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Development of optimized protocol for culturing African swine fever virus field isolates in MA104 cells
Hyeok-Il Kwon1, Duy Tien Do1, Hung Van Vo1
1ChoongAng Vaccine Laboratories, Daejeon, 34055, Republic of Korea (Hyeok-il Kwon, Seung-Chul Lee, Min Ho Kim, Joo Young Lee); Department of Infectious Diseases and Veterinary Public Health, Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Thu Duc District, Ho Chi Minh City, Vietnam (Duy Tien Do, Dung Thi Thuy Nguyen, Tram Thi Ngoc Ngo, Toan Tat Nguyen); Center for Veterinary Diagnostics, Regional Animal Health Office No. 6, Ho Chi Minh City, Department of Animal Health, Vietnam (Hung Van Vo, Tan Minh Tran, Quang Tin Vinh Le); Research Center for Genetics and Reproductive Health (CGRH), School of Medicine, National University HCMC, Vietnam, (Nam Minh Nguyen).
Abstract:
The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a mean cycle threshold (Ct) value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. African swine fever virus originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. When ASFV was cultured from tissue homogenates, however, there was no difference (P = 0.062) in ASFV growth between infectious media A and B. A model was developed to enhance ASFV replication through adaptation to MA104 cells. The lack of mutation at the genetic segments encoding p72, p54, p30, and the central hypervariable region (CVR) in serial culture passages is important in increasing the probability of maintaining immunogenicity when developing a vaccine candidate.
Insights
MA104 cells are ideal for African swine fever virus (ASFV) replication, especially when using blood samples and specific infectious media. This research aids in developing ASFV vaccines by ensuring viral genetic stability during culture.
Area of Science:
- Veterinary Virology
- Cell Biology
- Molecular Biology
Background:
- African Swine Fever Virus (ASFV) poses a significant threat to global swine populations.
- Effective replication of ASFV in cell culture is crucial for research and vaccine development.
- Identifying optimal cell lines and culture conditions is essential for consistent ASFV propagation.
Purpose of the Study:
- To identify a suitable commercial cell line for African swine fever virus (ASFV) replication.
- To compare the efficacy of different cell lines and infectious media for ASFV propagation.
- To establish a model for enhancing ASFV replication and maintaining viral genetic stability.
Main Methods:
- Comparative analysis of multiple cell lines (MA104, MARC-145) for ASFV sensitivity.
- Adaptation studies of ASFV from tissue homogenates and blood samples in various infectious media (A and B).
- Serial passage of ASFV in MA104 cells, monitoring viral load via cycle threshold (Ct) values.
- Genetic analysis of key ASFV genes (p72, p54, p30, CVR) to assess mutations.
Main Results:
- MA104 and MARC-145 cell lines demonstrated high potential for ASFV replication.
- ASFV from blood samples showed significantly higher viral loads than from tissue samples after 10 passages (Ct 20.39 vs 25.36).
- Infectious medium B supported more robust ASFV growth from blood samples compared to medium A (Ct 19.58 vs 21.20).
- ASFV from blood samples peaked in replication by the 15th passage in both media.
- No significant difference in ASFV growth was observed between media A and B when cultured from tissue homogenates.
- No mutations were detected in the analyzed genetic segments (p72, p54, p30, CVR) during serial passages.
Conclusions:
- MA104 cells are a promising candidate for ASFV replication, particularly with blood-derived isolates.
- The choice of infectious medium significantly impacts ASFV replication efficiency depending on the sample source.
- The developed model enhances ASFV replication in MA104 cells while maintaining genetic stability, crucial for vaccine development.
- Preservation of genetic integrity in key viral genes is vital for maintaining ASFV immunogenicity in vaccine candidates.
