Use of attenuated herpes simplex virus in preparation of neural circuit retrograde tracing tool virus

Abstract

The use of an attenuated herpes simplex virus in the preparation of a neural circuit retrograde tracing tool virus. The attenuated herpes simplex virus is prepared on the basis of a targeting vector, wherein 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 the vector pH129ΔRL1. It is found for the first time that the ltHSV-hUbC-tdT-WPRE virus can be used for neural circuit retrograde tracing, and that the virus enables efficient axonal terminal infection to label the upstream regulatory brain regions of the target brain region in a retrograde manner. Moreover, for up to two months of labeling, cell body signals can be observed only in the upstream brain regions that can directly project to the target brain region, without exhibiting anterograde transsynaptic spread. Therefore, the virus can be used as a new tracing virus system with a relatively stringent retrograde labeling ability and a broader spectrum of neural tropism.

Description

Application of an attenuated herpes simplex virus in the preparation of a tool virus for reverse tracing of neural circuits Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to neurobiology and molecular virology, and specifically relates to the use of an attenuated herpes simplex virus in the preparation of a neural circuit reverse tracing tool virus. Background Art

[0002] In recent years, viral transsynaptic tracing has been increasingly used in neural circuit analysis. Traditional neural network tracing methods, such as dyes, compound tracers, and protein peptides, can be transported along axons but cannot cross synapses, effectively marking only localized neuronal morphology. Compared to traditional neural network tracing methods, neurotropic viruses offer distinct advantages as tracing tools: 1) efficient infection of neurons; 2) ability to spread across synapses; 3) controllable transsynaptic direction, allowing for specific reverse or forward transmission; 4) viral self-replication after crossing synapses, without signal attenuation; and 5) the ability to carry complex regulatory elements and diverse markers.

[0003] Currently, the commonly used neurotropic viruses include pseudorabies virus (PRV) and herpes simplex virus (HSV) from the alphaherpesviridae family, and rabies virus (RV) from the rhabdoviridae family. Others include vesicular stomatitis virus (VSV). Among them, PRV Bartha strain and RV are retrograde transsynaptic infections, and VSV can be transmitted in both directions. The HSV-1 H129 strain can specifically transmit anterograde transsynaptically, which is consistent with the direction of nerve impulse transmission and is very suitable for labeling output neural networks.

[0004] Herpes simplex virus type 1 (HSV1) is a ubiquitous, conditionally pathogenic pathogen. It is a large, enveloped virus with a diameter of approximately 200 nm. Its core is a double-stranded DNA genome of approximately 153 kb, consisting of covalently linked long segments (UL) and short segments (US). Each segment contains an inverted repeat sequence at the end, so it can form four isomers.

[0005] The neurotropic virus strain HSV-1H129, due to its primary anterograde transsynaptic transmission within neural circuits and its large capacity for exogenous genes, has become one of the most promising tools for tracing anterograde neural circuits. After years of development as a neural tracing viral vector, HSV-1H129 has been genetically engineered to produce a diverse array of anterograde neural circuit tracing tools, essentially forming a comprehensive suite of anterograde tracing tools.

[0006] Summary of the Invention

[0007] In order to solve the above problems, the purpose of the present invention is to provide an attenuated herpes simplex virus for use in preparing a tool virus for reverse tracing of neural circuits.

[0008] The present invention provides an application of an attenuated herpes simplex virus in preparing a neural circuit reverse tracing tool virus. 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 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.

[0009] Furthermore, 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 transcription enhancing element.

[0010] Furthermore, the promoter is selected from one of the group consisting of hUbC promoter, CMV promoter, CAG promoter, EF1α promoter, pTH promoter, and pChAT promoter;

[0011] The fluorescent protein gene is tdTomato or EGFP.

[0012] Further, the promoter is selected from hUbC promoter;

[0013] The fluorescent protein gene is tdTomato.

[0014] Furthermore, the nucleotide sequence of the targeting vector is shown in SEQ ID NO.4.

[0015] Furthermore, retrograde tracing of neural circuits does not cross synapses.

