Application of attenuated herpes simplex virus in preparation of neural circuit reverse tracing tool virus

Abstract

The invention discloses an application of an attenuated herpes simplex virus in preparation of a neural circuit reverse tracing tool virus, and the attenuated herpes simplex virus is prepared based on a targeting vector. The targeting vector is a vector in which a complete fluorescent protein gene or other exogenous target gene expression cassettes are inserted between the upstream and downstream homologous arms of the neurovirulence factor gamma 34.5 gene of the vector pH129 delta RL1. It is found for the first time that the ltHSV-hUbC-tdT-WPRE virus can be used for neural circuit reverse tracing, efficient axonal ending infection can be carried out so as to reversely mark an upstream regulation and control brain region of a target brain region, and marking as long as two months only observes cellular signals in the upstream brain region which can be directly projected to the target brain region; and the system does not show cis-synaptic propagation, and can be used as a novel tracing virus system with relatively rigorous reverse marking capability and wider-spectrum neurotropism.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, involving neurobiology and molecular virology, and particularly relates to the application of an attenuated herpes simplex virus in the preparation of a neurocircuit retrograde tracing tool virus. Background Art

[0002] The virus trans-synaptic tracing technology has been increasingly widely used in the analysis of neural circuits in recent years. Traditional neural network tracing methods, such as dyes, compound tracers, protein peptides, etc., can be transported along axons but cannot cross synapses, and can only label the morphology of local neurons. Compared with traditional neural network tracing methods, neurotropic viruses have obvious advantages as tracing tools: 1) They can efficiently infect nerve cells; 2) They can spread across synapses; 3) The trans-synaptic direction is controllable, and they can specifically spread retrogradely or anterogradely; 4) After crossing synapses, the virus can self-replicate and the signal does not attenuate; 5) They can carry complex regulatory elements and diverse markers, etc.

[0003] Currently, the commonly used neurotropic viruses mainly include Pseudorabies virus (PRV) and Herpes simplex virus (HSV) from the family Alphaherpesviridae, as well as Rabies virus (RV) from the family Rhabdoviridae. There are also vesicular stomatitis virus (VSV), etc. Among them, the PRV Bartha strain and RV are retrograde trans-synaptic infections, VSV can spread in both directions, and the HSV-1 H129 strain that can specifically anterogradely cross synapses is consistent with the direction of nerve impulse transmission and is very suitable for labeling output neural networks.

[0004] Herpes simplex virus type 1 (HSV 1) is a widely distributed and conditionally pathogenic pathogen. It is a large enveloped virus with a diameter of about 200 nm. The core is a double-stranded DNA genome of about 153 kb, which consists of covalently linked long (UL) and short (US) segments. Each segment terminus contains inverted repeat sequences, so four isomers can be formed.

[0005] The neurotropic virus strain HSV-1 H129 has become one of the most promising anterograde neurocircuit tracing tool viruses due to its mainly anterograde trans-synaptic spread in neural circuits and large foreign gene capacity. As a neural tracing virus vector, HSV-1 H129 has developed through genetic engineering technology into a variety of neurocircuit anterograde tracing tool viruses, and has initially constituted a relatively complete anterograde tracing tool system. Summary of the Invention

[0006] To solve the above problems, the object of the present invention is to provide an application of an attenuated herpes simplex virus in preparing a viral tool for retrograde tracing of neural circuits.

[0007] The present invention provides an application of an attenuated herpes simplex virus in preparing a viral tool for retrograde tracing of neural circuits. The attenuated herpes simplex virus is prepared based on a targeting vector, and the targeting vector is a vector in which a complete fluorescent protein gene expression cassette is inserted between the upstream and downstream homologous arms of the neurovirulence factor γ34.5 gene of the vector pH129ΔRL1. The nucleotide sequence of the vector pH129ΔRL1 is shown in SEQ ID NO.1, the nucleotide sequence of the upstream homologous arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream homologous arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.3.

[0008] Further, the complete fluorescent protein gene expression cassette includes, from the 5' end to the 3' end: a promoter, a fluorescent protein gene, and a WPRE transcriptional enhancer element.

[0009] Further, the promoter is selected from one of the hUbC promoter, CMV promoter, CAG promoter, EF1α promoter, pTH promoter, and pChAT promoter;

[0010] The fluorescent protein gene is tdTomato or EGFP.

