Rabies Virus (RV)

The Rabies virus (RV) is used for trans-monosynaptic labeling in the retrograde direction. RV belongs to the single-stranded negative sense RNA virus of the genus Lyssavirus in the family Rhabdoviridae. After RV infects the central system, it mainly labels neurons and almost no glial cells. Infected neurons have little to no significant lesions and lysis for a certain period of time (7-12 days). The replication-defective recombinant RV based on the infectious clone of the RV vaccine strain Sad-B19 has very low toxicity and can clearly label the fine morphology of neurons. It realizes the reverse cross-single-level synaptic tracing of neural network connections through the reverse complementation strategy.

The envelope glycoprotein (glycoprotein G) of RV is an essential protein for its retrograde transsynaptic function, and its receptors are widely distributed at the axon terminals, which can retrograde along the axon into the neuronal cell body to open viral replication after infection. Rabies virus for retrograde monosynaptic labeling is genetically engineered to be replication-incompetent (RV-ΔG), meaning it lacks the glycoprotein (G) necessary for further transsynaptic spread. Instead, they are pseudotyped with the avian sarcoma and leukosis virus glycoprotein EnvA (RV-EnvA-ΔG). This modification restricts the virus's ability to infect neurons unless they express a specific receptor for EnvA known as TVA, which is normally absent in mammalian cells.

To enable the rabies virus to infect only specific neurons, the TVA receptor is introduced into the targeted "starter" neurons. This is typically done by co-injecting a second virus, such as a helper AAV, which expresses the TVA receptor under the control of a neuron-specific promoter. Once the TVA receptor is expressed in the starter neurons, the RV-EnvA-ΔG can selectively infect these neurons. This ensures that the virus only infects neurons pre-engineered to express TVA, providing a high degree of specificity in selecting the initial population of neurons for labeling. Since these "starter" neurons also express the rabies glycoprotein (G), the RV spreads retrogradely across one synapse to infect presynaptic neurons. However, because the virus is replication-incompetent and lacks its own glycoprotein, it cannot spread beyond the first synapse. This results in selective labeling of only the neurons that are directly synaptically connected to the starter cells.


Figure 1. A: Genetic engineering principle of trans-monosynaptic RV; B: Schematic diagram of RV retrograde trans-monosynaptic labeling (ArenkielBR, et al., Nature.2009.)