HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements, Production, and Applications


Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

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Herpes simplex virus 1


Herpes simplex virus 1 (HSV-1) strain H129




Fluorescence Micro-Optical Sectioning Tomography


Thymidine kinase


Adeno-associated virus


Cholera Toxin B


Multiplicity of infection


Cytopathic effects


Fetal bovine serum


Phosphate-buffered saline






Olfactory bulb


Primary motor cortex


Piriform cortex


Lateral entorhinal cortex


Entorhinal cortex


Field CA1 of the hippocampus


Basolateral amygdala


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We appreciate Prof. Bo Li (Cold Spring Harbor Laboratory, USA), Xiaohui Zhang (Beijing Normal University, China), Xiao-Min Wang (Capital Medical University, China), Jian-Zhi Wang (Huazhong University of Science and Technology, China), Zhi Zhang (University of Science and Technology of China, China), and Ping Zheng (Fudan University, China) for providing the tracing parameters and results. We would like to thank the Animal Experiment Center and Core Facilities and Analytical Center of the Wuhan Institute of Virology for their support in animal experiments and imaging. This work was supported by the National Key Basic Research Program of China (2015CB755601), the National Natural Science Foundation of China (81427801, 81571355, and 81601206), and the National Institutes of Health RF1 Funding of China (RF1MH120020-01).

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Corresponding authors

Correspondence to Wen-Bo Zeng or Fei Zhao or Min-Hua Luo.

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Conflict of interest

All authors claim that there are no conflicts of interest. The Wuhan Institute of Virology has filed a patent application for H129-G4 (US Patent No. 201615747742), and a patent application for H129-dTK-tdT and H129-dTK-T2 is still pending. The authors declare no other competing interests.

Ethics Approval and Consent to Participate

The standards of performance and animal studies were approved by the Institutional Review Board and Institutional Animal Welfare Committee (WIVA10201502), including intracerebral inoculation of mice and rats with viral tracers. All the experiments with viruses were performed in bio-safety level 2 laboratory and animal facilities.

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Yang, H., Xiong, F., Song, YG. et al. HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements, Production, and Applications. Neurosci. Bull. (2020). https://doi.org/10.1007/s12264-020-00614-3

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  • HSV-1 strain H129 (H129)
  • Anterograde transneuronal tracer
  • Polysynaptic tracer
  • Monosynaptic tracer
  • Production
  • Application
  • Neural circuit
  • Neural circuit tracing