Near-infrared fluorescent protein iRFP720 is optimal for in vivo fluorescence imaging of rabies virus infection.

In vivo imaging is a noninvasive method that enables real-time monitoring of viral infection dynamics in a small animal, which allows a better understanding of viral pathogenesis. In vivo bioluminescence imaging of virus infection is widely used but, despite its advantage over bioluminescence that no substrate administration is required, fluorescence imaging is not used because of severe autofluorescence. Recently, several far-red and near-infrared (NIR) fluorescent proteins (FPs) have been developed and shown to be useful for whole-body fluorescence imaging. Here, we report comparative testing of far-red and NIR FPs in the imaging of rabies virus (RABV) infection. Using the highly neuroinvasive 1088 strain, we generated recombinant RABV that expressed FPs such as Katushka2S, E2-Crimson, iRFP670 or iRFP720. After intracerebral inoculation to nude mice, the 1088 strain expressing iRFP720, the most red-shifted FP, was detected the earliest with the highest signal-to-noise ratio using a filter set for >700 nm, in which the background signal level was very low. Furthermore, we could also track viral dissemination from the spinal cord to the brain in nude mice after intramuscular inoculation of iRFP720-expressing 1088 into the hind limb. Hence, we conclude that the NIR FP iRFP720 used with a filter set for >700 nm is useful for in vivo fluorescence imaging not only for RABV infection but also for other virus infections. Our findings will also be useful for developing dual-optical imaging of virus–host interaction dynamics using bioluminescence reporter mice for inflammation imaging.