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1、388 JOURNAL OF INTERFERON reverse primer 5?-CACGAACGT- GACGAAT-3?) was used in the quantitative PCR assay.(6)Plas- mids containing the target sequence were used as positive controls. The viral titers were measured by back-titration ac- cording to standard protocols. IFN-? and IFN-? protected cells f
2、rom viral CPE. Preincu- bation for 1 h with 128 U/well IFN-? (Fig. 1A, top) and IFN- ? (Fig 1A, bottom) protected cells almost completely. The cell morphology was indistinguishable from that of normal, unin- fected cells. Marked protection was visible at concentrations as low as 16 U/ml IFN-? and IF
3、N-? (Fig. 1B, C). However, only A BC IFN (unit)0016128 CoV IFNa IFNa IFNb IFNb -+ Relative viral RNA copiesRelative viral RNA copies 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0 0 U 2.5 U20 U156 U 625 U 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0 0 U0.5 U4 U32 U 125 U 500 U FIG. 1.Inhibition of SCoV-medi
4、ated CPE and intracellular viral RNA accumulation by IFNs. The FRhk-4 cells were pretreated with various concentrations of different IFNs as indicated for 1 h and infected with SCoV. (A) CPE under phase-contrast mi- croscopy. ?400. (Top) IFN-? treated. (Bottom) IFN-? treated. Real-time RT-PCR was em
5、ployed to quantify the viral RNA of SCoV after the cells were treated with IFN and challenged with SCoV. (B and C) Reduction of intracellular viral RNA copies. weak protection was observed using IFN-? at a concentration of 1000 U/ml (data not shown). To further quantify the effects of IFNs on inhibi
6、tion of SCoV infection and replication in FRhk-4 cells, we measured SCoV intracellular viral RNA copies by quantitative real-time PCR using total cellular RNA as the template and viral titer by back-titration.(6,7)Our results showed that both IFN-? and IFN-? dose-dependently reduced SCoV viral RNA c
7、opies in cells and infectious titers in the con- ditioned medium. Approximately 50% reduction of intracellu- lar viral RNA and viral titers was observed at IFN-? and IFN- ? concentrations as low as 25 U/ml and 10 U/ml respectively, and 90% elimination of viral RNA or 98% of viral titers was seen at
8、about 3000 U/ml and 1800 U/ml, respectively. IFN-? did not exhibit significant antiviral activity. These results were, in general, consistent with the results conducted on Caco-2 cells,(7)although the concentrations of IFNs were miscalcu- lated.(8) Our results indicate that type I IFNs are much more
9、 effec- tive in inhibiting SCoV infection and replication than are type II IFNs. There is a wide variety of type I IFN subspecies, each of which may have different antiviral activities and specifici- ties.(35)For example, Cinatl et al.(7)reported that only IFN-? exhibited potent anti-SCoV activity i
10、n Vero cells and Caco2 cells. In fact, it has been show that pegylated IFN-? protected type 1 pneumocytes against SCoV in infection in macaques.(9) It will be important to identify the most potent IFN subspecies and conduct preclinical and clinical trials, as SARS is recurring in China. ACKNOWLEDGME
11、NTS This work was supported by a SARS grant (to BZ, MLH), CERG (to MLH, HFK) from the Research Grant Council of the Hong Kong Government. REFERENCES 1. KSIAZEK, T.G., ERDMAN, D., GOLDSMITH, C.S., ZAKI, S.R., PERET, T., EMERY, S., TONG, S., URBANI, C., COMER, J.A., LIM, W., ROLLIN, P.E., DOWELL, S.F.
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