In vitro antiviral activity of adenosine analog NITD008 against tick-borne flaviviruses

Abstract

There are currently no antiviral therapies available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). In this brief study, we describe the in vitro antiviral activity of adenosine analog NITD008 against KFDV, AHFV, OHFV, as well as Tick-borne Encephalitis virus (TBEV). Alongside the well-established activity of NITD008 against mosquito-borne flaviviruses, our results have demonstrated the feasibility of identifying nucleoside analog inhibitors that have pan-flavivirus activity.

Keywords: Antiviral; Flavivirus; NITD008; Nucleoside analog; Tick-borne.

Fig. 1

NITD008 inhibits flavivirus-induced cytopathic effect and also reduces levels of flavivirus antigen in infected cells. (A) Cytopathic Effect (CPE) Assay. Representative dose–response curves for AHFV (red), KFDV (green), OHFV (purple), and TBEV (blue) against NITD008. Tick-borne flavivirus Infected A549 cells were incubated with NITD008 at 3-fold serial dilutions for 72 h. Cell viability was measured using CellTiter-Glo 2.0 reagent and presented as a percentage of luminescence detected from the compound-treated cells compared with mock-treated cells. (B) Ebolavirus replication was measured in Vero cells treated with 3-fold serial dilutions of NITD008 by fluorescence levels emitted by the enhanced Green Fluorescent Protein at 48 h post-infection using a plate reader. (C) Cytotoxicity in mock-infected A549 and Vero cells. Mock-infected cells were incubated with NITD008 at 3-fold serial dilutions for 72 h. Cell viability was measured and presented in the same manner as the CPE assay. (D) Cell-based Flavivirus Immunodetection (CFI) assay. Dose-response curves for AHFV (red), KFDV (green), OHFV (purple), and TBEV (blue) against NITD008. Tick-borne flavivirus infected A549 cells were incubated with NITD008 at 3-fold serial dilutions for 24 h, and then fixed and stained with primary anti-flavivirus HMAF and secondary goat anti-mouse antibody conjugated with HRP, respectively. Levels of flavivirus antigen present in infected cells was measured by chemiluminescence, and were presented as a percentage of luminescence detected from the compound-treated cells compared with mock-treated cells. Error bars indicate standard deviation of the means.

Fig. 2

Immunofluorescence Assay (IFA). A549 cells were pretreated with NITD008, infected with indicated viruses for 24 h. Cells were fixed for 15 min in 10% formalin supplemented with 0.2% Triton-X detergent, then stained with a primary anti-flavivirus HMAF and a secondary donkey anti-mouse antibody conjugated with DyLight 488 (green). Cells were counterstained with DAPI (blue), and images were captured at 40 × magnification using a Nikon Eclipse Ti inverted fluorescence microscope. White bar indicates length of 100 μm.

Fig. 3

NITD008 inhibits tick-borne flavivirus replication. A549 cells were infected with (A) AHFV, (B) KFDV, (C) OHFV, or (D) TBEV at an MOI of 0.5 for 1 h before being treated with NITD008. Seventy-two h post-infection, the supernatants were harvested and were subjected to one freeze–thaw cycle before median tissue culture infective dose were determined (TCID₅₀/Ml). Mean titers from four biological replicates are depicted, and error bars indicate standard error of the means. Dotted lines indicate the limit of detection.