Development of methods for culture and titer determination of respiratory syncytial virus

Objective To determine the culture condition of respiratory syncytial virus(RSV), screen the formula of virus stabilizer and develop a micro-cytopathic effect(CPE)method for determination of virus titer. Methods Hep2, Vero and293 R cells were inoculated with RSV at a MOI of 0. 01 to optimize the cell substrate for virus culture. Hep2 cells were inoculated with RSV at MOIs of 0. 005 and 0. 02 respectively, while the virus liquid was harvested 4 ~ 9 d later and determined for titer to optimize the MOI and time for virus harvest. Virus liquid was added with stabilizers of six formula respectively, then subjected to seven cycles of freezing and thawing and determined for titer to optimize the formula. The virus was titrated at 33 and 37 ℃ respectively, of which the results were judged on days 7 and 9 to optimize the temperature for virus culture and time for result judgment in micro-CPE method. Results The optimal substrate for virus culture was Hep2 cells. RSV was inoculated at a MOI of 0. 02 and cultured at 37 ℃ for 7 ~ 9 d before harvest. The optimal stabilizer was of formula 1(0. 1% human serum albumin). In micro-CPE assay, RSV was titrated at 37 ℃, and the result was judged on day 7. Conclusion Stable and reliable methods for culture and titer determination of RSV were developed.