修 平 , 洪涛 . 轮 状 病 毒 致 病 机 制 的 研 究 机 制 .[J]. 世界 华 人 消 化 杂志,2003;11(11):1670~1673
[129] 于力,于康震等编著动物传染病学.[M].中国农业出版社.1997, 197~201
[130] 胥爱源,刘玑昌,丘富禧等.用葡萄球菌 A 蛋白固相免疫电镜法提高轮状病毒检出.[J].中华医学杂志 1983,62(2):73~76
[131] 于恩庶主编中国人兽共患病学[M].第二版.福建科学技术出版社,1996,672~683
[132] 张福萍.诊断 B 组轮状病毒的三种酶联免疫方案的建立[J].中国兽医科技,1995,5(5):3~5
[133] 王文洁,周明锦.应用金标免疫斑点法快速诊断轮状病毒感染及临床意义.[J].河南职工医学院学报 2002.14(2):164~165
[134] 吴亦伦,苏诚钦.轮状病毒快速 ELISA 诊断方法的研究和应用[J].安徽医学. 1998,19(2):1~2.
[135] 李艳萍,白植生.轮状病毒乳胶试剂盒的研制[J].微生物学免疫学进展. 1998,26(4):35~38.
[136] 何孔旺,吴纪棠,林继煌等.用反向间接血凝试验快速检测轮状病毒抗原和抗体[J].病毒学报. 1989,5(1):68~72.
[137] 王正党,王嘉福,张福萍.化学修饰法制备 B 组轮状病毒生物素核酸探针及应用研究[J].中国动物检验. 1995,12(3):5~7
[138] 刘明军,王正党.光敏生物素核酸探针检测 A 群轮状病毒的研究[J].中国畜禽传染病. 1998,20(3):171~172
[139] 傅万海,郝卫等.聚合酶链反应标记轮状病毒地高辛素探针的初步应用[J].中华实验和病毒学杂志. 1998,12(1):77~79
[140] 倪艳秀,林继煌,陆承平等.逆转录-聚合酶链反应(RT-PCR)检测轮状病毒[J].中国兽医学报. 1998,18(5):437~439
[141] 汪招雄,何启盖.检测伪狂犬病病毒双抗体夹心间接 ELISA 方法的建立与应用[J].中国预防兽医学报. 2006, 3 (2) 221~224
[142] 李昌文,刘怀然.应用间接 ELISA 方法检测兔轮状病毒抗体的初步研究[J].中国比较医学杂志.2003,12(6) 373~376
[143] 蔡雪晖,郭宝清,柴文君等,检测猪繁殖呼吸综合征病毒抗体ELISA方法的建立.中国预防兽医学报[J].2000,22(2):122~125
[144] 蒋正军,马世东,蔡丽娟等,非洲猪瘟间接ELISA诊断试剂盒的研究.中国预防兽医学报[J]. 2000,22(增刊),107~113.
[145] 庞其芳,丘福禧,愈富荣,等.婴幼儿秋季急性胃肠炎病原的轮状病毒的研究.[J].中华医学杂志,1979,59:589~591.
[2] Stannard L M. Observation on the morphology of two rotaviruses. [J].Gen.Virol. 1977, (37):435~441
[3] Prasad BV,Wang G J,Cler JPM. et al.Three-demessional structure of rotavirus. [J]. MolBiol, 1988, 199:269~275
[4] Hsu GG, Bellamy AR,Yeager M. Projection structure of VP6,the rotavirus inner capsidprotein,and comparison with bluetongue VP7.[J].Mol Biol. 1997,272(3):362~368.
[5] Espul C,Cuello H,Navarta L M, et al. Characterization of antigenic types of circulating rotaviruses in Mendoza,Argentina based on typing of the external VP7 capsid protein.[J].Acta Gastroenterol Latinoam. 1993, 23(4):211~216.
[6]Gorziglia M,Larralde G,kapikian AA Z, et al. Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4 .[J].Proc Natl Acad Sci U S A. 1990,87(18):7155~7159.