[0016] The beneficial effects of the present invention are:

[0017] The present invention constructs an attenuated herpes simplex virus by completely knocking out the double copies of the neurovirulence factor γ34.5 gene in the HSV-1H129 genome and inserting a complete fluorescent gene expression cassette into the knocked-out γ34.5 gene locus. It is discovered for the first time that the attenuated herpes simplex virus can be used for reverse tracing of neural circuits.

[0018] The present invention uses whole-cell patch clamp recordings of membrane voltage in pyramidal neurons labeled with ltHSV-hUbC-tdT-WPRE and control neurons under different current stimulations to demonstrate that ltHSV-hUbC-tdT-WPRE does not significantly change the physiological properties of the labeled neurons, thereby not interfering with normal neuroelectrophysiological mechanisms. Animal labeling experiments have found that ltHSV-hUbC-tdT-WPRE can efficiently infect axonal terminals, thereby retrogradely labeling the upstream regulatory brain regions of the target brain region. Furthermore, even after two months of labeling, cell body signals were only observed in the upstream brain regions that can directly project to the target brain region, with no evidence of anterograde transsynaptic transmission. Compared to the classic retrograde tracer virus RVdG-SAD19G, ltHSV exhibits a broader spectrum of neurotropic properties. Therefore, ltHSV can be used as a new tracer virus system with relatively rigorous retrograde labeling capabilities and a broader spectrum of neurotropic properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the recombinant, purified and molecularly identified ltHSV-hUbC-tdT-WPRE virus with HSVγ34.5 double-copy gene knockout; Figure 1A is a schematic diagram of the structure of the wild-type HSV-1H129 clinical strain genome; Figure 1B is a schematic diagram of the structure of the ltHSV-hUbC-tdT-WPRE virus genome with HSVγ34.5 double-copy gene knockout; Figure 1C is the molecular identification result of the ltHSV-hUbC-tdT-WPRE virus genome; Figure 1D is a fluorescence expression diagram of ltHSV-hUbC-tdT-WPRE virus-infected cells;

[0020] Figure 2 shows the evaluation of neurotoxicity in primary neurons infected with ltHSV-hUbC-tdT-WPRE;

[0021] Figure 3 is a flowchart of whole-cell electrophysiological recording in isolated brain slices;

[0022] FIG4 is a grayscale image and bright field image of fluorescently labeled pyramidal neurons recorded by whole-cell patch clamp;

[0023] FIG5 shows that ltHSV-hUbC-tdT-WPRE did not significantly change the physiological properties of labeled neurons;

[0024] Figure 6 shows that ltHSV-hUbC-tdT-WPRE can efficiently label the M1 brain region and its upstream brain regions over a long period of time;

[0025] Figure 7 shows that ltHSV does not exhibit anterograde transsynaptic transmission characteristics;

[0026] FIG8 compares the effects of retrograde non-transsynaptic labeling after VTA injection of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses;

[0027] Figure 9 compares the retrograde non-transsynaptic labeling effects of RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses after mixed injection into the VTA. DETAILED DESCRIPTION

[0028] 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 implementation of the present invention.

[0029] Example 1: Construction of a ltHSV-hUbC-tdT-WPRE viral targeting vector

[0030] The wild-type HSV-1H129 clinical strain was provided by Professor Lynn Enquist (Princeton University, Princeton, NJ, USA). The HSV-1H129 γ34.5 (also known as RL1) gene sequence and its flanking gene sequences were obtained from the GenBank database (GenBank: GU734772.1). The nucleotide sequence of the γ34.5 gene open reading frame (ORF) was 527 bp upstream and 547 bp downstream as the upstream homology arm (UHA) and downstream homology 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%. The present invention is designed to knock out the full-length γ34.5 gene (1007 bp), extract and purify the HSV-1H129 viral genomic DNA, and use it as a template to design primers to clone the upstream homology arm (UHA, 527 bp long, 80% GC content) and the downstream homology arm (DHA, 547 bp long, 66% GC content) of the γ34.5 gene. The cloned upstream and downstream homology arm fragments are digested with Hind III and BamH I, BamH I and XbaI, respectively, and then connected into the pcDNA3.1+ vector and named pH129ΔRL1. The nucleotide sequence of pH129ΔRL1 is shown in SEQ ID NO.1, and the nucleotide sequences of the upstream homology arm (UHA) and the downstream homology arm (DHA) are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The exogenous gene expression cassette hUbC-tdTomato-WPRE-PA was then inserted between the upstream and downstream homology arms of the γ34.5 gene to construct a ltHSV-hUbC-tdT-WPRE viral targeting vector named pH129ΔRL1-hUbC-tdTomato-WPRE-PA, the nucleotide sequence of which is shown in SEQ ID NO. 4.

