Method for constructing low-class multicellular mode animal infection model of vesicular stomatitis virus

Abstract

The invention discloses a method for constructing a model of vesicular stomatitis virus (VSV) infected low-class multicellular model animal caenorhabditis elegans. The method comprises the following steps: performing VSV virus infection on adult caenorhabditis elegans by using a microinjection mode; and the virus infection titer of the VSV is 10 < 10 >-10 < 12 > PFU. By optimizing conditions such as virus titer, culture temperature and the like, continuous cross-generation propagation of the VSV virus in caenorhabditis elegans is realized for the first time, existence of the VSV virus can still be detected in the 90th generation of progeny nematodes, and stable infection rate, fatality rate and virus load are presented in the cross-generation propagation process; by using the technology, the VSV can be infected with various tissues of the nematodes, and damage or dysfunction of various cells such as epithelium, muscle, nerve and the like can be caused; a fluorescence imaging and pathological detection system is established, so that virus infection dynamics and tissue damage conditions can be monitored in real time; a powerful research tool is provided for identification of virus-host interaction genes, analysis of various pathogenic mechanisms, screening of antiviral drugs and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing a lower multicellular model animal infection model of vesicular stomatitis virus. Background Art

[0002] Viral infections have now become a global problem seriously threatening human health. Viral infection models play a key role in studying the interaction mechanism between the host and the virus, evaluating the efficacy of drugs, and developing treatment methods. However, there are still many deficiencies in existing viral infection models: cell models cultured in vitro can be used to study the general process of viral infection of cells and screen gene products and drugs that interact with the virus, but they cannot simulate the complex physiological environment in vivo.

[0003] Although mammalian viral infection models can better simulate the clinical symptoms and pathological characteristics of human viral infections, due to their long reproductive cycle, limited number of offspring, difficult genetic manipulation, high maintenance cost, and ethical concerns, they are not suitable for large-scale screening of genes and drugs.

[0004] To make up for the deficiencies of mammalian viral infection models, researchers hope to use non-mammalian model organisms Drosophila melanogaster and Caenorhabditis elegans to construct viral infection models, so as to achieve large-scale gene and drug screening at a lower cost. Unfortunately, although Caenorhabditis elegans is significantly superior to Drosophila melanogaster in terms of the convenience of genetic editing, cryopreservation and experimental observation, due to the extremely small number of natural viruses that infect Caenorhabditis elegans in nature, the construction of a Caenorhabditis elegans viral infection model faces many challenges, which greatly hinders the wide application of Caenorhabditis elegans in the field of virology research.

[0005] Among the thousands of viruses existing in nature, so far, people have only discovered one virus that can infect Caenorhabditis elegans, called Orsay Virus (OV virus), which belongs to single-stranded positive-strand RNA virus. The OV virus can only infect 1 to 6 cells in the nematode intestine, cannot be vertically transmitted, and the OV virus infection is not fatal, making it difficult to evaluate the pathological characteristics of the infection. Secondly, after the OV virus infects Caenorhabditis elegans, it can only be detected by in situ hybridization experiments, QPCR or WB experiments, and it is impossible to directly label the virus with GFP. Most importantly, the OV virus is neither a representative model virus nor a virus related to human diseases, so the research value of the OV virus-infected Caenorhabditis elegans model is relatively limited.

[0006] In view of the many limitations of existing viral infection models, the present invention aims to develop a simple and easy-to-operate Caenorhabditis elegans viral infection model suitable for large-scale screening using the model virus with a wide host - VSV (vesicular stomatitis virus), so as to promote the application of Caenorhabditis elegans in the field of virology research. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to solve the disadvantages existing in the prior art, and to propose a method for constructing an infection model of a lower multicellular model animal with vesicular stomatitis virus. By only one microinjection combined with temperature conversion culture, a large number of nematodes infected with the virus can be obtained, which can provide a powerful research tool for the identification of virus-host interaction genes and the screening of antiviral drugs.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] A method for constructing an infection model of a lower multicellular model animal with vesicular stomatitis virus, the construction method comprising the following steps:

