Autophagy extracellular vesicles capable of swallowing viruses as well as preparation method and application of autophagy extracellular vesicles

Abstract

The invention relates to autophagy extracellular vesicles capable of swallowing viruses as well as a preparation method and application of the autophagy extracellular vesicles, and relates to the technical field of biological medicines. The preparation method of the high-purity autophagy extracellular vesicles is provided for the first time, the purity of the high-purity autophagy extracellular vesicles exceeds 85%, and the high-purity autophagy extracellular vesicles are rich in various protein markers. CVB4 virus is used for infecting the autophagy extracellular vesicles, it is found that compared with common extracellular vesicles, the autophagy extracellular vesicles contain complete CVB4 virus particles, and the virus particles carried by the autophagy extracellular vesicles can more effectively infect brain and spleen tissue of a mouse, so that the autophagy extracellular vesicles can be used for treating the brain and spleen tissue of the mouse; therefore, the autophagy extracellular vesicle disclosed by the invention can break through the limitation of a host virus specific receptor, efficiently infect corresponding tissues and enlarge the virus tissue tropism. Therefore, the autophagy extracellular vesicle disclosed by the invention can be used for screening antiviral drugs, and also has important significance for constructing a virus infected animal model.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to an autophagic extracellular vesicle that can phagocytose viruses, and a preparation method and application thereof. Background Art

[0002] The research field of extracellular vesicles has developed rapidly in the past decade, evolving from basic biological research to a discipline with important clinical significance. Extracellular vesicles are natural membrane-derived vesicles actively released by cells into the extracellular environment, encapsulating bioactive components such as proteins, nucleic acids, lipids, and small molecules, with a diameter ranging from 50 nm to 2,000 nm. They are widely involved in cell-to-cell signal transduction and material exchange, and thus have important application values in disease diagnosis, treatment, and biomarker research.

[0003] Autophagy, as a key mechanism for intracellular clearance and metabolic regulation, not only plays an important role in maintaining cell homeostasis but is also closely related to the generation and secretion of extracellular vesicles. Our research has found that autophagic extracellular vesicles (AEVs) are a special type of extracellular vesicles formed and released during autophagy, with unique biological characteristics and are interconnected with the classical autophagy pathway. The formation process is as follows: after autophagosomes fuse with multivesicular bodies (MVBs), they are released into the extracellular environment through exocytosis.

[0004] The isolation of extracellular vesicles is a process of efficiently enriching extracellular vesicles from samples based on their physical and chemical properties. After years of technological development, various isolation techniques have been developed, and the commonly used methods include ultracentrifugation, density gradient centrifugation, polymer precipitation, immunocapture, and size exclusion chromatography, etc.

[0005] Ultracentrifugation is the earliest classical method applied to the isolation of extracellular vesicles and is still the most commonly used separation method at present, often regarded as the "gold standard". This method separates by taking advantage of the difference in sedimentation coefficients between extracellular vesicles and impurity particles. Its advantage lies in being suitable for large-scale separation, but it has problems such as long operation time, low separation efficiency, and insufficient purity.

[0006] Density gradient centrifugation separates according to the density difference of vesicles by adding a density gradient medium to the sample. Although this method can improve the separation purity, the operation is complex and the sample recovery rate is relatively low.

[0007] The polymer precipitation method reduces the solubility of vesicles by using polymers such as polyethylene glycol, causing them to precipitate and separate. The advantage of this method is that it is simple to operate and has a low cost, making it suitable for processing large-volume samples. However, its non-specific precipitation easily leads to the mixing of impurities in the sample, and further purification is required subsequently to improve the purity of the sample.

[0008] The immunocapture method selectively captures specific types of extracellular vesicles by specifically recognizing vesicle surface markers with antibodies. Although this method has high specificity, the selection and optimization of antibodies are the keys to success, and there are certain limitations in large-scale applications. The immunocapture technique has a strong dependence on specific markers of autophagy-related extracellular vesicles.

[0009] Size exclusion chromatography separates vesicles of different sizes by using porous materials. This method can effectively separate particles of different sizes with relatively high purity, but its separation ability is limited, it is difficult to process large-volume samples simultaneously, and the separation effect on smaller vesicles is not ideal.

