Cell Culture for Production of Insecticidal Viruses

  • Steven Reid
  • Leslie C. L. Chan
  • Leila Matindoost
  • Charlotte Pushparajan
  • Gabriel Visnovsky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1477)

Abstract

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20–500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.

The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Key words

Insect cell technology Suspension culture Adherent culture Insecticidal viruses Bioreactors 

References

  1. 1.
    Reid S, Chan LCL, Van Oers MM (2014) Production of entomopathoginic viruses. In: Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI (eds) Mass production of beneficial organisms Invertebrates and entomopathogens. Elsevier, Amsterdam. ISBN 978-0-12-391453-8Google Scholar
  2. 2.
    Harrison R, Hoover K (2012) Baculoviruses and other occluded insect viruses. In: Vega F, Kaya H (eds) Insect pathology, 2nd edn. Elsevier, Amsterdam, pp 73–131CrossRefGoogle Scholar
  3. 3.
    Buerger P, Hauxwell C, Murray D (2007) Nucleopolyhedrovirus introduction in Australia. Virol Sin 22:173–179CrossRefGoogle Scholar
  4. 4.
    Ignoffo CM (1973) Development of a viral insecticide – concept to commercialization. Exp Parasitol 33:380–406CrossRefPubMedGoogle Scholar
  5. 5.
    Black BC, Brennan LA, Dierks PM, Gard IE (1997) Commercialisation of baculoviral insecticides. In: Miller LK (ed) The baculoviruses. Plenum, New York, NY, pp 341–388CrossRefGoogle Scholar
  6. 6.
    McIntosh AH, Ignoffo CM (1981) Replication and infectivity of the single-embedded nuclear polyhedrosis-virus, Baculovirus-heliothis, in homologous cell-lines. J Invertebr Pathol 37:258–264CrossRefGoogle Scholar
  7. 7.
    Lua LHL, Reid S (2000) Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture. J Gen Virol 81:2531–2543CrossRefPubMedGoogle Scholar
  8. 8.
    Nguyen Q, Qi YM, Wu Y, Chan LCL, Nielsen LK, Reid S (2011) In vitro production of Helicoverpa baculovirus biopesticides—automated selection of insect cell clones for manufacturing and systems biology studies. J Virol Methods 175:197–205CrossRefPubMedGoogle Scholar
  9. 9.
    Pedrini MRS, Reid S, Nielsen L, Chan LCL (2011) Kinetic characterization of the Group II Helicoverpa armigera nucleopolyhedrovirus propagated in suspension cell cultures: Implications for development of a biopesticides production process. Biotechnol Prog 27:614–624CrossRefPubMedGoogle Scholar
  10. 10.
    Mena JA, Kamen AA (2011) Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev Vaccines 10:1063–1081CrossRefPubMedGoogle Scholar
  11. 11.
    Almeida AF, Macedo GR, Chan LCL, Pedrini MRS (2010) Kinetic analysis of in vitro production of wild-type Spodoptera frugiperda nucleopolyhedrovirus. Braz Arch Biol Technol 53:285–291CrossRefGoogle Scholar
  12. 12.
    Micheloud GA, Gioria VV, Eberhardtb I, Visnovsky G, Claus J (2011) Production of the Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line in stirred reactor and airlift reactor. J Virol Methods 178:106–116CrossRefPubMedGoogle Scholar
  13. 13.
    Micheloud GA, Gioria VV, Perez G, Claus JD (2009) Production of occlusion bodies of Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line: influence of infection conditions and statistical optimization. J Virol Methods 162:258–266CrossRefPubMedGoogle Scholar
  14. 14.
    Crawford AM, Zelazny B, Alfiler RA (1986) Genotypic variation in geographical isolates of Oryctes baculovirus. J Gen Virol 67:949–952CrossRefGoogle Scholar
  15. 15.
    Zelazny B, Lolong A, Crawford AM (1990) Introduction and field comparison of baculovirus strains against Oryctes rhinoceros (Coleoptera: Scarabaeidae) in the Maldives. Environ Entomol 19:1115–1121CrossRefGoogle Scholar
  16. 16.
    Crawford AM (1982) A coleopteran cell line derived from Heteronychus arator (Coleoptera: Scarabaeidae). In Vitro 18:813–816CrossRefGoogle Scholar
