Recombinant herpes simplex virus, neural circuit tracing virus tool and application

Abstract

The invention discloses a recombinant herpes simplex virus, a neural circuit tracing virus tool and application. The invention discloses a clinical strain of a herpes simplex virus HSV1-CH001, and the collection number of the clinical strain is CCTCC (China Center For Type Culture Collection) NO: V202463. The invention also discloses a recombinant herpes simplex virus, which is obtained by recombining and inserting an exogenous gene expression cassette into the genome of the herpes simplex virus HSV1-CH001. The herpes simplex virus HSV1-CH001 is recombined and inserted into a fluorescent protein gene or other exogenous genes, a clinical strain virus is subjected to visual and other recombination transformation to construct a novel tool virus vector, and efficient tracing of neural circuit markers and exogenous gene delivery expression can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to neurobiology and molecular virology, and specifically relates to a recombinant herpes simplex virus, a neural circuit tracing virus tool and an application thereof. Background Art

[0002] At present, the widely used viral tools in neural circuit tracing research are mainly derived from the modification and recombination of neurotropic viruses. This type of virus has unique abilities. They can not only infect neurons, but also spread in different directions along synapses in neural pathways. For example, Herpes simplexvirus type (HSV), Yellow fever virus (YFV) attenuated live vaccine 17D, Pseudorabies virus (PRV), Rabies virus (RV) and Vesicular stomatitis virus (VSV). In addition, there are some viral tools that do not spread across synapses. They can efficiently mark the fine morphology of neurons in situ, such as Semliki Forest virus (SFV). Some viruses are used as auxiliary tools to help the expression of exogenous genes, such as adeno-associated virus (AAV). Other viruses can reveal upstream projections of local brain regions, such as canine adenovirus type 2 (CAV2). Neural circuit tracing tool viruses can be divided into five main categories according to their propagation direction and level. According to the specific needs of the research, these tracing tools can be further classified into three major systems: non-transsynaptic labeling systems, multi-level synaptic labeling systems, and single synaptic labeling systems. The classification and relationship of these systems provide important tools and methods for neuroscience research.

[0003] The neurotropic virus strain HSV-1H129 has become one of the most promising anterograde neural circuit tracing tool viruses because of its main anterograde transsynaptic transmission in neural circuits and large exogenous gene capacity. The virus particles of HSV-1 are spherical with a diameter of about 150-200nm. From the outside to the inside, the structure of HSV-1 is envelope, stroma, capsid shell and virus core. The outermost envelope is inlaid with about 12 glycoproteins, which play an important role in virus recognition of receptors, cell entry and budding. The stroma contains 26 proteins that play a key role in the virus life cycle. The nucleocapsid is a non-standard icosahedral structure composed of multiple capsid proteins, which encloses the HSV-1 genome of up to 152kbp. The genome of HSV-1 is a linear double-stranded DNA structure, consisting of a unique long (UL) segment and a unique short (US) segment covalently linked.

[0004] After years of development as a neural tracer virus vector, HSV-1H129 has been used to derive a variety of neural circuit anterograde tracing tool viruses through genetic engineering technology, initially forming a relatively complete anterograde tracing tool system.

[0005] At the same time, the genetic diversity of viruses is extremely rich and presents in various ways. Under suitable conditions, viruses can spread rapidly among many hosts, and their genetic mutations can expand within a specific population or evolve rapidly within an individual, competing with the host immune system. DNA viruses with polymerase proofreading capabilities are not prone to rapid base mutation errors. These viruses infect billions of human hosts worldwide and show unique genotypes in each randomly sampled infection, such as herpes simplex virus type 1 (HSV1 virus). During the infection process, viruses often show susceptibility to infection within a family or a certain group of people. This biogenomics study helps design specific vaccines, etc. And the mutations produced by the virus during the transmission process may be beneficial to the realization of certain research purposes. Therefore, the research, analysis and application of clinical strains of viruses are very promising. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a recombinant herpes simplex virus, a neural circuit tracing virus tool and an application.

[0007] The invention provides a clinical strain of herpes simplex virus HSV1-CH001, whose preservation number is CCTCC NO: V202463.

[0008] The present invention also provides a recombinant herpes simplex virus, which is obtained by recombinantly inserting an exogenous gene expression frame into the genome of the herpes simplex virus HSV1-CH001 according to claim 1.