[0031] Example 2: Recombination and plaque purification of attenuated herpes simplex virus

[0032] ① Viral recombination: The targeting vector pH129ΔRL1-hUbC-tdTomato-WPRE was extracted and transfected into 293T cells using lipofectamine. After 6 hours, the maintenance medium containing 2% FBS was replaced and the herpes simplex virus H129 strain was added for infection. The fluorescence expression and cytopathic effects were observed at different times. After all cells were diseased, the cell culture supernatant was collected and stored in a -80°C refrigerator.

[0033] ② Virus Purification: The collected viral supernatant was freeze-thawed three times and centrifuged at 6500g for 10 minutes to remove cell debris. 10 μL of the supernatant was then aspirated and used to infect Vero cells. One day later, the infected cells were observed for fluorescence expression to confirm successful recombinant virus. Later, the supernatant of the successfully recombined virus was serially diluted 10-fold and infected into Vero cells. After adsorption for 1 hour, the cells were plated with agar (a 1:1 mixture of DMEM medium containing 5% fetal bovine serum and 2% agar). After 48-72 hours, when viral plaques formed, the cells were picked under an inverted fluorescence microscope. After approximately six rounds of plaque purification, the wild-type virus was removed, resulting in the purified recombinant virus, ltHSV-hUbC-tdT-WPRE. The results of attenuated herpes simplex virus reconstitution, plaque purification, and fluorescence expression in infected cells are shown in Figure 1D. Cells infected with the low-virulence HSV lt exhibited strong fluorescence.

[0034] Example 3: Molecular identification of attenuated herpes simplex virus genome

[0035] A pure ltHSV-hUbC-tdT-WPRE monoclonal strain was obtained through homologous recombination and plaque purification. To verify that both copies of the γ34.5 gene were deleted in the obtained monoclonal strain, a 747-bp ORF fragment of the γ34.5 gene was amplified by PCR.

[0036] Concentrated and purified wild-type H129 and attenuated HSV (ltHSV-hUbC-tdT-WPRE) viruses were inactivated at 100°C for 10 minutes and subsequently used for molecular identification of the γ34.5 gene. Primers were designed based on the 747-bp γ34.5 ORF fragment. The primer sequences used for identification were: γ34.5-F: 5'-ATGGCCCGCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGG-3'; γ34.5-R: 5'-TTAGACCGAGTTCGCCGGGCCGGCTCCGCGGGCCAGGGCCCGGGC-3'. Because the γ34.5 gene has a high GC content of 82%, a high-GC buffer system was used to amplify the γ34.5 ORF: a 50 μL PCR reaction system was prepared, containing 1-5 μL of inactivated HSV virus sample, 25 μL of 2× PrimeStar high-GC buffer, 0.7 μL each of 20 μM / μL γ34.5-F and γ34.5-R, 5 μL of dNTPs, 0.6 μL of PrimeStar HS high-fidelity enzyme, and sterile water to 50 μL. PCR amplification conditions were: 98°C for 5 min, 32 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 1 min), extension at 72°C for 10 min, and 16°C for 30 min. The results of molecular identification are shown in Figure 1C. The negative control had no band; the positive control was the constructed plasmid expressing γ34.5 (pcDNA3.1-hUbC-γ34.5), which amplified a target band of about 700 bp by PCR; 1 μL and 3 μL of concentrated wild-type H129 virus supernatant samples amplified a target band of about 700 bp by PCR; while the low-toxicity HSV virus (ltHSV-hUbC-tdT-WPRE) was repeatedly tested, and no target band of about 700 bp was amplified by PCR regardless of whether 1 μL, 3 μL, or 5 μL of concentrated virus was used for PCR. The results indicate that the double copy γ34.5 gene in the ltHSV-hUbC-tdT-WPRE genome has been completely knocked out.

