Archives of Virology

, Volume 164, Issue 6, pp 1619–1628 | Cite as

Classical swine fever virus C-strain with eight mutation sites shows enhanced cell adaptation and protects pigs from lethal challenge

  • Tong Cao
  • Shengnan Zhang
  • Xiaoye Li
  • Yonghao Xu
  • Zuohuan Wang
  • Cong Chen
  • Narayan Paudyal
  • Xiaoliang Li
  • Jianhe Sun
  • Weihuan FangEmail author
Original Article


Control of classical swine fever (CSF) in developing countries is achieved by immunization with attenuated vaccines, such as the lapinized C-strain vaccine that has been widely used in China. However, C-strain has relatively low growth rate in cell cultures, thus affecting productivity of the vaccine for the industry. In this study, eight amino acid residues were mutated on the C-strain backbone, resulting in a cell-adapted strain Cmut8. The mutant strain exhibited rapid growth with titer of about 100 fold higher than its parental C-strain. The mutation sites located at structural proteins Erns and E2 contributed more to cell adaptation than those located in non-structural proteins. Sera collected from pigs inoculated with Cmut8 and C-strain at the same dose showed similar antibody levels and neutralization titers. Pigs inoculated with different doses of Cmut8 (low, medium and high) and with C-strain offered full protection against challenge with a virulent strain, shown as absence of fever and other symptoms, marginal low levels of viral load, and no obvious gross pathological changes in major organs. Unvaccinated control pigs challenged with the virulent strain showed high fever from day 2 post-challenge and apparent clinical symptoms with two deaths. Viral load were markedly elevated in these control pigs after challenge. The pigs inoculated with high dose of Cmut8 did not show fever or other typical CSF symptoms, and no apparent pathological changes were observed in major organs. Besides, the Cmut8 strain did not induce typical fever response in rabbits. These results demonstrate that the cell-adapted Cmut8 strain remains non-pathogenic to the weaned pigs, provides full protection and could be a good candidate vaccine strain for improved yield at lower cost.



This study is part of the work sponsored by Zhejiang Provincial Department of Science & Technology (2019C02043), Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai, China (KLAB201711) and Dabeinong Funds for Discipline Development and Talent Training in Zhejiang University (DB2018005).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal ethics

Animal experiments were conducted following the guidelines and approved protocols of the Laboratory Animal Management Committee of Zhejiang University (Approval No. ZJU20180190 and 20181061).


