Grass carp brain cell line sensitive to grass carp reovirus type II and application thereof

Abstract

The invention relates to the technical field of biology. The invention provides a grass carp brain cell line sensitive to grass carp reovirus type II, which is preserved in China Center for Type Culture Collection on October 19, 2024, and the preservation number is CCTCC NO: C2024101. The constructed grass carp brain cell line shows remarkable stability, and stable passage is successfully achieved for 60 times. And when GCRV-II is inoculated, an atypical cytopathic effect can be presented. In addition, the grass carp brain cell line has the capability of efficiently replicating GCRV-II, and even if blind passage is carried out for three generations, generated cytotoxicity can still cause morbidity and death of the grass carp. Therefore, a stable foundation is built for deeply exploring the pathogenic mechanism of the GCRV-II, developing vaccine development, screening antiviral drugs and the like, research in related fields is promoted to further progress, and a key cell model and a research basis are provided for development of related research and prevention and control strategies of the grass carp reovirus.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a grass carp brain cell line sensitive to grass carp reovirus type II and an application thereof. Background Art

[0002] Grass carp (Ctenopharyngodon idella, Cypriniformes, Cyprinidae, subfamily Leucophaginae) is an important freshwater aquaculture species in my country and is one of the "four major carps". Its aquaculture output often ranks first among freshwater fish. However, during the aquaculture process, diseases frequently occur, which seriously hinders the health, stability and sustainable development of grass carp aquaculture. Grass carp hemorrhagic disease caused by Grass Carp Reovirus (GCRV) is a common and highly contagious disease in grass carp aquaculture. It mainly harms the grass carp species of the year, with a high mortality rate and significant economic losses. Based on the gene sequence, researchers divided GCRV into three main genotypes: I, II and III. Among them, the GCRV-II strain has the strongest virulence, the highest pathogenicity and mortality rate, and is the main epidemic strain of grass carp hemorrhagic disease in my country. This strain has a wide range of prevalence, strong virulence, high pathogenicity and mortality rate, and is the main cause of frequent outbreaks of grass carp hemorrhagic disease, but its pathogenic mechanism remains to be clarified.

[0003] Compared with in vivo research, cell research has the advantages of small differences, good repeatability, and low cost, making cell culture an important research method in biology. Brain cells of grass carp (GCBr cells) are derived from grass carp brain tissue. In the physiological activities of grass carp, the brain plays a key role, participating in the perception of the external environment, the regulation of motor behavior, and the regulation of internal physiological processes. In the field of scientific research, GCBr cells are important materials for the study of fish neurobiology, neurodevelopment, neurological diseases, and environmental impacts. Research on them helps to deeply understand the operating mechanism of the fish nervous system and provide a basis for disease prevention and control and growth regulation in fish farming.

[0004] Virus-sensitive cells are indispensable in virology research and are widely used in virus replication, isolation and identification, pathogenic mechanism research, antibody and vaccine preparation, etc. After the virus infects cells, it usually produces cytopathic effect (CPE), which is crucial to understanding the characteristics of viral infection and the response of host cells to viral infection. Although the GCRV-Ⅱ strain has the highest pathogenicity and mortality rate at the in vivo level, its cell sensitivity is weak. It cannot produce obvious CPE when inoculated with existing fish cells, has low proliferation titers in existing cells, and its cytotoxicity cannot cause grass carp to become ill. This brings many difficulties to its infection and pathogenic mechanism research, diagnosis and prevention and control product development. Due to the lack of GCRV-Ⅱ sensitive cell line as a basic tool, the research on its pathogenic mechanism and immune prevention and control is greatly limited. Therefore, the establishment of sensitive cell lines has become a top priority and is a necessary prerequisite for in-depth exploration of viral invasion, infection and pathogenic mechanisms and the development of immune prevention and control. Summary of the invention

[0005] In view of this, the present invention proposes a grass carp brain cell line sensitive to grass carp reovirus type II and its application. The present invention takes grass carp, the natural host of grass carp reovirus type II (GCRV-II), as the core research object, and successfully constructs a grass carp brain cell line (GCBr cell line). The grass carp brain cell line has been stably passaged for 60 generations, and after inoculation with GCRV-II, it can show atypical cytopathic effect (CPE). The grass carp brain cell line constructed by the present invention can achieve efficient replication of GCRV-II. Even after three generations of blind transmission, the cytotoxicity produced can still cause grass carp to become ill and die. This shows that the virus replicated in the cell still maintains a high level of virulence, laying a solid foundation for subsequent related research and applications.

