High-brightness anterograde transneuronal HSV1 H129 tracer modified using a Trojan horse-like strategy

Abstract

Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adenoassociated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.

A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency

“…Neurotropic virus mediated anterograde trans-synaptic tracing is an important approach for mapping the output of brain circuits. Several viral tracers for anterogradely trans-synaptic tracing had been developed [20,48], but the rapid toxicity and lethality to the infected neurons and animals limited their (See figure on previous page.) Fig.…”

“…After 1 h of incubation at 35°C, the viral inoculum was removed, cells were washed 3 times with phosphate buffered saline (PBS), and 1.5 ml of fresh DMEM medium (containing 3% FBS) was added. Supernatants were harvested at different time points (12,24,36,48, 72 and 96 h post infection (hpi)), and virus titers were detected by plaque assay.…”

A Mutant Vesicular Stomatitis Virus with Reduced Cytotoxicity and Enhanced Anterograde Trans-synaptic Efficiency

“…Neurotropic virus mediated anterograde trans-synaptic tracing is an important approach for mapping the output of brain circuits. Several viral tracers for anterogradely trans-synaptic tracing had been developed [20,48], but the rapid toxicity and lethality to the infected neurons and animals limited their application in many areas of research that need dissecting the output network according to connection order within a longer time window, which were important to understand not only the anatomical mechanism of the given brain region, but also the variation of the circuits on various neurological and psychiatric disease. In order to overcome these defects, we introduced a replicationrelated mutation within the nucleoprotein, N R7A , into the genome of the VSV.…”

“…indicators to monitor or manipulate the network activity over a longer experimental window. With the canonical anterograde tracer, such as WT-VSV and HSV, the time window that ensures effectively trans-synaptic tracing has been reported to be between 3-5 days from the infection [7,48]. With Mutant-VSV, the useful temporal window for imaging can reach up to 10-15 days from the infection, which overcoming the shortage of relative short survival time of experimental animals.…”

“…After 1 h of incubation at 35 °C, the viral inoculum was removed, cells were washed 3 times with phosphate buffered saline (PBS), and 1.5 ml of fresh DMEM medium (containing 3% FBS) was added. Supernatants were harvested at different time points (12,24,36,48, 72 and 96 hours post infection (hpi)), and virus titers were detected by plaque assay.…”