[0010] The isolation of autophagic extracellular vesicles is similar to that of conventional extracellular vesicles. However, due to their unique biological characteristics and functions, more precise and specific isolation techniques are often required. Although existing ultracentrifugation and density gradient centrifugation methods can initially isolate autophagic extracellular vesicles, their purity and specificity are insufficient. Methods such as the immunocapture method and size exclusion chromatography have improved purity, but they have a strong dependence on markers of autophagic extracellular vesicles, and there is currently little research on specific markers of autophagic extracellular vesicles. Therefore, for autophagic extracellular vesicles, the existing technologies still have limitations such as lack of strong pertinence and unsatisfactory separation effects. Summary of the Invention

[0011] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of a method capable of preparing autophagic extracellular vesicles with high purity.

[0012] To solve the above technical problems, the present invention provides an autophagic extracellular vesicle that can phagocytose viruses, and its preparation method and application. The present invention increases the secretion of autophagic extracellular vesicles by inducing autophagy, and successfully isolates highly pure autophagic extracellular vesicles through multi-step filtration and differential centrifugation. The present invention first proposes a preparation method for highly pure autophagic extracellular vesicles, with a purity exceeding 85% and rich in various protein markers. After infecting the autophagic extracellular vesicles and ordinary extracellular vesicles of the present invention with CVB4 virus, it is found that ordinary extracellular vesicles cannot phagocytose CVB4 protein, while the autophagic extracellular vesicles of the present invention contain intact CVB4 virus particles (CVB4 virus has an escape mechanism and can escape being encapsulated into other extracellular vesicles within cells. Since the autophagy pathway itself can encapsulate and clear viruses, the autophagic extracellular vesicles contain virus particles), and the virus particles carried by the autophagic extracellular vesicles of the present invention can more effectively infect the brain and spleen tissues of mice, break through the restriction of host virus-specific receptors, and efficiently infect the corresponding tissues, expanding the tissue tropism of the virus. Therefore, the autophagic extracellular vesicles of the present invention can be used in the screening of antiviral drugs and are also of great significance for the construction of virus-infected animal models.

[0013] The first object of the present invention is to provide a preparation method for an autophagic extracellular vesicle that can phagocytose viruses, comprising the following steps:

[0014] S1. Subject the cells to stress treatment and centrifuge at 3000g - 10000g to obtain a supernatant. Filter the supernatant through a 0.7 - 0.9 μm filter membrane for the first time to obtain a first filtrate; filter the first filtrate through a 0.20 - 0.24 μm filter membrane for the second time to obtain a second filtrate; filter the second filtrate through a 0.09 - 0.11 μm filter membrane for several times to obtain a third filtrate;

[0015] S2. Centrifuge the third filtrate at 40000 - 60000g for the first time to obtain a first supernatant; centrifuge the first supernatant at 50000 - 70000g for the second time to obtain a second supernatant; centrifuge the second supernatant at 110000g - 130000g for the third time to obtain a precipitate; resuspend the precipitate with sterile PBS to obtain a resuspended solution, and centrifuge the resuspended solution at 110000g - 130000g for the fourth time. The obtained precipitate is the autophagic extracellular vesicle.

[0016] Further, the virus includes Coxsackievirus B.

[0017] Furthermore, the stress treatment in step S1 includes starvation induction of the cells, wherein the starvation induction is to culture the cells using a complete medium, and when the cell density reaches 80-99%, the complete medium is replaced with a serum-free medium and the culture is continued for 24 hours.

[0018] Furthermore, the number of filtering times in step S1 is greater than 3.

[0019] Furthermore, the cells are selected from one or more of 293T cells, THP-1 cells, and Neuro2a cells.

[0020] Furthermore, in step S2, the temperature of the first centrifugation is 0-10°C; the temperature of the second centrifugation is 0-10°C; the temperature of the third centrifugation is 0-10°C; and the temperature of the fourth centrifugation is 0-10°C.

[0021] The second object of the present invention is to provide an autophagic extracellular vesicle prepared by the above preparation method.

[0022] The third object of the present invention is to provide a use of the above-mentioned autophagic extracellular vesicles in screening antiviral drugs, wherein the action sites of the antiviral drugs include the brain and spleen infected by viruses.

