Antiviral activity research of artesunate against human cytomegalovirus by fractionation dosage method in vitro

Abstract

Objective To research the antiviral activity of artesunate(ART) in vitro fighting against both standard laboratory strains and ganciclovir(GCV)- resistance strains of human cytomegalovirus(HCMV) and to explore whether fractionation dosage method can obviously enhance the antiviral effect of ART. Methods 1.Cytotoxicity assay to ART was performed by the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) colo-rimetry.The 0%toxic concentration (TC0)were determined, and median cytotoxic concentration (TC50) was calculated with Probit regression method.2.Antiviral activity assays of ART against HCMV: human embryonic lung fibroblast cells(HELs) were infected with standard laboratory strains and GCV- resistance strains of HCMV, respectively, after which virus was removed and overlays of dulbecco's modified eagle medium(MEM) containing different antiviral drugs were added to the wells.All cells were cultured continuously at 37 ℃ in a 50 mL/L CO2 humidified atmosphere for 7-10 days and the cytopathic effect(CPE) was observed under a microscope.When the degree of CPE was clear(+ + + -+ + + + ), the values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The cell survival rate (CSR)and drug inhibitory rate (IR) for HCMV were calculated.By Probit regression method, the median inhibitory concentration (IC50) of 2 drugs was calculated respectively.3.To explore whether fractionation dosage method could obviously enhance the antiviral effect of ART against HCMV, the experiment was divided into 3 groups and compared with GCV group, respectively: Group 1: ART antiviral compounds were added to cell layers by one dosage.Group 2: Total drug dosage was divided into 3 parts, and each part was added to cell layers once a day for 3 days.Group 3: Total antiviral compounds were divided into 6 and delivery 2 times a day.The values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The CSR and viral inhibitory rates were calculated.All data were statistically analyzed by One-Way ANOVA analyzing using SPSS 18.0 statistical software.P value of <0.05 was considered to indicate statistical significance. Results 1.Cytotoxicity assay showed that cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.TC0 and TC50 value of ART were 62.5 μmol/L and 171.7 μmol/L.2.In concentration of 5 μmol/L, 15 μmol/L and 30 μmol/L, ART and GCV could obviously inhibit growth of HCMV AD169 strains.There was no significant difference between them.The value of GCV IC50 was 3.49 μmol/L, and the value of ART IC50 was 2.17 μmol/L.Treatment index (TI) of ART was 28.8, and GCV was 716.3.ART could still obviously inhibit growth of HCMV resistant strains, but GCV couldn't.Differences between them were statistically significant.The value of GCV IC50 to HCMV resistant strains was 44.4 μmol/L, and the value of ART IC50 was 2.5 μmol/L.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.Difference was statistically significant(P 0.05). Conclusions 1.Cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.2.ART could obviously inhibit growth of HCMV resistant strains and standard laboratory strains.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.4.Because the action mode of ART is different from other anti-HCMV drugs, and ART has a high biological activity and fewer side effects, it is expected to become a kind of new antiviral drugs for HCMV infections. Key words: Human cytomegalovirus; Artesunate; Antiviral activity