Rotavirus survival and stability in foods as determined by an optimised plaque assay procedure

Tissue culture adapted rotavirus strains were propagated in MA104 and CaCo2 cells using standard cell culture procedures. The progress of infection was monitored by examining for a cytopathic effect, and for the presence of viral RNA in the tissue culture supernatant as determined by a guanidinium- based method. Subsequently, an effective plaque assay for rotavirus was developed using MA104 cells by optimising the adsorption time (2 h) and the levels of fetal calf serum (2.5%) in the overlay medium. Tragacanth gum was used in the overlay medium to immobilise the virus, and plaques were subsequently stained with 1% crystal violet. Using this optimised plaque assay, the survival of rotavirus following exposure to heat and UV irradiation was evaluated by enumerating the clear plaques. It was shown that 60°C for 10 min was sufficient to reduce the viral titer by at least 7 logs, and 50 mJ of UV irradiation was sufficient to reduce the initial viral titer by >2.5 logs. This optimised plaque assay was also used to determine the survival and stability of rotavirus from a range of experimentally contaminated foods including fruit juice, formula milk and lettuce.