[0006] The technical solution of the present invention is achieved in this way:

[0007] In the first aspect, the present invention provides a grass carp brain cell line sensitive to grass carp reovirus type II, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit number CCTCC NO: C2024101.

[0008] Furthermore, the present invention provides a method for culturing the grass carp brain cell line, wherein the grass carp brain cell line is cultured in an L-15 culture medium containing 10-15% v / v fetal bovine serum at a culture temperature of 28°C.

[0009] Furthermore, the present invention provides the use of the grass carp brain cell line in aquatic virus culture.

[0010] Furthermore, the present invention provides the use of the grass carp brain cell line in the separation of aquatic viruses.

[0011] Furthermore, the present invention provides the use of the grass carp brain cell line in the preparation of aquatic virus vaccines or detection products.

[0012] Furthermore, the present invention provides the use of the grass carp brain cell line in the cultivation of grass carp reovirus.

[0013] Furthermore, the present invention provides the use of the grass carp brain cell line in the isolation of grass carp reovirus.

[0014] Furthermore, the present invention provides the use of the grass carp brain cell line in the preparation of grass carp reovirus vaccine or detection product.

[0015] Furthermore, the present invention provides an application of the grass carp brain cell line in drug screening, characterized in that the drug is used to prevent and treat grass carp reovirus infection, or to prevent and treat diseases caused by grass carp reovirus infection.

[0016] Furthermore, the present invention provides an application of the grass carp brain cell line in drug evaluation, characterized in that the drug is used to prevent and treat grass carp reovirus infection, or to prevent and treat diseases caused by grass carp reovirus infection.

[0017] The beneficial effects of the present invention include at least the following:

[0018] The present invention selects grass carp, the natural host of GCRV-II, to carry out research. By isolating grass carp brain tissue, a grass carp brain cell line (GCBr cell line) is successfully obtained. The grass carp brain cell line shows good stability and has successfully achieved stable passage to 60 generations, which provides a strong guarantee for its reliability in long-term research and application. After inoculation with GCRV-II, the cells show atypical CPE, which indicates the uniqueness of the interaction between the GCBr cells and GCRV-II, and provides important clues for in-depth exploration of the molecular mechanism between the two.

[0019] The grass carp brain cell line provided by the present invention has the ability to efficiently replicate GCRV-II. After three generations of blind propagation, the cytotoxicity produced can still cause the grass carp to become ill and die, which fully proves that the virus replicated in the cell can maintain a high level of virulence. This not only shows that the GCBr cell line can be used as an ideal GCRV-II amplification tool, providing an effective way for the large-scale preparation of the virus, but also lays a solid foundation for further research on the pathogenic mechanism, vaccine development, and antiviral drug screening of GCRV-II. In the process of vaccine development, the GCBr cell line can be used to efficiently amplify the virus, and then prepare a more immunogenic vaccine candidate strain; in terms of pathogenic mechanism research, by observing the details of the interaction between cells and viruses, it is helpful to reveal the key links and molecular targets of GCRV-II infection of grass carp, thereby providing a theoretical basis for the formulation of precise prevention and control strategies.

[0020]

Term Explanation

[0021] Cytopathic Effect (CPE): also known as typical CPE. It refers to a series of morphological changes in host cells that can be observed under an optical microscope, caused by the proliferation of the virus and its interaction with the host cells after the virus infects sensitive host cells. These morphological changes have typical characteristics and are one of the important bases for judging virus infection and preliminary identification of the virus.

[0022] Atypical CPE: Compared with typical CPE, atypical CPE refers to a series of unusual cell morphological changes that do not conform to the characteristics of conventional CPE after viral infection of cells. These changes may be subtle and difficult to directly identify through conventional microscopic observation; or they may be similar to cell morphological changes caused by other factors (such as natural cell aging process, drug toxicity, non-viral microbial infection, etc.), which increases the difficulty of accurate identification.

[0023] Blind virus transmission: The process of inoculating a sample suspected of containing a virus into sensitive host cells or host animals for serial subculture or inoculation in the absence of typical cytopathic effect (CPE) or virus detection confirmation.

