CYTOPATHIC EFFECTS OF WIDE-TYPE RUBELLA VIRUS STRAIN IN ECV304 ENDOTHELIAL-LIKE CELLS / 解剖学报

ABSTRACT

Objective To investigate the mechanisms for cytopathic effect(CPE) of wide-type rubella virus(RV)in ECV304 endothelial-like cells.Method ECV304 cells were cultured in MEM supplemented with 10% fetal calf serum,glutamine,sodium bicarbonate,penicillin and streptomycin at 37℃ with 5% CO-2 in a humidified incubator,and then grown in maintenance medium with 2% fetal calf serum to avoid overstimulating cell growth.Infected cells and control cells were harvested at day 2,4, and 6 after infection for following experiments.RT-PCR and indirect immunofluorescence were used to detect RV infection.Phase-contrast and electron microscopy were performed to analyze morphology changes in infected and uninfected groups.TUNEL assay was carried out to study wide-type RV-induced apoptosis.Result In RV-infected ECV304 cells,cytopathic effects were first observed approximately at 2-3 days post-infection.The majority of cells with CPE changes were of detachment from the monolayer,rounding of cells,cell shrinkage and membrane blebbing were seen.At 4-6 days post-infection,the detached cells increased clearly;the CPE regions on the monolayer were extended,and the infected cells in the CPE regions showed in spindly form with wide spacing.RT-PCR and indirect immunofluorescence results indicated RV infection in ECV304 endothelial-like cells.Using electron microscopy,RV particles and its cytopathic effects including the condensed chromatin,fragmented nuclei and clustering of mitochondria with dramatic changes around nuclear were further examined.TUNEL results showed that the difference of apoptosis index between the infected group and uninfected group was significant(P