Archives of Virology

, Volume 158, Issue 5, pp 993–1001

Development of a liquid-phase blocking ELISA based on foot-and-mouth disease virus empty capsid antigen for seromonitoring vaccinated animals

  • S. H. Basagoudanavar
  • M. Hosamani
  • R. P. Tamil Selvan
  • B. P. Sreenivasa
  • P. Saravanan
  • B. K. Chandrasekhar Sagar
  • R. Venkataramanan
Original Article


In foot-and-mouth disease (FMD) control programme, liquid-phase blocking ELISA (LPBE) is widely used to assay vaccine-induced seroconversion. Currently, the assay utilizes inactivated FMD virus antigen for the detection of antibodies in serum samples. To develop a non-infectious substitute for the antigen in LPBE, we expressed the structural polypeptide of FMDV (serotype A) using a baculovirus expression system, and show that inclusion of viral 3C with reduced protease activity resulted in a higher yield of structural proteins. Structural proteins expressed in insect cells assembled into empty virus-like particles (VLPs) and showed antigenicity comparable to chemically inactivated FMDV. Screening of serum samples from FMD-vaccinated cattle showed that the test performance of VLP-LPBE had a correlation of 0.89 with conventional inactivated virus antigen LPBE. The VLP-LPBE developed here demonstrates the diagnostic application of recombinant FMDV VLPs in monitoring seroconversion following FMD vaccination.