[0009] Furthermore, the exogenous gene expression frame is recombinantly inserted into the spacer sequence between UL26 and UL27 genes in the herpes simplex virus HSV1-CH001 genome.

[0010] Further, the exogenous expression cassette comprises a green fluorescent protein gene sequence and / or any other exogenous gene;

[0011] Preferably, the exogenous expression cassette contains three green fluorescent protein gene sequences.

[0012] The present invention also provides a method for preparing the recombinant herpes simplex virus, comprising the following steps: co-transfecting a targeting plasmid containing an exogenous gene expression frame and herpes simplex virus HSV1-CH001 into 293T cells and then performing recombination, recombining and inserting the exogenous gene expression frame into the genome of the herpes simplex virus HSV1-CH001, and then spot-picking and purifying the recombinant virus to obtain the recombinant herpes simplex virus.

[0013] Furthermore, the nucleotide sequence of the targeting plasmid is shown in SEQ ID NO.1.

[0014] The present invention also provides the use of the recombinant herpes simplex virus in preparing a neural circuit tracer or an exogenous gene delivery expression vector.

[0015] Furthermore, the neural circuit tracer is an anterograde trans-synaptic neural circuit tracer.

[0016] The present invention also provides a neural circuit tracing virus tool, which is the recombinant herpes simplex virus.

[0017] The present invention also provides the use of the neural circuit tracing virus tool in neural circuit tracing, anterograde transsynaptic labeling and tumor tracing labeling.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a Chinese clinical isolate of herpes simplex virus HSV1-CH001, and uses the Chinese clinical strain of HSV1-CH001 as a parent virus for gene recombination modification to obtain a novel virus vector and a neural circuit visualization virus tool: the HSV1 clinical strain carries and expresses green fluorescent protein to mark the neural circuit visualization, and utilizes the high specificity of the clinical strain virus to construct a more efficient, safer, and long-distance tracing stable tracing virus vector. In addition, since the clinical characterization of the clinical strain is determined, the modified clinical strain tool virus can also be used to simulate the natural transmission path of the virus in the body to analyze the pathological mechanism of the HSV clinical infection strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The construction process of HSV recombinant virus vector based on HSV1 Chinese clinical strain;

[0021] Figure 2 This is the plasmid map of pT2627U3GW;

[0022] Figure 3 This is a validation diagram of cells that have been successfully recombined with the virus after targeted plasmid transfection;

[0023] Figure 4 The results of agarose gel electrophoresis for purification and PCR gene identification of HSV1-CH001-3GFP; CH1-3GFP represents the recombinant virus HSV1-CH001-3GFP, CH1 represents the clinical strain HSV1-CH001, 2627-3GFP represents the targeting plasmid pT2627U3GW, and NC represents the negative control;

[0024] Figure 5 This is an in situ image of the CPU injection site for in vivo animal verification; CH1-3GFP represents the recombinant virus HSV1-CH001-3GFP;

[0025] Figure 6 This is a cross-synaptic brain region labeling diagram after animal in vivo verification of injection into the CPU brain region;

[0026] Figure 7 This is a diagram of transsynaptic brain region labeling after in vivo verification of injection into the V1 brain region.

[0027] Collection Instructions

[0028] The selected Chinese clinical isolate of herpes simplex virus HSV1-CH001, the low-passage wild-type virus after monoclonal purification was deposited in China Center for Type Culture Collection (CCTCC) according to the requirements and specified quantity on June 5, 2024, at Wuhan University, Wuhan, China. HSV1-CH001 was classified and named as Chinese clinical isolate of herpes simplex virus HSV1-CH001 (Herpes simplex virus), and the deposit number is CCTCC NO: V202463. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of the present invention. In the examples, each raw reagent material can be obtained commercially, and the experimental method without specifying the specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0030] The present invention provides a method for constructing a neural circuit tracing virus based on the Chinese clinical strain of HSV1. The construction process is as follows: Figure 1 shown.

