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Tejabhiram Yadavalli, Neel Thakkar, Dinesh Jaishankar, Alex Agelidis, Deepak Shukla; Antiviral activity of a 45 nucleotide DNA aptamer during HSV-1 infection of the cornea.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3599.
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© ARVO (1962-2015); The Authors (2016-present)
Herpes simplex virus type 1 (HSV-1) is the leading cause of infectious blindness in the United States. In this study, we use corneal model of HSV infection to report the high antiviral activity of a 45 nucleotide DNA aptamer directed against HSV-1 gD protein.
SYBR Green based ELISA was conducted to evaluate the specific binding affinity of the DNA aptamer by incubating 50 µL of multiple concentrations of soluble HSV-1 gD, gB proteins. MTT based cytotoxicity assay for 2 different aptamer concentrations were conducted along with a random DNA sequence and mock PBS. Viral entry and infectious spread were evaluated using β-galactosidase assay, immunoblotting and immunofluorescence assays. Freshly cut pig corneas were infected with HSV-1 GFP virus and its spread was monitored over a period of 10 days in the presence and absence of the aptamer. BALB/C mice (male and female, 3 weeks old) were anesthetized using ketamine (100 mg/kg) and xylazine (5 mg/kg) prior to scarring the right eye with a 30-G sterile needle in a 3 × 3 grid pattern. The scarred eyes were infected with GFP virus and the progress was monitored for a period of 10 days before sacrificing the mice and harvesting the tissue for analyzing the extent of virus infection and the role of cytokines through RT-PCR assay.
Our DNA aptamer showed specific binding affinity towards HSV-1 gD (Kd = 50 nM) and was able to restrict viral entry by 70% at a 2 µM concentration. Immunofluorescence and immunoblotting studies showed 40-50% inhibition of viral entry and viral replication in-vitro respectively. Ex-vivo studies on pig corneal models showed therapeutic efficacy of the aptamer in reducing productive infection over a period of 168 hours. In-vivo experiments on mice models exhibited significant decrease in ocular infection through prophylactic, therapeutic and neutralization treatment of corneal tissue with the DNA aptamer.
This is the first study to show a comprehensive decrease in HSV-1 infection using a DNA aptamer. Their role in inhibiting cell to cell fusion and consecutively restrict viral spread was also evaluated in this study. Future studies will shed more light on the clinical implications of this aptamer.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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