[0037] Based on the inventors' previous research results, the attenuated herpes simplex virus after double-copy γ34.5 gene knockout is replication-deficient and lacks the ability to spread across synapses, and cannot be used for transsynaptic tracing of output circuits.

[0038] Example 4: Amplification and preparation of novel attenuated HSV recombinant virus

[0039] After successful molecular identification, the purified ltHSV-hUbC-tdT-WPRE recombinant virus was mass-produced and purified by infecting Vero cells grown on 10 cm dishes. Vero cells were infected with the recombinant virus at an MOI of 0.01, and all cells showed obvious tdTomato fluorescence expression. After the cells showed obvious rounding lesions (approximately 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 and finally concentrated using a Beckman high-speed centrifuge (30,000 rpm, 3 hours). The concentrated recombinant virus pellet was resuspended in a small amount of PBS (pH = 7.4) and incubated at 4°C overnight with constant shaking. On the second day, the virus solution was mixed, and the resuspended recombinant virus solution was then added to the top layer of 20% sucrose solution for ultracentrifugation (30,000 rpm, 3 hours), concentrated and purified. Finally, the dissolved virus was aliquoted and frozen in a -80°C freezer. The titer of the concentrated HSV recombinant virus was determined by a standard plaque assay using Vero cells. The titer is expressed as plaque-forming units per milliliter (PFU / mL). The titer of the concentrated recombinant virus was determined to be approximately 3×10 9 This concentrated virus will be used for animal experiments such as in vivo neurotoxicity assays of the ltHSV-hUbC-tdT-WPRE virus, verification of its reverse labeling properties and its non-transsynaptic function, and demonstration of its in vivo neural circuit labeling applications.

[0040] Example 5: Characterization of low neurotoxicity of ltHSV-hUbC-tdT-WPRE virus

[0041] γ34.5 plays a crucial regulatory role in HSV replication and assembly. Complete gene knockout of both copies of γ34.5 significantly reduced the virulence of the virus. Animal brain infection experiments revealed that ltHSV-hUbC-tdT-WPRE infection in mice was nonlethal. Furthermore, the neurotoxicity of ltHSV was assessed by infecting primary neurons cultured in vitro in three chambers using a laboratory-developed microfluidic neuron culture chip. Specifically, primary mouse neurons were infected with ltHSV-hUbC-tdT-WPRE at an MOI of 1 on the cell body side (Figure 2A). Neurons survived up to 26 days post-infection and remained in good condition, with clear fluorescent marker expression and continued dendritic growth (Figure 2C). In contrast, neurons cultured in vitro infected with wild-type HSV gradually atrophied and disappeared after only two days, demonstrating that the toxicity of ltHSV-hUbC-tdT-WPRE was significantly reduced.

[0042] Example 6: Stereotaxic injection into mouse brain

[0043] Mice undergoing stereotaxic brain injections were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg body weight). After resting for approximately ten minutes to ensure deep anesthesia, the mouse's head was shaved and then secured to a stand provided with a Rayward stereotaxic apparatus. The mouse's scalp was then disinfected and cleaned with alcohol. A sagittal incision approximately 1 cm long was made using surgical scissors. The skull was exposed with forceps, and soft tissue removed using sterile cotton balls. The mouse stand was then securely fixed to the stereotaxic apparatus, and the pre-treated skull was adjusted to a three-dimensional horizontal plane. A glass capillary electrode, prepared for injection, was trimmed approximately 1-3 mm from the tip to prevent clogging. The capillary was then filled with liquid paraffin. Separately, a dedicated 10-µL microinjector was used. The plunger was removed, and the needle was filled with liquid paraffin, venting the air in the needle channel. Next, slowly place the glass electrode over the tip of the microinjector and secure the connection between the glass electrode and the syringe with hot melt adhesive. Multiple layers can be applied to ensure a seal. During the sealing process, hold the microinjector horizontally and slowly rotate it to evenly distribute the hot melt adhesive. Once the seal is established, connect the microinjector to a syringe pump and rapidly draw the required volume of virus at a rate of 999 nl / min. For injection, refer to Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates, 4th edition. Detailed coordinates of the injection area are shown in the table below. Using a 1 mm diameter skull drill, carefully drill through the skull at the predetermined coordinates, ensuring that the glass electrode can stably penetrate the designated brain region. Then, set the injection rate to 30 nl / min and inject the virus into the designated brain region (injection is usually completed within 5-10 minutes). To ensure accurate and reproducible injections, it is important to level the mouse skull before injecting the virus. After the virus injection, let the mouse sit for approximately 8-10 minutes to allow the virus to diffuse slowly and the pressure inside the injector to equalize, preventing viral backflow when the needle is removed. Observe the virus liquid level under the stereomicroscope to confirm that the virus injection is complete. Then, slowly rotate the Z-axis positioning knob to lift the glass electrode. Apply antibiotics to the surface of the animal's skull, suture the scalp, number the mouse, and record the experimental parameters. Return the mouse to its cage on a heating pad. Monitor the mouse's breathing throughout the procedure; slow and steady breathing is considered normal. After the experiment is complete and the mouse regains consciousness, return it to the breeding room for feeding. If the animal regains consciousness during the procedure, a small amount of isoflurane can be used to maintain anesthesia.