[0010] S1: Label the vesicular stomatitis virus with GFP green fluorescent protein, and introduce the GFP green fluorescent protein-labeled vesicular stomatitis virus into the body of adult Caenorhabditis elegans by microinjection;

[0011] S2: Place the adult Caenorhabditis elegans infected with the GFP green fluorescent protein-labeled vesicular stomatitis virus in a culture environment of 25±0.1°C;

[0012] S3: Transfer the Caenorhabditis elegans to 20±0.1°C for subculture;

[0013] S4: Through the amplification and concentration of the GFP green fluorescent protein-labeled vesicular stomatitis virus, the infection dynamics of the virus are monitored in real time.

[0014] Furthermore, the vesicular stomatitis virus is a continuously passaged vesicular stomatitis virus.

[0015] Furthermore, in step S1, the microinjection step is as follows:

[0016] A: Pick Caenorhabditis elegans at the L4 or young adult stage for microinjection experiments;

[0017] B: Take out the pre-packaged and cryopreserved concentrated solution of GFP green fluorescent protein-labeled vesicular stomatitis virus, thaw it at room temperature and then centrifuge it in a centrifuge at a rotation speed and time of 10000 rpm / 1 min to remove insoluble substances in the liquid;

[0018] C: Aspirate the GFP green fluorescent protein-labeled vesicular stomatitis virus and drop it to the tail of the syringe, and use the siphon effect to suck the virus solution to the needle part;

[0019] D: Using a microinjection instrument, inject 10 10 -10 12The concentrated solution of vesicular stomatitis virus labeled with GFP green fluorescent protein of PFU was injected into the gonad of nematodes. The femtojet Pc pressure and time used for injecting nematodes were 30 psi and 2 s, respectively.

[0020] Furthermore, the specific steps for subculture are as follows:

[0021] a: Place 5 microinjected Caenorhabditis elegans in each NGM petri dish.

[0022] b: Place the NGM petri dish containing Caenorhabditis elegans in a constant temperature incubator at 25 °C for 72 hours of culture.

[0023] c: Adjust the culture temperature to 20 °C and continue to culture for 24 hours.

[0024] d: When the first-generation develops to the adult stage, pick all the GFP-positive first-generation adults in the dish and place them on a new petri dish.

[0025] e: Place the petri dish in a constant temperature incubator at 20 °C for culture.

[0026] f: Observe the proportion of virus-carrying in each clone of the second-generation under a stereoscopic fluorescence microscope. Select GFP-positive second-generation nematodes from the clones with a higher VSV virus passaging rate. Place every three nematodes in an NGM petri dish and passage 10 dishes for large-scale culture.

[0027] Furthermore, the specific operating steps for the amplification and concentration of GFP green fluorescent protein-labeled vesicular stomatitis virus are as follows:

[0028] Step1. When VERO cells are cultured in 2 petri dishes until 100% cell adhesion, pour out the old culture medium, and add 9 ml of cell culture medium containing 10 μl of 10 7 PFU GFP green fluorescent protein-labeled vesicular stomatitis virus, that is, the concentrated solution of vesicular stomatitis virus expressing green fluorescent protein, to each petri dish.

[0029] Step2. After 24 hours of infection, use an inverted fluorescence microscope to observe the cell infection situation: When the cells become round and the whole dish of cells shows GFP fluorescence, seal the petri dish with plastic film, place it at -80 °C for freezing and then thaw on ice.

[0030] Step3. Repeatedly freeze-thaw according to Step2 to lyse the cells, collect all cell lysates, collect the virus supernatant at the same time, and filter it using a filter.

[0031] Step4. Concentrate the virus supernatant using the PEGeasy virus concentration reagent, and add pre-cooled DMEM serum-free medium according to a 200-fold concentration factor.

[0032] Step 5. Detect the TCID50 value of Vero cells infected with the virus, and obtain 10 10 -10 12 PFU of the concentrated vesicular stomatitis virus labeled with GFP green fluorescent protein, and store it at -80 °C.

[0033] Furthermore, the cell culture medium contains 88.995% DMEM, 10% serum, 1% double antibody, and 0.005% mycoplasma inhibitor.