[7] Johansen K, Svensson L. Neutralization of rotavirus and recognition of immunologically important epitopes on VP4 and VP7 by human IgA.[J].Arch Virol. 1997, 142(7):1491~1498
[8] Unicomb, L. E., G. Podder, et al. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995.[J]. Clin. Microbiol. 1999.37:1885~1891.
[9] Gouvea V, N. Santos. Rotavirus serotype G5: an emerging cause of epidemic childhood diarrhea.[J]. Vaccine 1999,17:1291~1292.
[10] Leite, J. ., A. A. Alfieri.Rotavirus G and P types circulating in Brazil: characterization by RT-PCR,probe hybridization, and sequence analysis.[J]. Arch. Virol. 1996,141:2365~2374.
[11] Santos, N., R. C. C. Lima, C. F. A. Pereira, et al. Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea.[J]. Clin. Microbiol. 1998, 36:2727~2729.
[12] Aijas, S., K. Gowda, H. V. Jagannath, et al.Epidemiology of symptomatic human rotaviruses in Bangladore and Mysore, India from 1988 to 1994 as determined by electropherotypes, subgroup and serotype analysis.[J]. Arch. Virol. 1996,141:715~726.
[13] Griffin, D. D, C. D. Kirkwood, U. D. Parashar, et al. The National Rotavirus Strain Surveillance System Collaborating Laboratories. Surveillance of rotavirus strains in the United States: identification ofunusual strains.[J]. Clin Microbiol. 2000, 38:2784~2787.
[14] Ramachandran M., J. R. Gentsch, U. D. Parashar, et al. TheNational Rotavirus Strain Surveillance System Collaborating Laboratories. Detection and characterization of novel rotavirus strains in the United States.[J]. Clin. Microbiol. 1998,36:3223~3229.
[15] Cunliffe, N. A., J. S. Gondwe, R. L. Broadhead, et al.Rotavirus Gand P types in children with acute diarrhea in Blantyre, Malawi, from 1997 to 1998: predominance of novel P[6]G8 strains.[J]. Med. Virol. 1999,57:308~312.
[16] Arista, S., E. Vizzi, D. Ferraro, et al. Distribution of VP7 and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea.[J]. Arch. Virol. 1997,142:2065~2071.
[17] Bon, F., C. Fromantin, S. Aho, P. Pothier, et al. G and P genotyping of rotavirus strains circulating in France over a three-year period: detection of G9 and P[6] strains at low frequencies.[J]. Clin. Microbiol. 2000,38:1681~1683.
[18]. Clark, H. F., Y. Hoshino, L. M. Bell, et al.Rotavirus isolate W161 representing a presumptive new human serotype.[J]. Clin. Microbiol. 1987,25:1757~1762.
[19] Cubitt, W. D., A. D. Steele, M. Iturrize. Characterisation of rotaviruses from children treated at a London hospital during 1996: emergence of strains G9P2A[6] and G3P2A[6].[J]. Med. Virol. 2000,61:150~154.
[20] Fang, Z.-Y., H. Yang, J. Qi, et al.Diversity of rotavirus strains among children with acute diarrhea in China: 1998–2001 surveillance study.[J]. Clin. Microbiol. 2002,40:1875~1878.
[21] Iturriza-Gomara, M.. J. Green, D. W. G. Brown, et al.Molecular epidemiology of human group A rotavirus infections in the United Kingdom between 1995 and 1998.[J]. Clin.Microbiol. 2000,38: 4394~4401.
[22] Masendycz, P., N. Bogdanovic-Sakran, C. Kirkwood, et al. Report of the Australian rotavirus surveillance program, 2000/2001.[J]. Commun Dis. Intell. 2001, 25:144~146.
[23] Oka, T., T. Nakagomi, and O. Nakagomi. Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis. [J],Microbiol. Immunol. 2000, 44:957~961.
[24] Palombo, P. J. Masendycz, H. C. Bugg, et al. Emergence of serotype G9 human rotaviruses in Australia. [J] .Clin. Microbiol. 2000,38:1305~1306.
[25] Ramachandran, B. K. Das, A. Vij, et al.Unusual diversity of human rotavirus G and P genotypes in India. [J]. Clin. Microbiol. 1996, 34:436~439.
