The inhibitory effect of Chinese herb on SARS virus infection

Min Zhuang, Peng Xiao

[Subject]

Severe acute respiratory syndrome (SARS) is a contagious atypical pneumonia with a high mortality rate. SARS coronavirus (SARS-CoV) is the pathogen of SARS. We established SARS-CoV S/HIV pseudo typed (SHP) virus system and the cell fusion assay system to screen inhibitors for entry of SARS-CoV.

[Materials and methods]

SHP or VSV-G pseudo type (VHP) virus was made by transfecting pCMVΔR8·2, pHR’CMV-Luc and pCMV/R-SARS-S or pMDG plasmids into 293T cells. 5ng p24 of SHP or VHP virus was added for each infection. Twelve Chinese herbs, which were reported to be effective against SARS were selected and their extracts were examined. The cytotoxic analysis of drugs was performed using Cell Counting Kit-8. For entry inhibition assays, the viruses was incubated with different concentrations of drugs at 37℃for 30 min or serum before adding to cells. We also treated the cells with these drugs before adding viruses. The cell fusion assay system was performed by incubation of transfected 293T cells with pCMV/R-SARS-S and Hela cells with pQACE2, 293Tcellswere labeled by Calcein-AM. After one hour

Incubation followed by washing, the cells were observed under an inverted fluorescence microscope.

[Results]

Among tested cell lines, HepG 2 was found to be the most sensitive for SHP virus and was used. The SARS patient serum blocked the SHP virus infection, confirming the involvement of S protein in infection. Nine out of 12 extracts showed inhibitory activities against both SHP and VHP viruses, however, 7 of them also showed apparent cytotoxic activities. IC50s of Cinnamomi Cortex extract (CCE) were 0·03mg/ml for SHPwhile no cytotoxicy was observed at these concentrations. Caryophylli Flos extract (CFE) also shon effect at low concentration (IC50 is 0·058 mg/ml) while CC50 of CCE and CFE were 0·2 mg/ml and 0·5 mg/ml, so selective indexes (SI) for SHP were 6·67 and 8·62. The firs tone inhibited the infection by pretreatment of target cells but the second one didn’t. We also examined 4 ethanol-aqueous fractions of CCE. BuOH fraction and AcOEt fraction apparently showed the inhibition at lower concentrations, however the latter fraction had strong cytotoxic effect, the SI of BuOHfraction for SHP was 4. The BuOH fraction dose dependently inhibited the cell fusion between the S protein andACE2 receptor, the concentrations 0·1 mg/ml completely inhibit the cell fusion.

[Discussion]

Inhibitory activities of on SARS virus infection were found in two Chinese herb extracts (CCE and CFE). They have different mechanisms of inhibition. BuOH fraction of CCE may contain the potent new inhibitor for entry of SARS-Cov.