臺灣博碩士論文加值系統

中文摘要

全球大部分國家都曾有口蹄疫發生，造成經濟上極大的損失，口蹄疫會造成偶蹄類動物嚴重急性的水泡性疾病，口蹄疫病毒(Foot-and-mouth disease virus;FMDV)屬小核醣核酸病毒科(Picornaviridae)的口瘡病毒屬(Aphthovirus)為最小的RNA病毒，有七種血清型分別為, O, A, C, SAT1, SAT2, SAT3以及Asia 1。口蹄疫病毒對舌、口腔、軟顎等上皮細胞具極強之親和性，在口蹄疫感染耐過後的牛羊可變成帶原者，只有反芻動物會保毒，帶原動物在初期感染後會持續帶毒幾個月，病毒會長期存在咽喉部位，但是豬隻不是本病毒的主要的保毒者。口蹄疫病毒抗體的檢測對於豬隻免疫狀態之評估或帶原者之偵測非常重要，因此開發一個快速、敏感和具有專一性的抗體檢測方法是相當重要的。
本研究將利用RT-PCR技術增幅口蹄疫病毒的VP1和VP2基因，將此二基因選殖至pET21d中，將建構好的重組載體分別命名為pET21d FMDV-VP1及pET21d FMDV-VP2。將pET21d FMDV VP1與 pET21d FMDV VP2重組表現載體分別轉形送入大腸桿菌BL21(DE3)，誘導表現FMDV VP1及FMDV VP2蛋白進行SDS-PAGE分析，結果可以發現FMDV VP1蛋白出現再約25.8 kDa大小的位置，FMDV VP2蛋白出現在約26.4 kDa大小的位置，證實成功表現出預期的蛋白，出現了預期的條帶。使用西方轉漬法（Western blot）利用豬隻感染血清來確認表現FMDV VP1以及VP2蛋白，將所表現的蛋白利用鎳離子親和性管柱純化所表現的蛋白。再將所表現出來所純化的蛋白作為抗原對感染病毒豬隻與免疫豬隻血清，進行酵素結合免疫吸附分析法(ELISA)偵測抗體力價時，結果發現當利用FMDV VP1表現當做抗原時會比利用FMDV VP2蛋白表現當抗原時，可以得到對陽性免疫血清抗體與疫苗血清抗體會有比較好的反應。

Abstract
Foot-and-mouth disease (FMD) outbreaks usually result in huge economic losses. FMD is a severe, clinically acute, vesicular disease of cloven-hoofed animals including domesticated ruminants and swine, FMDV belongs to the Picornaviridae family, and is the only member of the genus Aphthovirus, Serologically, FMDV can be classified into 7 antigenically distinct serotypes, O, A, C, SAT1, SAT2, and SAT3, and Asia 1. The virus has a predilection to replicate in epithelial cells including those lining the distal oropharynx and the dorsal surface of the soft palate. In these sites, infectious virus has been found for several months or even years in a proportion of recovered ruminants but not pigs. Detection of FMDV is important for monitoring vaccination status and detection of the carries. Therefore, it is important to develop diagnostic methods for the detection of FMDV antibody.
In this study, FMDV VP1 and VP2 gene of foot-and-mouth disease virus were amplified by RT-PCR. The DNA containing FMDV VP1 or VP2 gene sequences was further cloned into a prokaryotic expression vetor pET21d, respectively, named pET21d FMDV-VP1 or pET21d FMDV-VP2, respectively. The recombinant FMDV VP1 or VP2 protein was expressed by the host E. coli BL21(DE3). The protein with expected size about 25.8 kDa or 26.4 kDa was obviously observed in SDS-PAGE. The recombinant FMDV VP1 and VP2 proteins were identified by western blot assay with FMDV-infected swine serum. Expressed FMDV VP1 and VP2 proteins were purified by passing through the Ni-NTA Superflow column. Using the purified proteins as a coating antigen, FMDV-infected swine serum and vaccinated swine serum were used for ELISA test. Our results demonstrated that the recombinant FMDV VP1 protein has a better specificity than that of the VP2 recombinant protein.

