Viral titration and infectivity.

A 50% tissue culture infective dose (TCID₅₀) assay was performed to assess viral titration and infectivity. On day 0, MARC-145 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well. On day 1, the cells were inoculated with serially 10-fold diluted viruses at 37°C for 1 h. The excess virus inoculum was removed by washing the cells with PBS. Maintenance medium (2% FBS–DMEM; 200 μl) was added to each well, and the cells were cultured for a further 3 to 5 days. Cells showing the expected cytopathic effect were counted daily, and the TCID₅₀ value was calculated with the Reed-Muench method (75). Each assay was performed in triplicate.

(i) Intracellular infectivity. Cells were washed three times with PBS, scraped, and pelleted by centrifugation at 1,000 × g for 5 min. The cell pellets were subjected to three cycles of freeze-thawing with liquid nitrogen and a thermal block set to 37°C. The cell lysates were centrifuged at 10,000 × g for 10 min at 4°C to remove the cell debris. The titers of infectious virus were expressed as TCID₅₀ per milliliter after a limiting-dilution assay.

(ii) Extracellular infectivity. To determine the extracellular infectivity of the virus, the supernatants were harvested, filtered through a 0.45-μm-pore-size filter, and stored at 4°C. Their infectivity was determined in parallel with a limiting-dilution assay, as described above.