Engineering a low cytotoxicity recombinant rabies virus neurotracer via N-gene recoding

Viral-vector-based tracers have proven invaluable for mapping neural circuits. Rabies virus (RV)-based retrograde trans-monosynaptic tracers have been widely used for depicting the input neural circuit of targeted neuronal populations. However, the cytotoxicity of the RV tracer limits its application. Here, we employed the synthetic attenuated virus engineering technology to recode the N gene of the RV SAD-B19 strain by codon pair deoptimization to generate attenuated RV tracers. The characteristics of the recoded RV tracers were tested both in vitro and in vivo. The full-length recoded N gene abolished viral rescue, whereas the partial recoded N gene supported successful viral recovery. Compared to its parent, the recoded RV trans-monosynaptic tracer exhibited lower cytotoxicity and labeled more input neurons. It can also robustly express proteins to visualize the neuronal morphology and manipulate the neural circuit activity. Collectively, our work generated a new retrograde trans-monosynaptic tool, which complements existing neural circuit tracers.

Viral vectors play an important role across multiple fields in biomedicine. In the neuroscience community, rabies virus (RV)-based retrograde trans-monosynaptic tracers have been widely used to map the input neural circuits of targeted neuronal populations. However, the inherent cytotoxicity of RV-based tracers limits their broader application. In this study, we utilized synthetic attenuated virus engineering technology to recode the RV N gene, generating an attenuated neurotracer that will significantly benefit future neuroscience research.