[0031] Example 1: Isolation of Herpes Simplex Virus Clinical Strain HSV1-CH001

[0032] In collaboration with dermatologists from clinical hospitals such as the People's Hospital of Wuhan University and the General Hospital of the Central Theater Command of the Chinese People's Liberation Army, herpes blister fluid was collected from outpatients with oral and facial herpes, and 18 HSV1 clinical virus strains were isolated through culture, purification, and identification. By cloning and sequencing the viral gD and gB genes, there were certain base mutations in the gene sequences compared with the F strain and H129 clinical strains introduced from abroad, and there were also differences in the genes between the isolated clinical strains, which were reflected in the evolutionary biological characteristics (such as infectivity, virulence, replication efficiency, etc.). This shows that it is necessary to compare and screen the parent strains of oncolytic viruses from the HSV clinical strains isolated from the healthy population in China. The principles for screening the neural circuit tracing parent virus from clinical strains are as follows: ① The source of oral and facial herpes in healthy people; ② Genotype sequencing identified as HSV1; ③ High lytic activity of infected cells. By systematically comparing the replication efficiency, cytotoxicity, and lytic cell activity of clinical strains, the clinical strain CH001 was selected as the candidate parent strain. The deposit number of the Chinese clinical isolate of herpes simplex virus HSV1-CH001 is CCTCC NO: V202463.

[0033] Example 2: Viral recombination

[0034] The extracted targeting plasmid pT2627U3GW (nucleotide sequence as shown in SEQ ID NO.1, plasmid map as shown in Figure 2As shown), 293T cells cultured in a six-well plate were transfected by liposome transfection. After 6 hours of culture, the culture medium containing the transfection mixture was discarded, and the medium was replaced. 2 mL of culture medium containing 2% serum was added to each well, and 10 μL of HSV1-CH001 parent virus was added to each well. The mixture was gently shaken and mixed evenly. The cells were cultured in a 35°C, 3% CO2 incubator. After infection, the fluorescence ratio was observed under an inverted fluorescence microscope. The cells showed obvious lesions, particles appeared in the cells, and the cell morphology became round. After two or three days, all the cells were detached from the wall. At this time, the recombinant virus supernatant was aspirated into a 1.5 mL centrifuge tube, centrifuged at 6600 × g for 6 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube with a pipette. The adherent cells were blown off with PBS buffer and then collected in a 1.5 mL centrifuge tube. All centrifuge tubes were marked and placed in a -80°C refrigerator.

[0035] pT2627U3GW plasmid sequence (SEQ ID NO.1):

[0036]

[0037] Example 3: Recombinant virus purification

[0038] The virus supernatant collected in Example 2 was frozen and thawed once, centrifuged at 6500×g for 10 min to remove cell debris, and 10 μL to 50 μL of the virus supernatant was taken to infect BHK-21 cells. After 24 hours, the expression of the fluorescent reporter gene was observed under a fluorescence microscope to determine whether the virus was successfully recombined; later, the virus supernatant with successful recombinant was taken and diluted 10 times in a continuous gradient and infected BHK-21 cells. After adsorption for 1 hour, agar (DMEM medium containing 5% fetal bovine serum and 2% agar mixed in a ratio of 1:1) was spread. After 48-72 hours, the monoclonal plaques of the virus are marked under a fluorescent microscope, and then accurately picked out in a sterile clean bench using a white pipette tip, diluted and infected with cells in a new 6-well culture plate. This is called a round of plaque picking. The newly recombinant virus is generally purified through 5-6 rounds of plaque picking to remove the parent virus and obtain the desired progeny recombinant virus, until 100% of the field of view of each well plate is green fluorescent, and there is no non-fluorescent parent virus plaque, which means that the purified new recombinant virus HSV1-CH001-3GFP ( Figure 3 ).

[0039] Example 3: Identification of recombinant viruses

[0040] Take 100 μL of the purified recombinant virus HSV1-CH001-3GFP supernatant and put it into a 1.5 mL centrifuge tube, inactivate it in a 60°C metal bath for 30 minutes, and then heat it in a 95°C metal bath for 5 minutes. Then use the inactivated pure virus sample as a template and use the identification primers to amplify the target fragment. The specific steps are as follows:

[0041] (1) Using the purified virus HSVI-CH001-3GFP supernatant as a template, primers 1129-UL26arm-F and 1130-UL27arm-R were used to amplify the 3GFP fragment (4494 bp).