目錄
第一章 緒言……………………………………………………………………… 1
第二章 文獻探討‥……………………………………………………………… 3
第一節 豬口蹄疫病毒的歷史背景………………………………………… 3
第二節 豬口蹄疫病毒的分類、組成及特性……………………………… 4
第三節 豬口蹄疫病毒基因體的結構……………………………………… 6
第四節 豬口蹄疫病毒基因的表現、基因產物及生物功能‥…………… 6
第五節 豬口蹄疫病毒的病理學、免疫學、流行病學……………………10
第六節 致病機轉‥…………………………………………………………17
第七節 診斷方法‥…………………………………………………………18
第三章 材料與方法 …………………………………………………………… 23
（一）載體的建構 ………………………………………………………… 23
1.1原核表現質體建構利用聚合酵素連鎖反應 ………………………… 23
1.2 DNA片段回收(DNA fragment extraction)………………………… 23
1.3 接和反應(DNA ligation)……………………………………………… 24
1.4 勝任細胞(E. coli competent cells)的製備 ………………………… 25
1.5 轉形作用(Transformation)…………………………………………… 25
1.6 菌株之挑選與質體抽取……………………………………………… 25
1.7 限制酵素分析 …………………………………………………………26
1.8 轉形作用(Transformation)……………………………………………26
1.9 菌株之挑選及確認 ……………………………………………………27
（二）蛋白質膠體電泳分析…………………………………………………27
（三）西方轉漬法(Western blot) ………………………………………….28
（四）重組表現蛋白的純化…………………………………………………29
（五）濃度測定………………………………………………………………30
（六）酵素結合免疫吸附試驗………………………………………………31
第四章 結果………………………………………………………………………32
（一）重組基因再大腸桿菌之表現與確認…………………………………32
（二）表現蛋白的純化與確認………………………………………………32 （三）利用西方墨點法偵測重組表現蛋白…………………………………33 （四）利用酵素結合免疫吸附分析法(ELISA)偵測抗體力價 ……………34
第五章 討論………………………………………………………………………51

























圖次

圖一:口蹄疫病毒基因體結構………………………………………………………6
圖二：口蹄疫病毒結構蛋白VP1上的特異性βG-βH環卡通示意圖 …………9
圖三：FMDV VP1與VP2 之聚合酵素連鎖反應的結果………………………35
圖四：原核表現載體pET 21d FMDV –VP1之建構示意圖……………………36
圖五：原核表現載體pET 21d FMDV –VP2之建構示意圖……………………37
圖六：pET 21d FMDV-VP1重組載體在大腸桿菌BL21中，表現蛋白之SDS-
PAGE電泳分析……………………………………………………………38
圖七：pET 21d FMDV-VP2重組載體在大腸桿菌BL21中，表現蛋白之SDS-
PAGE 電泳分析……………………………………………………………39
圖八：重組載體pET 21d FMDV- VP1在大腸桿菌BL21中表現蛋白純化 SDS-
PAGE 電泳分析……………………………………………………………40
圖九：重組載體pET 21d FMDV- VP2在大腸桿菌BL21中表現蛋白純化 SDS-
PAGE 電泳分析……………………………………………………………41
圖十：重組載體pET 21d FMDV- VP1在大腸桿菌BL 21中表現蛋白純化以西方
墨點法分析…………………………………………………………………42
圖十一：重組載體pET 21d FMDV- VP2在大腸桿菌BL 21中表現蛋白純化以西方
墨點法分析 ………………………………………………………………43
圖十二：重組載體pET 21d FMDV- VP1在大腸桿菌BL 21中表現蛋白純化以西
方墨點法分析 …………………………………………………………44
圖十三：重組載體pET 21d FMDV- VP2在大腸桿菌BL 21中表現蛋白純化以西
方墨點法分析 ……………………………………………………………45
圖十四：口蹄疫表現蛋白VP1與VP2與豬隻血清關係圖 ……………………46
圖十五：口蹄疫蛋白VP1、VP2與豬隻稀釋免疫血清關係圖…………………47
圖十六：口蹄疫蛋白VP1、VP2與豬隻稀釋疫苗血清關係圖…………………48
圖十七：稀釋口蹄疫蛋白VP1、VP2與豬隻免疫血清關係圖…………………49
圖十八：稀釋口蹄疫蛋白VP1、VP2與豬隻疫苗血清關係圖…………………50