  1. 1.
    Anon (2012) Global Strategy for control of foot-and-mouth disease. In, Bangkok, Thailand, pp 27–29, June 2012Google Scholar
  2. 2.
    Curry S, Abu-Ghazaleh R, Blakemore W, Fry E, Jackson T, King A, Lea S, Logan D, Newman J, Stuart D (1992) Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus. J Mol Biol 228(4):1263–1268PubMedCrossRefGoogle Scholar
  3. 3.
    Fry EE, Stuart DI, Rowlands DJ (2005) The structure of foot-and-mouth disease virus. Curr Top Microbiol Immunol 288:71–101PubMedCrossRefGoogle Scholar
  4. 4.
    Palmenberg AC (1990) Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol 44:603–623PubMedCrossRefGoogle Scholar
  5. 5.
    Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, King A, Lea S, Newman J, Stuart D (1997) Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus. J Virol 71(12):9743–9752PubMedGoogle Scholar
  6. 6.
    Curry SR-RN, Zunszain PA, Leatherbarrow RJ (2007) Foot-and-mouth disease virus 3C protease: recent structural and functional insights into an antiviral target. Int J Biochem Cell Biol 39(1):1–6PubMedCrossRefGoogle Scholar
  7. 7.
    Rweyemamu MM, Terry G, Pay TW (1979) Stability and immunogenicity of empty particles of foot-and-mouth disease virus. Arch Virol 59(1–2):69–79PubMedCrossRefGoogle Scholar
  8. 8.
    Doel TR, Chong WK (1982) Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus. Arch Virol 73(2):185–191PubMedCrossRefGoogle Scholar
  9. 9.
    Grubman MJ, Morgan DO, Kendall J, Baxt B (1985) Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells. J Virol 56(1):120–126PubMedGoogle Scholar
  10. 10.
    Oem JK, Park JH, Lee KN, Kim YJ, Kye SJ, Park JY, Song HJ (2007) Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA. Vaccine 25(20):4112–4121PubMedCrossRefGoogle Scholar
  11. 11.
    Li Z, Yin X, Yi Y, Li X, Li B, Lan X, Zhang Z, Liu J (2011) FMD subunit vaccine produced using a silkworm-baculovirus expression system: protective efficacy against two type Asia1 isolates in cattle. Vet Microbiol 149(1–2):99–103PubMedGoogle Scholar
  12. 12.
    Li Z, Yi Y, Yin X, Zhang Z, Liu J (2008) Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine. PLoS One 3(5):e2273PubMedCrossRefGoogle Scholar
  13. 13.
    Lewis SA, Morgan DO, Grubman MJ (1991) Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs. J virol 65(12):6572–6580PubMedGoogle Scholar
  14. 14.
    Chung YC, Huang JH, Lai CW, Sheng HC, Shih SR, Ho MS, Hu YC (2006) Expression, purification and characterization of enterovirus-71 virus-like particles. World J Gastroenterol 12(6):921–927PubMedGoogle Scholar
  15. 15.
    Cao Y, Lu Z, Sun J, Bai X, Sun P, Bao H, Chen Y, Guo J, Li D, Liu X, Liu Z (2009) Synthesis of empty capsid-like particles of Asia I foot-and-mouth disease virus in insect cells and their immunogenicity in guinea pigs. Vet Microbiol 137(1–2):10–17PubMedCrossRefGoogle Scholar
  16. 16.
    Ko YJ, Choi KS, Nah JJ, Paton DJ, Oem JK, Wilsden G, Kang SY, Jo NI, Lee JH, Kim JH, Lee HW, Park JM (2005) Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 12(8):922–929PubMedGoogle Scholar
  17. 17.
    Pearson LD, Roy P (1993) Genetically engineered multi-component virus-like particles as veterinary vaccines. Immunol Cell Biol 71(Pt 5):381–389PubMedCrossRefGoogle Scholar
  18. 18.
    Roosien J, Belsham GJ, Ryan MD, King AM, Vlak JM (1990) Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors. J Gen Virol 71(Pt 8):1703–1711PubMedCrossRefGoogle Scholar
  19. 19.
    Maranga L, Cruz PE, Aunins JG, Carrondo MJ (2002) Production of core and virus-like particles with baculovirus infected insect cells. Adv Biochem Eng Biotechnol 74:183–206PubMedGoogle Scholar
  20. 20.
    OIE (2012) Foot-and-mouth disease. In: Manual of diagnostic tests and vaccines for terrestrial animals. World organization for animal health web. Accessed 1 Aug 2012
  21. 21.
    Hamblin C, Barnett IT, Hedger RS (1986) A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA. J Immunol Methods 93(1):115–121PubMedCrossRefGoogle Scholar
  22. 22.
    Grubman MJ, Moraes MP, Diaz-San Segundo F, Pena L, de los Santos T (2008) Evading the host immune response: how foot-and-mouth disease virus has become an effective pathogen. FEMS Immunol Med Microbiol 53(1):8–17PubMedCrossRefGoogle Scholar
  23. 23.
    Rowlands DJ, Sangar DV, Brown F (1975) A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus. J Gen Virol 26(3):227–238PubMedCrossRefGoogle Scholar
  24. 24.
    Ko YJ, Lee HS, Jeoung HY, Heo EJ, Ko HR, Chang BS, Joo HD, Gerelmaa U, Dashzeveg B, Tserendorj S, Sodnomdarjaa R, Park JH, Kweon CH, Cho IS, Paik SG (2010) Use of a baculovirus-expressed structural protein for the detection of antibodies to foot-and-mouth disease virus type A by a blocking enzyme-linked immunosorbent assay. Clin Vaccine Immunol 17(1):194–198PubMedCrossRefGoogle Scholar
  25. 25.
    Ko YJ, Lee HS, Park JH, Lee KN, Kim SM, Cho IS, Joo HD, Paik SG, Paton DJ, Parida S (2012) Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea. J Virol Methods 179(1):265–268PubMedCrossRefGoogle Scholar
  26. 26.
    Falk MM, Grigera PR, Bergmann IE, Zibert A, Multhaup G, Beck E (1990) Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3. J Virol 64(2):748–756PubMedGoogle Scholar
  27. 27.
    Belsham GJ, McInerney GM, Ross-Smith N (2000) Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells. J Virol 74(1):272–280PubMedCrossRefGoogle Scholar
  28. 28.
    Sweeney TR, Roque-Rosell N, Birtley JR, Leatherbarrow RJ, Curry S (2007) Structural and mutagenic analysis of foot-and-mouth disease virus 3C protease reveals the role of the beta-ribbon in proteolysis. J Virol 81(1):115–124PubMedCrossRefGoogle Scholar
  29. 29.
    Klopfleisch C, Minh LQ, Giesow K, Curry S, Keil GM (2010) Effect of foot-and-mouth disease virus capsid precursor protein and 3C protease expression on bovine herpesvirus 1 replication. Arch Virol 155(5):723–731PubMedCrossRefGoogle Scholar
  30. 30.
    Curry S, Abrams CC, Fry E, Crowther JC, Belsham GJ, Stuart DI, King AM (1995) Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids. J Virol 69(1):430–438PubMedGoogle Scholar
  31. 31.
    Wagner GG, Card JL, Cowan KM (1970) Immunochemical studies of foot-and-mouth disease. VII. Characterization of foot-and-mouth disease virus concentrated by polyethylene glycol precipitation. Arch Gesamte Virusforsch 30(4):343–352PubMedCrossRefGoogle Scholar
  32. 32.
    Wickham TJ, Nemerow GR (1993) Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system. Biotechnol Prog 9(1):25–30PubMedCrossRefGoogle Scholar
  33. 33.
    Frasca JM, Parks VR (1965) A routine technique for double-staining ultrathin sections using uranyl and lead salts. J Cell Biol 25:157–161PubMedCrossRefGoogle Scholar
  34. 34.
    Maradei E, La Torre J, Robiolo B, Esteves J, Seki C, Pedemonte A, Iglesias M, D’Aloia R, Mattion N (2008) Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina. Vaccine 26(51):6577–6586PubMedCrossRefGoogle Scholar
  35. 35.
    Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1(8476):307–310PubMedCrossRefGoogle Scholar
  36. 36.
    Grubman MJ, Zellner M, Bablanian G, Mason PW, Piccone ME (1995) Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus. Virology 213(2):581–589PubMedCrossRefGoogle Scholar
  37. 37.
    Huang X, Li Y, Fang H, Zheng C (2011) Establishment of persistent infection with foot-and-mouth disease virus in BHK-21 cells. Virol J 8:169PubMedCrossRefGoogle Scholar
  38. 38.
    Van Maanen C, Terpstra C (1989) Comparison of a liquid-phase blocking sandwich ELISA and a serum neutralization test to evaluate immunity in potency tests of foot-and-mouth disease vaccines. J Immunol Methods 124(1):111–119PubMedCrossRefGoogle Scholar
  39. 39.
    Hamblin C, Kitching RP, Donaldson AI, Crowther JR, Barnett IT (1987) Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. III. Evaluation of antibodies after infection and vaccination. Epidemiol Infect 99(3):733–744PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2012

Authors and Affiliations

  • S. H. Basagoudanavar
    • 1
  • M. Hosamani
    • 1
  • R. P. Tamil Selvan
    • 1
  • B. P. Sreenivasa
    • 1
  • P. Saravanan
    • 1
  • B. K. Chandrasekhar Sagar
    • 2
  • R. Venkataramanan
    • 1
  1. 1.Indian Veterinary Research InstituteBangaloreIndia
  2. 2.Department of NeuropathologyNIMHANSBangaloreIndia

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