[0044] Coordinates of brain regions tested with attenuated ltHSV-hUbC-tdT-WPRE virus

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

[0046] Developing a tool virus based on the ltHSV-hUbC-tdT-WPRE to express functional probes for analyzing the functional activity of neural circuits, or further optimizing it as a viral vector for gene delivery, requires that the attenuated HSV not only prevent neuronal apoptosis but also verify whether it perturbs the electrophysiological properties of labeled neurons. Whole-cell patch clamp techniques were used on brain slices obtained 18 days after injection of 200 nl of ltHSV-hUbC-tdT-WPRE into the lateral hypothalamic area (LH) of mice. Current clamp mode was used to record changes in membrane voltage in response to different current stimulations in pyramidal neurons in the anterior cingulate cortex (ACC) of mice. The experimental process is shown in Figure 3.

[0047] Figure 4 shows the morphological images of selected pyramidal neurons recorded using whole-cell patch clamp. The cell body diameter of a pyramidal neuron is approximately 20 to 40 μm. The images show that the pyramidal neurons fluorescently labeled with ltHSV-hUbC-tdT-WPRE still have clear cell bodies, and the morphology of the pyramidal neurons in the experimental recordings is consistent with that of uninfected control neurons. Whole-cell electrophysiological recordings in isolated brain slices involved depolarizing neurons with step stimulation (from -20pA to +300pA, in 20pA increments, for 2 seconds) and triggering a burst of action potentials after reaching the threshold potential. The results showed that pyramidal neurons labeled with ltHSV-hUbC-tdT-WPRE and uninfected control pyramidal neurons had the same baseline membrane potential before current stimulation, and the number and frequency of maximal action potentials evoked by stimulation were comparable for each stimulus amplitude (from -20pA to +300pA). Whole-cell patch-clamp recordings of membrane voltage in selected pyramidal neurons labeled with ltHSV-hUbC-tdT-WPRE and control neurons under different current stimulations were also performed. This suggests that ltHSV-hUbC-tdT-WPRE does not significantly alter the physiological properties of labeled neurons, thereby preventing interference with normal neurophysiological mechanisms (Figure 5). This result further demonstrates the low neurotoxicity of ltHSV-hUbC-tdT-WPRE. Subsequently, ltHSV-hUbC-tdT-WPRE will be used for long-term loop labeling and further transformed and optimized into a viral vector for gene delivery.

[0048] Example 8: Characterization of neural tracing properties of attenuated ltHSV-hUbC-tdT-WPRE

[0049] To evaluate the neural circuit tracing properties of ltHSV-hUbC-tdT-WPRE and investigate its potential for long-term circuit labeling, and to further validate its low neurotoxicity, the ltHSV-hUbC-tdT-WPRE virus was injected into the primary motor cortex (M1) of adult mice via stereotaxic injection. The experimental procedure is shown in Figure 6 . Perfusion sampling was performed on days 14, 30, and 64 postinjection (DPI), and slice imaging was performed.