[0024]

Biological Conservation

[0025] The grass carp brain cell line GCBr (Ctenopharyngodon idella) provided by the present invention has been deposited in the China Center for Type Culture Collection (abbreviated as CCTCC) on October 14, 2024, with a deposit number of CCTCC NO: C2024101, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 The observation results of cell morphology and growth status at different stages during the construction of the grass carp brain cell line of the present invention;

[0028] Figure 2 is the growth curve of the grass carp brain cell system of the present invention under different culture medium conditions;

[0029] Figure 3The growth curves of the grass carp brain cell system of the present invention at different culture temperatures;

[0030] Figure 4 This is the growth curve of the grass carp brain cell line of the present invention at different FBS concentrations;

[0031] Figure 5 The staining result of metaphase chromosomes of grass carp brain cells under an optical microscope of the present invention;

[0032] Figure 6 The diploid karyotype result of the grass carp brain cell of the present invention;

[0033] Figure 7 The statistical result of chromosome number distribution of grass carp brain cells of the present invention;

[0034] Figure 8 The electrophoresis result of the PCR amplification product of the grass carp brain cell 12S rRNA of the present invention;

[0035] Fig. 9 The sequence alignment result of the PCR amplification product sequencing result of the grass carp brain cell 12S rRNA of the present invention at NCBI;

[0036] Fig.10 The results are the observation results of blind propagation of GCRV-II virus for three generations in grass carp brain cells constructed by the present invention;

[0037] Fig.11 Transmission electron microscopy analysis results of GCRV-II virus blindly propagated for three generations in grass carp brain cells constructed by the present invention

[0038] Fig.12 is the replication curve of GCRV-II virus in grass carp brain cells constructed by the present invention;

[0039] Fig.13 The virulence detection results of the GCRV-II virus replicated in the brain cells of grass carp constructed by the present invention include A. survival curve; B. comparison results of hemorrhagic symptoms of dead grass carp; C. transmission electron microscopy analysis results of brain tissue of diseased grass carp. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1 Construction of grass carp brain cell line

[0042] The method for constructing the grass carp brain cell line provided by the present invention is as follows:

[0043] 1. Anesthesia and disinfection:

[0044] The grass carp was placed in water containing 30 mg / L MS-222 anesthetic (purchased from Sigma, catalog number A5040) until its activity was significantly slowed down. The anesthetized grass carp was then fished out and its body surface was thoroughly disinfected with 75% v / v alcohol. The dissection was performed on ice, and after the brain tissue was removed, it was immediately placed in a centrifuge tube containing 1 mL PBS.

[0045] 2. Tissue cleaning:

[0046] The specific operations in this step are all carried out in a biological safety cabinet to ensure a sterile environment.

[0047] Using sterile tweezers, immerse the brain tissue in the following solutions for cleaning, using 2 mL of solution each time for 1 min, and gently invert the centrifuge tube upside down: 1) 75% v / v alcohol, for initial disinfection; 2) 2% penicillin-streptomycin double antibody solution (purchased from Hyclone, product number SV30010), for further killing of bacteria; 3) PBS (purchased from Solarbio, product number P1020), for removing residual double antibodies and alcohol, in preparation for subsequent processing.

[0048] After each wash, use sterile forceps to remove the tissue from the current solution and immerse it in the next wash solution.

[0049] 3. Tissue mincing and centrifugation: In a 10 cm culture dish, use sterile scissors to mince the brain tissue until there are no obvious tissue pieces. Then add 10 mL of PBS and centrifuge at 1500 rpm for 5 minutes.

[0050] 4. Tissue digestion: After centrifugation, the pellet was suspended with 10 mL of 0.25% trypsin (purchased from Hyclone, catalog number SH30042.01) and digested at 28°C for 15 min. During this period, the pellet was mixed by inversion every 5 min.

[0051] 5. Preparation and culture of cell suspension:

[0052] After digestion, centrifuge at 1500 rpm for 5 min. Suspend the precipitate with 4 mL L-15 medium (purchased from EallBio, catalog number 03.7001C-PS) containing 10% (v / v) FBS (purchased from Gibco, catalog number 10099141) and 2% penicillin-streptomycin. Add the cell suspension to a sealed culture bottle and culture it in a CO2-free incubator at 28°C.

