Methylene Blue Inhibits Acute Hemorrhagic Conjunctivitis Virus Production and Induction of Caspase-3 Mediated Human Corneal Cell Cytopathy

Background: Acute hemorrhagic conjunctivitis (AHC) is a highly contagious eye disease caused by enterovirus type 70 (E70) and Coxsackievirus A24 variant (CA24v) with no clinically approved treatment. The antiviral activity of methylene blue (MB; a WHO essential medicine) against AHC viruses was investigated using human corneal epithelial cells (HCEC). Methods: Time and concentration-dependent MB accumulation by HCEC was determined colorimetrically and MB inhibition of virus production of 5 E70 and 3 CA24v AHC epidemic isolates in HCEC was determined by micro-plaque assay. AHC virus cytopathy inhibition by MB was detected by reductions in virus-induced caspase-3 activity and polymeric DNA fragments. Results: MB uptake by HCEC was rapid and concentration dependent. MB inhibition of E70 and CA24v production was concentration dependent. AHC virus yields were significantly lower (50 to >10,000 fold) in HCEC pre-treated with 0.25-1% MB than in placebo controls (p's <= 0.01). MB pre-treatment significantly inhibited virus-induced caspase-3 activation and DNA fragmentation (p's<0.01). Virus-infected cells accumulate oxidized MB and MB application up to 6 h after infection inhibited virus production and virusinduced HCEC cytopathy. Conclusion: The results suggest MB treatment prior to and shortly after infection can inhibit AHC virus production and caspase-mediated HCEC cytopathy. The results support the therapeutic potential of ophthalmic solutions containing MB against AHC virus infection during epidemics.