參考文獻

田蔚城。2000。生物技術的發展與應用。九洲。第三版。223-241。

朱瑞民、楊平政、鄭益謙。1997。綜說:口蹄疫。中華民國獸醫學會雜誌。
23:477-491。

黃千衿、賴貞利、徐翠君、張天傑、鍾明華。2002。口蹄疫病毒台灣分離株（O/Taiwan/97）之抗原性分析。台灣獸醫學雜誌28：47 - 53。

楊平政。1998。流行病學。台灣豬隻口蹄疫病理圖譜。中華民國獸醫病理學會編印。8 - 11。

鄭秀蓮。1998。病毒之特性。台灣豬隻口蹄疫病理圖譜。中華民國獸醫病理學會編印。5 - 8。

鍾明華、李淑慧。1998。台灣豬隻口蹄疫病毒特性疾病理變化。引自:口蹄疫及豬瘟診斷訓練班講義。台灣省家畜衛生所主編。p.l-13。

Berger HG, Straub OC, Ahl R, Tesar M and Marquadt O. 1990. Identification of foot and mouth disease virus replication in vaccinated cattle by antibodies to non-structural virus proteins. Vaccine, 8：213-216.

Bergmann IE, Malirat V, Neitzert E, Beck E, Panizzutti N, Sanchez C and Falczuk A. 2000. Improvement of serodiagnostic strategy for foot and mouth disease virus surveillance in cattle under systematic vaccination：a combined system of an inderct ELISA-3ABC with an enzyme-linked immunoelectrotransfer blot assay. Arch. Virol.,145：473-489.

Blood DC, Radostits. 1994. Viral disease characterized by alimentary tract
signsFoot-and-mouth disease. In Veterinary Medicine ed., pp824-831.
Bothwell, A., Yancopoulos, GD. and Alt, FW. 1990. Methods for cloning
and analysis of eukaryotic genes. 141-143.

Borman AM, Deliat FG and Kean KM. 1994. Sequence within the poliovirus internal ribosome entry segment control viral RNA sunthesis. EMBO J. 13:3149-3157.

Brown CC, Piccone ME, Mason PW, McKenna TSC, Grubman MJ. 1996. Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. J. Virol. Methods 67: 35-44.

Burness ATH and Clothier FW. 1970. Particle weight and other biophysic- cal properties of encephalomyocarditis virus. J. Gen. Virol. 6: 381-393.

Carrillo C, Plana J, Mascarella R, Bergada J, Sobrino F. 1990. Genetic and
phenotypicvariability during replication offoot-and-mouth disease vims in
swine. Virology 179:890-892

Clayton WB and Mason PW. 2000. Genetic determinants of altered virulence of Taiwan foot and mouth disease virus., J. Virol., 74：987-991

Chinsangaram J, Beard C, Mason PW, Zellner M K, Ward G, and
Grubman MJ. 1998. Antibody Response in Mice Inoculated with DNA Expressing Foot-and-Mouth Disease Virus Capsid Proteins, J. Virology. 72: 4454-4457

Crowther D, Melnick JL. 1961. Studies of the inhibitory action of guanidine on poliovirus multiplication in the cell culture. Virology 15:65-74.

Crowther D and Melnick J L. 1986. Studies of the inhibitory action of guanidine on poliovirus multiplication in cell culture. Virology 15: 65-74.

De Diego M, Brocchi E, Mackay D, De Simone F. 1997. The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Arch. Virol. 142:2021-2033.

Dı´az-San SF, Francisco JS, Ana D A, Ferna´ndez M,Miguel A. Sa´nchez- Martı´n, Noemı´ Sevilla.,2006. Selective Lymphocyte Depletion during the Early Stage of the Immune Response to Foot-and-Mouth Disease Virus Infection in Swine. J. Virol, p. 2369–2379

Domingo E, Baranowski E, Escarmis C and Sobrino F. 2002. Foot-and
-mouth disease virus. Comp Immun Microbiol Infect Dis. 25:297-308.

Donaldson AI, Kitching RP and Barnett PV. 1996. Foot-and-mouth disease. In:OIE manual of standards for diagnostic test and vaccines. Chapter 2. 1. 1., 47-56.