[0053] 6. Culture medium replacement: Replace half of the L-15 culture medium every 3 days.

[0054] 7. Cell passaging: After the cells grow into a monolayer, wash them with 1 mL of PBS, then digest them with 1 mL of trypsin. After the cells become round and slightly fall off, pass them on at a 1:1 ratio.

[0055] 8. Adjustment of culture medium and long-term culture: Since the 5th generation, double antibodies are no longer used in the culture medium. By continuing to subculture in this way, the cells have been stably subcultured to the 60th generation.

[0056] The results showed that the cell morphology and growth status at different stages during the construction of the grass carp brain cell line like Figure 1 Place Show Among them, in the early stage of culture (7th day, such as Figure 1 A) The cell structure is more complex and there is aggregation. At the third generation, Figure 1 B, the cells have begun to spread, the morphology is relatively simple, and the distribution is relatively uniform. Figure 1 C, the cell morphology is more regular, the cells are arranged neatly, and a more obvious cell layer is formed. Figure 1 D. The thickness and density of the cell layer reached a high level, indicating that the cells had formed a mature and stable cell line at this time. The cell line was deposited in the China Center for Type Culture Collection (abbreviated as CCTCC) on October 14, 2024, with a collection number of CCTCC NO: C2024101, and the collection address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0057] Example 2

[0058] In order to determine the most suitable culture conditions, the following comparative tests were performed in this example:

[0059] 1. Determination of the optimal culture medium: The grass carp brain cells constructed in Example 1 were cultured at 3×10 4 Each well was inoculated in a 96-well plate and cultured at 28°C. Four wells of cells were selected from each group every day for trypsin digestion and counting, and culture was continued for 7 days. The growth curve was drawn with the time point as the horizontal axis and the number of cells as the vertical axis. like Figure 2 As shown, The results showed that L-15 medium was the optimal medium for grass carp brain cells.

[0060] 2. Determination of the optimal culture temperature: The cells were cultured at 3×10 4Each well was inoculated in a 96-well plate and cultured at 23°C, 28°C and 33°C. The subsequent method was the same as step 1 (i.e., digestion, counting and plotting growth curve every day), but the culture temperature was changed. like Figure 3 As shown, The results showed that the optimal culture temperature for grass carp brain cells was 28℃.

[0061] 3. Determination of the optimal FBS concentration: Cells were cultured at 3×10 4 Each well was inoculated in a 96-well plate and cultured at 28° C. The subsequent method was the same as step 1 (i.e., digestion, counting and drawing growth curve every day), but the FBS concentration was changed. like Figure 4 Shown The optimal FBS concentration for the growth of grass carp brain cells is 10%-15% (v / v). In order to reduce the experimental cost, the subsequent culture stage can be carried out using L-15 medium containing 10% (v / v) FBS concentration.

[0062] Example 3 Chromosome Karyotype Analysis of Grass Carp Brain Cell Line

[0063] 1. Reagents and cell treatment:

[0064] The 50th generation grass carp brain cells constructed in Example 1 were treated with colchicine (purchased from Sangon, item number A600322) at a final concentration of 1 μg / mL for 24 h. The cells were then centrifuged at 1500 rpm for 5 min and collected. The collected cells were resuspended in 5 mL of 0.075 M potassium chloride hypotonic solution and allowed to stand at 28° C. for 30 min.

[0065] 2. Cell Fixation:

[0066] Add 1 mL of freshly prepared pre-cooled fixative (methanol: acetic acid = 3:1 v / v) and pre-fix the cells at 28°C for 5 min. After centrifugation (same steps as above), the cell pellet is gently resuspended with 3 mL of fixative and fixed at 28°C for 30 min. Repeat the centrifugation, resuspension and fixation steps twice more.

[0067] 3. Staining and observation:

[0068] The treated cells were dropped onto a cold slide and stained with Wright-Giemsa staining solution (purchased from Solarbio, catalog number G1020) for 3 min. The number of metaphase chromosomes of 100 cells was analyzed under an optical microscope.