[0011] Further, the promoter is selected from the hUbC promoter;

[0012] The fluorescent protein gene is tdTomato.

[0013] Further, the nucleotide sequence of the targeting vector is shown in SEQ ID NO.4.

[0014] Further, the retrograde tracing of neural circuits does not cross synapses.

[0015] The beneficial effects of the present invention are as follows:

[0016] In the present invention, the two copies of the neurovirulence factor γ34.5 gene in the HSV-1 H129 genome are completely knocked out, and a complete fluorescent gene expression cassette is inserted into the knocked-out γ34.5 gene locus to construct an attenuated herpes simplex virus. It is first discovered that the attenuated herpes simplex virus can be used for retrograde tracing of neural circuits.

[0017] The present invention shows, through the whole-cell patch-clamp recording results of the membrane voltages of ltHSV-hUbC-tdT-WPRE-labeled pyramidal neurons and control neurons under different current stimulations, that ltHSV-hUbC-tdT-WPRE does not significantly change the physiological properties of the labeled neurons, thus not interfering with the normal neuroelectrophysiological mechanism. Animal labeling experiments found that ltHSV-hUbC-tdT-WPRE can efficiently infect axon terminals for retrograde labeling of upstream regulatory brain regions of the target brain region, and somatic signals were only observed in the upstream brain regions that can directly project to the target brain region during the two-month labeling, without showing anterograde trans-synaptic transmission. Compared with the classical retrograde tracer virus RVdG-SAD19G, it shows a broader neurotropism. Therefore, ltHSV can be used as a new tracer virus system with relatively rigorous retrograde labeling ability and broader neurotropism. Description of the Drawings

[0018] Figure 1 Recombination, purification and molecular identification of the ltHSV-hUbC-tdT-WPRE virus with double-copy knockout of HSVγ34.5; among them Figure 1 A is a schematic diagram of the genome structure of the wild-type HSV-1 H129 clinical strain; Figure 1 B is a schematic diagram of the genome structure of the ltHSV-hUbC-tdT-WPRE virus with double-copy knockout of HSVγ34.5; Figure 1 C is the molecular identification result of the ltHSV-hUbC-tdT-WPRE virus genome; Figure 1 D is the fluorescence expression map of cells infected with the ltHSV-hUbC-tdT-WPRE virus;

[0019] Figure 2 Evaluation of neurotoxicity of primary neurons infected with ltHSV-hUbC-tdT-WPRE;

[0020] Figure 3 Flow chart of whole-cell electrophysiological recording of ex vivo brain slices;

[0021] Figure 4 Gray-scale and bright-field images of fluorescence-labeled pyramidal neurons selected for whole-cell patch-clamp recording;

[0022] Figure 5 ltHSV-hUbC-tdT-WPRE did not significantly change the physiological properties of the labeled neurons;

[0023] Figure 6 ltHSV-hUbC-tdT-WPRE can efficiently and long-term label the M1 brain region and its upstream brain regions;

[0024] Figure 7ltHSV did not exhibit anterograde trans-synaptic propagation characteristics;

[0025] Figure 8 Comparison of the retrograde non-trans-synaptic labeling effects after separate injections of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses into the VTA;

[0026] Figure 9 Comparison of the retrograde non-trans-synaptic labeling effects after co-injection of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses into the VTA. Detailed implementation manners

[0027] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description is made of the specific implementation manners of the present invention, but it should not be construed as a limitation on the implementable scope of the present invention.

[0028] Example 1: Construction of a targeting vector for ltHSV-hUbC-tdT-WPRE virus