[0034] Furthermore, during the collection of the virus supernatant, take the cell lysate and centrifuge it at 2200 g for 10 min at 4 °C, collect the virus supernatant, and filter it using a 0.45 μm filter.

[0035] A lower multicellular model animal infection model of vesicular stomatitis virus, which is constructed by the method for constructing a lower multicellular model animal infection model of vesicular stomatitis virus described above.

[0036] The above-mentioned lower multicellular model animal infection model of vesicular stomatitis virus is applied to the research in the field of virology.

[0037] Advantages of the present invention:

[0038] 1. The integrated innovation of high-titer virus microinjection and temperature conversion culture method: The present invention combines microinjection of high-titer virus with the temperature conversion culture method, and for the first time realizes the continuous transgenerational transmission of the model virus VSV in Caenorhabditis elegans, solving the problem that the virus cannot be passaged in Caenorhabditis elegans.

[0039] 2. The batch culture of Caenorhabditis elegans virus infection: Utilize the vertical transmission of VSV virus in C. elegans to achieve the batch culture of model virus-infected C. elegans, thus meeting the needs of large-scale genetic screening and drug screening.

[0040] 3. The visualization of virus detection: Caenorhabditis elegans is transparent all over, and the VSV virus used in this model is labeled with green fluorescent protein GFP. Therefore, the change in virus load and the passage situation can be conveniently observed through a low-magnification stereomicroscope, providing great convenience for subsequent screening of genes, drugs, and environmental factors that regulate virus replication and vertical transmission. Brief Description of the Drawings

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0042] Figure 1 This is the overall flowchart of the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention.

[0043] Figure 2 This is a schematic diagram of the continuous transgenerational transmission of VSV virus in Caenorhabditis elegans by microinjection combined with temperature shift culture method in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention;

[0044] Figure 3 This is a schematic diagram of the VSV virus in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention that can infect various tissue cells during the transgenerational transmission process;

[0045] Figure 4 This is a schematic diagram of the necrosis of the epidermal epithelial cells of Caenorhabditis elegans caused by VSV virus infection in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention;

[0046] Figure 5 This is a schematic diagram of the dysfunction of body wall muscles and motor nerves of Caenorhabditis elegans caused by VSV virus infection in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention;

[0047] Figure 6 This is a schematic diagram of the dysfunction of gustatory nerve cells of Caenorhabditis elegans caused by VSV virus infection in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention;

[0048] Figure 7 This is a schematic diagram of the relatively constant viral load, infection rate and lethality rate of VSV virus during the transgenerational transmission process in the method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals proposed by the present invention; Detailed implementation manners

[0049] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] Example 1

[0051] The present invention provides a method for constructing an infection model of vesicular stomatitis virus in lower multicellular model animals, as Figure 1As shown, the model introduced a high titer of VSV virus into Caenorhabditis elegans by microinjection to increase the initial exposure of the virus.

[0052] Subsequently, the virus-infected Caenorhabditis elegans were placed in a culture environment at 25°C to accelerate the replication and diffusion process of the VSV virus. When the virus replication reached a certain level, the Caenorhabditis elegans were transferred to 20°C for subculture to promote the survival rate of the virus-infected nematodes and their offspring, thus achieving stable and continuous transgenerational transmission of the VSV virus in nematodes.

[0053] In addition, the VSV virus labeled with GFP green fluorescent protein was used in this model, enabling researchers to monitor the infection dynamics of the virus in real time.

[0054] The operation of this invention is simple. Only one microinjection combined with temperature-converted culture is required to obtain a large number of virus-infected nematodes. Therefore, it can provide a powerful research tool for the identification of virus-host interaction genes and the screening of antiviral drugs.

[0055] Example 2

[0056] Unless otherwise specified, the experimental methods in the following implementation cases are all conventional methods.

[0057] Implementation Case 1: Amplification and concentration of vesicular stomatitis virus labeled with GFP green fluorescent protein.