[0023] Furthermore, the virus includes CVB4 virus.

[0024] Furthermore, the antiviral drug has the brain and spleen infected by CVB4 virus as the sites of action. When CVB4 virus infects cells or tissues, the viral capsid needs to bind to the specific receptor protein CAR on the cell membrane before entering the cell, while the spleen and brain tissue do not express CAR protein, and theoretically cannot be infected by CVB4. The CVB4 virus particles contained in the autophagic extracellular vesicles do not need the viral capsid protein to bind to the specific receptor when infecting the cell, but rely on the endocytosis pathway of the autophagic extracellular vesicle to enter the cell. The mechanism of autophagic extracellular vesicles entering the cell through the endocytosis pathway currently includes the clathrin-caveolin-dependent pathway, the phagocytic pathway, and the lipid raft-mediated endocytosis pathway, so it breaks through the limitation of specific receptors.

[0025] The fourth object of the present invention is to provide an application of the above-mentioned autophagic extracellular vesicles in preparing a virus-infected animal model.

[0026] Furthermore, the virus-infected animal model includes a virus-infected animal model in which the brain and spleen are infected with CVB4 virus.

[0027] Beneficial effects of the present invention:

[0028] The present invention increases the secretion of autophagic extracellular vesicles by inducing autophagy, and successfully isolates highly pure autophagic extracellular vesicles through multi-step filtration and differential centrifugation. The present invention first proposes a method for preparing highly pure autophagic extracellular vesicles, with a purity exceeding 85% and rich in various protein markers. After infecting the autophagic extracellular vesicles of the present invention with CVB4 virus, it is found that compared with ordinary extracellular vesicles, the autophagic extracellular vesicles of the present invention contain complete CVB4 virus particles, and the virus particles carried by the autophagic extracellular vesicles can more effectively infect the brain and spleen tissues of mice, break through the host virus-specific receptor restriction, efficiently infect the corresponding tissues, and expand the viral tissue tropism. Therefore, the autophagic extracellular vesicles of the present invention can be used in the screening of antiviral drugs and are also of great significance for the construction of virus-infected animal models. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in conjunction with the drawings, where

[0030] Figure 1 is a schematic diagram of the isolation of autophagic extracellular vesicles;

[0031] Figure 2 is the particle size of autophagic extracellular vesicles obtained after preprocessing cells by differential centrifugation at different speeds;

[0032] Figure 3 is the analysis result of nanoparticles;

[0033] Figure 4 is the analysis result of the particle size of autophagic extracellular vesicles;

[0034] Figure 5 is the distribution result diagram of the particle size of autophagic extracellular vesicles;

[0035] Figure 6 is the Western blot analysis result diagram of autophagic extracellular vesicles secreted by HP-1 and Neuro2a cells;

[0036] Figure 7 is the electron microscopy analysis result diagram of autophagic extracellular vesicles extracted from wild-type 293T cells and Rab11-KO cells after infecting with CVB4 virus;

[0037] Figure 8 is the CVB4 content diagram in different tissues of mice after autophagic extracellular vesicles extracted from wild-type 293T cells and Rab11-KO cells infect CVB4 virus and then infect mice;

[0038] Figure 9The survival experiment results of autophagic extracellular vesicles extracted from wild-type 293T cells and Rab11-KO cells after infecting mice with CVB4 virus are shown below. Detailed implementation mode

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0040] Example 1: Preparation of autophagic extracellular vesicles using 293T cells

[0041] Culture 293T cells using 10% DMEM medium. When the cell density reaches about 90%, replace it with serum-free medium and continue to culture for 24 hours to induce autophagy in cells and promote the secretion of autophagic extracellular vesicles.

[0042] Collect the cell supernatant after stress treatment and extract autophagic extracellular vesicles. First, remove large particles and cell debris by low-speed centrifugation (3000g - 10000g), remove cell debris through a 0.8μm filter membrane, then filter through a 0.22μm filter membrane to remove large vesicles, and finally use a 0.1μm filter membrane to filter multiple times (>3 times) to ensure that only smaller vesicles are retained.