[26] Ramachandran, J. R. Gentsch, U. D. Parashar, et al.Detection and characterization of novel rotavirus strains in the United States. [J]. Clin. Microbiol. 1998, 36:3223~3229.
[27] Steele, A. D., L. Nimzing, I. Peenze, et al. Circulation of the novel G9 and G8 rotavirus strains in Nigeria in 1998/1999. [J]. Med. Virol. 2002, 67:608~612.
[28] Unicomb, G. Podder, J. R. Gentsch, et al. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. [J]. Clin. Microbiol. 1999, 37: 1885~ 1891 .
[29] Zhou, Y., J. Supawadee, C. Khamwan, et al. Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997. [J]. Med. Virol. 2001,65:619~628.
[30] Estes M K.Cohen J, et al.Rotavirus gene structure and function.[J].Microbiol Rev.1989,53(4):410~449.
[31] Racz M L,KroeffS S,Munford V, et al.Molecular characterization of porcine rotaviruses from the sounthern region og Brazil:characterization of an atypical genotype G [9] strain.[J] Clin Microbiol. 2000.38(6): 2443~2446.
[32] Chen D,Zeng CQY,Wentz.M J.Template-dependent in vitro replication of rotavirus RNA.[J ]. Virology. 1994,68: 7030~7039
[33] Desslberger U. Microbiology Immunol .1994.185:31~66.
[34] DuboisE,Le Guyader F, Haugarreau L.Molecular epidemiological survey of rotavirus in sewage by reversetranscriptase seminested PCR and restriction fragment length polymorphism assay.[J]. Appl Environ Microbiol.1997.63(5):1794~1800.
[35] Rao C D,Gowda K,Reddy B S. Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses[J].Virology. 2000,276(1):104~113
[36] Piec T L,Palombo E A. Sequence comparison of the VP7 of serotype G2 rotaviruses from diverse geographical locations[J].DNA Seq. 1998,9(5-6):369~373.
[37] Valenzuela S,Pizarro J,Sandino A M, et al. Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP:identification of the viral RNA polymerase[J].Gen Virol. 1991,65:3964~3967
[38] Liu M,Offit P A,Estes M K. Idetification of simian rotavirus SA11 genome segment 3 product[J].Virol.1988,163:26~32
[39] Hsu GG,Bellamy AR,Yeager M. Projection structure of VP6,the rotavirus inner capsid protein,and comparison with bluetongue VP7.[J].Mol Biol. 1997,272(3):362~368.
[40] Sandino A M,Jashes M,Faundex G, et al.Role of the inner protein capsid on invitro human rotavirus transcription.[J].Gen Virol. 1985,60:797~802
[41] Eequivel F R,Lopez S,Arias C. The internal rotavirus protein VP6 primes for an enhanced neutralizing antibody response.[J].Arch Virol. 2000,145(4):813~25.
[42] Rao C D,Gowda K,Reddy B S. Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses.[J].Virology. 2000,276(1):104~113
[43] Wang L,Huang J A,Nagesha H S. Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice.[J].Vaccine. 1999,17(20~21):2636~2645.
[44] Johnson M A,Misra R M,Lardelli M. Synthesis in Escherichia coli of the major glycoprotein of human rotavirus:analysis of the antigenic regions.[J].Gene. 1989,84(1):73~81
[45] Mendez E,Arias CF,Lopez S.Interactions between the two surface protein of rotavirus may alter the receptor-binding specificity of the virus.[J].Virol.1996,70(2):1218~1222.
[46] Graham KL, Halasz P, Tan Y, et al.Integrin-using rotaviruses bin alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognizalphaXbeta2 and alphaVbeta3 by using VP7 during cell entry.[J].J Virol.2003,77(18):9969~9978.
[47] Chemello ME, Aristimuno OC, Michelangeli F, et al.Requirement for vacuolarH+-ATPase activity and Ca2+ gradient during entry of rotaviruinto MA104 cells.[J].J Virol.2002,76(24):13083~13087.
