Human norovirus (NoV) is a leading cause of viral gastroenteritis globally and infections cause more than 220,000 deaths every year. Hence, there is an urgent need for a vaccine or antiviral therapy to reduce the burden of NoV infections. A promising area in antiviral research includes the use of Toll-like receptor (TLR) agonists to stimulate the production of pro-inflammatory cytokines and interferon to promote viral clearance from the host. TLR7 agonists are a group of compounds progressing through clinical trials for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections, either alone or in combination with other antivirals. However, the effects of TLR7 agonists against caliciviruses have not been studied. We used the murine norovirus (MNV) model to investigate whether a range of these compounds could inhibit NoV infection and replication, and thus show promise as a therapeutic for NoV-associated gastroenteritis.
The TLR7 agonists R-848, Gardiquimod, GS-9620, R-837 and Loxoribine were screened against MNV using a plaque reduction assay and each displayed marked inhibition of plaque formation with EC50 values of 23.5 nM, 134.4 nM, 0.59 µM, 1.5 µM and 79.4 µM, respectively. We also performed RNA-sequencing of TLR7 agonist stimulated cells to measure the gene expression changes that could lead to an antiviral state. We found that genes involved in the innate immune response and cellular antiviral responses were significantly upregulated, including interferon stimulated genes (ISGs). In addition, we show that TLR7 agonists trigger cells to secrete soluble antiviral molecules that can inhibit MNV infection. Furthermore, the combination of R-848 and polymerase nucleoside inhibitor 2’C-methylcytidine elicited an additive antiviral effect against MNV. Together, these findings suggest that TLR7 agonists could be used alone or in combination with direct acting antivirals (DAAs) as a therapeutic option to combat NoV infections.