[0043] Separate the samples by differential ultracentrifugation. First, centrifuge the filtered supernatant at 40000g, 4°C for 90 minutes, discard the precipitate, and collect the supernatant; centrifuge the supernatant at 60000g, 4°C for 60 minutes, discard the precipitate, and collect the supernatant; centrifuge the supernatant at 120000g, 4°C for 90 minutes; resuspend the precipitate in sterile PBS and centrifuge again at 4°C for 90 minutes. The resulting precipitate is the autophagic extracellular vesicle.

[0044] Characterize the isolated autophagic extracellular vesicles. Use a nanoparticle tracking analyzer to measure the particle size distribution of the vesicles. The particle size analysis results are as Figure 2 and Figure 5 shown. The results show that the particle size of the isolated vesicles is mainly particles of 60 - 100nm, and the purity exceeds 85%.

[0045] The isolated vesicles were analyzed by Western blot, and autophagy-related protein markers such as LC3-II, p62, and NBR1 were detected. Further, proteomic analysis of the isolated vesicles was performed using mass spectrometry, and core autophagy-related proteins and potential functional molecules were detected. As shown in Table 1, after autophagy was induced by starvation in 293T cells, the protein markers of autophagic extracellular vesicles (starv-AEVs) secreted were analyzed by proteomic analysis. It was found that these proteins in Table 1 were highly specifically enriched in autophagic extracellular vesicles, and their expression levels were significantly higher than those of the basal autophagic extracellular vesicles secreted by cells cultured under normal conditions (without autophagy induction, nm-AVEs) (the calculation formula for relative abundance is: (starv-AEVs) / (nm-AVEs)).

[0046] Table 1 Analysis results of protein markers of autophagic extracellular vesicles

[0047] Protein Relative abundance Change RAB13 114.7 Increase TSG101 30.1 Increase CHMP2A 23.3 Increase CHMP1A 22.2 Increase VPS4A 20.5 Increase VPS4B 18 Increase VTA1 13.6 Increase MVB12A 13.5 Increase CHMP2B 13.3 Increase PDCD6IP / Alix 8.5 Increase

[0048] Example 2: Effect of centrifugation speed on purity

[0049] 293T cells were cultured in 10% DMEM medium. When the cell density reached about 90%, the medium was replaced with serum-free medium, and the cells were further cultured for 24 hours to induce autophagy and promote the secretion of autophagic extracellular vesicles.

[0050] The cell supernatant after stress treatment was collected and autophagic extracellular vesicles were extracted. First, large particles and cell debris were removed by low-speed centrifugation (3000g - 10000g), cell debris was removed by a 0.8 μm filter membrane, large vesicles were removed by filtering through a 0.22 μm filter membrane, and finally, the sample was filtered through a 0.1 μm filter membrane multiple times (>3 times) to ensure that only smaller vesicles were retained.

[0051] The sample was separated by differential ultracentrifugation. First, the filtered supernatant was centrifuged for the first time at 4°C for 90 min, the precipitate was discarded, and the supernatant was collected; the supernatant was centrifuged for the second time at 60,000 g and 4°C for 60 min, the precipitate was discarded, and the supernatant was collected; the supernatant was centrifuged for the third time at 120,000 g and 4°C for 90 min to obtain a precipitate; the precipitate was resuspended with sterile PBS and then centrifuged at 120,000 g and 4°C for 90 min, and the resulting precipitate was the autophagy extracellular vesicles. The rotation speeds of the first centrifugation were respectively selected from 20,000 g, 30,000 g, 40,000 g, 50,000 g or 60,000 g to explore the effect of different rotation speeds of the first centrifugation on the purity of autophagy extracellular vesicles. The results of nanoparticle tracking showed that autophagy extracellular vesicles with higher purity could be obtained at a centrifugation speed of 40K g (40,000 g), and increasing the centrifugation speed (50K - 60K g) did not significantly increase its purity.

[0052] Example 3: Preparation of autophagy extracellular vesicles using THP-1 cells

[0053] THP-1 cells were cultured using 10% DMEM medium. When the cell density reached about 90%, the medium was replaced with serum-free medium and cultured for another 24 hours to induce autophagy in cells and promote the secretion of autophagy extracellular vesicles.

