Substitutions accrued on Foot-and-mouth disease virus capsid during propagation in cell culture

  • Laxmi N. Sarangi
  • Jajati K. Mohapatra
  • Saravanan Subramaniam
  • Biswajit Das
  • Aniket Sanyal
  • Bramhadev Pattnaik
Research Article


Three lineages of serotype O of foot-and-mouth disease virus were passaged serially in BHK-21 cell culture without application of any immune pressure, to study the frequency, nature and location of the substitutions accruing on the virus capsid. The viruses showed unusual stability as only 12 substitutions were observed in 13 different regimens and the majority of the substitutions reverted back to the parental genotype very soon after their appearance. Of the 12 substitutions, a maximum of 8 were found in the VP1 region. Some substitutions (81, 147, 152, 203 and 210 in VP1 and 50 in VP3) were observed at the established antigenic sites suggesting that antigenic diversification can occur in the absence of immune selection. The viruses after serial cytolytic infection of BHK-21 cells, demonstrated an ability to infect the integrin-deficient CHO-K1 cell line suggesting an expansion in their receptor usage potential. Even after 25–50 passages in BHK-21 cell system no histidine to arginine switch was observed at the 56th residue of VP3. Amino acid sequence analysis of 141 Indian field isolates for the residues involved in heparin binding sites suggest the importance of net positive charge in the HS-binding pocket or elsewhere on the capsid for interaction with the alternative receptors and cell culture adaptation rather than acquisition of positive charge at any particular position for all serotype O strains.


Foot-and-mouth disease virus Serotype O virus Serial cytolytic infection Antigenic sites Heparin binding sites 



