Inhibition of Coxsackievirus A24 in permissive Hep2 cells by small interfering RNA (siRNA)

Abstract

RNA interference is a sequence specific post transcriptional gene silencing mechanism which works through cleaving of nucleic acids by small RNA molecules of 19-21 mers. RNA interference tool found very effective to destroy the pathogenicity of several viruses. The molecular scissor activity of small interfering RNA (siRNA) was applied for inhibition of CoxsackievirusA24 (CA24), an Enterovirus of family Picornaviridae responsible for acute haemorrhagic conjunctivitis in humans. Four different siRNA molecules were used to target 5’ untranslated region (si5U), cis acting replication element of 2C (siCre), RNA dependent RNA polymerase enzyme coding region (si3D pol ) and 3’ untranslated region of CA24. Virus inhibition study by siRNA was done in Hep2 cells. Cytopathic effect, immunofluorescence assay, 50% tissue culture infectious dose (TCID50), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and virus copy number in Real-time PCR assays were used for the validation of virus inhibition potential of designed siRNA. Analysis of cumulative data reflected that si5U and si3D pol are highly efficient to stop CA24 propagation in Hep2 cells. Transfected Hep2 cells with any of these siRNA found refrained for CA24 infection in cell culture, immunofluorescence assay, TCID50 and Real Time PCR assay (p<0.05).