[0054] Large particles and cell debris were removed by low-speed centrifugation (3000 g - 10000 g), cell debris was removed by a 0.8 μm filter membrane, large vesicles were removed by filtering through a 0.22 μm filter membrane, and finally, a 0.1 μm filter membrane was used for multiple filtrations (>3 times) to ensure that only smaller vesicles were retained.

[0055] The sample was separated by differential ultracentrifugation. First, the filtered supernatant was centrifuged at 40,000 g and 4°C for 90 min, the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged at 60,000 g and 4°C for 60 min; the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged at 120,000 g and 4°C for 90 min; the precipitate was resuspended with sterile PBS and centrifuged again at 4°C for 90 min, and the resulting precipitate was the autophagy extracellular vesicles.

[0056] The isolated autophagy extracellular vesicles were characterized. Western blot was used to analyze the isolated vesicles, and autophagy-related protein markers such as LC3-II, p62, and NBR1 were detected.

[0057] Example 4: Preparation of autophagy extracellular vesicles using Neuro2a cells

[0058] Neuro2a cells were cultured in 10% DMEM medium. When the cell density reached approximately 90%, the serum-free medium was replaced and the cells were further cultured for 24 hours to induce autophagy and promote the secretion of autophagic extracellular vesicles.

[0059] Large particles and cell debris were removed by low-speed centrifugation (3000g - 10000g), cell debris was removed by passing through a 0.8μm filter membrane, large vesicles were removed by passing through a 0.22μm filter membrane, and finally the sample was filtered multiple times (>3 times) through a 0.1μm filter membrane to ensure that only smaller vesicles were retained.

[0060] The samples were separated by differential ultracentrifugation. First, the filtered supernatant was centrifuged at 40000g, 4°C for 90 min, the pellet was discarded, and the supernatant was collected. The supernatant was then centrifuged at 60000g, 4°C for 60 min; the pellet was discarded, and the supernatant was collected. The supernatant was further centrifuged at 120000g, 4°C for 90 min; the pellet was resuspended in sterile PBS and centrifuged again at 4°C for 90 min. The resulting pellet was the autophagic extracellular vesicles.

[0061] The isolated autophagic extracellular vesicles were characterized. By Western blot analysis, the specific biomarkers of the autophagic extracellular vesicles secreted by THP-1 and Neuro2a cells extracted by the methods of Examples 3 and 4 were shown to be NBR1, p62, LC3-II, CHMP2A, VPS4A / B, Rab13, CD81, etc.

[0062] Example 5

[0063] Wild-type 293T cells (WT cells) and 293T cells with Rab11 gene knockout (Rab11-KO, whose secretion of autophagic extracellular vesicles was blocked) were respectively infected with CVB4 virus, and autophagic extracellular vesicles were extracted by the method of Example 1. Electron microscopy revealed that the autophagic extracellular vesicles secreted by wild-type cells contained intact CVB4 virus particles (about 30nm), but no virus particles were detected in the extracellular vesicles secreted by Rab11-KO cells ( Figure 7 ). When these two types of extracellular vesicles were used to infect mice, it was found that the virus particles carried by autophagic extracellular vesicles could more effectively infect the brain and spleen tissues of mice, which are tissues negative for the CVB4 virus-specific receptor CAR, indicating that the virus particles carried by autophagic extracellular vesicles could break through the restriction of the host virus-specific receptor and efficiently infect the corresponding tissues, expanding the tissue tropism of the virus ( Figure 8 ). The mouse survival experiment confirmed that the virus carried by autophagic extracellular vesicles could cause a higher mortality rate ( Figure 9 ).

[0064] For tissues and organs such as the heart and lungs, when extracting autophagic extracellular vesicles and extracellular vesicles from cell supernatants, a centrifugation speed of 120,000g is used. This speed can also centrifuge down CVB4 virus particles, resulting in the precipitate containing not only autophagic extracellular vesicles but also virus particles. Therefore, in both wild-type cells and Rab11-KO cells, the precipitate after ultracentrifugation of the supernatant contains not only extracellular vesicles but also CVB4 virus particles, and thus can infect tissues and organs such as the heart and lungs of mice that contain CVB4-specific receptors.

