Aqueous sodium deoxycholate

The virus titers were enumerated using the Reed and Muench method [12 ]. Briefly, HEp-2 cells (1×10⁴ cells per well) were seeded overnight in 96-well plates. Frozen virus stocks were diluted (10⁻¹) in sterile 1% aqueous sodium deoxycholate (Sigma Aldrich) and mixed for 15 min to disaggregate virus. The virus was then subjected to 10-fold serial dilution in maintenance medium (DMEM, 2% FBS) and 100 μL of the diluted virus from the 10⁻³ dilution onwards was added into each well of HEp-2 cells. Plates were incubated at 37°C and observed daily using an inverted phase contrast microscope for the appearance of distinct CPE. Virus titers were reported as 50% cell culture-infectious doses per volume (CCID₅₀/mL).
HEp-2 cells in DMEM containing 2% FBS were infected with viruses at an 100 CCID₅₀ and incubated at 37°C. Supernatants and cells were harvested at 1, 3, 5 and 7 d post-infection. The intracellular and extracellular viral levels were determined by real-time quantitative PCR (qPCR) assays.

Virus supernatants were subjected to endpoint titration and assayed in both NIH/3T3 and Vero cells. The virus titer was enumerated using the Reed and Muench method [61] and the Reed and Muench calculator [62] (link). Briefly, NIH/3T3 (1×10⁴cells per well) and Vero cells (4×10³ cells per well) were seeded overnight in a 96-well plate. Frozen virus thawed to room temperature were diluted (10⁻¹) in sterile 1% aqueous sodium deoxycholate (Sigma Aldrich, USA), and vigorously mixed for 15 minutes to disaggregate virus. Disaggregated virus was subjected to ten-fold serial dilution in maintenance medium, and 100 μl diluted virus from 10⁻³ dilution onwards was added onto each well of cells. Plates were incubated at 37°C and observed daily under inverted light microscopy for the appearance of distinct CPE. Virus titer was reported as 50% cell culture-infectious doses per volume (CCID₅₀/ml).
To assess the degree of adaptation of EV71:TLLm and EV71:TLLmv in NIH/3T3 cells, virus supernatants harvested from previously infected primate and rodent cell lines were subjected to virus titer assay using both NIH/3T3 and Vero cells. The titer ratio used to measure relative replication rates (RRR) in NIH/3T3 and Vero cells was calculated using the following formula: where A is the virus titer assayed in NIH/3T3 cells, and B is the virus titer assayed in Vero cells.