Donaldson AI and Kihm U. 1997. Research and technological developments required for more rapid control and eradication of foot and mouth disease. Rev. Sci. Tech. Oof. Int. Epiz., 15：863-873.

Donaldson AI. 1997. Foot-and-mouth disease in Taiwan. Vet. Rec. 140: 407.

Dunn CS and Donaldson AI. 1997. Natural adaption to pig of a Taiwanese isolte of foot and mouth disease virus. Vet. Rec., 16：174-175.

Erickson JW, Frankenberger EA, Rossmann MG, Fout GS, Medappaand KC Rueckert RR. 1983. Crystallization of a common cold virus, human rhinovirus14: isomorphism with poliovirus crystals. Proc.
Natl. Acad. Sci. USA 80: 931-934.

Falk MM, Sobrino F and Beck E. 1992. VPg amplification correlates with infective particle formation in foot and mouth disease virus. J. Virol., 66：2251-2260.
Flanegan J B, Pettersson RF, Ambrose V, Hewlett MJ and Baltimore D. 1977 .Covalent linkage of a protein to a defined nucleotide sequence at the 5 terminus of the virion and replicative intermediate RNAs of poliovirus. Proc. Natl. Acad. Sci.USA 74: 961-965.

Forss S and Schaller HA. 1982. A tandem repeat gene in a picornavirus.
Nucleic Acids Res. 10: 6441-6450.

Fox G, Parry NR, Barnett PV, Mcginn B, Rowlands DJ and Brown F. 1989. The Cell Attachment Site on Foot-and-Mouth Disease Virus Includes the Amino Acid Sequence RGD(Arginine-Glycine-Aspartic Acid) J. Gen. Virol. 70:625-637.

Fraenkel CH, Kimball PC and Levy JA. 1988. Picornavirus. Virology 2nd ed：83-93.

Francis MJ, Ouldridge EJ and Black L. 1983. Antibody response in bovine pharyngeal fluid following foot and mouth disease vaccination and, or, exposure to live virus. Res. Vet. Sci., 35:206-210.

Funner FJ, Gibbs EPJ, Murphy FA,Rott R, Studdert MJ, White DO. 1993. Foot-and-mouth disease. In: Veterinary Virology. 2nd ed., Academic Press, Inc., San Diego, USA, p408-416.

Haydon D, Lea S, Fry L, Knowles N, Samuel AR, Stuart D, Woolhouse ME.
1998. Characterizing sequence variation in the VPI capsid proteins of foot and mouth disease virus(serotype 0) with respect to virion structure. J Mol Evol. 46:465-75

Huang CC, Jong MH. and Lin SY. 2000. Characteristics of foot and mouth disease virus in Taiwan. Virology, 59：677-679.

Huang CC, Lin YL, Huang TS, Tu WJ, Lee SH, Jong MH and Lin SY. 2001. Molecular characterization of foot and mouth disease virus isolated from ruminants in Taiwan in 1999-2000. Vet. Microbiol., 81：193-205.

Hruby DE and Roberts WK. 1957. Variations in polyadenylic acid content
and biological activity. J. Virol. 19: 325-330.

Jordi XF, Antoni B, Baldomero O, Francesc XA and Antonio V. 1998. Conform ational Flexibility in a Highly Mobile Protein Loop of Foot-and-Mouth Disease Virus: Distinct Structural Requirements for Integrin and Antibody Binding. J. Mol. Biol. 283, 331±338

Klug A and Caspar DLD. 1960. The structure of small viruses. Adv. Virus
Res. 7:225-325.

Kitching RP, 1992. The application of biotechnology to the control of foot-and -mouth diseasevirus. J. Gen. Virol. 45:579-590.

Kitching R P.1998. A recent history of foot-and-mouth disease. J. Camp. Path. 118:89-108.

Kiston JDA, McCahon D, Belsham GJ. 1990. Sequence analysis of monoclonal antibody mutants of type 0 foot and mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites. Virology 179:26-34.

Leippert M, Beck E, Weiland F and Pfaff E. 1997. Point mutations within the βG-ΒH loop of foot and mouth disease virus O1K affect virus attachment to Target cells. J. Virol., 71:1046-1051.