[48] Ball JM,Tian P.Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.Science. 1996,272:101~104
[49] Sasaki S,Horie Y. Group C rotavirus NSP4 induces diarrhea in neonatal mice.[J].Arch Virol .2001,146:801~806
[50] Mori Y,Borgan MA, et al.Diarrhea inducing activity of avian rotavirus NSP4 glycoproteins,which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice.[J].Virol. 2002,76:5829~5834
[51] Horie Y,Nakagomi O, et al.Diarrhea inductionby rotavirus NSP4 in the homologous mouse model system.[J].Virology .1999,262:398~407
[52] Mirazimi A,Magnusson KE,Svens,son L.A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retentionin the endoplasmic reticulum.[J]. Gen Virol .2003,84:875~883
[53] Ruiz MC,Cohen J,Michelangeli F.Role of Ca2+ from the replication and pathogenesis of rotavirus and other viral infections.[J].Ceff Calcium .2000,28:137~149
[54] Tian P,Estes MK.The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+from the endoplasmic reficulum.[J] Virol .1995,69:5763~5772
[55] Dong V, Zeng CQ.The rotavirusenterotoxin NSP4 mobilizes intracellular calcium in humanintestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production.[J].Proc Natl Acad Sci USA .1997,94:3960~3965
[56] Tian P,Ball JM,Zeng CQ Estes MK.The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.[J]. Virol .1996,70:6973~6981
[57] Browne EP,Bellamy AR,Taylor JR.Membrane-destabilizingactiviW of rotavirus NSP4 is mediated by a membrane-proxi—mal amphipathic domain.[J]. Gen Virol .2000,81:1955~1959
[58] Zhang M,Zeng CQ,et al.Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence.[J]. Virol .1998,72:3666~3672
[59] Zhang M,Zeng CQ,Moms AP,Estes MK.A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.[J] Virol .2000,74:11663~11670
[60] Huang H,Schroeder F,Zeng C, et al.Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.[J].Biochemistry. 2001,40:4169~4180
[61] Jourdan N,Brunet JP.Rotavirus infection re-duces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization ofmicrovillar cytoskeleton.[J] Virol. 1998,72:7228~7236
[62] Halaihel N,Lievin V, et al.Direct inhibitory effect of rotavirus NSP4(114~135)peptide on the Na(+)-D-glucose symporter of rabbit intestinalbrush border membrane.[J] Virol .2000,74:9464~9470
[63] Halaihel N,Lievin V, et al.Rotavirus infection impairs intestinal brush-border membrane Na(+)-solutecotransport activities in young rabbits.[J].Am Physiol Gastrointest Liver Physiol. 2000, 279: G587~G596
[64]Ashby MC,Tepikin AV.ER calcium and the functions ofintracellular organelles.[J].Semin Cell Dev Biol .2001,12:1 1~17
[65] COrbett EF,Michalak M.Calcium, a signaling molecule in theendoplasmic reticulum.[J].Trends Biochem Sci. 2000,25:307~311
[66] Brunet JP,Cotte-Laffitte J, et al.Rotavirus infection induces an inherem intracellular calcium concentration in human intestinal epi-thelial cells:role in microvillar actin alteration.[J] Virol .2000,74:2323~2332
[67] Ma n,Tran D.Mechanism of extracellular calcium reffulation of intestinal epithelial tight iunction perrneability:role of cytoskeletal involvement.[J].Microsc Res tech. 2000,51:156~168
[68] Obeft G,Peiffer I,Servin AL.Rotavirus-induced structuraland functional alterations in tight iunctions of polarized intestinal Caco-2 cell monolayers.[J]. Virof .2000,74:4645~4651
[69] Dickman KG,Hempson SJ, et al.Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells.[J].Am Physiol Gastrointest Liver Physiol. 2000,279:G757~G766
[70] Bowman GD,Nodelman IM.Crystal structure of the oligomerization domain of NSP4 from rotavirusreveals a core metalbinding site.[J ].Mol Biol. 2000,304:861~871