[0069] 4 Results Analysis

[0070] Figure 5 for Wright-Giemsa staining of metaphase chromosomes of GCBr cells (grass carp brain cells). Figure 6 The diploid karyotype analysis results of GCBr cells. The chromosomes are marked as m (metacentric chromosome), sm (sub-metacentric chromosome) and st (sub-telocentric chromosome). like Figure 7 Shown The statistical results of the chromosome number distribution in the metaphase showed that about 67% of the cells had a chromosome number of 48. The chromosome number of normal grass carp somatic cells is 2n = 48. This indicates that the cell line still maintains the original chromosome number of grass carp as a whole, without large-scale mutation, and is more suitable for simulating the response of grass carp cells to viruses in the natural state.

[0071] Example 4 Identification of 12S rRNA in Grass Carp Brain Cells

[0072] The 12S rRNA identification of the grass carp brain cells constructed in Example 1 was performed, and the specific operation steps were as follows:

[0073] 1. Cell processing and DNA extraction: When 25cm 2 When the cell confluence of the 50th generation in the cell bottle reached 90%, the cells were digested with trypsin and then centrifuged. The centrifugation condition was 1500 rpm for 5 minutes, and the supernatant was discarded after the centrifugation. The EE101 kit of Transgen was used to extract genomic DNA from the cell pellet.

[0074] 2. PCR amplification: Specific primers were designed based on the grass carp 12S rRNA sequence with GenBank accession number AY897013.1. (Upstream primer: 5'-TTAGATACCCCACTATGCTC-3', downstream primer: 5'-ACTAAATCCTCCTTCAAGCA-3'). The reaction system was 50 μL, including 25 μL of 2×mix (purchased from Transgen, item number AS231), 1 μL of upstream and downstream primers, 1 μL of DNA, and 22 μL of sterile water. Amplification conditions were: 94°C pre-denaturation for 3 min; 40 cycles: 94°C denaturation for 40 s, 55°C annealing for 40 s, 72°C extension for 40 s; and finally 72°C extension for 7 min. After the reaction, the reaction was stored at 4°C.

[0075] 3. Product detection and analysis: The PCR product was detected by 1.5% agarose electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared on NCBI.

[0076] Electrophoresis results ( Figure 8 ) showed that the band size of the amplified product was between 300-400 bp, which was consistent with the expected size of 336 bp. Fig. 9 ) showed that the product sequence had 99% identity with the known grass carp 12S rRNA sequence.

[0077] Example 5 Cryopreservation and revival of grass carp brain cells

[0078] The grass carp brain cells constructed in Example 1 were cryopreserved and revived, and the specific steps were as follows:

[0079] 1. Cell freezing: The cells in the cell bottle with a confluence of 90% were digested with trypsin and centrifuged. Centrifuge at 1500rpm for 5 minutes, discard the supernatant, suspend the cell pellet with 1mL of complete culture medium containing 10% DMSO (purchased from Solarbio, item number D8371), and transfer it into a cryopreservation tube. Place the cryopreservation tube in a program cooling box and place it in an ultra-low temperature refrigerator overnight. Transfer the cryopreservation tube to a liquid nitrogen tank the next day and record the storage location.

[0080] 2. Cell recovery: Take out the cryotubes stored in the liquid nitrogen tank, quickly place them in a 28℃ water bath, and shake them back and forth to thaw quickly. Centrifuge at 1500rpm for 5 minutes, discard the supernatant, add complete culture medium, and place them in the incubator for normal culture.

[0081] Example 6 Verification of sensitivity of grass carp brain cell line to GCRV-II virus

[0082] In this example, the sensitivity of the grass carp brain cell line constructed by the present invention to GCRV-II (HN14) virus, that is, to the grass carp reovirus type II (GCRV-II) strain HN14 was verified, and the specific operation was as follows:

[0083] Blind virus transfection: cells were cultured at 4×10 5 / mL inoculated at 25cm 2 in a culture flask. After overnight, the cell medium was changed, and grinding solution of diseased fish tissue infected with GCRV-II (MOI=1) was added and placed at 28°C. After 2 hours, the virus solution was removed, the cells were washed 3 times with PBS, and the cells were replaced with L-15 medium containing 5% FBS and continued to be cultured. Thereafter, the cell pathological changes were observed every day to determine whether CPE occurred. After 12 days, the cell supernatant and precipitate were collected separately. The cell supernatant was used to infect a new round of blind cells; the cell precipitate was used to prepare electron microscope sections to observe intracellular virus particles. According to this step, the virus derived from the diseased fish tissue was blindly propagated in the cells for three consecutive generations.