  17. 17.
    Jackson TA, Crawford AM, Glare TR (2005) Oryctes virus—time for a new look at a useful biocontrol agent. J Invertebr Pathol 89:91–94CrossRefPubMedGoogle Scholar
  18. 18.
    Bedford GO (2014) Advances in the control of rhinoceros beetle, Oryctes rhinoceros. In oil palm—review article. J Oil Palm Res 26:183–194Google Scholar
  19. 19.
    Manjeri GR, Muhamad R, Tan SG (2014) Oryctes rhinoceros beetles, an oil palm pest in Malaysia. Ann Res Rev Biol 4:3429–3439CrossRefGoogle Scholar
  20. 20.
    Portner R (2014) Animal cell biotechnology, 3rd edn. Humana, New York, NY. ISBN 978-1-62703-732-7CrossRefGoogle Scholar
  21. 21.
    Murhammer DW (2007) Baculovirus and insect cell expression protocols, 2nd edn. Humana, Totowa, NJ. ISBN 978-1-59745-457-5CrossRefGoogle Scholar
  22. 22.
    Drugmand J-C, Schneider Y-J, Agathos SN (2012) Insect cells as factories for biomanufacturing. Biotechnol Adv 30:1140–1157CrossRefPubMedGoogle Scholar
  23. 23.
    Felberbaum RS (2015) The baculovirus expression vector system: a commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnol J 10:702–714CrossRefPubMedGoogle Scholar
  24. 24.
    Meghrous J, Mahmoud W, Jacob D, Chubet R, Cox M, Kamen AA (2010) Development of a simple and high-yielding fed-batch process for the production of influenza vaccines. Vaccine 28:309–316CrossRefGoogle Scholar
  25. 25.
    Chan LCL, Greenfield PF, Reid S (1998) Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system. Biotech Bioeng 59:178–188CrossRefGoogle Scholar
  26. 26.
    Matindoost L, Hu H, Chan LCL, Nielsen LK, Reid S (2014) The effect of cell line, phylogenetics and medium on baculovirus budded virus yield and quality. Arch Virol 159:91–102CrossRefPubMedGoogle Scholar
  27. 27.
    Tran TTB, Dietmair S, Chan LCL, Huynh HT, Nielsen LK, Reid S (2012) Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insectcells. Methods 56:396–407CrossRefPubMedGoogle Scholar
  28. 28.
    Nguyen Q, Nielsen LK, Reid S (2013) Genome scale transcriptomics of baculovirus-insect interactions. Viruses 5:2721–2747. doi: 10.3390/v5112721 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Monteiro F, Carinhas N, Carrondo MJ, Bernal V, Alves PM (2012) Toward system-level understanding of baculovirus-host cell interactions: from molecular fundamental studies to large-scale proteomics approaches. Front Microbiol 3:391CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chen YR, Zhong S, Fei Z, Gao S, Zhang S, Li Z, Wang P, Blissard GW (2014) Transcriptome responses of the host Trichoplusia ni to infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus. J Virol 88:13781–13797CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Visnovsky G, Claus J (1994) Influence of the time and multiplicity of infection on the batch production of Anticarsia gemmatalis nuclear polyhedrosis virus in lepidopteran insect cell cultures. Adv Bioproc Eng 1994:123–128CrossRefGoogle Scholar
  32. 32.
    Pushparajan C, Claus JD, Marshall SDG, Visnovsky G (2013) Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line. Cytotechnology 65:1003–1016CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Nielsen LK, Smyth GK, Greenfield PF (1991) Hemacytometer cell count distributions-implications of non-poisson behavior. Biotechnol Prog 7:560–563CrossRefGoogle Scholar
  34. 34.
    Pijlman GP, Van Schijndel JE, Vlak JM (2003) Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J Gen Virol 84:2669–2678CrossRefPubMedGoogle Scholar
  35. 35.
    Lua LHL, Pedrini MRS, Reid S, Robertson A, Tribe DE (2002) Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. J Gen Virol 83:945–955CrossRefPubMedGoogle Scholar
  36. 36.
    Lynn DE (1994) Enhanced infectivity of occluded virions of the gypsy-moth nuclear polyhedrosis-virus for cell-cultures. J Invertebr Pathol 63:268–274CrossRefGoogle Scholar
  37. 37.
