Herpes Simplex Virus (HSV)

Herpes simplex virus (HSV), a member of the herpesviridae family in the α-herpesvirus subfamily, exists in two subtypes: HSV-1 and HSV-2. Its genome consists of approximately 152 kb of linear double-stranded DNA, organized into covalently linked long (UL) and short (US) segments, each containing unique sequences and inverted repeats. Upon infection of host cells, the HSV genome circularizes. Nearly half of the HSV genome comprises non-essential genes, providing ample space for genetic modification. This allows for the insertion of large foreign genes or multiple transgenes into the viral genome simultaneously.

HSV is capable of infecting humans and is widely prevalent globally, with approximately 70% to 90% of adults having been exposed to the virus. HSV infections can result in conditions such as gingivostomatitis (e.g., cold sores) and keratoconjunctivitis, although these occur in only a small proportion of cases. Due to the potential risks associated with HSV, all neural circuit tracing experiments utilizing this virus must be conducted in a biosafety level 2 laboratory. Proper protective measures should be strictly followed, and all procedures should be performed with caution.

Depending on the experimental objectives, HSV can be divided into three categories: HSV amplicons, HSV H129, and HSV TK-deleted (HSV-∆TK).

1) HSV amplicons
HSV-1 amplicon vectors are pseudoviral vectors that lack viral structural proteins, containing only the HSV-1 origin of replication (ori) and packaging signal (pac) along with an exogenous gene expression cassette. These vectors offer several key advantages:

1) Large capacity: Capable of carrying up to 150 kb of genetic material;
2) Broad host range: Can infect both dividing and non-dividing cells, including those in the central nervous system;
3) No viral structural genes: The vector does not carry genes that encode viral structural proteins;
4) Non-integrative: The HSV-1 amplicon vector does not integrate into the host cell genome;
5) High safety profile: It lacks the pathogenic genes of HSV-1, containing only the replication origin (ori) and packaging signal (pac), ensuring safety in use;
6) Natural tropism: The vector retains the HSV-1 capsid, allowing it to leverage the virus’s natural tropism for specific tissues.

These properties make the HSV-1 amplicon vector a highly effective tool for gene therapy, particularly for neurological diseases, and open avenues for treating a wide range of systemic diseases, including those affecting the central nervous system.

 2) HSV H129
H129 is a clinical isolate of HSV-1 strain with an anterograde trans-polysynaptic characteristic. After extensive testing in animal models, it has been shown to trace neural connections not only between different brain regions in the CNS, but also between peripheral nervous systems and central brain regions. After the virus infects neurons, it replicates and expresses the target gene in the cell, and the progeny virus is transported to the presynapse through the axon, and enters the downstream neurons across the synapse, starting a new round of replication and transsynaptic transmission. Engineered with fluorescent proteins, H129 enables efficient, stable anterograde labeling of neuronal output circuits, and its wide range of infection host makes it ideal for neural tracing in various species, including rodents, non-human primates, and other mammals and birds.



Figure 7.  Anterograde trans-polysynaptic neuronal tracing by HSV H129.

3) HSV TK-deleted (HSV-∆TK)
Herpes simplex virus thymine kinase (HSV-TK) plays an important role in acute ganglion replication and recurrent latent ganglion infections following HSV infection of neurons (Coen et al., PNAS, 1989). When HSV-TK is deleted, HSV-ΔTK can infect neurons but cannot replicate, and thus loses the ability to cross synapses. To enable the HSV-ΔTK to cross a synapse, a helper AAV that expresses TK is co-injected to the target regions. Once TK is expressed, the HSV-ΔTK can spread anteriorly across a single synapse. 


Figure 8.  Anterograde trans-monosynaptic neuronal tracing by HSV-ΔTK.