, Volume 27, Issue 1, pp 84–90 | Cite as

Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae

  • Manoj Kumar
  • P. SaravananEmail author
  • S. K. Jalali
Original Article


Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7 days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47 kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30–2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.


FMDV Virus like particles (VLPs) Eri silkworm larva Hemolymph Baculovirus expression Purification of VLPs 



The authors thank Director, IVRI, Izatnagar, Uttar Pradesh, India and Joint Director, IVRI Campus, Hebbal, Bangalore, Karnataka, India, for providing the necessary facilities to carry out the research work. Authors are also grateful to the assistance provided by laboratory staff of FMD Vaccine Production Laboratory and Isolation unit of Yelahanka, IVRI Campus, Bangalore, India. The financial assistance granted to Dr. Manoj Kumar, in the form of Institute Fellowship for Master degree from IVRI is gratefully acknowledged.


  1. 1.
    Baek JO, Seo JW, Kim IH, Kim CH. Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography. Protein Expr Purif. 2011;75(2):211–7.CrossRefPubMedGoogle Scholar
  2. 2.
    Bhat SA, Saravanan P, Hosamani M, Basagoudanavar SH, Sreenivasa BP, Tamilselvan RP, Venkataramanan R. Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge. Res Vet Sci. 2013;95(3):1217–23.CrossRefPubMedGoogle Scholar
  3. 3.
    Biswal JK, Sanyal A, Rodriguez LL, Saravanan S, Artz J, Sharma GK, Hammond JM, Parida P, Mohapatra JK, Mathapati BS, Dash BB, Ranjan R, Rout M, Venketaramanan R, Misri J, Krishna L, Prasad G, Pathak KML, Pattnaik B. Foot-and-mouth disease: global status and Indian perspective. Indian J Anim Sci. 2012;82(2):109–31.Google Scholar
  4. 4.
    Cao Y, Lu Z, Sun J, Bai X, Sun P, Bao H, Chen Y, Guo J, Li D, Liu X, Liu Z. 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. 2009;137(1):10–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris. Protein Expr Purif. 2011;75(2):218–24.CrossRefPubMedGoogle Scholar
  6. 6.
    Grubman MJ, Baxt B. Foot-and-mouth disease. Clin Microbiol Rev. 2004;17(2):465–93.PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Hu YC, Hsu JT, Huang JH, Ho MS, Ho YC. Formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing P1 and 3CD in insect cells. Biotechnol Lett. 2003;25:919–25.CrossRefPubMedGoogle Scholar
  8. 8.
    Ko YJ, Kang SC, Nah JJ, Paton DJ, Oem JK, Wilsden G, Kang SY, Jo NI, Lee JH, Kim JH, Lee HW, Park JM. Non infectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 2005;12(8):922–9.PubMedCentralPubMedGoogle Scholar
  9. 9.
    Kushnir N, Streatfield S, Yusibov V. Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine. 2012;31(1):58–83.CrossRefPubMedGoogle Scholar
  10. 10.
    Lee CD, Yan YP, Liang SM, Wang TF. Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli. J Biomed Sci. 2009;11(16):69.CrossRefGoogle Scholar
  11. 11.
    Li Z, Yi Y, Yin X, Zhang Z, Liu J. Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine. PLoS ONE. 2008;3(5):E2273. doi: 10.1371/journal.pone.0002273.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Li Z, Yin X, Yi Y, Li X, Li B, Lan X, Zhang Z, Liu J. FMD subunit vaccine produced using a silkworm-baculovirus expression system: protective efficacy against two type Asia 1 isolates in cattle. Vet Microbiol. 2011;149(1–2):99–103.PubMedGoogle Scholar
  13. 13.
    Li Z, Yi Y, Yin X, Zhang Y, Liu M, Liu H, Li X, Li Y, Zhang Z, Liu J. Development of a foot-and-mouth disease virus serotype a empty capsid subunit vaccine using silkworm (Bombyx mori) pupae. PLoS ONE. 2012;7(8):e43849. doi: 10.1371/journal.pone.0043849.PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Nerome K, Sugita S, Kuroda K, Hirose T, Matsuda S, Majima K, Kawasaki K, Shibata T, Poetri ON, Soejoedono RD, Mayasari NL, Agungpriyono S, Nerome R. The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae. Vaccine. 2015;33(1):117–25.CrossRefPubMedGoogle Scholar
  15. 15.
    Rodriguez LL, Gay CG. Development of vaccines toward the global control and eradication of foot-and-mouth disease. Expert Rev Vaccines. 2011;10(3):377–87.CrossRefPubMedGoogle Scholar
  16. 16.
    Rueckert R. Picornaviruses and their replication. In: Fields B, editor. Virology. New York: Raven Press; 1985. p. 705.Google Scholar
  17. 17.
    Singh B, Prasad S, Sinha DK, Verma MD. Estimation of economic losses due to foot-and-mouth disease in India. Indian J Anim Sci. 2013;83(9):964–70.Google Scholar
  18. 18.
    Urakawa T, Ferguson M, Minor PD, Cooper J, Sullivan M, Almond JW, Bishop DHL. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J Gen Virol. 1989;70(6):1453–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Valarcher JF, Leforban Y, Rweyemamu M, Roeder PL, Gerbier G, Mackay DK, Sumption KJ, Paton DJ, Knowles NJ. Incursions of foot-and-mouth disease virus into Europe between 1985 and 2006. Transbound Emerg Dis. 2008;55(1):14–34.CrossRefPubMedGoogle Scholar
  20. 20.
    Xiang J, Wunschmann S, George SL, Klinzman D, Schmidt WN, LaBrecque DR, Stapleton JT. Recombinant hepatitis C virus-like particles expressed by baculovirus utility in cell-binding and antibody detection assays. J Med Virol. 2002;68(4):537–43.CrossRefPubMedGoogle Scholar

Copyright information

© Indian Virological Society 2015

Authors and Affiliations

  1. 1.Foot-and-Mouth Disease Vaccine CentreIndian Veterinary Research InstituteBangaloreIndia
  2. 2.National Bureau of Agricultural Insect ResourcesBangaloreIndia

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