EVALUATION OF THE BIOLOGICAL ACTIVITY OF ORTHOPOXVIRUS STRAINS IN PRIMARY CHICKEN EMBRYO FIBROBLASTS
Main Article Content
Authors
B.S. Myrzakhmetova
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
G.A. Zhapparova
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
L.B. Kutumbetov
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
T.M. Tlenchieva
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
A.B. Zhumabek
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
A.M. Tuyakov
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
A.V. Azanbekova
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
K.D. Zhugunissov
Research Institute for Biological Safety Problems, Gvardeiskiy, 080409, Kazakhstan
Abstract
Understanding the biological features of the genus, Orthopoxvirus, including their replicative activity, is essential for their application in virology and vaccinology. Comparative analysis of biological activity in cell cultures makes it possible to evaluate the replicative potential of strains and the degree of their adaptation to the cellular substrate.
The aim of the study was to standardize a method for obtaining primary chicken embryo fibroblasts (CEF) and to use them as an in vitro substrate for assessing the biological activity of various Orthopoxvirus strains, with an emphasis on cytopathic effect (CPE) and infectious activity. Vaccinia virus, cowpox virus, and camelpox virus were used in the study. Primary CEF were obtained from 9-11-day embryonated chicken eggs (ECE), the optimal seeding concentration for the formation of a monolayer substrate was determined, and the cultures were infected with the viruses under study; CPE and virus titer were assessed during sequential passages. The results showed that a seeding concentration of 5×105 cells/mL ensured the formation of a uniform CEF monolayer within 24 h. Upon infection, all three viruses induced similar CPE, characterized by cell rounding, loss of elongated morphology, and partial destruction of the monolayer, with the main difference being the timing of CPE onset. Vaccinia virus strains induced CPE within 24 h, whereas cowpox virus and camelpox virus induced CPE after 36 and 48 h, respectively. Both vaccinia virus strains exhibited high infectious activity and rapid adaptation (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), whereas cowpox and camelpox viruses showed a lower and stable titer (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), and delayed CPE development.
The results emphasize the significance of CPE onset time and infectious titer level as key parameters for evaluating replicative capacity and strain adaptation, which is important for laboratory studies and the selection of vaccine candidates.
Keywords
Orthopoxvirus, chicken embryo fibroblasts, cytopathic effect, infectious titer
Article Details
References
Kaynarcalidan O., Moreno Mascaraque S., Drexler I. Vaccinia Virus: From Crude Smallpox Vaccines to Elaborate Viral Vector Vaccine Design // Biomedicines. – 2021. – № 9(12). – Article 1780. Crossref
Jacobs B.L., Langland J.O., Kibler K.V., Denzler K.L., White S.D., Holechek S.A., Wong S., Huynh T., Baskin C.R. Vaccinia virus vaccines: past, present and future // Antiviral Research. – 2009. – № 84(1). – P. 1–13. Crossref
Щелкунов С.Н., Сергеев А.А., Кабанов А.С., Кабанов С.Н., Якубицкий С.Н., Бауэр Т.В., Пьянков С.А. Патогенность и иммуногенность вариантов вируса коровьей оспы, вводимых различными путями у мышей // Инфекция и иммунитет. – 2021. – № 11(2). – С. 357–364.
Moss B. Poxviridae: The Viruses and Their Replication // Fields Virology. – 6th ed. – Lippincott Williams & Wilkins, 2013.
Essbauer S., Pfeffer M., Meyer H. Zoonotic poxviruses // Veterinary Microbiology. – 2010. – № 140(3–4). – P. 229–236. Crossref
Tulman E.R., Delhon G., Afonso C.L., et al. Genome of horsepox virus // Journal of Virology. – 2006. – № 80(18). – P. 9244–9258. Crossref
Shchelkunova G.A., Shchelkunov S.N. Smallpox, Monkeypox and Other Human Orthopoxvirus Infections // Viruses. – 2023. – № 15(1). – Article 103. Crossref
Minhaj F.S. Poxvirus Epidemiology // Methods in Molecular Biology. – 2025. – № 2860. – P. 1–14. Crossref
Manuka A., Mehmeti I., Radaelli A., Hashorva E., Zanotto C., De Giuli Morghen C. Insights into poxviruses: virology and vaccines // Acta Virologica. – 2025. – № 69. – Article 14597. Crossref
Freshney R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. – 7th ed. – Wiley-Blackwell, 2015.