[0042] 1029UL26arm-F: 5'-AAATCGGCACTGACCAAGGGGGTCCG-3'SEQ ID NO.2;

[0043] 1030UL27arm-R: 5'-TGTCCGCGGCCTTGTTTCCTCTTTCC-3'SEQ ID NO.3.

[0044] The PCR reaction system (50 μL) is shown in the following table.

[0045] PCR reaction system for purification and identification of recombinant virus HSV1-CH001-3GFP

[0046]

[0047] Place the prepared system in a PCR gene amplifier. The PCR reaction procedure is as follows:

[0048]

[0049] After the reaction was completed, the sample was subjected to 1% agarose gel electrophoresis, and then the band size was observed by gel imaging. There was a band at 3000-5000 bp, and it was compared with the target fragment cloned from plasmid pT2627U3GW. The results showed that the size was correct, see Figure 4 , indicating that the virus was recombined and purified successfully. The 3GFP fragment was recovered by EZNAGel Extraction Kit and sent for sequencing, and the sequencing was correct.

[0050] Example 5: Recombinant virus concentration

[0051] The HSV1-CH001-3GFP virus was inoculated with an MOI of 0.01 and infected with baby hamster kidney cells (BHK cells) grown to appropriate confluence in a T75 culture flask or culture dish. During the virus amplification process, the cell culture medium used was DMEM containing 2% FBS and 1% double antibody. After inoculation of the virus, the cells were placed in a constant temperature incubator at 35°C and 3% CO2 for culture.

[0052] Every 24 hours, the cell status needs to be observed under a microscope. When cells are observed to generally express fluorescence and show pathological changes, the supernatant can be collected. The supernatant is collected in a 50 mL centrifuge tube and temporarily stored in a -80°C refrigerator.

[0053] 10 to 16 hours before the virus concentration operation, the collected virus supernatant needs to be taken out of the -80℃ refrigerator and placed in a 4℃ refrigerator for thawing. After the supernatant is completely thawed, centrifuge at 6500×g for 10 minutes to remove cell debris. After centrifugation, collect the supernatant in a T75 culture flask.

[0054] Next, the supernatant was filtered using a 0.22 μm filter and centrifuged at 50,000 × g for 3 hours. After centrifugation, the precipitate was gently blown off with PBS and centrifuged again at 50,000 × g for 3 hours with 20% sucrose solution. After centrifugation, the supernatant was discarded and the virus was carefully resuspended with sterile PBS.

[0055] Finally, the resuspended virus was dispensed into small tubes with a volume of 3 μL per tube and stored in a -80°C refrigerator. Repeated freezing and thawing should be avoided during storage to ensure the stability and activity of the virus.

[0056] Example 6: Stereotaxic injection of viral vectors into mouse brain

[0057] For experimental mice that need to undergo brain stereotaxic injection, they are first anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dose of 50 mg / kg body weight). After the mice enter a deep anesthesia state, they are left to rest for about ten minutes, then the hair on their heads is shaved and the mice are fixed on the supporting bracket of the Reward stereotaxic instrument. Then, the mouse scalp is disinfected and cleaned with alcohol.

[0058] Then, a 1 cm long incision was made along the sagittal plane with surgical scissors, the skull was gently exposed with tweezers, and the attached soft tissue was removed with a sterile cotton ball. After that, the mouse holder was stably fixed on the stereotaxic instrument, and the processed mouse skull was adjusted to a three-dimensional horizontal position.

[0059] Next, take a pre-prepared glass capillary electrode, trim its tip by about 1 to 3 mm to prevent clogging, and fill the inside of the capillary with liquid paraffin. At the same time, take a dedicated 10-microliter range microinjector, remove the injector push rod, fill it with liquid paraffin, and exhaust the gas in the injector needle channel. Then slowly put the glass electrode on the tip of the microinjector and use hot melt glue to fix the connection part to ensure sealing. During the sealing process, the microinjector needs to be held horizontally and rotated slowly to evenly distribute the hot melt glue.

[0060] After sealing, connect the microinjector to the syringe pump and set the virus aspiration parameter to 999 nL / min to quickly extract the required volume of virus solution. When injecting, refer to the coordinate information of "Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates, 4th Edition" as shown in the following table.

