Real-Time RT-PCR for Quantitative Detection of Bovine Viral Diarrhoea Virus during Manufacture of Biologics

생물의약품 제조공정에서 Bovine Viral Diarrhoea Virus 정량 검출을 위한 Real-Time RT-PCR

  • Cho, Hang-Mee (Department of Biological Sciences, Hannam University) ;
  • Lee, Dong-Hyuck (Department of Biological Sciences, Hannam University) ;
  • Kim, Hyun-Mi (Department of Biological Sciences, Hannam University) ;
  • Kim, In-Seop (Department of Biological Sciences, Hannam University)
  • 조항미 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 이동혁 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김현미 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명과학과)
  • Published : 2008.03.28


Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologics using bovine materials have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine viral diarrhoea virus (BVDV) is the most common bovine pathogen and has widely been known as a contaminant of biologics. In order to establish the validation system for the BVDV safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BVDV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BVDV RNA was selected, and BVDV RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1 $TCID_{50}/mL$. The rent-time RT-PCR method was validated to be reproducible and very specific to BVDV. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BVDV. BVDV RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect $10TCID_{50}/mL$ of BVDV artificially contaminated in bovine collagen.


  1. Adamson, S. R. 1999. Experiences of virus, retrovirus and retrovirus-like particles in chinese hamster ovary (CHO) and hybridoma cells used for production of protein therapeutics. Dev. Biol. Stand. 93: 89-96
  2. Bhudevi, B. and D. Weinstock. 2003. Detection of bovine viral diarrhoea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman). J. Virol. Methods 109: 25-30
  3. Bolin, S. R., J. F. Ridpath, J. Black, M. Macy, and R. Roblin. 1994. Survery of cell lines in the American Type Culture Collection for bovine viral diarrhea virus. J. Virol. Methods 48: 211-221
  4. Bruhn, S., L. Bruckner, and H. P. Ottiger. 2005. Application of RT-PCR for the detection of avian reovirus contamination in avian viral vaccines. J. Virol. Methods 123: 179-186
  5. Celis, P. and G. Silvester. 2004. European regulatory guidance on virus safety of recombinant proteins, monoclonal antibodies and plasma derived medicinal products. Dev. Biol. Stand. 118: 3-10
  6. Choi, Y.-M., J.-K. Kim, J.-I. Park, and S.-W. Jeong. 2006. Evaluation of bovine amniotic membrane for the treatment of superficial canine corneal ulcer. J. Vet. Clinics 23: 334-336
  7. Code of federal regulation 9 (9CFR), animal and animal products. 1996. part 113.53. Requirement for ingredients of animal origin used for production of biologics
  8. Darling, A. 2002. Validation of biopharmaceutical purification process for virus clearance evaluation. Mol. Biotechnol. 21: 57-83
  9. Eloit, M. 1999. Risks of virus transmission associated with animal sera or substitutes and methods of control. Dev. Biol. Stand. 99: 9-16
  10. Erickson, G. A., S. R. Bolin, and J. G. Landgraf. 1991. Viral contamination of fetal bovine serum used for tissue culture: risks and concerns. Dev. Biol. Stand. 75: 173-175
  11. Faraj, K. A., T. H. van Kuppevelt, and W. F. Daamen. 2007. Construction of collagen scaffolds that mimic the threedimensional architecture of specific tissues. Tissue Eng. 13: 2387-2394
