Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Continuous Passaging of a Recombinant C-Strain Virus in PK-15 Cells Selects Culture-Adapted Variants that Showed Enhanced Replication but Failed to Induce Fever in Rabbits

  • Tong, Chao (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Chen, Ning (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Liao, Xun (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Yuan, Xuemei (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Sun, Mengjiao (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Li, Xiaoliang (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine) ;
  • Fang, Weihuan (Zhejiang University Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine)
  • Received : 2017.05.02
  • Accepted : 2017.07.11
  • Published : 2017.09.28
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Abstract

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that $RecCpp40_{+10}$ failed to induce fever in rabbits, whereas $RecCpp80_{+10}$ caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

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References

  1. Floegel-Niesmann G, Blome S, Gerss-Dulmer H, Bunzenthal C, Moennig V. 2009. Virulence of classical swine fever virus isolates from Europe and other areas during 1996 until 2007. Vet. Microbiol. 139: 165-169. https://doi.org/10.1016/j.vetmic.2009.05.008
  2. Schweizer M, Peterhans E. 2014. Pestiviruses. Annu. Rev. Anim. Biosci. 2: 141-163. https://doi.org/10.1146/annurev-animal-022513-114209
  3. Lamp B, Riedel C, Roman-Sosa G, Heimann M, Jacobi S, Becher P, et al. 2011. Biosynthesis of classical swine fever virus nonstructural proteins. J. Virol. 85: 3607-3620. https://doi.org/10.1128/JVI.02206-10
  4. Suradhat S, Damrongwatanapokin S, Thanawongnuwech R. 2007. Factors critical for successful vaccination against classical swine fever in endemic areas. Vet. Microbiol. 119: 1-9. https://doi.org/10.1016/j.vetmic.2006.10.003
  5. Huang YL, Deng MC, Wang FI, Huang CC, Chang CY. 2014. The challenges of classical swine fever control: modified live and E2 subunit vaccines. Virus Res. 179: 1-11. https://doi.org/10.1016/j.virusres.2013.10.025
  6. Luo Y, Li S, Sun Y, Qiu HJ. 2014. Classical swine fever in China: a minireview. Vet. Microbiol. 172: 1-6. https://doi.org/10.1016/j.vetmic.2014.04.004
  7. Xia H, Wahlberg N, Qiu HJ, Widen F, Belak S, Liu L. 2011. Lack of phylogenetic evidence that the Shimen strain is the parental strain of the lapinized Chinese strain (C-strain) vaccine against classical swine fever. Arch. Virol. 156: 1041-1044. https://doi.org/10.1007/s00705-011-0948-5
  8. Wu SC, Liau MY, Lin YC, Sun CJ, Wang CT. 2013. The feasibility of a novel bioreactor for vaccine production of classical swine fever virus. Vaccine 31: 867-872. https://doi.org/10.1016/j.vaccine.2012.12.017
  9. Moormann RJ, van Gennip HG, Miedema GK, Hulst MM, van Rijn PA. 1996. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J. Virol. 70: 763-770.
  10. van Gennip HG, van Rijn PA, Widjojoatmodjo MN, Moormann RJ. 1999. Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase. J. Virol. Methods 78: 117-128. https://doi.org/10.1016/S0166-0934(98)00171-2
  11. Hu W, Zhang H, Han Q, Li L, Chen Y, Xia N, et al. 2015. A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages. Vaccine 33: 374-381. https://doi.org/10.1016/j.vaccine.2014.11.007
  12. Biswal JK, Mohapatra JK, Bisht P, Subramaniam S, Sanyal A, Pattnaik B. 2015. A positively charged lysine residue at VP2 131 position allows for the enhanced adaptability of footand- mouth disease virus serotype A in BHK-21 cells. Biologicals 43: 71-78. https://doi.org/10.1016/j.biologicals.2014.07.001
  13. Berryman S, Clark S, Kakker NK, Silk R, Seago J, Wadsworth J, et al. 2013. Positively charged residues at the five-fold symmetry axis of cell culture-adapted foot-andmouth disease virus permit novel receptor interactions. J. Virol. 87: 8735-8744. https://doi.org/10.1128/JVI.01138-13