[0065] In addition, cells themselves can secrete various extracellular vesicles. For Rab11-KO cells, although the secretion of autophagic extracellular vesicles is blocked, the secretion of other subtypes of extracellular vesicles is normal, such as exosomes and microvesicles. Figure 8 Electron micrographs can show vesicles of other subtypes secreted by Rab11-KO cells. Due to the existence of the CVB4 virus evasion mechanism, these subtypes of vesicles cannot phagocytose CVB4 virus, so CVB4 cannot enter the spleen and brain without CVB4-specific receptors through the endocytic pathway of these vesicles, and thus cannot infect the spleen and brain.

[0066] Comparative Example 1

[0067] 293T cells were cultured in 10% DMEM medium. When the cell density reached about 90%, the medium was replaced with serum-free medium and cultured for another 24 hours to induce autophagy and promote the secretion of autophagic extracellular vesicles.

[0068] The cell supernatant after stress treatment was collected and autophagic extracellular vesicles were extracted. First, large particles and cell debris were removed by low-speed centrifugation (3000g - 10000g), cell debris was removed by a 0.8μm filter membrane, large vesicles were removed by passing through a 0.22μm filter membrane, and finally the sample was filtered multiple times (>3 times) through a 0.1μm filter membrane to ensure that only smaller vesicles were retained.

[0069] The sample was separated by differential ultracentrifugation. First, the filtered supernatant was centrifuged at 120,000g at 4°C for the first time for 90 minutes, the precipitate was discarded, and the supernatant was collected; the supernatant was centrifuged at 120,000g at 4°C for the second time for 60 minutes, and the resulting precipitate was autophagic extracellular vesicles. Detection by nanoparticle tracking analysis showed that the proportion of autophagic extracellular vesicles was approximately 25% - 30%.

[0070] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill 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 enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for preparing autophagic extracellular vesicles for engulfing viruses, characterized in that: The following steps are involved: S1. Stress-treating the cells and centrifuging at 3000g-10000g to obtain a supernatant, filtering the supernatant for the first time under a 0.7-0.9 μm filter membrane to obtain a first filtrate; filtering the first filtrate for the second time under a 0.20-0.24 μm filter membrane to obtain a second filtrate; filtering the second filtrate for several times under a 0.09-0.11 μm filter membrane to obtain a third filtrate; S2, centrifuging the third filtrate for the first time at 40000-60000g to obtain a first supernatant; Centrifuging the first supernatant for a second time at 50,000-70,000 g to obtain a second supernatant; The second supernatant is centrifuged for the third time at 110000g-130000g to obtain a precipitate; the precipitate is resuspended and centrifuged for the fourth time at 110000g-130000g to obtain the autophagic extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: Such viruses include Coxsackievirus B.

3. The preparation method according to claim 1, characterized in that: The stress treatment in step S1 includes starvation induction of the cells, wherein the starvation induction is to culture the cells using a complete medium, and when the cell density reaches 80-99%, the complete medium is replaced with a serum-free medium and the culture is continued.

4. The preparation method according to claim 1, characterized in that: The number of filtering times in step S1 is greater than 3.

5. The preparation method according to claim 1, characterized in that: The cells are selected from one or more of 293T cells, THP-1 cells, and Neuro2a cells.

6. The preparation method according to claim 1, characterized in that: In step S2, the temperature of the first centrifugation is 0-10°C; the temperature of the second centrifugation is 0-10°C; the temperature of the third centrifugation is 0-10°C; and the temperature of the fourth centrifugation is 0-10°C.

7. The autophagic extracellular vesicles prepared by the preparation method according to claims 1-6.

8. The use of the autophagic extracellular vesicles according to claim 7 in screening antiviral drugs, characterized in that: The sites of action of the antiviral drugs include the brain and spleen infected by the virus.

9. The use according to claim 8, characterized in that: Such viruses include Coxsackievirus B.

10. The use of the autophagic extracellular vesicles according to claim 7 in preparing a virus-infected animal model, characterized in that: The virus infection animal model includes a virus infection animal model in which the brain and spleen are infected with Coxsackie B virus.

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