[71] Angel J,Tang B.Studies of therole for NSP4 in the pathogenesis of homologous murinerotavirus diarrhea.[J].Infic Dis .1998,177:455~458
[72] Horie Y,Masamune O,Nakagomi 0.rnuee major alleles ofrotavirus NSP4 proteins identified by sequence analysis.[J]. GeH Virol. 1997,78:2341~2346
[73] Mori Y,Borgan MA.Sequential analysis of nonstructural protein NSP4s derived from Group A avian rotaviruses.[J].Virus Res .2002,89:145~151
[74] Mohan KV,KDlkarni S.Ahuman vaccine strain of lamb rotavirus(Chinese)NSP4 gene:complete nucleotide sequence and phylogenetic analyses.[J].Vi-rus GeHEs .2003,26:1R5~192
[75] Ciadet M,Liprandi F,Conner ME,Estes MK.Species specificity and interspecies relatedness of NSP4 genetic groups bycomparative NSP4 sequence analyses of animal rotaviruses.[J].Arch Virol. 2000,145: 371~383
[76] Lee CN,Wang YL,Kao CL, et al.NSP4 gene analysis of rotaviruses recovered from infected childrenwith and without diarrhea.[J] .Clin Microbiof .2000,38:4471~4477
[78] Kirkwood CD,Gentsch JR,Glass RI.Sequence analvsis of theNSP4 gene from human rotavirus strains isolated in the United States.[J].Virus Genes. 1999,19:113~122
[79] Jaup EA,Timar Peregrin A,Jodal M, et al.Nervouscontrol of alkaline secrefion in the duodenum as studied bythe use of cholera toxin in the anaesthetized rat.[J].Acta Physiol Scand. 1998,162:165~174
[80] Lundgnm O,Peregrin AT,Person K, et al.Role of the enteric nervous systern in the fluid andelectrolytesecretion of rotavirus diarrhea,Science 2000,287:491~495
[81] Ltmdgren 0,Jodal M.The entericnervous system and choleratoxin induced secretion.[J].Comp Biochem Physiol A Phvsiol 1997,118:319~327
[82] Kagnoff MF,Eckmann L.Epithelial cells as sensors for micro-bial infection.[J].Clin Invest 1997,100:6~10
[83] Sansonetti P.Host-pathogen interactions:the seduction ofmolecular cross talk.Gut. 2002,50(Suppl 3):IIl2~IIl8
[84] Neish AS.The gut microflora and intestinal epithelial cells:a continuing dialogue.[J].Microbes Infect 2002,4:309~317
[85] Philpott DJ,Girardin SE,Sansonetti PJ.Innate immune re.sponses of epithelial cells following infection with bacterialpathogens.CUTT Opin ImmunoI. 2001,13:410~416
[86] Rollo EE,KDmar KP,Reich NC, et al.The epithelial cell response to rotavirus infection.[J] Immunol 1999,163:4442~4452
[87] Casola A,Garofalo RP,Crawford SE, et al.Intedeukin-8 gene regulation in intesti-nal epithelial cells infected with rotavirus:role of viral-irI.duced IkappaB kinase activation.[J].ViroloKg. 2002,298:8~19
[88] Kim J,Sanders SP,Siekierski Es, et al.Role of NF-kappa B in cytokine production induced from human airway epithelial cells by rhinovirus infection.[J] Immunol 2000,165:3384~3392
[89] Sugawara T,Yamabe H,Osawa H, et al.way inhibitor production by human proximal tubular epithe-lial cells in culture.[J].Thromb Res .2003,110:141~147
[90] KirKDp Al,Brunsden AM,Grundy D.Receptors and trans.mission in the brain-gut axis:potential for novel therapies.[J].Am J Physiol Gastrointest LiverPhysiol. 2001,280:G787~G794
[91] Booth CE,KirKDp AJ,Hicks GA, et al.Somatostatin sst(2) receptor-mediated inhibition of mesen.teric afferent nerves of the iejunum in the anesthetized rat.[J]. GastroenteroloKg .2001,121:358~369
[92] KirKDp AJ,Booth CE,Chessell IP, et al.Excitatory effect of P2xreceptor activation on mesenteric after-ent nerves in the anaesthetised rat.[J] PhlJsiol .1999,520:551~563
[93] AzimT,Zaki MH,Podder G, et al.Rotavirus-specific subclass antibody ancytokine responses in Bangladeshi children with rotavirus diarrhoea.[J].Med Virol,2003,69(2):286~295.
[94] CuadrasMA,Feigelstock DA,An S, et al.Gene expression pattern in Caco-cells following rotavirus infection.[J].J Virol,2002,76(9):4467~4482.
