A grass carp brain cell line sensitive to grass carp reovirus type II and its application

Abstract

The present invention relates to the field of biotechnology. 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 19, 2024, with a deposit number of CCTCC NO: C2024101. The grass carp brain cell line constructed by the present invention shows remarkable stability and successfully achieved 60 stable passages. When inoculated with GCRV‑II, an atypical cytopathic effect can be exhibited. In addition, the grass carp brain cell line has the ability to efficiently replicate GCRV‑II, and even after three generations of blind transmission, the generated cytotoxin can still cause grass carp to become ill and die. Therefore, the present invention has laid a solid foundation for in-depth exploration of the pathogenic mechanism of GCRV‑II, vaccine development, and antiviral drug screening, which will help promote further progress in related research and provide a key cell model and research basis for the formulation of grass carp reovirus-related research and prevention and control strategies.

Description

Technical Field

[0001] The present 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 applications thereof. Background Art

[0002] Grass carp (Ctenopharyngodon idella, Cypriniformes, Cyprinidae, Leucineae) is an important freshwater aquaculture species in my country and one of the "four major carps." Its aquaculture output often ranks first among freshwater fish. However, diseases frequently occur during aquaculture, seriously hindering the health, stability, and sustainable development of the grass carp aquaculture industry. Grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV), is a common and highly contagious disease in grass carp aquaculture. It primarily affects the current-season grass carp, resulting in high mortality and significant economic losses. Based on genetic sequences, researchers have classified GCRV into three major genotypes: I, II, and III. GCRV-II is the most virulent, causing the highest morbidity and mortality rates, and is the predominant strain of grass carp hemorrhagic disease in my country. This strain, with its widespread prevalence, high virulence, pathogenicity, and high mortality rate, is the primary cause of frequent outbreaks of grass carp hemorrhagic disease, but its pathogenic mechanism remains to be elucidated.

[0003] Compared with in vivo research, cell-based research has the advantages of small differences, good reproducibility, and low cost, making cell culture an important research method in biology. Grass carp brain cells (GCBr cells) are derived from grass carp brain tissue. The brain plays a key role in the physiological activities of grass carp, participating in the grass carp's 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, neural development, neurological diseases, and environmental impacts. Studying them helps to deepen our understanding of 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 virological research and are widely used in aspects such as viral replication, isolation and identification, pathogenic mechanism research, and antibody and vaccine preparation. After the virus infects cells, it usually produces a cytopathic effect (CPE), which is crucial for understanding the characteristics of viral infection and the host cell response to viral infection. Although the GCRV-Ⅱ strain has the highest pathogenicity and mortality rate at the in vivo level, its cell sensitivity is relatively weak. It cannot produce obvious CPE when inoculated into existing fish cells, has low proliferation titers in existing cells, and its cytotoxicity cannot cause grass carp to become ill. This poses many difficulties for the study of its infection and pathogenic mechanism, diagnosis, and the development of prevention and control products. The lack of a basic tool, the GCRV-Ⅱ sensitive cell line, has greatly limited the research on its pathogenic mechanism and immune prevention and control. Therefore, the establishment of a sensitive cell line has become a top priority and is a necessary prerequisite for in-depth research on the virus invasion, infection and pathogenic mechanism, as well as the implementation 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 exhibit 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 as follows:

[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 using 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 isolation of aquatic viruses.

[0011] Furthermore, the present invention provides the use of the grass carp brain cell line in preparing 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 preparing a 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 selected grass carp, the natural host of GCRV-II, for research. By isolating grass carp brain tissue, a grass carp brain cell line (GCBr cell line) was successfully obtained. The grass carp brain cell line exhibited good stability and had successfully achieved stable passage to 60 generations, which provided a strong guarantee for its reliability in long-term research and application. After inoculation with GCRV-II, the cells showed atypical CPE, a characteristic that indicates the uniqueness of the interaction between the GCBr cells and GCRV-II, providing 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 grass carp to become ill and die, which fully confirms 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 of GCRV-II, vaccine development, and antiviral drug screening. 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, refers to a series of morphological changes observed under an optical microscope after a virus infects susceptible host cells, caused by viral proliferation and interaction with the host cells. These morphological changes are characteristic and serve as a key indicator of viral infection and preliminary identification.

[0022] Atypical CPE: In contrast to typical CPE, atypical CPE refers to a series of unusual cellular morphological changes that occur after viral infection and do not conform to the typical CPE characteristics. These changes may be subtle, making them difficult to identify directly through conventional microscopy; or they may mimic cellular morphological changes caused by other factors (such as natural cell aging, drug toxicity, and non-viral microbial infection), making accurate identification more difficult.