  1. 1.
    Becher P, Avalos Ramirez R, Orlich M, Cedillo Rosales S, König M, Schweizer M, Stalder H, Schirrmeier H, Thiel H-J (2003) Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311(1):96–104CrossRefGoogle Scholar
  2. 2.
    Moormann RJ, Warmerdam PA, van der Meer B, Schaaper WM, Wensvoort G, Hulst MM (1990) Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E1. Virology 177(1):184–198CrossRefGoogle Scholar
  3. 3.
    Rumenapf T, Meyers G, Stark R, Thiel HJ (1991) Molecular characterization of hog cholera virus. Arch Virol Suppl 3:7–18CrossRefGoogle Scholar
  4. 4.
    Collett MS, Moennig V, Horzinek MC (1989) Recent advances in pestivirus research. J Gen Virol 70(Pt 2):253–266CrossRefGoogle Scholar
  5. 5.
    Thiel HJ, Stark R, Weiland E, Rumenapf T, Meyers G (1991) Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65(9):4705–4712Google Scholar
  6. 6.
    Qiu HJ, Tong GZ, Shen RX (2005) The lapinized Chinese strain of classical swine fever virus: a retrospective review spanning half a century. J Integr Agric 38:1675–1685 (Chinese) Google Scholar
  7. 7.
    Terpstra C, Wensvoort G (1988) The protective value of vaccine-induced neutralising antibody titres in swine fever. Vet Microbiol 16(2):123–128CrossRefGoogle Scholar
  8. 8.
    Kaden V, Lange B (2001) Oral immunisation against classical swine fever (CSF): onset and duration of immunity. Vet Microbiol 82(4):301–310CrossRefGoogle Scholar
  9. 9.
    Graham SP, Everett HE, Haines FJ, Johns HL, Sosan OA, Salguero FJ, Clifford DJ, Steinbach F, Drew TW, Crooke HR (2012) Challenge of pigs with classical swine fever viruses after C-strain vaccination reveals remarkably rapid protection and insights into early immunity. PLoS One 7(1):e29310CrossRefGoogle Scholar
  10. 10.
    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(2):763–770Google Scholar
  11. 11.
    Luo Y, Li S, Sun Y, Qiu HJ (2014) Classical swine fever in China: a minireview. Vet Microbiol 172(1–2):1–6CrossRefGoogle Scholar
  12. 12.
    Tong C, Chen N, Liao X, Yuan X, Sun M, Li X, Fang W (2017) 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. J Microbiol Biotechnol 27(9):1701–1710Google Scholar
  13. 13.
    Chen N, Tong C, Li D, Wan J, Yuan X, Li X, Peng J, Fang W (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 7:378CrossRefGoogle Scholar
  14. 14.
    Liao X, Wang Z, Cao T, Tong C, Geng S, Gu Y, Zhou Y, Li X, Fang W (2016) Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization. Vaccine 34(33):3723–3730CrossRefGoogle Scholar
  15. 15.
    Mittelholzer C, Moser C, Tratschin JD, Hofmann MA (2000) Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet Microbiol 74(4):293–308CrossRefGoogle Scholar
  16. 16.
    Cao T, Wang Z, Li X, Zhang S, Paudyal N, Zhang X, Li X, Fang W (2019) E2 and E(rns) of classical swine fever virus C-strain play central roles in its adaptation to rabbits. Virus Genes. Google Scholar
  17. 17.
    Koenig P, Lange E, Reimann I, Beer M (2007) CP7_E2alf: a safe and efficient marker vaccine strain for oral immunisation of wild boar against Classical swine fever virus (CSFV). Vaccine 25(17):3391–3399CrossRefGoogle Scholar
  18. 18.
    Chinese Ministry of Agriculture (2001) Quality Standards for Veterinary Biological Products of People’s Republic of China. Chinese Agricultural Science and Technology Press, Beijing, pp 84–85 (Chinese) Google Scholar
  19. 19.
    Blome S, Staubach C, Henke J, Carlson J, Beer M (2017) Classical swine fever-an updated review. Viruses 9(4):86CrossRefGoogle Scholar
  20. 20.
    Ji W, Guo Z, Ding NZ, He CQ (2015) Studying classical swine fever virus: making the best of a bad virus. Virus Res 197:35–47CrossRefGoogle Scholar
  21. 21.
    Dahle J, Liess B (1995) Assessment of safety and protective value of a cell culture modified strain “C” vaccine of hog cholera/classical swine fever virus. Berl Munch Tierarztl Wochenschr 108(1):20–25Google Scholar
  22. 22.
    Kaden V, Riebe B (2001) Classical swine fever (CSF): a historical review of research and vaccine production on the Isle of Riems. Berl Munch Tierarztl Wochenschr 114(7–8):246–251Google Scholar
  23. 23.
    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 E(rns). J Virol 74(20):9553–9561CrossRefGoogle Scholar
  24. 24.
    Wu R, Li L, Zhao Y, Tu J, Pan Z (2016) Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus. Virus Res 211:79–85CrossRefGoogle Scholar
  25. 25.
    Li Y, Xie L, Zhang L, Wang X, Li C, Han Y, Hu S, Sun Y, Li S, Luo Y, Liu L, Munir M, Qiu H (2018) The E2 glycoprotein is necessary but not sufficient for the adaptation of classical swine fever virus lapinized vaccine C-strain to the rabbit. Virology 519:197–206CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Tong Cao
    • 1
  • Shengnan Zhang
    • 1
  • Xiaoye Li
    • 1
  • Yonghao Xu
    • 1
  • Zuohuan Wang
    • 1
  • Cong Chen
    • 1
  • Narayan Paudyal
    • 1
  • Xiaoliang Li
    • 1
  • Jianhe Sun
    • 2
  • Weihuan Fang
    • 1
    • 2
    Email author
  1. 1.Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary MedicineZhejiang UniversityHangzhouChina
  2. 2.Shanghai Key Laboratory of Veterinary BiotechnologyShanghaiChina

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