[0058] When VERO cells were cultured in 2 100-mm culture dishes until 100% cell adhesion, the old culture medium was poured off. 9 ml of cell culture medium (high-glucose DMEM + 10% serum + 1% double antibody + mycoplasma inhibitor) containing 10 μl of 10^7 PFU VSV-GFP (VSV expressing green fluorescent protein) virus concentrate was added to each culture dish. After 24 hours of infection, the cell infection situation was observed using an inverted fluorescence microscope. When the cells were basically all rounded and the whole dish of cells showed GFP fluorescence, the culture dish was sealed with plastic film and placed at -80°C for freezing and then thawed on ice. This freezing and thawing process was repeated three times to lyse the cells, and all cell lysates were collected. Centrifugation was performed at 2200 g for 10 minutes at 4°C, the virus supernatant was collected, and filtered using a 0.45-μm filter. Then, the virus supernatant was concentrated using the PEGeasy virus concentration reagent, and pre-cooled serum-free DMEM medium was added according to a 200-fold concentration factor. The TCID50 value of the virus infecting VERO cells was detected to obtain a GFP green fluorescent protein-labeled vesicular stomatitis virus concentrate of 10^10 - 10^12 PFU. It was stored at -80°C.

[0059] Implementation Case 2: Microinjection of virus-infected Caenorhabditis elegans.

[0060] Pick about 50 Caenorhabditis elegans at the L4 or young adult stage for microinjection experiments. Take out the pre-aliquoted and cryopreserved concentrated vesicular stomatitis virus labeled with GFP green fluorescent protein, thaw it at room temperature and then centrifuge it in a centrifuge at a speed and time of 10000 rpm / 1 min to remove insoluble substances in the liquid to avoid clogging the needle. Aspirate 1 μl of VSV-GFP virus and add it to the tail of the syringe, and use the siphon effect to suck the virus solution to the needle part. To avoid the harm of VSV-GFP virus to the human body, wipe off the virus solution at the tail of the needle after each addition of the virus to prevent the virus from remaining on the microinjector. Use a microinjector to inject 1010 - 1012 PFU of concentrated vesicular stomatitis virus labeled with GFP green fluorescent protein into the gonad of the nematode. The nematode microinjection technique is described in full in the following literature: Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. (by Mello et al., 1991). The pressure and time of the femtojet Pc used for injecting nematodes are 30 psi and 2 s respectively.

[0061] Example 3: Passage of virus-infected nematodes.

[0062] Place the microinjected nematodes at 5 per dish in a 6-cm diameter NGM culture dish. Then, place these culture dishes in a constant temperature incubator at 25 °C for 72 hours of culture. After that, adjust the culture temperature to 20 °C and continue to culture for 24 hours. When the first-generation (F1) develops to the adult stage, pick all the GFP-positive F1 adults in the dish and place them on a new culture dish, with only one nematode per dish. Then, place these culture dishes in a constant temperature incubator at 20 °C for culture. Observe the proportion of virus-carrying in each clone of the second-generation (F2) under a stereoscopic fluorescence microscope, select the GFP-positive F2-generation nematodes from the clones with a higher VSV virus passage rate, and place every three nematodes in a 6-cm NGM culture dish for about 10 dishes of subculture for expansion.

[0063] The experimental results are as follows:

[0064] Stereoscopic fluorescence pictures show that the VSV virus can infect the parental generation, the first-generation (F1), the second-generation (F2) to the sixth-generation (F6) Figure 2A) in the previous text. So far, the C. elegans strain infected with the VSV virus has been cultured to about the 90th generation, and basically achieved the infinite transgenerational transmission of the VSV virus in C. elegans and the acquisition of a large number of infected individuals. Stereo fluorescence images show that there is almost no difference in the overall fluorescence signal among different offspring ( Figure 2 B) in the previous text, indicating that the virus does not reduce the viral load of nematode individuals with the increase of the number of generations during transgenerational transmission. Confocal imaging results show that the VSV virus can completely infect the whole body tissues of C. elegans among different offspring, including the head central nervous tissue, epidermis, muscle, intestine, vulva and other major organs ( Figure 3 ).

