Establishment of a longitudinal pre-clinical model of lyssavirus infection

•Luciferase-expressing reporter virus used to study lyssavirus pathogenesis in mice.•In vivo longitudinal imaging of Australian bat lyssavirus (ABLV) infection.•ABLV infections anatomically traced from inoculation-site replication to death.•Method enables quantification of relative viral load at distinct anatomical sites.•Method should facilitate in vivo analyses of candidate lyssavirus therapeutics. Traditional mouse models of lyssavirus pathogenesis rely on euthanizing large groups of animals at various time points post-infection, processing infected tissues, and performing histological and molecular analyses to determine anatomical sites of infection. While powerful by some measures, this approach is limited by the inability to monitor disease progression in the same mice over time. In this study, we established a novel non-invasive mouse model of lyssavirus pathogenesis, which consists of longitudinal imaging of a luciferase-expressing Australian bat lyssavirus (ABLV) reporter virus. In vivo bioluminescence imaging (BLI) in mice revealed viral spread from a peripheral site of inoculation into the central nervous system (CNS), with kinetically and spatially distinct foci of replication in the footpad, spinal cord, and hindbrain. Detection of virus within the CNS was associated with onset of clinical disease. Quantification of virus-derived luminescent signal in the brain was found to be a reliable measure of viral replication, when compared to traditional molecular methods. Furthermore, we demonstrate that in vivo imaging of ABLV infection is not restricted to the use of albino strains of mice, but rather strong BLI signal output can be achieved by shaving the hair from the heads and spines of pigmented strains, such as C57BL/6. Overall, our data show that in vivo BLI can be used to rapidly and non-invasively identify sites of lyssavirus replication and to semi-quantitatively determine viral load without the need to sacrifice mice at multiple time points.