Mahy B.W.J., Van Regenmortel M.H.V., editors. Desk Encyclopedia of General Virology. – Academic Press, 2010.
Earl P.L., Cooper N., Wyatt L.S., Moss B., Carroll M.W. Preparation of cell cultures and vaccinia virus stocks // Current Protocols in Protein Science. – 2001. – Chapter 5. – Unit 5.12. Crossref
Earl P.L., Americo J.L., Moss B. Development and Use of a Vaccinia Virus Neutralization Assay Based on Flow Cytometric Detection of Green Fluorescent Protein // Journal of Virology. – 2003. – № 77(19). – P. 10684–10688. Crossref
Hornemann S., Harlin O., Staib C., et al. Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L // Journal of Virology. – 2003. – № 77(15). – P. 8394–8407. Crossref
Gilhare V.R., Hirpurkar S.D., Kumar A., Naik S.K., Sahu T. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture // Veterinary World. – 2015. – № 8(3). – P. 245–250. Crossref
Кузнецова Т.А., Береснева Н.Н., Алиев М.Р., Щелканов М.Ю. Клеточные культуры в вирусологии: от прошлого к будущему // Журнал микробиологии, эпидемиологии и иммунобиологии. – 2024. – № 101(1). – С. 143–153. Crossref
Huo S., Wu C., Qi Z., Sun J., Meng X., Song J., Zhang Z., Jin L., Shu C., Lin Z., Huo W., Deng Y., Zhao L., Li J., Tan W. Identification of a novel ectromelia virus from rodent: Implications for use as an in vivo infection model for vaccine and antiviral research // Virologica Sinica. – 2025. – № 40(4). – P. 601–612. Crossref
Cotter C.A., Earl P.L., Wyatt L.S., Moss B. Preparation of Cell Cultures and Vaccinia Virus Stocks // Current Protocols in Microbiology. – 2015. – № 39(1). – P. 14A.3.1–14A.3.18. Crossref
Remichkova M., Petrov N., Galabov A.S. Synergistic combination effect of cidofovir and idoxuridine on vaccinia virus replication // Antiviral Chemistry & Chemotherapy. – 2006. – № 17(2). – P. 53–58. Crossref
Ghiselli F., Rossi B., Felici M., Parigi M., Tosi G., Fiorentini L., Massi P., Piva A., Grilli E. Isolation, culture, and characterization of chicken intestinal epithelial cells // BMC Molecular and Cell Biology. – 2021. – № 22(1). – Article 12. Crossref
Dayı B., Akkoç A., Yumuşak E., Sevimli A. Isolation and culture of chicken embryonic synovial fibroblasts // Veterinaria Italiana. – 2023. – № 59(4). Crossref
Guan W., He X., Li L., Liang H., Zhao Q., Pu Y., Ma Y.H. Establishment and biological characterization of fibroblast cell line from the Langshan chicken // Cell Proliferation. – 2010. – № 43(2). – P. 157–163. Crossref
Yuan C., He Q., Li J.M., Azzam M.M., Lu J.J., Zou X.T. Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro // Animal Science Journal. – 2015. – № 86(6). – P. 588–594. Crossref
Культуры тканей [Электронный ресурс]. – URL: URL (дата обращения: ДД.ММ.2024).
Ma A., Langer J., Hanson K.E., Bradley B.T. Characterization of the Cytopathic Effects of Monkeypox Virus Isolated from Clinical Specimens and Differentiation from Common Viral Exanthems // Journal of Clinical Microbiology. – 2022. – № 60(12). – Article e0133622. Crossref
McFadden G. Poxvirus tropism // Nature Reviews Microbiology. – 2005. – № 3(3). – P. 201–213. Crossref
Hsu S.-C., Liu P.-C., Tsai S.-K., Chen A.-Y., Tsai H.-P., Sun J.-R., Li T.-Y., Hsieh P.-Y., Yang J.-Y., Chang T.-Y. Evaluating the susceptibility of various common cell lines and assessing inactivation conditions to Mpox virus // Microbiology Spectrum. – 2025. – № 13(12). – Article e0080325. Crossref
Rahman M.M., McFadden G. Role of cytokines in poxvirus host tropism and adaptation // Current Opinion in Virology. – 2022. – № 57. – Article 101286. Crossref
Reynolds M.G., Damon I.K. Outbreaks of human monkeypox after cessation of smallpox vaccination // Trends in Microbiology. – 2012. – № 20(2). – P. 80–87. Crossref