[0084] The results show that ( Fig.10 ), atypical CPE was observed in cells 4 days after virus inoculation. Compared with control cells, cells treated with virus had a higher number of cell droplets. like Fig.11 Shown Transmission electron microscopy (TEM) analysis of cell samples that were blindly passaged for three generations showed the presence of a large number of viral inclusion bodies in the cells, indicating that GCRV-II was able to replicate in the GCBr cells.

[0085] Example 7 Virus copy number detection:

[0086] The cells were plated at 4 × 10 5 / mL was inoculated in a 24-well plate. After overnight, the cell medium was changed, the virus was inoculated (MOI = 1), and it was placed at 28°C. After 2 hours, the virus solution was removed, PBS was washed 3 times, and the cells were replaced with L-15 medium with 5% FBS, and cultured for another 12 days. During this period, the cell supernatant and cells from 4 wells were collected every two days for RNA extraction and detection of changes in the virus content in the cell supernatant and cells. The quantitative primer sequence of the HN14 S6 fragment is: upstream 5'-GAACGCACGGAGAAGAAG-3', downstream 5'-AGCCCCTGAGTAAGCATC-3'). The reaction system is 20μL, including 10μL of 2×mix (Transgen, AQ601), 0.5μL of upstream and downstream primers, 1μL of cDNA, and 8μL of sterile water. Amplification conditions were as follows: pre-denaturation at 94°C for 3 min; 40 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s; and finally extension at 72°C for 5 min.

[0087] The results of virus copy number detection (virus replication curve) (such as Fig.12 ) showed that as time went on, the content of GCRV-II in the cell supernatant and in the cells increased, indicating that the virus was able to reproduce in the GCBr cells and had a time effect.

[0088] Example 8 Virus Virulence Detection

[0089] In order to verify whether the virus replicated in the GCBr cells provided by the present invention is still pathogenic, a live virus challenge experiment was carried out. The specific steps are as follows:

[0090] Healthy grass carp weighing 1.15±0.07g and 4.83±0.14cm were selected and randomly divided into two groups. The grass carp in the treatment group were injected with the supernatant of cells cultured for three generations blindly (the virus concentration was 2.8×10 5 The grass carp in the control group were injected with L-15 medium containing 5% FBS. Each group contained 40 fish, and each fish was injected with 25 microliters of solution. The status and survival of the grass carp were observed and recorded every day.

[0091] like Fig.13 ShownAfter injection, no grass carp in the control group died, with a mortality rate of 0. However, grass carp in the treatment group began to die from the 4th day, and all died on the 11th day, with a mortality rate of 100%. The dead grass carp showed typical muscle bleeding symptoms of grass carp hemorrhagic disease, and viral inclusion bodies were found in the brain tissue of the dead grass carp. These results show that GCRV-II replicated in GCBr cells is still infectious and can cause healthy grass carp to become ill.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A grass carp brain cell line susceptible to grass carp reovirus type II, characterized in that: It was deposited in the China Center for Type Culture Collection on October 19, 2024, with the deposit number being CCTCC NO: C2024101.

2. The method for culturing grass carp brain cell line according to claim 1, characterized in that: The grass carp brain cell line is cultured using L-15 culture medium, wherein the L-15 culture medium contains 10-15% v / v fetal bovine serum and the culture temperature is 28°C.

3. Use of the grass carp brain cell line according to claim 1 in aquatic virus culture.

4. Use of the grass carp brain cell line according to claim 1 in the isolation of aquatic viruses.

5. Use of the grass carp brain cell line according to claim 1 in the preparation of aquatic virus vaccines or detection products.

6. Use of the grass carp brain cell line according to claim 1 in the cultivation of grass carp reovirus.

7. Use of the grass carp brain cell line according to claim 1 in the isolation of grass carp reovirus.

8. Use of the grass carp brain cell line according to claim 1 in the preparation of grass carp reovirus vaccine or detection product.

9. The use of the grass carp brain cell line according to claim 1 in drug screening, characterized in that: The medicine is used for preventing and treating grass carp reovirus infection, or preventing and treating diseases caused by grass carp reovirus infection.

10. The use of the grass carp brain cell line according to claim 1 in drug evaluation, characterized in that: The medicine is used for preventing and treating grass carp reovirus infection, or preventing and treating diseases caused by grass carp reovirus infection.

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