[0029] The wild-type HSV-1 H129 clinical strain was provided by Professor Lynn Enquist (Princeton University, Princeton, NJ, USA). The γ34.5 (also known as RL1) gene sequence of HSV-1 H129 and its flanking gene sequences were obtained from the GenBank database (GenBank: GU734772.1). The nucleotide sequence of 527 bp upstream of the open reading frame of the γ34.5 gene and the sequence of 547 bp in length downstream of it were selected as the upstream homologous arm (UHA) and the downstream homologous arm (DHA), respectively. The γ34.5 gene has two copies in the HSV genome, located in the terminal repeat sequences TRL and IRL of the ML long fragment respectively. The full length of the gene is 1007 bp, the coding frame ORF is 747 bp long, and the GC content is as high as 80%; in the present invention, the full-length γ34.5 gene (1007 bp) was designed to be knocked out. The genomic DNA of HSV-1 H129 was extracted and purified, and using it as a template, primers were designed to clone the upstream homologous arm of the γ34.5 gene (UHA, 527 bp long, GC content 80%) and the downstream homologous arm (DHA, 547 bp long, GC content 66%). The cloned upstream and downstream homologous arm fragments were respectively ligated into the pcDNA3.1+ vector after digestion with Hind III and BamH I, BamH I and XbaI, and named pH129ΔRL1. The nucleotide sequence of pH129ΔRL1 is shown in SEQ ID NO.1, and the nucleotide sequences of the upstream homologous arm (UHA) and the downstream homologous arm (DHA) are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. Then, the foreign gene expression cassette hUbC-tdTomato-WPRE-PA was inserted between the upstream and downstream homologous arms of the γ34.5 gene to construct the targeting vector of the ltHSV-hUbC-tdT-WPRE virus, named pH129ΔRL1-hUbC-tdTomato-WPRE-PA, and its nucleotide sequence is shown in SEQ ID NO.4.

[0030] Example 2: Attenuated herpes simplex virus recombination and plaque purification

[0031] ① Recombination of the virus: The targeting vector pH129ΔRL1-hUbC-tdTomato-WPRE was extracted and transfected into 293T cells by liposome transfection. After 6 hours, the maintenance medium containing 2% FBS was replaced and the herpes simplex virus H129 strain was added for infection; the expression of fluorescence and the cytopathic effect were observed at different times. After all the cells showed cytopathic changes, the cell culture supernatant was collected and placed in a -80°C refrigerator.

[0032] ② Purification of the virus: The collected virus supernatant was subjected to three cycles of repeated freezing and thawing, centrifuged at 6500 g for 10 minutes to remove cell debris completely. 10 μL of the virus supernatant was taken to infect Vero cells, and after 1 day, the fluorescence expression of the infected cells was observed to determine whether the novel virus was successfully recombined. In the later stage, the virus supernatant with successful recombination was serially diluted 10-fold and then used to infect Vero cells. After adsorption for 1 hour, agar (a 1:1 mixture of DMEM medium containing 5% fetal bovine serum and 2% agar) was overlaid. After 48 - 72 hours, when virus plaques formed, plaque picking was carried out under an inverted fluorescence microscope. The wild-type virus was removed after about 6 rounds of plaque purification of the novel recombinant virus, and the purified novel recombinant virus ltHSV-hUbC-tdT-WPRE virus was obtained. The situation of the recombinant, plaque purification, and fluorescence expression of the attenuated herpes simplex virus in infected cells is shown in Figure 1 D. It can be seen that the fluorescence expression of the cells infected with the low-toxic HSVLT is very bright.

[0033] Example 3: Molecular identification of the genome of the attenuated herpes simplex virus

[0034] Through methods such as homologous recombination and plaque purification, a pure ltHSV-hUbC-tdT-WPRE monoclonal strain was obtained. To verify that the two copies of the γ34.5 gene were indeed deleted in the obtained monoclonal strain, a 747-bp ORF fragment of the γ34.5 gene was amplified by PCR.

[0035] The concentrated and purified wild-type H129 and the attenuated HSV (ltHSV-hUbC-tdT-WPRE) virus were inactivated at 100 °C for 10 minutes and were subsequently used for the molecular identification of the γ34.5 gene. Primers were designed for the 747-bp ORF fragment of γ34.5, and the primers and sequences used for identification were γ34.5-F: 5'-ATGGCCCGCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGG-3'; γ34.5-R: 5'-TTAGACCGAGTTCGCCGGGCCGGCTCCGCGGGCCAGGGCCCGGGC-3'. Since the GC content of the γ34.5 gene is as high as 82%, a high-GC buffer system was selected to amplify the γ34.5 ORF: The PCR reaction system was 50 μL, in which the dosage of the inactivated HSV virus-like was 1 - 5 μL, 2×PrimeStar high-GC buffer 25 μL, 0.7 μL each of 20 μM / μL γ34.5-F and γ34.5-R, 5 μL of dNTPs, 0.6 μL of Prime star HS high-fidelity enzyme, and sterile water was added to make up to 50 μL. The PCR amplification conditions were: 98 °C for 5 min, (98 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min) for 32 cycles, 72 °C for 10 min, and 16 °C for 30 min. The results of the molecular identification are as Figure 1As shown in C, there was no band in the negative control; the positive control was the constructed plasmid expressing γ34.5 (pcDNA3.1-hUbC-γ34.5), and a target band about 700 bp long was amplified by PCR; a target band about 700 bp long was amplified by PCR from 1 μL of concentrated wild-type H129 virus and 3 μL of inactivated virus supernatant sample; however, no target band about 700 bp long was amplified by PCR for the low-toxic HSV virus (ltHSV-hUbC-tdT-WPRE) in multiple repeated experiments regardless of whether 1 μL, 3 μL, or 5 μL of concentrated virus was used. The results indicated that the two copies of the γ34.5 gene in the genome of ltHSV-hUbC-tdT-WPRE had been completely knocked out.