[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 Collection

[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 paying any creative work.

[0027] Figure 1 These are 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 This is the growth curve of the grass carp brain cell line of the present invention under different culture medium conditions;

[0029] Figure 3The growth curves of the grass carp brain cell line 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 is shown;

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

[0033] Figure 7 The statistical results of chromosome number distribution of grass carp brain cells of the present invention are as follows;

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

[0035] Figure 9 The 12S rRNA amplified product of grass carp brain cells of the present invention is sequenced and compared with the sequence in NCBI.

[0036] Figure 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] Figure 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] Figure 12 is the replication curve of GCRV-II virus in grass carp brain cells constructed by the present invention;

[0039] Figure 13 The virulence test results of the GCRV-II virus that replicates in grass carp brain cells 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 diseased grass carp brain tissue. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort 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] Grass carp were placed in water containing 30 mg / L MS-222 anesthetic (Sigma, Catalog No. A5040) until their movements were noticeably slowed. The anesthetized grass carp were then removed and their body surfaces thoroughly disinfected with 75% v / v alcohol. Dissection was performed on ice, and the brain tissue was immediately removed and placed in a centrifuge tube containing 1 mL of PBS.

[0045] 2. Tissue cleaning:

[0046] The specific operations in this step are all performed in a biosafety cabinet to ensure a sterile environment.

[0047] Using sterile forceps, the brain tissue was washed by immersing it in the following solutions, 2 mL each time for 1 min, and gently inverting the centrifuge tube: 1) 75% v / v alcohol for initial disinfection; 2) 2% penicillin-streptomycin double antibody solution (purchased from Hyclone, product number SV30010) for further killing bacteria; 3) PBS (purchased from Solarbio, product number P1020) for removing residual double antibody 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 no obvious tissue fragments remain. Then, add 10 mL of PBS and centrifuge at 1500 rpm for 5 minutes.

[0050] 4. Tissue Digestion: After centrifugation, the pellet was suspended in 10 mL of 0.25% trypsin (Hyclone, Cat. No. 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 minutes. Resuspend the pellet in 4 mL of L-15 medium (EallBio, Catalog No. 03.7001C-PS) containing 10% (v / v) FBS (Gibco, Catalog No. 10099141) and 2% penicillin-streptomycin. Add the cell suspension to a sealed culture flask and culture in a 28°C, CO2-free incubator.

[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 and then digest them with 1 mL of trypsin. After the cells become round and slightly fall off, subculture them at a 1:1 ratio.

[0055] 8. Medium adjustment and long-term culture: From the fifth generation onwards, double-antibody was no longer used in the culture medium. By continuing to subculture according to this method, the cells have been stably subcultured to the 60th generation.

[0056] The results showed that during the construction of grass carp brain cell line, the cell morphology and growth status at different stages 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, 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 deposit number of CCTCC NO: C2024101, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0057] Example 2

[0058] In order to determine the optimal culture conditions, the following comparative experiments 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 a rate of 3×10 4 Cells were seeded into 96-well plates at 100 cells / well and incubated at 28°C. Four wells of cells were selected from each group daily for trypsin digestion and counting. Cultures were continued for 7 days. Growth curves were plotted with time as the horizontal axis and cell number as the vertical axis. like Figure 2 As shown, The results showed that L-15 medium was the optimal culture medium for grass carp brain cells.

[0060] 2. Determination of the optimal culture temperature: Cells were cultured at a rate of 3 × 10 4Each well was inoculated into 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 growth curve drawing 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 a rate of 3×10 4 Each well of the plate was inoculated with 100 cells / well and incubated at 28°C. The subsequent steps were the same as in step 1 (i.e., digestion, counting, and growth curve drawing every day), except that the FBS concentration was changed. like Figure 4 shown The optimal FBS concentration for grass carp brain cell growth is 10%-15% (v / v). To reduce experimental costs, subsequent culture stages can be carried out using L-15 medium containing 10% (v / v) FBS.

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

[0063] 1. Reagents and cell treatment:

[0064] Grass carp brain cells from passage 50, constructed in Example 1, were treated with colchicine (available from Sangon, Catalog No. A600322) at a final concentration of 1 μg / mL for 24 hours. The cells were then centrifuged at 1500 rpm for 5 minutes and harvested. The harvested cells were resuspended in 5 mL of a 0.075 M potassium chloride hypotonic solution and allowed to stand at 28°C for 30 minutes.