[0065] This result indicates that the virus can be effectively diffused to multiple organs throughout the body, including the reproductive system, during transgenerational transmission, providing an explanation at the tissue level for its stable continuous transgenerational transmission phenomenon. At the same time, this result also shows that this model can be applied to study the systemic and whole-body antiviral immune response of C. elegans, thus making more academic contributions to revealing the complete antiviral immune strategy of C. elegans.

[0066] It should be noted that the staining results of propidium iodide (PI) for labeling necrotic cells show that VSV virus infection can cause necrosis of the epidermal epithelial cells of C. elegans, but does not cause obvious necrosis of other tissue cells ( Figure 4 ). This result shows that similar to the situation in higher animals, the VSV virus also has obvious epitheliotropism in C. elegans.

[0067] In addition, quantitative analysis of the movement speed, body bending angle and frequency of C. elegans individuals shows that VSV infection can cause functional defects in the body wall muscles and motor nervous system of C. elegans ( Figure 5 ). The results of chemotaxis experiment analysis show that VSV virus infection can cause damage to the gustatory nervous system of C. elegans ( Figure 6 ).

[0068] The infection rate of C. elegans infected with the virus by microinjection in the F2 generation is about 30%, and the average infection rate of its subsequent offspring is close to 10%, and gradually stabilizes with the increase of the number of infectious generations ( Figure 7 A) in the previous text. The average viral load of the VSV virus in C. elegans individuals of each generation basically remains constant ( Figure 7 B) in the previous text. The detection of the survival rate of nematode individuals carrying the virus shows that the VSV virus can cause lethality in about 60% of the positive individuals ( Figure 7 C) in the previous text. The highest infection rate of the offspring of a single virus-carrying individual can reach about 40% ( Figure 7 D) in the previous text.

[0069] The above experimental results indicate that this technology has successfully achieved the continuous trans - generational transmission of VSV virus in Caenorhabditis elegans, and has a relatively constant infection rate, viral load, and lethality rate, providing a simple and practical model for in - depth study of novel antiviral mechanisms in nematodes.

[0070] Compared with the existing non - nematode virus infection models, the advantages of the present invention are as follows:

[0071] (1) Greatly reduce the experimental cost and time:

[0072] Defects of the prior art: The current non - nematode experimental animal models used for virus infection research have the problems of high feeding cost and long experimental period, so they are not suitable for large - scale gene and drug screening.

[0073] Advantages and benefits of the present invention: Through single - time microinjection and simple culture temperature conversion operations, the present invention has achieved the infinite vertical transmission of GFP - labeled vesicular stomatitis virus in Caenorhabditis elegans. At the same time, due to the strong reproductive ability and fast life cycle of Caenorhabditis elegans, this invention can obtain a large number of nematode samples infected with GFP - labeled vesicular stomatitis virus in a short time, thus providing sufficient experimental materials for large - scale screening experiments.

[0074] (2) Simplify the monitoring means:

[0075] Defects of the prior art: The methods for monitoring virus infection in existing virus - infected animal models are complex and often rely on means such as histological staining and molecular biology detection.

[0076] Advantages and benefits of the present invention: The VSV virus used in the present invention is self - labeled with green fluorescent protein (GFP), and combined with the transparent body of Caenorhabditis elegans itself, enabling researchers to real - time monitor the replication level and distribution of the virus through a stereoscopic fluorescence microscope. Therefore, this model not only greatly facilitates the dynamic analysis of virus infection, but also provides an intuitive and efficient reporting system for large - scale screening experiments.

[0077] (3) Reduce the difficulty of genetic manipulation:

[0078] Defects of the prior art: The genetic manipulation of non - nematode experimental animals used in existing virus infection models is difficult and costly.

[0079] Advantages and benefits of the present invention: The model organism Caenorhabditis elegans selected in the present invention has strong technical advantages in genetic screening and can efficiently perform forward and reverse genetic screening at low cost and technical difficulty. Therefore, the present invention can provide a powerful research tool for large - scale screening of virus - interacting genes.

[0080] Compared with the existing Caenorhabditis elegans virus infection model, the advantages of the present invention are as follows:

[0081] (1)Enhance the generality of research results:

[0082] Defects of the prior art: The commonly used Caenorhabditis elegans virus infection model in the prior art uses the nematode-specific pathogenic virus OV, which limits the generality and comparability of research findings.