[95] Colomina J,GilMT,Codoner P, et al.Viral proteinsVP2,VP6 and NSP2 arstrongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection.[J]. Med Virol.1998,56(1):58~65.
[96] Iosef C,Chang KO,Azevedo MS, et al.Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pimodel of human rotavirus disease.[J]. Med Virol,2002.68(1):119~128.
[97] Blutt SE,Warfield KL,Lewis DE, et al.Early response to rotavirus infectio involves massive B cell activation.[J].J Immunol.2002,168(11):5716~5721.
[98] KDshnir N,Bos NA,Zuercher AW, et al.B2 but not B1 cells can contribute to CD4+T-cell-mediated clearance of rotavirus in SCID mice.[J].J Virol. 2001,75(12):5482~5490.
[99] Flores J.Boggeman E,Purcall R A, et al.A dot hybridization assay for detection of rotavirus[J].Lancet. 1983,1:555~558
[100] K.O.Chang. Detection of Group B Rotaviruses in Fecal Samples from Diarrheic Calves and Adult Cows and Characterization of Their VP7 Genes.[J]. JOURNAL OF CLINICAL MICROBIOLOGY Aug. 1997, 2107~2110
[101] Manuel G.Claros. RNA isolation from plant tissues: a practical experience for biological undergraduates.[J]. Biochemical Education 27,1999,110~113
[102] Fedorcsak I, Ehrenberg L.Effects of diethyl pyrocarbonate and methyl methanesulfonate on nucleic acids and nucleases.[J]. Acta Chem Scand 1966,20: 107~112
[103] Maria Marone. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.[J]. Biol.Proced ,Online 2001,3(1):19~25
[104] Gouvea. V, R. I. Glass, P. Woods, et al.Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.[J].J Clin Microbiol, 1990, 28:276~282.
[105] Acres SD, Babiuk LA.Studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay.[J]. J Am Vet Med Assoc. 1978, 1:173(5 Pt 2):555~9
[106] A H Hussein, E Cornaglia, M S Saber, et al.Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec .[J]. Can J Vet Res. 1995 July; 59(3): 235–237.
[107] Niedrig.M., Sonnenberg.K, Steinhagen.K, et al.Comparison of ELISA and immunoassays formeasurement of IgG and IgM antibody to West Nile virus in human sera against virus neutralization. [J].Virol, 2007, 103~105.
[108] Thurber ET, Bass EP, Beckenhauer WH. Field trial evaluation of a reo-coronavirus calf diarrhea vaccine. [J].Can J Comp Med.1977,41:131~136.
[109] Waltner-Toews D, Martin SW, Meek AH, et al. A field trial to evaluate the efficacy of a combined rotavirus-coronavirus/Escherichia coli vaccine in dairy cattle.[J].Can Comp Med. 1985,49:1~9
[110] Collins JK, Riegel CA, Olson JD, et al.Shedding of enteric coronavirus in adult cattle. [J].Am J Vet Res. 1987,48:361~365
[111]Myers H, Snodgrass DR. Colostral and milk antibody titers in cows vaccinated with a modified live-rotavirus –coronavirus vaccine.[J]. Am Vet Med Assoc. 1982,181:289~298.
[112] Estes M K,Cohen J. Rotavirus gene structure and function.[J].Microbiol Rev. 1989,53(4):410~449
[113] 张礼壁等,ELISA方法用于诊断病毒病的进展.病毒学报.[J].1988,4(1)91~95.
[114] 殷震,刘景华.动物病毒学(第二版).[M].北京:科学出版社,1997.562~571.
[115] 蔡宝祥.家畜传染病(第四版).[M].北京:中国农业出版社,2001,401~410.
[116] 何孔旺,林继煌,江杰元,等.牛轮状病毒性腹泻的研究.[J].中国兽医学报,1998,18(2):224~227.
[145] 庞其芳,丘福禧,愈富荣,等.婴幼儿秋季急性胃肠炎病原的轮状病毒的研究.[J].中华医学杂志,1979,59:589~591.
