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 (adeno-associated 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.

Keywords: Adeno-associated virus; Anterograde tracer; H129; Herpes simplex virus; High-brightness; Neural circuit tracing; Trojan horse-like strategy.

Fig. 1

Construction and validation of H1 and H8 viruses in vitro. a The Trojan horse-like strategy of GOI amplification by HSV/AAV chimaera. AAV replicase expression cassette and ITR flanked GOI (gene of interest) cassette were inserted into H129 genome to construct the chimaera virus. The genome of the HSV chimaera enters the nucleus following infection of cells. With the assistance of HSV, the Rep replicase specifically recognizes GOI flanked by ITRs and replicates it, resulting in amplification of the copy numbers of the GOI. b Schematic of H1 and H8 genome. IRL and IRS indicate long and short inverted repeats, respectively. c Western blot validation of rep protein expression. H129, H1 and H8 infected BHK cells were detected using the anti-replicase antibody. d Single-step growth curves for H1 and H8 (each point represents the mean of triplicate assays). e Fluorescent protein expression level of H1 and H8 infected BHK cells at 24 h post infection, MOI = 5. Scale bar, 100 μm. f H8 showed significant higher DNA level of egfp than H1. g, h, i H8 showed significant higher protein level of GFP than H1. The genomes and proteins of H1 and H8 infected BHK cells were extracted 48 hpi

Fig. 2

Validation of high fluorescence intensity of H8 in vivo. a The schematic diagram of virus injection and sample treatment. H1 or H8 virus was injected into midbrain VTA region of mice, respectively. All brains were taken 72 hpi and ground evenly in liquid nitrogen and then 100 μl lysate of each brain was used to extract genome or protein. b-e H8 showed significant higher EGFP DNA copies and protein levels than that of H1 in vivo

Fig. 3

Validation of anterograde transport phenotype of H8 in vivo. a Left, GFP expression in the injection site following injection of H1 into V1 of C57BL/6 mice. Blue was DAPI staining. Right three panels, GFP fluorescence in the regions downstream of V1 (SC, LGN, and CPU). High-magnification images (bottom) showed GFP-labeled cell bodies in the region indicated by the white box. b Left, GFP expression in the injection site following injection of H8 into V1 region of mice. Right three panels, GFP fluorescence in the regions downstream of V1 (SC, LGN, and CPU). High-magnification images (bottom) showed GFP-labeled cell bodies in the region indicated by the white box. Scale bars, 200 μm, left and right top panels; 20 μm, right bottom panels

Fig. 4

Labeling efficiency of H8 in tracing NAC output pathway at different timeframe. Three early infection stages were chose to show the labeling performance of H8. H8 was injected into the accumbens nucleus (NAc) of adult C57BL/6 mice and collected samples at 24, 36, and 48 h post-injection. Scale bar, 1000 μm. anterior olfactory nucleus (AON), orbital cortex (ORB), agranular insular cortex (AI), piriform cortex (Pir), substantia innominata (SI), paraventricular thalamic nucleus (PVN), amygdala (Amy), entorhinal cortex (ENT), hippocampus (HIP), ventral tegmental area (VTA), lateral hypothalamus (LHA), dorsal raphe nuclei (DRN)

Fig. 5

Comparison of labeling performance of H8 and H1 at 36 h post infection. H8 and H1 were injected into the accumbens nucleus (NAc) of C57BL/6 mice respectively and the brain samples were collected at 36 h post-injection. Serial coronary slices were displayed to show the differences between H8 and H1 (Scale bar =1000 μm) and smaller areas in dashed boxes were magnified (Scale bar = 100 μm). prefrontal cortex (PFC), anterior cingulate cortex (Acc), substantia innominata (SI), paraventricular thalamic nucleus (PVN), ventral tegmental area (VTA), dorsal raphe nuclei (DRN)

Fig. 6

The structural neural networks labeled by H8 in the NAc output pathway. Medial-sagittal and lateral-sagittal plates and labeled structures are from the Franklin and Paxinos Atlas (Franklin and Paxinos, 2001). Light green circles mark GFP-labeled structures by H8 tracer at both 36 hpi and 48 hpi; dark green circles mark GFP-labeled structures at 48 hpi