# Anti-orthopoxvirus drugs inhibit lumpy skin disease virus replication by targeting viral DNA polymerase

## TL;DR

Researchers identified six drugs that inhibit lumpy skin disease virus replication by targeting its DNA polymerase, offering potential treatments for cattle.

## Contribution

A recombinant LSDV model and six repurposed anti-orthopoxvirus drugs with antiviral activity against LSDV were identified.

## Key findings

- Six nucleotide analogs showed high antiviral activity against LSDV with low cytotoxicity.
- AraC specifically inhibits viral DNA synthesis and late gene expression by targeting the viral DNA polymerase.
- AraC induces minimal host DNA damage and is a promising candidate for repurposing as an LSDV therapy.

## Abstract

Lumpy skin disease (LSD) is an emerging transboundary disease caused by lumpy skin disease virus (LSDV), posing significant threats to global cattle health in the absence of validated drugs. Here, we constructed a recombinant LSDV (rLSDV) expressing both mCherry and luciferase reporters for high-throughput drug screening, and the rLSDV retained virological characteristics phenotypically indistinguishable from the parental strain, with the reporter genes serving as precise and sensitive quantitative indicators for viral replication. Leveraging this platform, we identified six candidates from a library of anti-orthopoxvirus compounds, namely cytarabine (AraC), enrofloxacin (ENR), idoxuridine (IDU), fialuridine (FIAU), ribavirin (RBV), and vidarabine (AraA), demonstrating high antiviral activity concomitant with low cytotoxicity. Time-of-addition experiments revealed that all identified candidates primarily inhibited the viral replication phase. Mechanistical analysis revealed that anti-LSDV agents suppressed synthesis of both viral and host DNA and/or RNA. In particular, AraC markedly blocked viral DNA synthesis and prevented activation of viral late gene promoters, thereby arresting the replication cycle at an early stage. Structural alignment data suggested that AraC may bind to the viral DNA polymerase at residues D554, R639, K666, N670, and D758 to inhibit its activity. Notably, AraC induced only minimal host DNA damage and apoptosis, and host DNA synthesis gradually recovered during treatment, although these residues are conserved in bovine DNA polymerase. Hence, the mechanistic landscape delineated herein, together with the established clinical availability of the anti-orthopoxvirus agents, underscore their potential as repurposable therapeutics for LSDV infection.

Lumpy skin disease virus (LSDV) threatens cattle production worldwide, yet limited knowledge of its pathogenic mechanisms has hindered the development of effective antiviral therapies. To address the lack of approved drugs, we established a sensitive real-time high-throughput screening platform and identified six nucleotide analogs from an anti-orthopoxvirus compound library that interfere with viral nucleotide synthesis, highlighting viral DNA or RNA polymerase as viable antiviral targets. Among them, AraC showed exclusively high antiviral activity with a wider safe concentration range. Mechanistically, AraC is predicted to target the viral DNA polymerase, leading to suppressed viral DNA synthesis and the consequent completely blockade of viral late protein expression. Given their existing clinical use in human and veterinary medicine, these compounds represent promising lead candidates for developing effective therapies against LSD.