Real-time visualization of infection dynamics using a bioluminescent reporter-expressing recombinant Getah virus

Getah virus (GETV), an arthropod-borne alphavirus, has emerged as a significant pathogen responsible for reproductive failure and systemic disease in swine, incurring substantial economic losses within the livestock industry and presenting a potential zoonotic threat. Currently, no approved vaccines or antiviral therapies exist. Here, we employed reverse genetics to engineer a recombinant GETV (rGECNLuc) expressing a Nanoluciferase (NLuc) reporter gene. The reporter was inserted at the C-terminus of the viral capsid (Cap) protein via a T2A self-cleavage peptide to facilitate co-translational release. In vitro characterization demonstrated that rGECNLuc replicated efficiently in BHK-21 and Vero cells, with plaque morphology and multi-step growth kinetics comparable to the parental virus, rGETV-GX. The NLuc insertion was genetically stable over six consecutive passages without deletion. In vivo assessment in a 10-day-old suckling mouse model revealed a significant attenuation of virulence for rGECNLuc compared to the parental strain, despite efficient viral replication in joints, lungs, and brain tissues. Using in vivo imaging (IVIS), we established a strong positive correlation between bioluminescent signal intensity and viral load in 3-week-old and 5-week-old mice. Signals disseminated systemically from the inoculation site, with pronounced enrichment in joints and lungs, and exhibited greater intensity in younger animals. Collectively, rGECNLuc enables real-time, non-invasive visualization of GETV infection and provides a tractable platform for quantifying viral dissemination, tissue tropism, and antiviral activity in vivo.