Engineering recombinant replication-competent bluetonge viruses expressing reporter genes for in vitro and non-invasive in vivo studies

Sergio Utrilla-Trigo, Luis Jeménez-Cabello, Alejandro Marín-López, Miguel Illescas-Ammo, Germán Andrés, Eva Calvo-Pinilla, Gema Lorenzo, Piet A. van Rijn, Javier Ortego*, Aitor Nogales

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(−/−) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (−/−) mice using BTV/NLuc and non-invasive in vivo imaging systems.
Original languageEnglish
JournalMicrobiology Spectrum
Issue number3
Early online date14 Feb 2024
Publication statusPublished - 5 Mar 2024


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