    Nielsen J, Villadsen J, Liden G (2003) Bioreaction engineering principles. Kluwer, New York, NYCrossRefGoogle Scholar
  38. 38.
    Zhu H, Nienow AW, Bujalski W, Simmons MJH (2009) Mixing studies in a model aerated bioreactor equipped with an up- or a down-pumping ‘Elephant Ear’ agitator: power, hold-up and aerated flow field measurements. Chem Eng Res Des 87:307–317CrossRefGoogle Scholar
  39. 39.
    Visnovsky G, Claus J, Merchuk JC (2003) Airlift reactors as a tool for insect cells and baculovirus mass production. LA Appl Res 33:117–121Google Scholar
  40. 40.
    Louis KS, Seigel AC (2011) Cell viability analysis using Trypan blue: manual and automated methods. Mammalian cell viability. Methods Mol Biol 740:7–12CrossRefPubMedGoogle Scholar
  41. 41.
    Phillips HJ (1973) Dye exclusion tests for cell viability. In: Kruse PF, Patterson MK (eds) Tissue culture. Academic, London, pp 406–408CrossRefGoogle Scholar
  42. 42.
    Vaughn JL (1976) The production of nuclear polyhedrosis viruses in large-volume cell cultures. J Invertebr Pathol 28:233–237CrossRefGoogle Scholar
  43. 43.
    Pushparajan C (2015) Development and optimization of an in vitro process for the production of Oryctes nudivirus in insect cell cultures. Ph.D. thesis, University of Canterbury, ChristchurchGoogle Scholar
  44. 44.
    Ikonomou L, Drugmand J-C, Bastin G, Schneider Y-J, Agathos SN (2002) Microcarrier culture of lepidopteran cell lines: implications for growth and recombinant protein production. Biotechnol Prog 18:1345–1355CrossRefPubMedGoogle Scholar
  45. 45.
    Lazar A, Silberstein L, Reuveny S, Mizrahi A (1987) Microcarriers as a culturing system of insect cells and insect viruses. Dev Biol Stand 66:315–323PubMedGoogle Scholar
  46. 46.
    Sandford KK, Earle WR, Evans JE, Waltz HK, Shannon JE (1951) The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J Natl Cancer Inst 11:773–795Google Scholar
  47. 47.
    Van Wezel AL (1973) Microcarrier cultures of animal cells. In: Kruse PF, Patterson MK (eds) Tissue culture: methods and applications. Academic, New York, NY, p 372CrossRefGoogle Scholar
  48. 48.
    Reed LJ, Muench H (1938) A simple method of estimating 50% endpoints. Am J Epidemiol 27:493–497Google Scholar
  49. 49.
    Huynh HT, Tran TTB, Chan LCL, Nielsen LK, Reid S (2013) Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression. Appl Microbiol Biotech 97:5245–5257CrossRefGoogle Scholar
  50. 50.
    Huynh HT, Chan LCL, Tran TTB, Nielsen LK, Reid S (2012) Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine. Biotechnol Prog 28:788–802CrossRefPubMedGoogle Scholar
  51. 51.
    Agathos SN (2007) Development of serum free media for lepidopteran insect cell lines. In: Murhammer DW (ed) Baculovirus and insect cell expression protocols, 2nd edn. Humana, Totowa, NJ. ISBN 978-1-59745-457-5Google Scholar
  52. 52.
    Matindoost L, Chan LCL, Qi YM, Nielsen LK, Reid S (2012) Suspension culture titration: a simple method for measuring baculovirus titers. J Virol Methods 183:201–209CrossRefPubMedGoogle Scholar
  53. 53.
    Jorio H, Tran R, Kamen A (2006) Stability of serum-free and purified baculovirus stocks under various storage conditions. Biotechnol Prog 22:319–325CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Steven Reid
    • 1
  • Leslie C. L. Chan
    • 2
  • Leila Matindoost
    • 3
  • Charlotte Pushparajan
    • 4
  • Gabriel Visnovsky
    • 5
  1. 1.School of Chemistry & Molecular BiosciencesThe University of QueenslandBrisbaneAustralia
  2. 2.Patheon Biologics Australia Pty LtdWoolloongabbaAustralia
  3. 3.Australian Institute for Bioengineering and NanotechnologyThe University of QueenslandBrisbaneAustralia
  4. 4.Laboratory for Evolution and Development, Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  5. 5.Chemical & Process Engineering DepartmentUniversity of CanterburyCanterburyNew Zealand

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