[0061] Coordinates of brain regions tested for HSV1-CH001-3GFP

[0062]

[0063] Use an animal skull drill with a diameter of 1 mm to carefully drill through the skull at the predetermined coordinates to ensure that the glass electrode can stably reach the designated brain area. Then set the injection speed to 30 nL / min and inject the virus liquid into the designated brain area of ​​the mouse.

[0064] To ensure the accuracy and repeatability of the injection, it is important to adjust the mouse skull to a horizontal position before injecting the virus. After the virus injection, let it stand for about 8 to 10 minutes to allow the virus to slowly diffuse and balance the pressure inside the injector to prevent virus backflow when the needle is removed. Observe whether the virus liquid level drops under a stereo microscope to confirm that the virus has been injected. Then slowly rotate the Z-axis positioning knob to lift the glass electrode.

[0065] Finally, antibiotics were applied to the surface of the animal's skull, the scalp was sutured, and the mice were numbered and the experimental parameters were recorded. The mice were returned to the cage on the heating pad, and their breathing was monitored at all times to keep them stable and normal. After the experiment was completed, when the mice woke up, they were returned to the breeding room for feeding.

[0066] In each experiment, at different times after injection according to specific needs, the mice were anesthetized and perfused with PBS and 4% paraformaldehyde through the heart, and then the scalp and skull were peeled off and the brain tissue was collected.

[0067] Example 7: Brain tissue slice sample preparation and imaging

[0068] The mouse brain tissues collected in Example 6 were first immersed in a 4% paraformaldehyde solution for subsequent fixation. After being placed at room temperature for about 16 hours, the brain tissues were transferred to a 30% sucrose solution to complete the dehydration process. After sucrose dehydration, the brain tissue samples were embedded in a freezing embedding medium (SAKURA, 4583) and prepared for sectioning.

[0069] Next, the embedded brain tissues were sectioned coronally or sagittally using a cryostat (Thermo Fisher Scientific), with the thickness of each slice set to 40 μm. After sectioning, they were carefully collected in a 24-well plate containing antifreeze and stored at -20°C for subsequent experimental needs.

[0070] For those slices that do not need to be subjected to immunofluorescence imaging, we selected the required brain slices and washed them with PBS (phosphate buffered saline). These brain slices were stained with 4',6-diamidino-2-phenylindole (DAPI) to stain the nuclei for easy observation. After staining, the brain slices were carefully attached to the slides and washed three times with PBS on a shaker, each washing time was 5 minutes.

[0071] Finally, after being mounted with 70% glycerol, the brain slices can be subjected to fluorescent imaging for further observation and analysis.

[0072] Taking Caspase3 immunohistochemical staining as an example, the immunohistochemical and imaging process of brain slices is as follows:

[0073] ① First, select the target brain slice from the sample and place it on a shaker. Wash it three times with PBS (phosphate buffered saline), each wash lasting 5 minutes to ensure the cleanliness of the brain slice.

[0074] ②After washing, add pre-prepared 10% goat serum (containing 1×PBS and 0.3% Triton) to immerse and incubate the brain slices. This step is carried out at room temperature for 1 hour, and 150ul of goat serum is used per well.

[0075] ③After the sheep serum incubation is completed, remove the excess sheep serum (note that no additional washing is required), and then add the pre-diluted Caspase-3 mouse monoclonal antibody (primary antibody). The primary antibody is diluted 1000 times using 1×PBS and 0.3% Triton, 120ul is added to each well, and incubated overnight at 4°C.

[0076] ④ On the next day, remove the primary antibody solution and wash the brain slices three times with 1×PBS, each wash for 5 minutes.

[0077] ⑤After washing, add the pre-prepared secondary antibody solution. Here we use goat anti-mouse FITC (fluorescein-labeled secondary antibody), which is diluted 400 times with 1×PBS and 0.3% Triton. Add 120uL of secondary antibody solution to each well and incubate in a constant temperature incubator at 37°C for 1 to 1.5 hours.

[0078] ⑥After the secondary antibody incubation is completed, remove the excess secondary antibody solution and wash the brain slices three times with 1×PBS, each wash for 5 minutes.

[0079] ⑦ Next, perform nuclear staining. Add DAPI (4',6-diamidino-2-phenylindole) diluted 5000 times with 1× PBS. Place in room temperature away from light for 7 to 10 minutes for nuclear staining.

