| hand-foot and mouth disease（HFMD）is a children’s infectious disease,also known as exanthematous vesicular stomatitis. It occurs in children underage of5, and can cause herpes in hand, foot, mouth and other parts. A smallnumber of children can get myocarditis, pulmonary edema, asepticmeningoencephalitis and other complications. Individual children with severeprogression of the disease lead to death quickly. Hand, foot and oral herpes orulceration after the formation of ulcers is the main clinical symptoms. HFMD isan infectious disease caused by enterovirus, HFMD caused by more than20types of enterovirus, including Coxsackie virus type A16(Cox A16) andenterovirus71(EV71),which are the most common ones. Enterovirus71(EV71) is one of the hand, foot and mouth disease pathogens. It is reported inrecent years in the Asia-Pacific region there was a large outbreak of hand, footand mouth disease in children under the age of six, but unfortunately, there isnot yet any effective anti-EV71virus treatment or vaccine right now.Polyoxometalates (POMs) are metal oxides with anionic groups. Thiscompound has demonstrated broad-spectrum antiviral activity. The goal of thisstudy was to evaluate the biological anti-EV71virus activity of the POMs. Thetest compounds were marked as4#,6#and12#, we first examined thecytotoxicity of this group. After adding the three compounds for2days, thenumbers of viable cells with and without test compounds are not very different.The compounds in the concentration of500μM co-cultured with cells for2days, did not have an impact on cell growth and cell toxicity was very weak.Subsequently, we tested in vitro anti-virus activity: in the CPE inhibition assay,the cells occurred CPE after virus inoculation and died which could not becrystal violet stained, while adding the POM compounds into the positive control group, the compounds in concentration of100μM, cell viabilities weresignificantly increased with obviously stained by crystal violet, and we foundthat4#,6#and12#compounds have the CPE inhibitory effects. In the plaqueassay, POMs show a dose-dependent inhibition of EV71infection, the IC50values is about100μM. We did an adding time assay to study the POMsantiviral mechanism: virus infected cells one hour so that EV71could enterinto cells completely, and then washed away the virus solution, and cultured forone hour after adding the compounds, and washed it away, then added a coverlayer. Put it into the incubator for two days before the observation. We foundthat after adding the three compounds, the plaque number is far greater than300, compared with only infected group without POMs addition positivecontrol group, the plaque number has no statistical significance, but the12#compound’s plaque inhibition rate was significantly higher than the4#and6#compounds, with a visible50%inhibition rate. Pretreat cells with POMsfor1hour and washed away, then infected cells with the virus for one hour.After washing with PBS we added a cover layer, and put the plate intoincubator for two days. We found that12#compound in the concentration of100μM inhibited plaque formation evidently, but in concentration of10μM,thethree compounds did not show any inhibition of the plaques. The virussolution mixed with compounds for1hour at37℃, CO2incubator, and addedthe mixture into the cells, then washed, and then added the overlayer afterincubated for2days. Under this reaction conditions, the three compoundsshowed plaque inhibitory effects obviously, and in the concentration of100μM,three compounds inhibited in rate of more than90%, Meanwhile in4#and12#compounds groups we even did not observe any plaque-forming, and12#compound in20μM showed inhibition rate of nearly90%. Antiviral activity ofthree POM compounds after pre-incubation with the virus showed the mostsignificant anti-EV71effect, it suggests that the POMs inhibit EV71infection at an early stage, there may be a direct effect passivation or inactivation of thevirus rather than acting on the host cell. In EV71RNA quantificationexperiments, the mixture of compounds and viruses were added into cells,4#did not significantly inhibited the EV71RNA release.6#compound inconcentration of10μM and100μM did not show great difference in inhibitionof viral RNA release with a rate of declination of about40%.12#in aconcentration of100μM decreased more than90%while in the concentrationof10μM decreased by about70%. The PCR products were detected in proteingel electrophoresis, and we found that the productions located between100~250bp in length which is the length between the two EV71primers genesequence of the interval. We conducted the results in the Blast Hits on theQuery Sequence, the results were consistent with the human EV71highly,showing that the extraction product was the target viral gene instead of otherinterference and pollution.12#compound obviously declined the EV71RNAafter mixed with the virus before infected the cells and it actually reduced thevirus RNA quantification. Our results gave a possibility for the anti-EV71drugs discovery. |
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