Standardization of C6/36 cells for DENV-2 virological assays: isolation, TCID₅₀ and serum neutralization

This study aimed the standardization of detection, isolation, titration, replication kinetics, and virus neutralization assay protocols for DENV-2 (New Guinea C strain) with the C6/36 mosquito-derived cell line. DENV-2 was isolated, titrated by TCID₅₀, and quantified via molecular assays using serially diluted viral RNA to generate a standard curve using the C6/36 cell line. The serum neutralization assay was optimized in 96-well plates based on visual scoring of cytopathic effect (CPE). Optimized serum dilutions effectively reduced nonspecific cytotoxicity caused by residual complement, preventing misinterpretation and enabling reliable detection of serum neutralizing activity in C6/36 cells. RT-qPCR assays calibrated using a TCID₅₀-titrated DENV-2 viral stock showed high amplification efficiency (90–100%) with R² ≥ 0.98. The results demonstrate that the standardized workflow enables proof-of-principle DENV-2 neutralization testing in C6/36 cells, yielding neutralization patterns consistent with those observed in Vero cells, and supports the use of this platform as an alternative for resource-limited laboratories.