ABSTRACT
Objective To comparatively analyze the in vitro antiviral mechanism( s) of eugeniin and quercetin against varicella-zoster virus ( VZV) by using a novel antiviral assay based upon a reporter cell line (MV9G cells) for VZV. Methods Selection indexes (SIs) of potential antiviral compounds extracted from Chinese herbs or plants including eugeniin, eugenol, morin, curcumin, myricetin and quercetin for in vitro inhibition of VZV were calculated. The compounds with relatively higher SIs were screened out for fur-ther investigation of their in vitro inhibitory mechanisms with a cell-free virus ( CFVs) direct-infection assay and a cell-associated virus (CAVs) co-culture assay established with MV9G cells in our previous study. The inhibitory mechanisms analyzed in this study included direct inactivation of CFVs, inhibition of the adhesion and/or penetration capabilities of CFVs to MV9G cells, inhibition of the intracellular replication of CAVs and inhibition of the transcription and / or expression of viral immediate early gene 62 ( IE62 ) . Results Among the tested compounds, eugeniin and quercetin showed relatively higher SIs of 5. 82 and 8. 97, respec-tively. Eugeniin rather than quercetin directly but partly inactivated CFVs and inhibited their attachment to and penetration into MV9G cells in a concentration-dependent manner. Both eugeniin and quercetin revers-ibly inhibited the intracellular replication of CAVs and the transcription and expression of viral IE62 gene, for which eugeniin needed to be added within 12 hours after infection. Conclusion Eugeniin and quercetin had different in vitro inhibitory mechanisms against VZV, but inhibiting the transcription and expression of viral IE62 gene was a common mechanism shared by both of them.
ABSTRACT
Objective To investigate mutations in immediate early ( IE) gene ORF62 of three var-icella vaccine Oka strains ( vOka ) including two strains produced in China and their parental Oka strain (pOka), and then to further elucidate its possible roles in attenuation mechanism by comparing their ORF 62 promoter sequences and its activities , ORF62 coding regions and its transactivities .Methods ORF62 pro-moter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains ( vOka-BK from Changchun BCHT Biotechnology Co ., vOka-SH from Shanghai Institute of Biological Product Co .Ltd., and vOka-GSK from GlaxoSmithKline plc, as control) were constructed, respectively.ORF62 promoter regions and coding regions of the four strains were sequenced and then compared with each other .Differences of ac-tivities of the ORF62 promoter, and transactivities of the ORF62-encoded IE62 upon immediate early (ORF4), early (ORF28) and late (ORF67) gene promoters between pOka and vOka strains were assayed with transient transfection technique .Results Compared with pOka strain , three vOka strains had a con-sistent T deletion mutation at site 110 050 in ORF62 promoters, which did not result in any change of tran-scription factor binding motif .However , activities of ORF62 promoters from three vOka strains were signifi-cantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 cod-ing regions of three vOka strains and three new enzyme restriction sites including SmaⅠ, NaeⅠand BssHⅡwere generated, respectively.Transactivities of IE62 from three vOka strains upon ORF4, ORF28 and ORF67 promoters were significantly higher than those of pOka both in CV-1 and MeWo cells , except that vOka-SH IE62 showed significantly lower transactivities upon ORF 4 promoter than those of pOka strain in CV-1 cells.Conclusion Consistent T deletion mutation at site 110 050 in ORF62 promoters of three vOka strains might be responsible for the reduced promoter activities and the changes of IE 62 transactivities .How-ever , it seemed that cell types have no significant effect on ORF 62 promoter activity or IE 62 transactivity be-tween pOka and vOka strains .