The authors are thankful to Indian Council of Agricultural Research for providing necessary facilities to carry out this work. The first author is thankful to ICAR for providing senior research fellowship during his doctoral degree.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Sobrino F, Dávila M, Ortín J, Domingo E (1983) Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture. Virology 128:310–318CrossRefPubMedGoogle Scholar
  2. 2.
    Arias A, Lazaro E, Escarmis C, Domingo E (2001) Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning. J Gen Virol 82:1049–1060CrossRefPubMedGoogle Scholar
  3. 3.
    Domingo E, Escarmis C, Baronowski E et al (2003) Evolution of foot-and-mouth disease virus. Virus Res 91:47–63CrossRefPubMedGoogle Scholar
  4. 4.
    Domingo E, Escarmis C, Martinez MA, Martinez-Salas E, Mateu MG (1992) Foot-and-mouth disease virus populations are quasispecies. Curr Top Microbiol Immunol 176:33–47PubMedGoogle Scholar
  5. 5.
    Borrego B, Novella IS, Andreu D, Giralt E, Domingo E (1993) Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence and absence of immune selection. J Virol 67:6071–6079PubMedPubMedCentralGoogle Scholar
  6. 6.
    Domingo E, Díez J, Martínez MA et al (1993) New observations on antigenic diversification of RNA viruses. Antigenic variation is not dependent on immune selection. J Gen Virol 74(10):2039–2045CrossRefPubMedGoogle Scholar
  7. 7.
    ManojKumar R, Sanyal A, Tosh C et al (2003) Mutation in the 1D gene (VP1) of foot-and-mouth disease virus serotype Asia1 during serial cytolytic infections in cell culture. Arch Virol 148:1815–1825CrossRefGoogle Scholar
  8. 8.
    Sarangi LN, Mohapatra JK, Subramaniam S, Sanyal A, Pattnaik B (2013) Antigenic site variation in foot-and-mouth disease virus serotype O grown under vaccinal serum antibodies in vitro. Virus 176:273–279CrossRefGoogle Scholar
  9. 9.
    Domingo E, Holland JJ (1997) RNA virus mutations and fitness for survival. Annu Rev Microbiol 51:151–178CrossRefPubMedGoogle Scholar
  10. 10.
    Domingo E (1999) Quasispecies. In: Granoff A, Webster RG (eds) Encyclopedia of virology. Academic Press, London, pp 1431–1436CrossRefGoogle Scholar
  11. 11.
    Haydon DT, Woolhouse ME (1998) Immune avoidance strategies in RNA viruses: fitness continuums arising from trade-offs between immunogenicity and antigenic variability. J Theor Biol 193:601–612CrossRefPubMedGoogle Scholar
  12. 12.
    Domingo E, Biebricher C, Eigen M, Holland JJ (2001) Quasispecies and RNA virus evolution: principles and consequences. Landes Bioscience, AustinGoogle Scholar
  13. 13.
    Mateu MG, Martinez MA, Capucci L et al (1990) A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus serotype C. J Gen Virol 71:629–637CrossRefPubMedGoogle Scholar
  14. 14.
    González M, Mateu M, Martínez MA, Carrillo C, Sobrino F (1992) Comparison of capsid protein VP1 of the viruses used for the production and challenge of foot-and-mouth disease vaccines in Spain. Vaccine 10:731–734CrossRefPubMedGoogle Scholar
  15. 15.
    Feigelstock DA, Mateu MG, Valero ML, Andreu D, Domingo E, Palma EL (1996) Emerging foot-and-mouth disease virus variants with antigenically critical amino acid substitutions predicted by model studies using reference viruses. Vaccine 14:97–102CrossRefPubMedGoogle Scholar
  16. 16.
    Holgui´n A, Herna´ndez J, Marti´nez MA, Mateu MG, Domingo E (1997) Differential restrictions on antigenic variation among antigenic sites of foot-and-mouth disease virus in the absence of antibody selection. J Gen Virol 78(3):601–609CrossRefGoogle Scholar
  17. 17.
    Jackson T, Ellard FM, Ghazaleh RA et al (1996) Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J Virol 70:5282–5287PubMedPubMedCentralGoogle Scholar
  18. 18.
    Baranowski E, Sevilla N, Verdaguer N, Ruiz-Jarabo CM, Beck E, Domingo E (1998) Multiple virulence determinants of foot-and-mouth disease virus in cell culture. J Virol 72:6362–6372PubMedPubMedCentralGoogle Scholar
  19. 19.
    Baranowski E, Ruiz-Jarabo CM, Sevilla N, Andreu D, Beck E, Domingo E (2000) Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage. J Virol 74:1641–1647CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Fry EE, Lea SM, Jackson T et al (1999) The structure and function of a foot-and-mouth disease virus- oligosaccharide receptor complex. EMBO J 18:543–554CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Sa-Carvalho D, Rieder E, Baxt B, Rodarte R, Tanuri A, Mason PW (1997) Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. J Virol 71:5115–5123PubMedPubMedCentralGoogle Scholar
  22. 22.
    Escarmís C, Carrillo EC, Ferrer M et al (1998) Rapid selection in modified BHK-21 cells of a foot and-mouth disease virus variant showing alterations in cell tropism. J Virol 72:10171–10179PubMedPubMedCentralGoogle Scholar
  23. 23.
    Sobrino F, Sáiz M, Jiménez-Clavero MA et al (2001) Foot-and-mouth disease virus: a long known virus, but a current threat. Vet Res 32:1–30CrossRefPubMedGoogle Scholar
  24. 24.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sevilla N, Domingo E (1996) Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification. J Virol 70:6617–6624PubMedPubMedCentralGoogle Scholar
  26. 26.
    Pandey LK, Mohapatra JK, Subramaniam S, Sanyal A, Pande V, Pattnaik B (2014) Evolution of serotype A foot-and-mouth disease virus capsid under neutralizing antibody pressure in vitro. Virus Res 181:72–76CrossRefPubMedGoogle Scholar
  27. 27.
    Airaksinen A, Pariente N, Menendez-Arias L, Domingo E (2003) Curing of foot and mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis. Virology 311:339–349CrossRefPubMedGoogle Scholar
  28. 28.
    Rieder-Rojas E, Carrillo EC, Schiappacassi M, Campos R (1992) Modification of foot-and-mouth disease virus O1 Caseros after serial passages in the presence of antiviral polyclonal sera. J Virol 66:3368–3372Google Scholar
  29. 29.
    Fry EE, Newman JWI, Curry S et al (2005) Structure of foot and-mouth disease virus serotype A1061 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J Gen Virol 86:1909–1920CrossRefPubMedGoogle Scholar
  30. 30.
    Mohapatra JK, Pandey LK, Rai DK et al (2015) Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions. J Gen Virol 96(3):553–564CrossRefPubMedGoogle Scholar

Copyright information

© The National Academy of Sciences, India 2018

Authors and Affiliations

  • Laxmi N. Sarangi
    • 1
    • 2
  • Jajati K. Mohapatra
    • 1
  • Saravanan Subramaniam
    • 1
  • Biswajit Das
    • 1
  • Aniket Sanyal
    • 1
  • Bramhadev Pattnaik
    • 1
  1. 1.ICAR- Directorate of Foot-and-Mouth DiseaseMukteswar-Kumaon, NainitalIndia
  2. 2.National Dairy Development Board Research and Development LaboratoryHyderabadIndia

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