Generation and characterization of replication-competent rBTV-3 expressing fluorescent and luminescent reporter genes using a reverse genetics system

Bluetongue virus (BTV) is a widespread vector-borne pathogen of major concern for animal health, mainly affecting both wild and domestic ruminants. This virus stands out for its ability to cause significant disease and its impact on livestock production. In 2023, BTV serotype 3 was detected in the Netherlands and spread rapidly to neighbouring countries. Compared to other serotypes circulating in Europe, BTV-3 shows greater virulence and transmissibility. The aim of this study is the design and rescue of reporter-expressing BTV-3 viruses using a BTV-1 (ALG2006/01) genetic backbone and the VP2 and VP5 outer capsid proteins from BTV-3 (SPA/2024). Using a plasmid-based reverse genetic system, we have designed and rescued rBTV-3 reporter-expressing viruses encoding NanoLuc luciferase (Nluc) or the fluorescent proteins Venus and mCherry. Rescue was achieved by transfecting BSR-T7 cells with plasmids encoding BTV genome segments. The reporter-expressing viruses were generated by chaining segment 5, which encodes the NS1 protein, with a Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site, followed by the reporter genes. Consequently, fluorescence and luminescence signals are only detected following viral replication and expression of non-structural proteins. BSR cells were mock-infected or infected (MOI 0.1) with rBTV-3, rBTV-3/Venus, rBTV-3/mCherry or rBTV-3/Nluc. Fluorescence was detected in the cytoplasm of the cells infected with rBTV-3/Venus or rBTV-3/mCherry but not in those infected with rBTV-3 or rBTV-3/Nluc. Moreover, Nluc activity was only detected in rBTV-3/Nluc infected cells. The use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein may be promising tools for studying BTV infectivity, transmissibility, pathogenesis, diagnostics, and vector competence.