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@#Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r = 0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.
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Objective:To observe the stability of severe acute respiratory syrdrome coronavirus （SARS-CoV-2） in cell cultures at different temperatures so as to provide basic data and scientific basis for the research and control of COVID-19 epidemic. Methods:The Vero E6 cells inoculated with SARS-CoV-2. According to TCID50， SARS-CoV-2 with different dilution （10-1， 10-3， 10-5， 10-6）were stored at 37 °C， 22.5 °C， and 4 °C for one to seven days， and then infectious titer was determined by micro cytopathogenic effect assay， observing cytopathic effect （CPE）， and real-time fluorescence quantitative testing. Results:SARS-CoV-2 was stable under 4 °C. The infectivity of high concentration （10-1 dilution） under 22.5 °C for seven days gradually decreased， while lower concentration completely lost infectivity after one day. The virus lost infectivity when stored at 37 °C for more than one day. Conclusion:SARS-CoV-2 is highly stable at 4 °C， sensitive to heat， and related to virus concentration.
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Objective To investigate the inactivating effect of heat and ultraviolet(UV) light on HCV JFH-1 strain using the cell culture system. Methods The HCV JFH-1 virus stock, with an initial titer of 2.5 × 104 FFU/ml, was exposed in 56℃ water bath or to UV light for varying durations of time for explo-ring their inactivating effects on the virus. The kinetics of virus titer reduction was determined by an indirect immuno-fluorescence assay (IFA). If the cells infected with the exposed virus stock were IFA negative after three blind passages, the virus stock was considered to be inactivated completely. Results After incubation of the HCV JFH-1 virus stock (2.5 × 104 FFU/ml)in 56℃ water bath for 10 min, 20 min and 30 min, the virus titers were reduced to 1.6 × 103 FFU/ml, 3.1 × 102 FFU/ml and 3.3 × 10 FFU/ml, respectively. The exposure of the virus stock to UV light (wavelength 253.7 nm, intensity ≥60 μW/cm2, 30 cm below the UV lamp) for 15 s, 30 s and 45 s resulted in virus fiter reduction to 1.0 × 103 FFU/ml, 1.1 × 102 FFU/ml and 2.7 × 10 FFU/ml, respectively. After 40 min incubation of the virus stock at 56℃, or 1 min exposure to UV light (wavelength 253.7 nm, intensity ≥60 μW/cm2) the virus infectious titer was reduced below the detection limit of IFA, and the IFA was still negative even after three blind passages, indicating that the virus was inactivated completely. Conclusion HCV is sensitive to heat and UV light treatment. For HCV JFH-1 virus stock containing 2.5 × 104 FFU/ml virus, heat treatment at 56℃ for 40 min, or UV light expo-sure at an intensity of ≥60 μW/cm2 for 1 min, resulting in complete virus inactivation.