[0080] ⑧After nuclear staining, the DAPI solution was aspirated and the brain slices were washed three times with 1×PBS, each wash for 5 minutes.

[0081] ⑨ Carefully place the brain slice on the anti-slip slide and seal it with 70% glycerol. To ensure the seal between the cover glass and the slide, apply nail polish on the edge of the cover glass to seal it.

[0082] ⑩Finally, the brain slices can be observed using a fluorescence microscope. To obtain clearer images, a slide scanner (such as Olympus VS120) or a confocal microscope (such as Leica TCS SP8) can be used to perform fluorescence imaging of the brain slices, and the images can be exported and saved in Tiff format.

[0083] The brain region labeling results of the experimental results are shown in the figure Figure 5 , Figure 6 As shown, the clinical strain tracer virus exhibited efficient trans-synaptic labeling ability. Figure 5 The CPU brain area is the injection site. In addition to the original area a, the M2 brain area b can also be labeled; Figure 6 Area a is the CL brain area, area b is the DM brain area, and area c is the PRh brain area.

[0084] In order to further determine the direction of virus tracing, the virus was mixed and injected into the brain area V1 using the same experimental scheme. The results of virus transsynaptic labeling are shown in Figure 7 As shown. In the figure, A shows the transsynaptic labeling results in the MVePC region after HSV1 CH001-3GFP and H129-hUbC-tdTomato were mixed and injected into V1, and in the figure, B shows the transsynaptic labeling of the cortex and other related areas after HSV1 CH001-3GFP and PRV-CAG-3Ms were mixed and injected. Scale bar = 2mm. According to the results, the two clinical strains of tracer viruses showed highly consistent co-labeling of the labeled brain areas in the comparison of the transsynaptic labeling effects of mixed injection with H129-hUbC-tdTomato for anterograde tracing, and the red fluorescent protein expression overlapped with the green fluorescent protein expression and appeared yellow. In the comparison of the transsynaptic labeling effects of mixed injection with PRV-CAG-3Ms for retrograde tracing, it was found that the brain area labeling results were significantly different, and the red fluorescence and green fluorescence were expressed in different brain areas. From the experimental results, it is believed that the recombinant virus HSV1CH001-3GFP tends to be a new type of anterograde transsynaptic tracer.

[0085] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A clinical strain of herpes simplex virus HSV1-CH001, characterized in that: Its deposit number is CCTCC NO: V202463.

2. A recombinant herpes simplex virus, characterized in that: The recombinant herpes simplex virus is obtained by recombinantly inserting an exogenous gene expression frame into the genome of the herpes simplex virus HSV1-CH001 according to claim 1.

3. The recombinant herpes simplex virus according to claim 2, characterized in that The exogenous gene expression frame is recombinantly inserted into the spacer sequence between UL26 and UL27 genes of the herpes simplex virus HSV1-CH001 genome.

4. The recombinant herpes simplex virus according to claim 2, characterized in that The exogenous expression frame comprises a green fluorescent protein gene sequence and / or any other exogenous gene; Preferably, the exogenous expression cassette contains three green fluorescent protein gene sequences.

5. A method for preparing the recombinant herpes simplex virus according to claim 2, characterized in that: The method comprises the following steps: co-transfecting a targeting plasmid containing an exogenous gene expression frame and herpes simplex virus HSV1-CH001 into 293T cells and then performing recombination, recombining and inserting the exogenous gene expression frame into the genome of herpes simplex virus HSV1-CH001, and then plaque-picking and purifying the recombinant virus to obtain the recombinant herpes simplex virus described in claim 2.

6. The preparation method according to claim 5, characterized in that: The nucleotide sequence of the targeting plasmid is shown in SEQ ID NO.

1.

7. Use of the recombinant herpes simplex virus according to claim 2 in the preparation of a neural circuit tracer or an exogenous gene delivery expression vector.

8. The use according to claim 7, characterized in that: The neural circuit tracer is an anterograde transsynaptic neural circuit tracer.

9. A neural circuit tracing virus tool, characterized in that: It is the recombinant herpes simplex virus as described in claim 2.

10. Use of the neural circuit tracing virus tool according to claim 9 in neural circuit tracing, anterograde transsynaptic labeling and tumor tracing labeling.

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