2.9. Recombinant HAV Amplification and Titer Determination

The purified HAV-HEp148 virus was diluted 1:100 in MEM to infect confluent monolayers of Vero cells in 175 cm³ flasks for 6 h at 37 °C. Maintenance medium with 2% newborn calf serum was added, and infected cells were incubated at 35 °C in a CO₂ incubator for 3 weeks. The virus was harvested on day 21 after infection, and then extracted with chloroform and filtered through a 0.22 µm filter. HAV titers were determined by inoculating the confluent monolayer of cells cultured in 25 cm³ culture flasks, and infection was verified by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, four replicate flasks were inoculated with 1 mL of 10-fold dilutions of HAV for 2 h and cultured in MEM with 2% newborn calf serum for 3 weeks at 35 °C. After virus harvest, a commercial ELISA kit (qualitative manual method; WNT Biological Co., Ltd., Kunming, China) was used to determine the 50% cell culture infectious titers (CCID₅₀/mL) according to the method previously described by Reed and Muench [30,31,32]. The infectious titer of the HAV-HEp148 viruses was found to be 7.5 log CCID₅₀/mL as determined by the ELISA.