  14. Liu S, Xiao L, Nelson C, Hagedorn CH. 2012. A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses. PLoS One 7: e44965. https://doi.org/10.1371/journal.pone.0044965
  15. Kaul A, Woerz I, Meuleman P, Leroux-Roels G, Bartenschlager R. 2007. Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J. Virol. 81: 13168-13179. https://doi.org/10.1128/JVI.01362-07
  16. Tong C, Chen N, Liao X, Xie W, Li D, Li X, Fang W. 2015. The epitope recognized by monoclonal antibody 2B6 in the B/C domains of classical swine fever virus glycoprotein E2 affects viral binding to hyperimmune sera and replication. J. Microbiol. Biotechnol. 25: 537-546. https://doi.org/10.4014/jmb.1407.07073
  17. Chen N, Tong C, Li D, Wan J, Yuan X, Li X, et al. 2010. Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China. Virol. J. 7: 378. https://doi.org/10.1186/1743-422X-7-378
  18. McHugh ML. 2011. Multiple comparison analysis testing in ANOVA. Biochem. Med. (Zagreb) 21: 203-209.
  19. Tews BA, Meyers G. 2007. The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix. J. Biol. Chem. 282: 32730-32741. https://doi.org/10.1074/jbc.M706803200
  20. Hulst MM, Moormann RJ. 2001. Erns protein of pestiviruses. Methods Enzymol. 342: 431-440.
  21. Hulst MM, van Gennip HG, Moormann RJ. 2000. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J. Virol. 74: 9553-9561. https://doi.org/10.1128/JVI.74.20.9553-9561.2000
  22. Li C, Li Y, Shen L, Huang J, Sun Y, Luo Y, et al. 2014. The role of noncoding regions of classical swine fever virus Cstrain in its adaptation to the rabbit. Virus Res. 183: 117-122. https://doi.org/10.1016/j.virusres.2014.02.003
  23. Graham SP, Haines FJ, Johns HL, Sosan O, La Rocca SA, Lamp B, et al. 2012. Characterisation of vaccine-induced, broadly cross-reactive IFN-gamma secreting T cell responses that correlate with rapid protection against classical swine fever virus. Vaccine 30: 2742-2748. https://doi.org/10.1016/j.vaccine.2012.02.029
  24. Graham SP, Everett HE, Johns HL, Haines FJ, La Rocca SA, Khatri M, et al. 2010. Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses. Vet. Microbiol. 142: 34-40. https://doi.org/10.1016/j.vetmic.2009.09.040
  25. Dong XN, Chen YH. 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine 25: 205-230. https://doi.org/10.1016/j.vaccine.2006.07.033
  26. Jiang J, Luo G. 2012. Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus. J. Virol. 86: 8987-8997. https://doi.org/10.1128/JVI.00004-12
  27. Koutsoudakis G, Perez-del-Pulgar S, Coto-Llerena M, Gonzalez P, Dragun J, Mensa L, et al. 2011. Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation. PLoS One 6: e23587. https://doi.org/10.1371/journal.pone.0023587
  28. Kang JI, Kim JP, Wakita T, Ahn BY. 2009. Cell cultureadaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity. Virus Res. 144: 107-116. https://doi.org/10.1016/j.virusres.2009.04.002
  29. van Rijn PA, Miedema GK, Wensvoort G, van Gennip HG, Moormann RJ. 1994. Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J. Virol. 68: 3934-3942.
  30. Vanderhallen H, Mittelholzer C, Hofmann MA, Koenen F. 1999. Classical swine fever virus is genetically stable in vitro and in vivo. Arch. Virol. 144: 1669-1677. https://doi.org/10.1007/s007050050622
  31. Ferrari M. 1992. A tissue culture vaccine with lapinized Chinese (LC) strain of hog cholera virus (HCV). Comp. Immunol. Microbiol. Infect. Dis. 15: 221-228. https://doi.org/10.1016/0147-9571(92)90095-9
  32. Gualandi GL, Ferrari M, Cardeti G, Boldini M, Buonavoglia C. 1991. Protection tests in pigs vaccinated with the lapinized Chinese strain of hog cholera virus (HCV) previously adapted in a minipig kidney (MPK) cell line, to challenge infection with virulent HCV. Microbiologica 14: 213-217.
  33. Xiao M, Gao J, Wang Y, Wang X, Lu W, Zhen Y, et al. 2004. Influence of a 12-nt insertion present in the 3' untranslated region of classical swine fever virus HCLV strain genome on RNA synthesis. Virus Res. 102: 191-198. https://doi.org/10.1016/j.virusres.2004.01.029

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