[0065] 2. Cell Fixation:

[0066] Add 1 mL of freshly prepared, pre-chilled fixative (methanol:acetic acid = 3:1 v / v) and pre-fix the cells at 28°C for 5 minutes. After centrifugation (same steps as above), gently resuspend the cell pellet in 3 mL of fixative and fix at 28°C for 30 minutes. Repeat the centrifugation, resuspension, and fixation steps two more times.

[0067] 3. Staining and observation:

[0068] Treated cells were dropped onto cold slides and stained with Wright-Giemsa staining solution (purchased from Solarbio, Cat. No. G1020) for 3 min. Metaphase chromosome counts of 100 cells were analyzed under a light microscope.

[0069] 4 Results Analysis

[0070] Figure 5 for Wright-Giemsa staining of metaphase chromosomes in GCBr cells (grass carp brain cells). Figure 6 The diploid karyotype analysis results of GCBr cells are shown in Figure 2. The chromosomes are labeled as m (metacentric chromosome), sm (submetacentric chromosome), and st (subtelocentric chromosome). like Figure 7 shown Statistical results of metaphase chromosome number distribution showed that approximately 67% of cells had a chromosome number of 48. Normal grass carp somatic cells have a chromosome number of 2n = 48. This indicates that the cell line generally retains the original grass carp chromosome number and has not undergone large-scale mutations, making it more suitable for simulating the natural response of grass carp cells to viruses.

[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 steps were as follows:

[0073] 1. Cell processing and DNA extraction: When 25cm 2 At passage 50, when the cell confluency reached 90%, the cells were digested with trypsin and then centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. Genomic DNA was extracted from the cell pellet using the Transgen EE101 kit.

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

[0075] 3. Product detection and analysis: PCR products were detected by 1.5% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared at 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. Sequence alignment analysis ( Figure 9 ) showed that the product sequence was 99% identical to the known grass carp 12S rRNA sequence.

[0077] Example 5 Cryopreservation and thawing 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 Cryopreservation: Trypsinize cells in a flask that have reached 90% confluency and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and suspend the cell pellet in 1 mL of complete medium containing 10% DMSO (Solarbio, Cat. No. D8371). Transfer the cell pellet to a cryovial. Place the cryovial in a programmed cooling box and freeze overnight. The next day, transfer the cryovial to a liquid nitrogen tank and record the storage location.

[0080] 2. Cell Thawing: Remove the cryovials from the liquid nitrogen tank and quickly place them in a 28°C water bath, shaking them back and forth to rapidly thaw. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add complete culture medium, and incubate in an incubator for normal growth.

[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 in the present invention to GCRV-II (HN14) virus, that is, to grass carp reovirus type II (GCRV-II) strain HN14, was verified by the following specific procedures:

[0083] Blind virus transfection: cells were plated 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 3 consecutive generations.

[0084] The results showed that ( Figure 10 ), atypical CPE was observed in cells 4 days after virus inoculation. Compared with control cells, virus-treated cells showed a higher number of cell droplets. like Figure 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 Cells were inoculated at 100 μg / mL in a 24-well plate. After overnight, the cell culture medium was changed, the virus was inoculated (MOI = 1), and the cells were incubated at 28°C. After 2 hours, the virus solution was removed, the cells were washed three times with PBS, and the culture medium was replaced with 5% FBS-containing L-15 medium. Culture was continued for 12 days. During this period, the cell supernatant and cells from four wells were collected every two days for RNA extraction and detection of changes in viral content in the cell supernatant and cells. The quantitative primer sequences for the HN14 S6 fragment are: upstream 5'-GAACGCACGGAGAAGAAG-3', downstream 5'-AGCCCCTGAGTAAGCATC-3'). The reaction system was 20 μL, including 10 μL of 2×mix (Transgen, AQ601), 0.5 μL each of upstream and downstream primers, 1 μL of cDNA, and 8 μL of sterile water. The 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 Figure 12 ) showed that as time went on, the content of GCRV-II in the cell supernatant and cells showed an upward trend, which indicated that the virus could 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 in length 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 μL of the solution. The condition and survival of the grass carp were observed and recorded daily.

[0091] like Figure 13 shownAfter injection, no grass carp in the control group died, resulting in a mortality rate of 0%. However, grass carp in the treated group began to die on the fourth day, and all died by the eleventh day, with a mortality rate of 100%. The dead grass carp displayed typical muscle hemorrhages characteristic of grass carp hemorrhagic disease, and viral inclusion bodies were found in their brain tissue. These results indicate that GCRV-II replicating within GCBr cells remains infectious and capable of causing disease in healthy grass carp.

[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 scope of protection 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 CCTCC NO: C2024101.

2. The method for culturing the 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 containing 10-15% v / v fetal bovine serum at a culture temperature of 28°C.

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

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

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

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