[0036] Based on the previous research results of the inventors, the attenuated herpes simplex virus after knocking out the two copies of the γ34.5 gene was replication-defective and lacked the ability of trans-synaptic transmission, and could not be used for trans-synaptic tracing of the output circuit.

[0037] Example 4: Amplification and preparation of a novel attenuated HSV recombinant virus

[0038] After successful molecular identification, the purified ltHSV-hUbC-tdT-WPRE recombinant virus was mass-produced and purified by infecting Vero cells grown on a 10-cm petri dish. The recombinant virus was infected with Vero cells at an MOI of 0.01, and obvious tdTomato fluorescence expression was observed. After the cells showed obvious rounding lesions (about 3 days), the supernatant containing the recombinant virus was collected into a 50-mL centrifuge tube, and cell debris was removed by centrifugation (6400 rpm, 10 minutes). The supernatant was filtered through a 0.22-μm filter membrane, and finally concentrated using a Beckman high-speed centrifuge (30000 rpm, 3 hours); the concentrated recombinant virus precipitate was resuspended with a small amount of PBS (pH = 7.4), incubated overnight at 4°C with continuous shaking; on the second day, the virus solution was mixed, and then the resuspended recombinant virus solution was added on top of a 20% sucrose solution for ultracentrifugation (30000 rpm, 3 hours) and concentration and purification; finally, the dissolved virus was aliquoted and stored in an -80°C refrigerator. The titer of the concentrated HSV recombinant virus was determined using a standard plaque assay with Vero cells, and the titer was expressed as plaque-forming units per milliliter (PFU / mL). The titer of the concentrated recombinant virus was determined to be approximately 3×10 9 PFU / mL. This concentrated virus will be used for in vivo neurotoxicity determination, retrograde labeling characteristics and non-trans-synaptic verification of the ltHSV-hUbC-tdT-WPRE virus, and in vivo neural circuit labeling application demonstration and other animal experiments.

[0039] Example 5: Characterization of low neurotoxicity of the ltHSV-hUbC-tdT-WPRE virus

[0040] γ34.5 plays an important regulatory role in the processes of HSV virus replication and assembly. After completely knocking out the gene of double-copy γ34.5, the virulence of the virus is significantly reduced. In the animal brain infection experiment, it was found that ltHSV-hUbC-tdT-WPRE did not cause death in infected mice, and the neurotoxicity of ltHSV was evaluated by using the microfluidic neuron culture chip established in the laboratory to infect primary neuron cells cultured in vitro in a three-chamber culture. Specifically, ltHSV-hUbC-tdT-WPRE was infected with primary neurons of mice at the soma side at an MOI of 1 ( Figure 2 A), and it was observed that the neurons could survive for up to 26 days after infection, and the neuron state was good, the fluorescence expression was clearly marked, and continuously growing dendritic fibers could be observed ( Figure 2 C). The wild-type HSV-infected neurons in vitro gradually atrophied and disappeared only on the second day, indicating that the toxicity of ltHSV-hUbC-tdT-WPRE was indeed significantly reduced.