[117] 于恩庶等主编,中国人畜共患病学(第二版).[M],福建科学技术出版社.1996.672~683
[118] 宣长和,任凤兰,孙富先.猪病学(第一版).[M],北京,中国农业出版社,1996.69~71.
[119] 庞昕,柴家前,赵宏坤.猪轮状病毒感染研究进展.[J].吉林农业大学学报. 1999,21(增刊),77~79
[120] 斯特劳B E,阿莱尔S D,蒙加林W L主编2000,猪病学.[M].第八版.中国农业大学出版社,263~276
[121] 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
[123] 李明锐,李春玲,李浩等.牦犊牛流行性腹泻病院的调查,中国兽医科技,199,2:12~13.
[124] 黄文林主编 M 分子病毒学(供研究生用).[M].人民卫生出版社. 2002,84~85、321~333
[125] 温乐英.轮状病毒蛋白的研究进展[J].国外医学病毒学分册. 1996,3(3):65~67
[126] 陈军华,刘作义.轮状病毒感染发病机制研究进展.[J].国外医学儿科学分册 2004.9.31(5)237~239.
[127] 王大燕 , 王 健 伟 , 于 修 平 , 洪涛 . 轮 状 病 毒 致 病 机 制 的 研 究 机 制 .[J]. 世界 华 人 消 化 杂志,2003;11(11):1670~1673
[129] 于力,于康震等编著动物传染病学.[M].中国农业出版社.1997, 197~201
[130] 胥爱源,刘玑昌,丘富禧等.用葡萄球菌 A 蛋白固相免疫电镜法提高轮状病毒检出.[J].中华医学杂志 1983,62(2):73~76
[131] 于恩庶主编中国人兽共患病学[M].第二版.福建科学技术出版社,1996,672~683
[132] 张福萍.诊断 B 组轮状病毒的三种酶联免疫方案的建立[J].中国兽医科技,1995,5(5):3~5
[133] 王文洁,周明锦.应用金标免疫斑点法快速诊断轮状病毒感染及临床意义.[J].河南职工医学院学报 2002.14(2):164~165
[134] 吴亦伦,苏诚钦.轮状病毒快速 ELISA 诊断方法的研究和应用[J].安徽医学. 1998,19(2):1~2.
[135] 李艳萍,白植生.轮状病毒乳胶试剂盒的研制[J].微生物学免疫学进展. 1998,26(4):35~38.
[136] 何孔旺,吴纪棠,林继煌等.用反向间接血凝试验快速检测轮状病毒抗原和抗体[J].病毒学报. 1989,5(1):68~72.
[137] 王正党,王嘉福,张福萍.化学修饰法制备 B 组轮状病毒生物素核酸探针及应用研究[J].中国动物检验. 1995,12(3):5~7
[138] 刘明军,王正党.光敏生物素核酸探针检测 A 群轮状病毒的研究[J].中国畜禽传染病. 1998,20(3):171~172
[139] 傅万海,郝卫等.聚合酶链反应标记轮状病毒地高辛素探针的初步应用[J].中华实验和病毒学杂志. 1998,12(1):77~79
[140] 倪艳秀,林继煌,陆承平等.逆转录-聚合酶链反应(RT-PCR)检测轮状病毒[J].中国兽医学报. 1998,18(5):437~439
[141] 汪招雄,何启盖.检测伪狂犬病病毒双抗体夹心间接 ELISA 方法的建立与应用[J].中国预防兽医学报. 2006, 3 (2) 221~224
[142] 李昌文,刘怀然.应用间接 ELISA 方法检测兔轮状病毒抗体的初步研究[J].中国比较医学杂志.2003,12(6) 373~376
[143] 蔡雪晖,郭宝清,柴文君等,检测猪繁殖呼吸综合征病毒抗体ELISA方法的建立.中国预防兽医学报[J].2000,22(2):122~125
[144] 蒋正军,马世东,蔡丽娟等,非洲猪瘟间接ELISA诊断试剂盒的研究.中国预防兽医学报[J]. 2000,22(增刊),107~113.
[145] 庞其芳,丘福禧,愈富荣,等.婴幼儿秋季急性胃肠炎病原的轮状病毒的研究.[J].中华医学杂志,1979,59:589~591.