[0050] After injection of ltHSV-hUbC-tdT-WPRE into the primary motor cortex, mice remained in good condition and showed no symptoms for up to two months. The fluorescent protein expressed by ltHSV-hUbC-tdT-WPRE was not eliminated even two months after infection, and labeling was effective. Imaging of the M1 and upstream regions projecting to M1 at 14, 30, and 64 days per iod (dpi) revealed that ltHSV-hUbC-tdT-WPRE efficiently labeled the injection site, M1, at different time points, as well as brain regions directly projecting to M1, including the contralateral primary motor cortex (Cont-M1), the bilateral primary somatosensory area (S1), and the bilateral perirhinal cortex (PRh), without any decrease in fluorescence expression (Figure 6). However, due to the bidirectional projection between M1 and Cont-M1, S1, and PRh, it is not yet clear whether the efficient fluorescent labeling effect of ltHSV-hUbC-tdT-WPRE is achieved by infecting axonal terminals and then reversely labeling the upstream brain areas.

[0051] However, at 14, 30, and 64 days per iod (dpi), cell body signals were only observed in brain regions that directly project to the M1 region. No fluorescent signal expression was observed in other brain regions with multi-level projections to the M1 region over the long term, indicating that ltHSV-hUbC-tdT-WPRE did not replicate or proliferate after infecting neurons. Furthermore, no tdTomato fluorescent reporter gene expression was observed in neuronal cell bodies in downstream brain regions known to directly project to the M1 region, including the caudate nucleus (CPu), thalamus (TH), and substantia nigra (SN) (Figure 7), indicating that ltHSV-hUbC-tdT-WPRE did not exhibit anterograde transsynaptic spread in the M1 region. These results indicate that ltHSV-hUbC-tdT-WPRE is a relatively rigorous retrograde labeling tool virus that does not proliferate in infected highly differentiated neurons. This makes ltHSV a promising new addition to the arsenal of retrograde labeling tools for studying the structure and function of neural circuit networks.

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

[0053] Two commonly used retrograde labeling tools for projecting neural circuits, rAAV2-retro and RV-SAD19, have different neurotropisms. These two tracers were injected into the lateral geniculate nucleus (DLG) of the thalamus. RV-SAD19G labeled layer VI of the primary visual cortex (V1), while rAAV2-retro retrogradely labeled layer V of V1. rAAV2-retro and RV-SAD19 differ in their efficiency and range of retrograde labeling, with RV-SAD19 having a more localized viral spread and a broader spectrum of neurotropism. To evaluate the neurotropism of ltHSV retrograde labeling, RV-SAD19G and ltHSV-hUbC-tdT-WPRE viruses were mixed with appropriate amounts of CTB and injected into the lateral geniculate nucleus (DLG) in a 200-nl injection volume. Samples were collected 7 days after infection. The DLG is a neural nucleus located in the thalamus. It is the first stop for visual signals in the brain and is responsible for receiving and processing information from the retina. The lateral geniculate nucleus 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, thereby realizing the interaction and integration of visual information between different levels.

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

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

[0056] Given that rAAV2-retro and RV-SAD19 label different layers of the DLG input circuit but fail to label multiple layers of neurons in V1, researchers can choose more retrograde labeling viruses to achieve broader labeling, depending on their research objectives. If the goal is to study specific layers of V1, rAAV2-retro and RV-SAD19G may be better choices. However, if the goal is to retrogradely label neural circuits more broadly, ltHSV-hUbC-tdT-WPRE may be more appropriate.

[0057] In summary, the present invention constructs an attenuated ltHSV-hUbC-tdT-WPRE virus by knocking out the neurovirulence factor γ34.5 gene, which exhibits efficient retrograde labeling and low toxicity. The inventors have constructed a series of low-toxicity ltHSV recombinant virus tools and verified that ltHSV with γ34.5 knockout possesses relatively rigorous retrograde labeling capabilities and a broader spectrum of neurotropism, becoming an important new addition to the arsenal of retrograde labeling tools for studying 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. 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 application 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 application according to claim 2, wherein 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 application according to claim 2, characterized in that The promoter is selected from the hUbC promoter; The fluorescent protein gene is tdTomato.

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

4.

6. The application according to claim 1, wherein Retrograde tracing of neural circuits does not cross synapses.

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