[0041] Example 6: Stereotactic injection into the mouse brain

[0042] For experimental mice that need to be injected by stereotaxic injection into the brain, an intraperitoneal injection of 1% sodium pentobarbital (dosage: 50 mg / kg body weight) was given for anesthesia. After leaving the mice to rest for about ten minutes to ensure they entered a deep anesthesia state, first, the hair on the scalp of the mice was shaved off. Then, the mice were fixed on the bracket equipped with the Stoelting stereotaxic apparatus. Next, the scalp of the mice was surface-disinfected and cleaned with alcohol. Subsequently, a cut about 1 cm long was made along the sagittal plane using surgical scissors, and the skull was exposed with forceps and the soft tissues on the skull were removed with sterilized cotton balls. After that, the mouse bracket was stably fixed to the stereotaxic apparatus and the processed mouse skull was adjusted to the three-dimensional horizontal plane. A pre-prepared glass capillary electrode for injection was taken, and the tip was trimmed by about 1 - 3 mm to prevent blockage, and then the inside of the capillary was filled with liquid paraffin. Additionally, a special 10-μl range microsyringe was taken, the syringe plunger was removed, filled with liquid paraffin, and the air in the syringe needle channel was discharged. Then, the glass electrode was slowly sleeved on the tip of the microsyringe, and the connection part between the glass electrode and the syringe was fixed with hot melt adhesive. It could be done multiple times and in multiple layers to ensure sealing. During the sealing process, the microsyringe needed to be held horizontally and slowly rotated so that the hot melt adhesive could be evenly distributed for sealing. After completion of the sealing, the microsyringe was connected to the injection pump, and the required volume of virus solution was quickly aspirated with a virus aspiration parameter of 999 nl / min. During injection, refer to *Paxinos and Franklin’s The Mouse Brain in Stereotaxic Coordinates*, 4th edition. The detailed coordinate information of the injection brain regions is shown in the following table. Using an animal skull drill with a diameter of 1 mm, the skull was carefully drilled at the predetermined coordinates to ensure that the glass electrode could be stably inserted into the designated brain region. Then, the virus liquid was injected into the designated brain region of the mice with a parameter of an injection speed generally of 30 nl / min (usually completed within 5 - 10 minutes). To ensure the accuracy and repeatability of the injection, it was very important to adjust the mouse skull to the horizontal position before injecting the virus. After the virus injection was completed, the mice were left to rest for about 8 - 10 minutes to allow the virus to slowly spread and to balance the internal pressure of the syringe to prevent virus reflux when pulling out the needle. Whether the virus liquid level dropped was observed under a stereomicroscope to confirm that the virus injection was completed. Subsequently, the Z-axis positioning knob was slowly rotated to lift the glass electrode. Antibiotic drugs were applied to the surface of the animal skull, the scalp was sutured, and the mice were numbered to record the experimental parameters, and then they were put back into the mouse cage on the heating pad. During the whole process, the breathing condition of the mice was constantly noted. Slow and steady breathing was normal. After the mice woke up after the experiment was completed, they were put back into the breeding room for feeding. If the animals woke up during the operation, a small amount of isoflurane could be used to keep them under anesthesia.

[0043] Coordinates of the brain regions tested with the attenuated ltHSV-hUbC-tdT-WPRE virus

[0044]

[0045] Example 7: Analysis of the effect of attenuated ltHSV-hUbC-tdT-WPRE tool virus infection on neuronal activity

[0046] The tool virus developed based on ltHSV-hUbC-tdT-WPRE to express functional probes for analyzing the functional activities of neural circuits, or further modified and optimized into a viral vector for gene delivery, requires that the attenuated HSV not only does not cause apoptosis of neuronal cells, but also needs to verify whether it disturbs and changes the electrophysiological properties of the labeled neuronal cells. For brain slices sampled 18 days after injecting 200 nl of ltHSV-hUbC-tdT-WPRE into the lateral hypothalamic area (LH) of mice, the whole-cell patch-clamp method was used to record the changes in membrane voltage of pyramidal neurons in the anterior cingulate cortex (Acc) of mice under different current stimulations in the current-clamp mode. The experimental procedure is shown in Figure 3 .

[0047] The morphological images of the pyramidal neurons selected for whole-cell patch-clamp recording are shown in Figure 4 . The soma diameter of the pyramidal neurons is about 20-40 μm. It can be found in the figure that the pyramidal neurons fluorescently labeled by ltHSV-hUbC-tdT-WPRE still have clear somas, and the pyramidal neurons in the experimental records are consistent in morphology with the control neurons not infected with the virus. The procedure for whole-cell electrophysiological recording of ex vivo brain slices depolarizes the neurons with step stimuli (from -20 pA to +300 pA, with an increment of 20 pA and a duration of 2 s), and action potentials burst after reaching the threshold potential. The results show that the pyramidal neurons labeled by ltHSV-hUbC-tdT-WPRE and the control pyramidal neurons not infected with the virus have the same baseline membrane potential before the current stimulation, and for each stimulation amplitude (from -20 pA to +300 pA), the number and frequency of the maximum action potentials induced by the stimulation are comparable. The whole-cell patch-clamp recording results of the membrane voltage of the pyramidal neurons labeled by ltHSV-hUbC-tdT-WPRE and the control neurons under different current stimulations are selected. This indicates that ltHSV-hUbC-tdT-WPRE does not significantly change the physiological properties of the labeled neurons, thus not interfering with the normal neuroelectrophysiological mechanism ( Figure 5 ). This result further proves that ltHSV-hUbC-tdT-WPRE has low neurotoxicity. Subsequently, ltHSV-hUbC-tdT-WPRE will be used for long-term circuit labeling and further modified and optimized into a viral vector for gene delivery.

