Characterisation of new animal cell cultures’ sensitivity to Coxsackievirus B5 and Herpes simplex virus‑1

The increase in the number of cell cultures for virology and biotechnology enhances the chances of a successful response to threats related to outbreaks of well-known and new human infectious diseases. It is a vital task to search for cell cultures sensitive to a wide spectrum of viruses.

The aim of the study was to investigate the sensitivity of new diploid animal cell cultures (fibroblasts of a foetal pig’s kidneys and larynx) to Coxsackievirus B5 (CVB5) and Herpes simplex virus-1 (HSV-1).

Materials and methods. The cultures of porcine foetal kidney fibroblasts (PFKF) and porcine foetal larynx fibroblasts (PFLF) were derived from a foetus of a healthy pig by mild trypsinisation. The study determined the sensitivity of these new PFKF and PFLF cultures to the above-mentioned viruses by the cytopathic effect (CPE) expressed as a percentage. The infectious activity of CVB5 was studied using real-time polymerase chain reaction (PCR) with the determination of amplification cycle threshold values (C_t); that of HSV-1 was studied using quantitative titration of the virus-containing liquid (VCL). Infectious activity values were expressed as tissue culture 50% infective doses (TCID₅₀).

Results. The authors developed diploid PFKF and PFLF cell cultures. PFKF cells demonstrated high sensitivity to CVB5, with a CPE of 87.5±3.3% after passage 3 and a satisfactory concentration of enterovirus RNA in the VCL of 22–24 C_t . The sensitivity of PFKF cells to HSV-1 corresponded to a CPE of 92.1±5.5%. In these cells, the infectious activity of HSV-1 corresponded to 10^4.25 TCID₅₀/0.2 mL. The experiments with PFLF cells showed low CPE and infectious activity values for both viruses.

Conclusions. The study demonstrated high CPE values with the CVB5 (CB5-8100) and HSV-1 (HSV-1/L-2) strains as examples and confirmed the sensitivity of the new diploid PFKF cell culture to these test viruses. Thus, the PFKF cell culture offers potential applications in virology and biotechnology and may be a candidate for testing other strains of CVB5 and HSV-1.

1. Ogilvie M. Molecular techniques should not now replace cell culture in diagnostic virology laboratories. Rev Med Virol. 2001;11(6):351–4. https://doi.org/10.1002/rmv.335

2. Leland DS, Ginocchio CC. Role of cell culture for virus detection in the age of technology. Clin Microbiol Rev. 2007;20(1):49–78. https://doi.org/10.1128/CMR.00002-06

3. Genzel Y. Designing cell lines for viral vaccine production: where do we stand? Biotechnol J. 2015;10(5):728–40. https://doi.org/10.1002/biot.201400388

4. Aubrit F, Perugi F, Léon A, Guéhenneux F, Champion-Arnaud P, Lahmar M, Schwamborn K. Cell substrates for the production of viral vaccines. Vaccine. 2015;33(44):5905–12. https://doi.org/10.1016/j.vaccine.2015.06.110

5. Chen P, Wu X, Su Y, Hao X, Mao Q, Liang Z. Development of a pseudovirus based assay for measuring neutralizing antibodies against Coxsackievirus B5. J Virol Methods. 2017;246:21–6. https://doi.org/10.1016/j.jviromet.2017.04.005

6. Dolskiy AA, Grishchenko IV, Yudkin DV. Cell cultures for virology: usability, advantages, and prospects. Int J Mol Sci. 2020;21(21):7978. https://doi.org/10.3390/ijms21217978

7. Hematian A, Sadeghifard N, Mohebi R, Taherikalani M, Nasrolahi A, Amraei M, Ghafourian S. Traditional and modern cell culture in virus diagnosis. Osong Public Health Res Perspect. 2016;7(2):77–82. https://doi.org/10.1016/j.phrp.2015.11.011

8. Glinskikh NP, Kolesnikova GG, Ustyantsev VP, Zakirova SF, Vlasova LV, Stanislavskaya VK. Diploid human embryo lung cell strain, LECh-4(81), for diagnosis of viral infections. Patent of the USSR No. SU 1147748 A1; 1985 (In Russ.).

9. Noort WA, Oerlemans MI, Rozemuller H, Feyen D, Jaksani S, Stecher D, et al. Human versus porcine mesenchymal stromal cells: phenotype, differentiation potential, immunomodulation and cardiac improvement after transplantation. J Cell Mol Med. 2012;16(8):1827–39. https://doi.org/10.1111/j.1582-4934.2011.01455.x

10. Schweizer R, Waldner M, Oksuz S, Zhang W, Komatsu C, Plock JA, et al. Evaluation of porcine versus human mesenchymal stromal cells from three distinct donor locations for cytotherapy. Front Immunol. 2020;11:826. https://doi.org/10.3389/fimmu.2020.00826

11. Vikulov GH. URTI, influenza and herpes: common aspects and differences in diagnosis and therapy. A clinical immunologist’s and infectologist’s view-point. Russian Medical Journal. 2015;23(17):1032–7 (In Russ.).

12. Alimov AV, Fedotova OS, Shmelyova NA, Bakharev AA, Rezaykin AV, Usoltseva PS, et al. Determining sensitivity of novel animal derived cell cultures to clinical isolates of human enterovirus Echovirus 11 and Coxsackievirus B5. Medical alphabet. 2020;(18):17–9 (In Russ.). https://doi.org/10.33667/2078-5631-2020-18-17-19

13. Glinskikh NP, Bakharev AA, Ustyantsev PV, Ustyantsev IV. A method for obtaining stable cell cultures. Patent of the Russian Federation No. 2392318; 2008 (In Russ.).

14. Adams RLP. Cell culture for biochemists. Amsterdam: Elsevier/North-Holland; 1980.

15. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res. 1960;20:613–6. https://doi.org/10.1016/0014-4827(60)90138-5

16. Husson-van Vliet J, Roussel P. Pipetting errors in viral titrations: a useful approach. J Virol Methods. 1988;22(2–3):183–90. https://doi.org/10.1016/0166-0934(88)90101-2