Logan D, Abu GR, Blakemore W, Curry S, Jackson T, King A, Lea S, Lewis R, Newman J, Parry N. 1993.Structure of a major immunogenic site on foot-and-mouth disease virus. Nature. Apr 8: 362(6420): 566-8.

Lubroth J and Brown F. 1995. b. Identification of native foot and mouth disease virus non-structural protein 2C as a serological indicator to differentiate infected from vaccinated livestock. Vet. Sci., 59:70-75.

Mackay DK, Forsyth MA, Davies PR, Berlinzani A, Belsham GJ, Flint M, Ryan MD. 1997. Differentiating infection from vaccination in foot-and- mouth disease using a panel of recombinant, non-structural proteins in ELISA.Vaccine. Mar;16(5): 446-59.

Mason P, Rieder E and Baxt B.1994. RGD sequence of foot and mouth disease virus is essential for infecting cell via the natural receptor but can be bypassed by an antibody dependent enhancement pathway. Proc. Natl. Acad. Sci. USA., 91:1932-1936.

Mackay DK, Forsyth MA, Davies PR, Berlinzani A, Belsham GJ, Flint M, Ryan MD. 1997. Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA.Vaccine. Mar;16(5):446-59.

Mc Cllough KC, Simone FD, Brocchi E, Capucci L, Crowther JR, Kihm U. 1992. Protective immune response against foot-and-mouth disease. J. Virol. 66:1835-1840

Meyer F, Abcock GD, Newman JFE, Burrage TG, Toohey K, Lubroth J and Brown F. 1997. Baculovirus expressed 2C of food-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals. J. Virol. Methods. 65:33-43.

Murphy FA, Gibbs EPJ, Horzinek MC and Studdert MJ. 1999. Picrornaviridae. In: Veterinary Virology, 3th edition, Academic Press, London, 517-532

Nicholson R, Pelletier J, Le SY and Sonenberg N. 1991. Structural and
functionalanalysis of the ribosome landing pad of poliovirus type 2: in
vivo translation studies. J.Virol. 65: 5886-5894.

Pallansch MA, Kew OM, Palmenberg AC, Golini F, Wimmer E and
Ruecker RR. 1980. Picornaviral VPg sequences are contained in the replicase precursor. J.Virol. 35: 414-419.

Parry N, Fox G, Rowland D, Brown F, Fry E, Acharya R, Logan D and
Stuart D.1990. Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus. Nature. 347:569-572.

Perez FM, Wigdorovitz A, Romera A, Zamorano P, Borea MV, Sadir AM. 2000. Detection and characterization of functional T-cell epitopes on the structural proteins VP2, VP3, and VP4 of foot-and-mouth disease virus 01 campos. Virology. 271:234-9.

Piccone ME, Rieder E, Mason PW and Grubman MJ. 1995. The foot-and mouth disease virus leader proteinase gene is not required for viral replication. J. Virol.69: 5376-5382.

Porter AG. 1993. Picornavirus nonstructural proteins：emerging roles in virus replication and inhibition of host cell function. J. Virol., 67：6917-6921.

Reid SM, Forsyth MA, Hutchings GH. and Ferris NP. 1998. Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot and mouth disease . J. Virol. Methods., 70：213-217.

Reid SM, Hutchings GH, Ferris NP and Clercq KD. 1999. Diagnosis of foot and mouth disease by RT PCR：evaluation of primers for serotypic characterization of viral RNA in clinical samples. J. Virol. Methods., 83：113-123.

Reid SM, Ferris NP, Hutchings GH and Samuel AR. 2000. Primary diagnosis of foot and mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods., 89：167-176.

Reid SM, Ferris NP, Bruning A, Hutchings GH, Kowalska Z and Akerblom L. 2001. Development of a novel real time RT-PCR assay for quantitation of foot and mouth disease virus in diverse porcine tissues. J. Virol. Methods., 92:23-35.

Roland RR. 1996. Picornaviridae: The virus and their replication. In Fields Virology.3rd ed., Lippincott-Raven publishers, New York, USA pp. 609-654.
Salt JS, 1993. The carrier state in foot and mouth disease –an immunological review. Br. Vet. J., 149:207-223.