[0048] Example 8: Characterization of the neurotracing properties of attenuated ltHSV-hUbC-tdT-WPRE

[0049] To evaluate the neurocircuit tracing properties of ltHSV-hUbC-tdT-WPRE, study whether it can be used for long-term circuit labeling, and further verify its low neurotoxicity, the ltHSV-hUbC-tdT-WPRE virus was injected into the primary motor cortex (M1) of adult mice by stereotaxic injection. The experimental procedure is as Figure 6 , and perfusion sampling and section imaging were observed at 14, 30, and 64 days postinjection (DPI) respectively after injection.

[0050] After observing for up to two months after injecting ltHSV-hUbC-tdT-WPRE into the primary motor cortex, the mice were in good condition and showed no signs of infection. The fluorescent protein expressed by ltHSV-hUbC-tdT-WPRE was not cleared after 2 months of infection, and the labeling effect was good. Imaging was performed on the M1 of mice sampled at 14, 30, and 64 DPI and the upstream regions that can project to M1. The results showed that ltHSV-hUbC-tdT-WPRE could efficiently label the injection site M1 in situ at different time periods, as well as brain regions with direct projections to M1, such as the contralateral primary motor cortex (Cont-M1), bilateral primary somatosensory areas (S1), and bilateral perirhinal cortices (PRh), etc., and there was no attenuation of the fluorescence expression abundance( Figure 6 ). However, since there are bidirectional projections between M1 and Cont-M1, S1, and PRh, it was not yet possible to explain that the efficient fluorescence labeling effect of ltHSV-hUbC-tdT-WPRE was due to retrograde labeling of the upstream brain regions after infection of axon terminals.

[0051] However, cell body signals were only observed in brain regions that can directly project to the M1 brain region at 14, 30, and 64 DPI, and no fluorescent signal expression was seen in other brain regions with multi-level projections to M1 in the whole brain over a long period, indicating that ltHSV-hUbC-tdT-WPRE did not replicate and proliferate after infecting neurons. And no expression of the tdTomato fluorescent reporter gene was seen in the cell bodies of neurons in the downstream brain regions known to have direct projections from M1, including the caudate-putamen (CPu), thalamus (TH), and substantia nigra (SN), etc.( Figure 7), indicating that ltHSV-hUbC-tdT-WPRE did not show anterograde trans-synaptic propagation in the M1 brain region. These results suggest that ltHSV-hUbC-tdT-WPRE is a relatively strict retrograde labeling tool virus that does not proliferate in highly differentiated infected neurons. This enables ltHSV to become a new member of the retrograde labeling tool library for studying the structure and function of neural circuit networks.

[0052] Example 9: Comparison of the neurotropic properties of attenuated ltHSV-hUbC-tdT-WPRE and RV-SAD19G retrograde labeling

[0053] There are two commonly used tool viruses, rAAV2-retro and RV-SAD19, that can retrogradely label projection neural circuits and have different neurotropisms. These two tracers were respectively injected into the dorsal lateral geniculate nucleus (DLG) of the thalamus. RV-SAD19G labeled the sixth layer of the primary visual cortex (V1), while rAAV2-retro retrogradely labeled the fifth layer of V1. rAAV2-retro and RV-SAD19 differ in retrograde tracing efficiency and range. RV-SAD19 has a more limited virus diffusion range and a broader spectrum of neurotropism. To evaluate the neurotropic properties of ltHSV retrograde labeling, RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses were respectively mixed with an appropriate amount of CTB and injected into the DLG. The injection volume was 200 nl, and samples were taken 7 days after infection. The DLG is a nuclear group located in the thalamus. It is the first station of visual signals in the brain and is responsible for receiving and processing information from the retina. The lateral geniculate body is not only a relay station for visual information but also a dynamic information processing center. While receiving input from the retina, it is also affected by feedback from the cortex, thus realizing the interaction and integration of visual information at different levels.

