Viability of dengue virus in culture stocks is efficiently preserved by storage in diluted forms

Storage of dengue virus (DENV) culture stocks in -80°C is a common laboratory practice to maintain the viability of the virus for long-term usage. However, the efficiency of this method could still be hindered by multiple factors. In our laboratory, we observed a constant and substantial deterioration in the titer of DENV in Vero culture supernatant stored in -80°C. Such incident had badly hampered the laboratory work and prompted an investigation to determine the cause. DENV isolates representing all four serotypes were propagated and the culture supernatants were harvested and stored in aliquots of original stock and 10 fold dilutions (10^-1 -10^-4). DENV titer in these stocks was determined prior to storage and reassessed on the third and sixth month of storage by focus forming unit assay (FFUA). The result demonstrated a constant preservation of titer ranging from 10⁴ ffu/ml to 10⁵ ffu/ml in the diluted DENV virus culture stocks of 10^-1, and 10^-2 of DENV1-4, a minor reduction of titer from 10³ ffu/ml to 10² ffu/ml at dilution 10^-3 for DENV4 only and complete deterioration in undiluted culture stock and lower dilution (10^-4) within 6 months of storage in -80°C for all serotypes. It is recommended that propagated DENV in Vero cells are stored in 10 fold dilutions as compared to the original form to preserve the titer for long-term usage.