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Introduction: Epidemic Japanese encephalitis is one of the most important zoonotic diseases that cause central nervous system damage. The vaccination has become the most effective and economical measure for its control. Hence, real-time monitoring of Japanese encephalitis virus (JEV) proliferation is crucial to optimize virus inoculation, culturing conditions, and virus harvest time. Methods: The proliferation dynamics of JEV in BHK-21 cells was studied by combining the established quantitative PCR method with the conventional TCID50 assay in this study. Results: The proliferation curve determined by the 2 methods has a definite parallel relationship, but the quantitative real-time PCR method (4 h) is faster and more sensitive than the TCID50 method (3–4 days). The determination results of TCID50 showed that the highest viral titer was 105.44 TCID50/0.1 mL and 104.86 TCID50/0.1 mL in cell suspension and culture supernate, respectively, while the virus RNA copies reached the peak at 1.0 × 107.5 copies/µL and 1.0 × 105.6 copies/µL in cell suspension and culture supernate, respectively. Conclusion: The comprehensive analysis showed that the best time for JEV proliferation in BHK-21 cell was 60 h post infection. [ABSTRACT FROM AUTHOR]