Identification of bovine viral diarrhea virus variants — contaminants of industrial cell lines

Relevance. Contamination with bovine BVDV virus remains a threat to the quality of biological products. The most common factor of contamination is fetal bovine serum, the main factor supporting growth when cultivating cell lines, the use of which makes the risk of spreading the bovine BVDV virus almost inevitable.

The aim of the study was to identify variants of bovine viral diarrhea virus, which are contaminants of production cell lines.

Methods. The cell lines MDBK, BHK-21/13-02, PK-15, Vero and the production strains of the bovine HD virus NADL, VK-1 were used To amplify the target regions of the viral genome, oligonucleotide primers flanking the highly conserved untranslated region 5`UTR with a length of 289 bp were used. Comparative analysis of nucleotide sequences determined by sequencing was carried out using standard methods of molecular phylogenetics.

Results. The fact of chronic infection with non-cytopathogenic bovine BVDV virus was confirmed in relation to the cell lines MDBK, BHK-21/13-02 and PK-15. Based on sequencing results, the contaminating virus variants were identified as representatives of subgenotype 1a. Phylogenetic analysis revealed their significant similarity between themselves and a number of strains isolated from persistently infected llamas, alpacas and roe deer with an identity of 96.8–99.2%. The presented data demonstrate the need for ongoing such screenings, which will improve the quality of technological raw materials used for the production of biological products, as well as improve existing means of serological diagnosis of infections caused by representatives of the Pestivirus genus.

1. Hou P., Xu Y., Wang H., He H. Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay. BMC Veterinary. Research. 2020; 16: 114. https://doi.org/10.1186/s12917-020-02330-6

2. Wang Y., Pang F. Diagnosis of bovine viral diarrhea virus: an overview of currently available methods. Frontiers in Microbiology. 2024; 15: 1370050. https://doi.org/10.3389/fmicb.2024.1370050

3. de Oliveira P.S.B., Silva Júnior J.V.J., Weiblen R., Flores E.F. Subtyping bovine viral diarrhea virus (BVDV): Which viral gene to choose?. Infection, Genetics and Evolution. 2021; 92: 104891. https://doi.org/10.1016/j.meegid.2021.104891

4. Khafizova A.M. et al. Viral diarrhea is a disease of the mucous membranes of cattle: an analysis of the epizootic situation and antiepizootic measures. Scientific notes Kazan Bauman State Academy of Veterinary Medicine. 2024; 258: 214–221 (in Russian). https://www.elibrary.ru/dbwgnw

5. Abd El Fadeel M.R., Soliman E.M., Allam A.M., ElKersh M.F., Abd El-Baky R.M., Mustafa A. Efficacy and durability of bovine virus diarrhea (BVD) virus killed vaccine adjuvanted with monolaurin. PLoS.ONE. 2022; 17(7): e0269031. https://doi.org/10.1371/journal.pone.0269031

6. Gómez-Romero N., Velazquez-Salinas L., Ridpath J.F., VerdugoRodríguez A., Basurto-Alcántara F.J. Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and fetal bovine serum lots originating in Mexico. Archives of Virology. 2021; 166(7): 1999–2003. https://doi.org/10.1007/s00705-021-05089-9

7. Gauvin G., Nims R. Gamma-Irradiation of Serum for the Inactivation of Adventitious Contaminants. PDA Journal of Pharmaceutical Science and Technology. 2010; 64(5): 432–435.

8. Uryvaev L.V. et al. About the contamination of cell cultures with the virus of diarrhea — diseases of the mucous membranes of cattle (BVDV). Bulletin of Experimental Biology and Medicine. 2012; 153(1): 77–81. https://doi.org/10.1007/s10517-012-1648-1

9. Glotov A.G., Glotova T.I., Koteneva S.V. On contamination of imported fetal blood serum of cattle with pestiviruses as a factor in the spread of viral diarrhea in the context of globalization: a mini-review. Agricultural Biology. 2018; 53(2): 248–257. https://doi.org/10.15389/agrobiology.2018.2.248eng

10. Alekseenkova S.V., Yurov G.K., Galnbek T.V., Kalita K.I.A., Yurov K.P. Testing of cell cultures for contamination with the bovine diarrhea virus is a necessary condition for the production of biological preparations. Russian veterinary journal Productive animals. 2013; (1): 15–18 (in Russian). https://www.elibrary.ru/pyednf

11. Pastoret P.-P. Human and animal vaccine contaminations. Biologicals. 2010; 38(3): 332–334. https://doi.org/10.1016/j.biologicals.2010.02.015

12. Semenova O.V., Glotova T.I., Glotov A.G., Nefedchenko A.V. Frequency detection by RT-PCR and BVDV isolation in cell culture in Siberia. Russian veterinary journal.2017; (1): 24–27 (in Russian). https://www.elibrary.ru/xxyqwb

13. Spetter M.J. et. al. Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period. Brazilian Journal of Microbiology. 2020; 51(4): 2077–2086. https://doi.org/10.1007/s42770-020-00296-z

14. Aguirre I.M., Fuentes R., Celedón M.O. Genotypic characterization of Chilean llama (Lama.glama) and alpaca (Vicugna.pacos) pestivirus isolates Veterinary Microbiology. 2014; 168(2–4): 312–317. https://doi.org/10.1016/j.vetmic.2013.11.031

15. Harasawa R., Giangaspero M., Ibata G., Paton D.J. Giraffe Strain of Pestivirus: Its Taxonomic Status Based on the 5′-Untranslated Region. Microbiology and Immunology. 2000; 44(11): 915–921. https://doi.org/10.1111/j.1348-0421.2000.tb02583.x

16. Lamothe-Reyes Y., Figueroa M., Sánchez O. Host cell factors involved in classical swine fever virus entry. Veterinary Research. 2023; 54: 115. https://doi.org/10.1186/s13567-023-01238-x

17. Choe S. et al. Prevalence of Bovine Viral Diarrhea Virus Infections in Pigs on Jeju Island, South Korea, from 2009–2019 and Experimental Infection of Pigs with BVDV Strains Isolated from Cattle. Veterinary Sciences. 2022; 9(3): 146. https://doi.org/10.3390/vetsci9030146

18. Van Rijn P.A. A common neutralizing epitope on envelope glycoprotein E2 of different pestiviruses: Implications for improvement of vaccines and diagnostics for classical swine fever (CSF)? Veterinary Microbiology. 2007; 125(1–2): 150–156. https://doi.org/10.1016/j.vetmic.2007.05.001

19. Schmitz-Dräger B.J. et al. Molecular Markers for Bladder Cancer Screening, Early Diagnosis, and Surveillance: The WHO/ICUD Consensus. Urologia Internationalis. 2015; 94(1): 1–24. https://doi.org/10.1159/000369357