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For establishment of plaque assay on BHK-21(Cl-13) cell line, different parameters were standardized. A cell count of 3-4 X 10 5 /ml was found suitable when grown in Eagle's minimum essential medium (EMEM) containing 5% foetal bovine serum (FBS) whereas a concentration of 2% FBS was optimum for the overlay media containing 0.2% final conc. of carboxymethyl cellulose (CMC); a pH value of 7. 8 was suitable for normal growth of BHK-21 cells as well as for the development of plaques; an inoculum size of 0.2ml per well of a 6-well TC plate at 10 -8 dilution of the Japanese Encephalitis (JE) virus was found to be sufficient to give the optimum plaque count. A 90 minutes adsorption time was adequate for the virus to adsorb onto the BHK-21 cells. Under these conditions the development of plaques occurred in five days at 36 + 1 0 C, 5% CO2 and >90% Relative Humidity (RH). Repeatability of the assay was tested and found to be good (Mean + SD= 90 + 6.6; CV=7.2%). A comparison between the JE virus plaques on BHK-21 cells and Chick Embryo Fibroblast (CEFB) cells showed comparable results. Thus, it was concluded that the continuous cell line such as BHK-21 can be used in place of CEFB cells for plaque assay of JE virus and this system can be used as an alternative approach for the potency assay of JE vaccine.

Standardization of Plaque Assay of Japanese Encephalitis Virus (Nakayama NIH Strain) on BHK-21 (Cl-13) Cell Line