Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

The condensed JEV was titrated by plaque formation assay with a semi-solid overlay. BHK-21 cells (ATCC ® CCL-10™) were obtained from Ms. L. Jia (The National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). BHK-21 cells were used in JEV titration and JEV progeny determination in RNAi-treated Vero cells, while attempt to identify molecules capable of binding JEV by VOPBA using BHK-21 cells failed (unpublished data). Briefly, BHK-21 cells (1x10 5 cells/well) were grown to a confluent monolayer in a 6-well plate in culture medium [containing 60% RPMI-1640, 30% 0.5% LH, 10% FCS, 1% penicillin, 100 IU/ml streptomycin, 0.75% NaHCO 3 (1% in total) NaHCO 3 and 3% glutamine (1% in total)] and infected with 200 µl of 10-fold serially diluted JEV aliquots dissolved in virus dilution solution [containing 96% LH, 2% FCS, and 0.75% NaHCO 3 (2% in total)]. The cells absorbed the virus at 37˚C for 1 h with gentle circular tilting every 15 min. The fluids were then pipetted out, and the monolayers were washed 3 times with RPMI-1640 without serum and finally covered with a semi-solid overlay [containing 1% methyl cellulose, 1/10 volume 10X RPMI-1640, 25% distilled water, 10% FCS, 1% antibiotics (penicillin and streptomycin), 0.75% NaHCO 3 (3% in total) and 3% glutamine (1% in total)]. Following incubation at 37˚C for 96 h, the overlay was withdrawn, and the cell monolayer was stained with a 1% crystal violet solution containing 0.2% formaldehyde. All plaque formation assay experiments were performed in triplicate, and the plaques were counted and calculated in PFU/ml.