OPTIMIZATION OF INFLUENZA A VIRUS ISOLATION ON MDCK-SIAT1 CELLS: INFLUENCE OF CELL CONFLUENCE ON VIRAL PRODUCTION

Introduction: Influenza A virus is a major cause of viral respiratory infections and a significant global public health concern. Isolation and propagation of the virus in cell culture are crucial steps for diagnosis, epidemiological surveillance, and research. Madin-Darby Canine Kidney (MDCK) cells are considered the gold standard for influenza virus culture. However, several experimental parameters, such as cell confluence at the time of inoculation, can affect the efficiency of viral replication.
Methods: This study aimed to optimize the inoculation of Influenza A/H1N1 virus onto MDCK cells by examining how different cell confluences influence viral production After amplification and maintenance of the MDCK-SIAT1 cells, four cell densities were prepared in T25 flasks: 6 × 10⁵, 1.2 × 10⁶, 2.5 × 10⁶, and 3 × 10⁶ cells per flask. For each condition, one non inoculated flask and one inoculated flask with the A/H1N1 virus were used. The cultures were monitored by microscopic observation of the cytopathic effect (CPE) and then analyzed by RT-qPCR to assess viral replication.
Results: Microscopic observations showed that CPE was absent at low confluences (6 × 10⁵ and 1.2 × 10⁶ cells/flask), while massive CPE, characterized by cell rounding, loss of adhesion, and detachment of the cell mat, was observed at high confluences (2.5 × 10⁶ and 3 × 10⁶ cells/flask). RT-qPCR results revealed a progressive decrease in Ct values with increasing cell density, reflecting a growing viral load. The condition corresponding to 3 × 10⁶ cells per flask exhibited the lowest Ct values (Flu A1 = 17.4; Flu A2 = 19.1), suggesting maximum viral genome production. These results highlight that cell confluence is a determining factor in the efficiency of Influenza A/H1N1 virus isolation and propagation on MDCK-SIAT1 cells. Under the tested conditions, a density of 3 × 10⁶ cells per T25 flask appears to be the most favorable for viral production. 
Conclusion: This study contributes to optimizing viral culture protocols and can serve as a basis for future work aimed at improving the reproducibility and standardization of virological techniques in cell culture.