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Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. In this chapter we describe the use of a replication-competent recombinant (r)RVFV expressing NanoLuc Luciferase (Nluc) for in vitro studies. The determination of parameters such as neutralizing antibodies in serum samples, or the antiviral activity of drugs is usually carried out using standard assays based on the assessment of cytopathic effect on cell cultures. The use of a virus encoding a traceable reporter protein allows to correlate the presence or absence of infection with the detection of the product in the infected cultures, thus tracking the level of RVFV infection in an objective, quantitative manner. In addition to this quantitative measurement of results, our protocol offers two other advantages, such as a shorter time to read, given that 48 h post-infection the production of the reporter protein is enough to give an accurate result, and the use of an attenuated virus, which reduces the risk of exposure., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments. Recently, we described two reporter rVACV expressing luciferase (Nluc) and fluorescent (GFP or Scarlet) proteins to easily track viral infections in different systems, overcoming the limitations associated with the use of rVACV expressing a single luciferase or fluorescent reporter gene. Here, we describe the experimental procedures to carry out in vitro, in vivo and ex vivo studies using these novel bireporter-expressing rVACV, which also represent an excellent option to study the biology of VACV, including the use of these reporter viruses for testing new antivirals and vaccines, using cultured cells and/or well-characterized animal models of infection., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)