Samina I, Rones ZZ. and Peleg BA. 1997. Homologous and heterologous antibody response of cattle and sheep after vaccination with foot and mouth disease and influenza viruses. Vaccine, 16: 551-557.
Samuel AR and Knowles NJ. 2001. Foot and mouth disease type O virus exhibit genetically and geographically distinct evolutionary lineages. J. Virol., 82:609-621.
Sangar DV, Rowlands DJ, Harris TJR. and Harris F. 1977. Protein covalently linked to foot and mouth disease virus RNA. Nature., 268: 648-650.

Sarnow P, Bernstein HD and Baltimore D. 1986. A poliovirus temperature-s ensitive RNA synthesis mutant located in a noncoding region of the genome. Proc. Natl. Acad. Sci. USA 83: 571-575.

Sarnow P. 1989. Role of the 3 end sequences in infectivity of poliovirus transcripts made in vitro. J. Virol. 63: 467-470.

Saiz JC, Sobrinao F, Dopazo J. 1993. Molecular epidemiology of foot-and-mouth disease virus type 0. J. Gen. Virol. 74: 2281-2285.

Shen F, Chen PD, Walfield AM, House J, Ye J, Brown F. and Wang CY. 1999. Differentiation of convalescent animals from those vaccinated against foot and mouth disease by a peptide ELISA. Vaccine, 17: 3039-3049

Shi J P and Fersht AR. 1984. Fidelity of DNA replication under conditions
used for oligodeoxynucleotide-directed mutagenesis. J. Mol. Biol. 177:
269-278.

Shieh HK.1997. The FMD situation in Taiwan. J. Chin. Soc. Vet. Sci., 23: 395-402.

Semler BL, Anderson CW, Kitamura N, Rothberg PG, Wishart WL and
Wimmer E. 1981.Poliovirus replication proteins:RNA sequence encoding
P3-1b and the sites of proteolytic processing. Proc. Natl. Acad. Sci. USA
78: 3464-3468.

Singh M, Mohan BM, Suryanarayana WS. 1996. Serological and molecular analysis of serotype 0 FMDV isolated form disease outbreaks in India during 1987-91. Virus Res. 43: 48-55.

Sobrino F, Saiz M, Jimenez CMA, Nunez J , Rosas MF,Baranowski E and Ley V. 2001. Foot-and-mouth disease virus:a long known virus, but acurrent threat. Vet Res. 32:1-30

Sorensen KJ, Madsen KG, Madsen ES, Salt JS, Nqindi J and Mackay DKJ. 1998. Different of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D,3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch.Virol.,143:1461-1476.

Svitkin YV, Pestova TV, Maslova SV and Agol VI. 1988. Point mutations modify the response of poliovirus RNA to a translation initiation factor: a comparison of neurovirulent and attenuated strains. Virology 166: 394-404.
.
Tsai CP, Pan CH, Liu MY, Lin YL, Chen CM, Huang TS, Cheng IC, Jong MH, Yang PC. 2000. Molecular epidemiological studies on foot-and-mouth disease type 0 Taiwan viruses form the 1997 epidemic. Vet Microbiol. 74: 207-216.

Weber S, Granzow H, weiland F and Marquardt O. 1996. Intracellular membrane proliferation in E.coli induced by foot and mouth diease virus 3A gene products. Virus Genes, 12: 5-14.

Wimmer E. 1982. Genome-linked proteins of viruses. Cell 28: 199-201.

Viswanathan S, Ratish G, Reddy GR, Suryanarayana W. 1999. Comparative
studies on immunoreactivity of truncated recombinant proteins of foot and
mouth disease virus (FMDV) produced in E. coli and insect cells. Indian J
Exp Biol. 37: 536-540.

VaMateu MG, Camarero JA, Giralt E, Andreu D, Domingo E. 1995. Direct
evaluation of the immunodominance of a major antigenic site of
foot-and-mouth disease virus in a natural host.Virology 206: 298-306.

Zamorano P, Wigdorovitz A, Chaher MT, Femandez FM, Carrillo C,
Marcovecchio FE, Sadir AM, Borca MV. 1994. Recognition of B and T cell
epitopes by cattle immunized with a synthetic Virology 201: 383-387.