[0054] From the localization of the mixed injection of CTB, it can be observed that the grouped experiments of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses were accurately injected into the target brain region DLG. Judging from the results, both RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses were able to efficiently label the V1 and SC brain regions ( Figure 8 ). This means that both retrograde labeling viruses have a high infection efficiency. However, there were differences when labeling the V1 brain region. RV-SAD19G mainly labeled the neurons in the 6th layer of V1, which is consistent with previous research results. It is worth noting that the ltHSV-hUbC-tdT-WPRE virus labeled multiple cortical layers from the outside to the inside of V1.

[0055] After characterizing the labeling properties of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses in the VTA respectively, a mixed injection strategy was further adopted to explore the labeling dynamics of the two viruses under mixed labeling. A sample of the equal proportion mixture of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses was mixed with CTB647 and then injected into the DLG. The injection volume was 200 nl. Samples were still taken 7 days after infection. The labeled brain slices were imaged using a two-photon microscope. CTB647 was shown as purple pseudocolor. The localization of CTB showed that both viruses were accurately injected into the target brain region DLG after mixing. The labeling results in the upstream brain regions V1 and SC of the DLG showed that both retrograde labeling viruses had a high infection efficiency, which was consistent with the results of single labeling of the two viruses, indicating that there was no interaction or synergistic effect between them. However, differences were also shown when labeling the V1 brain region. RV-SAD19G still only labeled the neurons in layer 6 of V1, while the ltHSV-hUbC-tdT-WPRE virus also labeled multiple layers of neurons in V1( Figure 9 ).

[0056] In view of the fact that rAAV2-retro and RV-SAD19 labeled different layers when labeling the input circuit of the DLG, but failed to label multiple layers of neurons in V1, researchers can choose more retrograde labeling viruses for a broader spectrum of labeling according to different research purposes. If the goal is to study specific layers of V1, rAAV2-retro and RV-SAD19G may be better choices. But if the purpose is to more widely retrogradely label neural circuits, ltHSV-hUbC-tdT-WPRE may be more suitable.

[0057] In summary, the present invention constructs an attenuated ltHSV-hUbC-tdT-WPRE virus by knocking out its neurovirulence factor γ34.5 gene, which has the characteristics of efficient retrograde labeling and low toxicity. The inventor constructs a series of low-toxicity ltHSV recombinant virus tools, verifies that the ltHSV with γ34.5 knocked out has a relatively rigorous retrograde labeling ability and a broader neurotropism, and becomes an important new member of the retrograde labeling tool library for the study of the structure and function of neural circuit networks.

Claims

1. Use of an attenuated herpes simplex virus in the preparation of a virus for retrograde tracing of neural circuits, characterized in that, The attenuated herpes simplex virus is prepared based on a targeting vector, and the targeting vector is a vector in which a complete fluorescent protein gene or other exogenous target gene expression cassette is inserted between the upstream and downstream homologous arms of the neurovirulence factor γ34.5 gene of vector pH129ΔRL1. The nucleotide sequence of vector pH129ΔRL1 is as shown in SEQ ID NO.1, the nucleotide sequence of the upstream homologous arm of the neurovirulence factor γ34.5 gene is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream homologous arm of the neurovirulence factor γ34.5 gene is as shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that, The complete fluorescent protein gene expression cassette includes, from the 5' end to the 3' end: a promoter, a fluorescent protein gene, and a WPRE transcriptional enhancer element.

3. The use according to claim 2, characterized in that, The promoter is selected from one of the hUbC promoter, CMV promoter, CAG promoter, EF1α promoter, pTH promoter, and pChAT promoter; The fluorescent protein gene is tdTomato, EGFP, or a fluorescent protein gene of other colors.

4. The use according to claim 2, characterized in that, The promoter is selected from the hUbC promoter; The fluorescent protein gene is tdTomato.

5. The use according to claim 1, characterized in that, The nucleotide sequence of the targeting vector is as shown in SEQ ID NO.

4.

6. The use according to claim 1, characterized in that, Neural circuit retrograde tracing does not cross synapses.

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