[0083] Advantages and benefits of the present invention: The present invention uses the model virus VSV with a wide range of hosts, making the research results easier to compare with other biological systems and helping to reveal the general laws of the interaction between viruses and multicellular organisms. Using this model to deeply explore the ancient and conserved antiviral innate immune strategies contained in Caenorhabditis elegans is expected to provide a new perspective and approach for the prevention and treatment of viral diseases.

[0084] (2)Suitable for the study of antiviral immune mechanisms in multiple tissues:

[0085] Defects of the prior art: The OV virus used in the existing Caenorhabditis elegans virus infection model can only infect the intestinal cells of nematodes, which limits the comprehensive study of the body's antiviral immune response.

[0086] Advantages and benefits of the present invention: The present invention realizes the spread of the VSV virus in all tissue cells of different generations of nematodes, which is suitable for studying the systemic and whole-body antiviral immune response of nematodes, thus helping to reveal the complexity and tissue specificity of the nematode antiviral immune network.

[0087] (3)Vertical transmission from infected individuals to offspring can be achieved

[0088] Defects of the prior art: The OV virus used in the existing Caenorhabditis elegans virus infection model can only infect 1 to 6 cells in the nematode intestine and cannot be vertically transmitted, which limits the feasibility of large-scale screening.

[0089] Advantages and benefits of the present invention: The present invention realizes the continuous transgenerational transmission of the VSV virus in nematodes. Using this vertical transmission, the batch infection of Caenorhabditis elegans by the model virus is achieved, thus meeting the needs of large-scale genetic screening and drug screening.

[0090] (4)Can be used to deeply study tissue and cell damage and pathogenic mechanisms caused by virus infection:

[0091] Defects of the prior art: The OV virus infection is not fatal, making it difficult to evaluate the pathological characteristics of the infection, which limits the research on virus pathogenic mechanisms and the assessment of the severity of the infection.

[0092] Advantages and benefits of the present invention: VSV virus infection can cause necrosis of the epidermal epithelial cells of nematodes, and VSV virus can cause mortality in about 60% of positive individuals. The pathological characteristics of these infections are conducive to a deep understanding of the viral pathogenic mechanism, promote in-depth virological research, and provide a basis for vaccine and drug research and development.

[0093] (5) Intuitive and clear detection methods:

[0094] Deficiencies of existing technologies: After OV virus infects nematodes, it can only be detected through in situ hybridization experiments, QPCR or WB experiments, which are complex, time-consuming and labor-intensive.

[0095] Advantages and benefits of the present invention: The VSV virus used in the present invention carries a green fluorescent protein (GFP) marker, which enables researchers to conveniently monitor the changes in virus load and distribution in real time through a low-magnification fluorescence microscope, which not only greatly facilitates the dynamic analysis of viral infection, but also provides an intuitive and efficient reporting system for subsequent large-scale screening experiments.

[0096] (6) Higher potential clinical practical value:

[0097] Deficiencies of the existing technology: The OV virus used in the existing C. elegans virus infection model has very low virological similarity to human pathogenic viruses, which limits the practical value of the model.

[0098] Advantages and benefits of the present invention: The vesicular stomatitis virus (VSV) used in the present invention is a model virus with a wide range of hosts including humans. The model virus is widely used in the study of human disease pathology related to various viral infections, antiviral drug screening, and vaccine development. Therefore, the research results obtained using the nematode infection model of the virus are easier to be transformed into clinical applications and have more practical value.

[0099] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0100] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus, characterized in that: The construction method comprises the following steps: S1: Vesicular stomatitis virus was labeled with green fluorescent protein GFP and introduced into adult Caenorhabditis elegans by microinjection; S2: Adult Caenorhabditis elegans infected with vesicular stomatitis virus labeled with green fluorescent protein (GFP) were placed in a culture environment at 25±0.1℃; S3: Transfer C. elegans to 20 ± 0.1°C for subculture; S4: Real-time monitoring of viral infection dynamics by amplifying and concentrating vesicular stomatitis virus labeled with green fluorescent protein (GFP).