  12. Gajiwala, K. and A. L. Gajiwala. 2004. Evaluation of lyophilised, gamma-irradiated amnion as a biological dressing. Cell Tissue Bank 5: 73-80
  13. Garnick, R. L. 1998. Raw materials as a source of contamination in large-scale cell culture. Dev. Biol. Stand. 93: 21-29
  14. Harasawa, R. and H. Mizusawa. 1995. Demonstration and genotyping of pestivirus RNA from mammalian cell lines. Microbiol. Immunol. 39: 979-985
  15. Harasawa, R. and T. Tetsuo. 1994. Evidence of pestivirus RNA in human virus vaccines. J. Clin. Microbiol. 32: 1604-1605
  16. Horaud, F. 1991. Introductory remark: viral safety of biologicals. Dev. Biol. Stand. 75: 3-7
  17. International Conference on Harmonisation. 1998. Guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin; availability. Federal Resister 63: 51074-51084
  18. Jennings, A. 1999. Detecting viruses in sera: methods used and their merits. Dev. Biol. Stand. 99: 51-59
  19. Jeong, H.-S., J,-H. Shin, Y.-N. Park, J.-Y. Choi, Y.-L. Kim, B.-G. Kim, S.-R. Ryu, S.-Y. Baek, S.-H. Lee, and S.-N. Park. 2003. Development of real-time RT-PCR for evaluation of JEV clearance during purification of HPV type 16 L1 viruslike particles. Biologicals 31: 223-229
  20. Kil, T. G., W. J. Kim, D. H. Lee, Y. Kang, H. M. Sung, S. H. Yoo, S.-N. Park, and I. S. Kim. 2005. Quantitative real-time PCR of porcine parvovirus as a model virus for cleaning validation of chromatography during manufacture of plasma derivatives. Kor. J. Microbiol. 41: 216-224
  21. Letellier, C. and P. Kerkhofs. 2003. Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhoea virus. J. Virol. Methods 114: 21-27
  22. Makoschey, B., P. T. van Gelder, V. Keijsers, and D. Goovaerts. 2003. Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production. Biologicals 31: 203-208
  23. Park, J. S., H. J. Moon, B. C. Lee, W. S. Hwang, H. S. Yoo, D. Y. Kim, and B. K. Park. 2004. Comparative analysis on the 5'-untranslated region of bovine viral diarrhes virs isolated in Korea. Res. Vet. Sci. 76: 157-163
  24. Parkman, P. D. 1996. Safety of biopharmaceuticals: a current perspective. Dev. Biol. Stand. 88: 5-7
  25. Ridpath, J. F. and S. R. Bolin. 1998. Differentiation of types 1a, 1b and 2 bovine viral diarrhoea virus (BVDV) by PCR. Mol. Cell. Probes 12: 101-106
  26. Rudnick, A. 2006. Advances in tissue engineering and use of type 1 bovine collagen particles in wound bed preparation. J. Wound Care 15: 402-404
  27. Ryu, S.-R., J.-H. Shin, S.-Y. Baek, J.-O. Kim, K.-I. Min, B.- S. Min, B.-G. Kim, D.-K. Kim, M.-K. Park, M.-J. Ahn, K.-S. Chae, H.-S. Jeong, S.-H. Lee, and S.-N. Park. 2003. Evaluation of limit of detection and range of quantitation for RT-PCR, real-time RT-PCR and RT-PCR-ELISA detection of bovine viral diarrhea virus contamination in biologics derived from cell cultures. J. Bacteriol. Virol. 33: 161-168
  28. Schneider, G. 2003. Bioimplants-characteristics and use. Laryngorhinootologie 82: 839-852
  29. Silva, R. F., A. M. Fadly, and S. P. Taylor. 2007. Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminant in commercial Marek's disease vaccines. Avian Dis. 51: 663-667[663:DOAPCR]2.0.CO;2
  30. Studer, E., B. Guiseppe, and C. Urs. 2002. Detection and characterization of pestivirus contamination in human live viral vaccines. Biologicals 30: 289-296
  31. Warrener, P. and M. C. Collett. 1995. Pestivirus NS3(p80) protein possess RNA helicase activity. J. Virol. 69: 1720-1726
  32. Yarlagadda, P. K., M. Chandrasekharan, and J. Y. Shyan. 2005. Recent advances and current developments in tissue scaffolding. Biomed. Mater. Eng. 15: 159-177