2. The method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus according to claim 1, characterized in that: The vesicular stomatitis virus is a continuously passaged vesicular stomatitis virus.

3. The method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus according to claim 1, characterized in that: In step S1, the microinjection step is: A: Select L4 or young adult stage C. elegans for microinjection experiments; B: Take out the pre-packaged frozen GFP green fluorescent protein labeled vesicular stomatitis virus concentrate, thaw it at room temperature, and centrifuge it at a speed and time of 10000rpm / 1min to remove the insoluble matter in the liquid; C: Pipette GFP green fluorescent protein-labeled vesicular stomatitis virus and drop it into the tail of the syringe, and use the siphon effect to suck the virus liquid to the needle tip; D: Using a microinjection instrument, 10 10 -10 12 PFU of GFP green fluorescent protein labeled vesicular stomatitis virus concentrate was injected into the nematode gonads. The pressure and time of the femtojet PC used for injection of nematodes were 30 psi and 2 s, respectively.

4. The method for constructing a model of vesicular stomatitis virus infection in Caenorhabditis elegans according to claim 1, characterized in that: In step S3, the specific steps of subculturing are: a: Microinjected Caenorhabditis elegans were placed in NGM culture dishes at a rate of 5 per dish; b: The NGM culture dish containing Caenorhabditis elegans was placed in a constant temperature incubator at 25°C for 72 hours; c: Adjust the culture temperature to 20°C and continue culturing for 24 hours; d: When the first generation develops to the adult stage, pick all the GFP-positive first generation adults in the dish and put them into a new culture dish; e: Place the culture dish in a constant temperature incubator at 20°C for culture; f: Observe the proportion of virus-carrying second-generation offspring of each clone under a stereofluorescence microscope, select GFP-positive second-generation nematodes from clones with a higher VSV virus passage rate, place every three nematodes in an NGM culture dish, and subculture 10 dishes for expansion.

5. The method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus according to claim 1, characterized in that: In step S4, the specific operation steps of amplifying and concentrating vesicular stomatitis virus labeled with GFP green fluorescent protein are as follows: Step 1. Cultivate VERO cells in two culture dishes until the cells are 100% attached to the wall. Pour out the old culture medium and add 10 μl of 10 7 PFU GFP green fluorescent protein labeled vesicular stomatitis virus, i.e. 9 ml cell culture medium of vesicular stomatitis virus concentrate expressing green fluorescent protein; Step 2. 24 hours after infection, use an inverted fluorescence microscope to observe the cell infection: when the cells become round and the entire dish of cells shows GFP fluorescence, seal the culture dish with plastic film and freeze it at -80℃ and then thaw it on ice; Step 3, repeat step 2 and freeze-thaw to lyse the cells, collect all the cell lysate, collect the virus supernatant at the same time, and filter it through a filter; Step 4, use PEGeasy virus concentration reagent to concentrate the virus supernatant, and add pre-cooled DMEM serum-free medium according to the concentration factor of 200; Step 5, detect the TCID50 value of virus-infected VERO cells and obtain 10 10 -10 12 PFU of GFP green fluorescent protein-labeled vesicular stomatitis virus concentrate and stored at -80°C.

6. The method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus according to claim 5, characterized in that: The cell culture medium contains 88.995% DMEM, 10% serum, 1% double antibody and 0.005% mycoplasma inhibitor.

7. The method for constructing a low-level multicellular model animal infection model of vesicular stomatitis virus according to claim 5, characterized in that: Collect the virus supernatant, take the cell lysate and centrifuge it at 2200g at 4°C for 10 min, collect the virus supernatant and filter it using a 0.45 μm filter.

8. A low multicellular animal infection model of vesicular stomatitis virus, characterized in that: The lower multicellular model animal infection model of vesicular stomatitis virus is constructed by the method for constructing a lower multicellular model animal infection model of vesicular stomatitis virus according to any one of claims 1-7.

9. The low multicellular animal infection model of vesicular stomatitis virus as claimed in claim 8 is applied to research in the field of virology.

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