Pelteobagrus fulvidraco brain tissue cell line PYCB sensitive to viruses and application thereof

Abstract

The invention discloses a pelteobagrus fulvidraco brain tissue cell line PYCB sensitive to viruses and application thereof, and belongs to the technical field of aquatic organism cell culture and disease prevention and control. The preservation number of the Pelteobagrus fulvidraco brain tissue cell line PYCB is CCTCC (China Center For Type Culture Collection) NO: C2025148. The Pelteobagrus fulvidraco brain tissue cell line PYCB is successfully constructed by taking the Pelteobagrus fulvidraco brain tissue as an object, and the cell line can be continuously and stably subcultured, is high in proliferation speed and simple in culture method, is sensitive to Pelteobagrus fulvidraco small RNA viruses and Pelteobagrus fulvidraco stem casing viruses, and can be used for isolated culture of viruses. The invention provides an important research platform and an experimental material for separating and identifying fish viruses and virus vaccines and developing medicines.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism cell culture and disease control technology, and in particular to a virus-sensitive yellow catfish brain tissue cell line PYCB and its applications. Background Technology

[0002] Yellow catfish (Pelteobagrus fulvidraco) has delicious flesh, is rich in nutrients, and has a high calcium and phosphorus content, leading to a gradual increase in its farming volume in recent years. Yellow catfish diseases are mainly viral, bacterial, and parasitic, with viral diseases being the most serious. Among them, yellow catfish small RNA virus and yellow catfish bacillus virus are the most impactful viral pathogens in recent years. Diseases caused by pathogens such as yellow catfish small RNA virus and yellow catfish bacillus virus have resulted in significant economic losses to the yellow catfish farming industry.

[0003] However, there is currently a lack of cell lines on the market that are highly sensitive to small RNA viruses and barnacle viruses in yellow catfish. According to reports, cell lines such as channel catfish ovary cells (CCO), grass carp ovary cells (GCO), carp epithelioma cells (EPC), rainbow trout gonadal cells (RTG-2), bighead carp cells (FHM), silver carp brain cells (GiCB), and yellow eel kidney cells (CrEK) are all insensitive to small RNA viruses in yellow catfish. Furthermore, only three passaged cell lines and two primary cell lines of yellow catfish have been reported to have been successfully constructed so far: patented technologies disclose passaged yellow catfish embryonic cell lines and kidney cell lines, and primary cultured hepatocytes; literature reports snout cells (passaged to only 8 generations) and fin cells (primary culture). This severely restricts research on diseases affecting yellow catfish and the development of vaccines.

[0004] Fish cell lines have been widely used in virology, genetics, gene regulation and functional analysis, physiology, endocrinology, and resource conservation. Their primary application is in the isolation, purification, and identification of fish viruses, as well as in the study of cell function. While fish cell line establishment technology is relatively mature, challenges remain in constructing cell lines for certain fish with specific functions. For example, the construction of yellow catfish brain cell lines and cells sensitive to both yellow catfish small RNA viruses and bacillus subtilis viruses has not been successfully achieved. Therefore, establishing yellow catfish brain tissue cell lines sensitive to yellow catfish small RNA viruses plays a crucial role in the prevention and treatment of yellow catfish viral diseases and in improving the economic benefits of yellow catfish aquaculture. Summary of the Invention

[0005] The purpose of this invention is to provide a virus-sensitive yellow catfish brain tissue cell line, PYCB, and its applications, to address the problems existing in the prior art. This invention successfully constructed the yellow catfish brain tissue cell line PYCB using yellow catfish brain tissue. This cell line can be continuously and stably passaged and is sensitive to yellow catfish small RNA virus and yellow catfish bacillus virus, and can be used for virus isolation and culture. This invention provides an important research platform and experimental materials for the isolation and identification of fish viruses and the development of viral vaccines and drugs.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a yellow catfish brain tissue cell line PYCB (Pelteobagrus fulvidraco), which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2025148 and deposit date of May 8, 2025.

[0008] The present invention also provides a method for constructing the above-mentioned yellow catfish brain tissue cell line PYCB, comprising the following steps:

[0009] (1) Brain tissue from healthy yellow catfish was obtained, and single cells were obtained through washing and digestion;

[0010] (2) The single cells are cultured in a proliferation medium for primary culture;

[0011] (3) Primary culture until the cell confluence reaches 60%-90%, digest and then passage culture in subculture medium to obtain the yellow catfish brain tissue cell line PYCB;

[0012] The proliferation medium was M199 medium containing 15% fetal bovine serum, 2% yellow catfish serum, 1% MEM non-Essential Amino Acids, 100 U / mL penicillin and 100 U / mL streptomycin;

[0013] The passage medium was M199 medium containing 15% fetal bovine serum, 1% yellow catfish serum, 1% MEM non-essential Amino acids, 100 U / mL penicillin, and 100 U / mL streptomycin.

[0014] Optionally, the rinsing solution used during rinsing is an HBSS solution containing 500 U / mL penicillin and 500 U / mL streptomycin.

[0015] Optionally, the digestive fluid used during digestion is pancreatic enzyme digestive fluid.

[0016] Optionally, the primary culture temperature is 28℃; the subculture temperature is 28℃, and subculture is performed every 3-5 days.

[0017] The present invention also provides the application of the above-mentioned yellow catfish brain tissue cell line PYCB in the culture of yellow catfish small RNA virus and / or yellow catfish baculovirus.

[0018] The present invention also provides the application of the above-mentioned yellow catfish brain tissue cell line PYCB in constructing a yellow catfish small RNA virus and / or yellow catfish baculovirus infection cell model.

[0019] The present invention also provides the application of the above-mentioned yellow catfish brain tissue cell line PYCB in screening drugs for the prevention and treatment of yellow catfish small RNA virus and / or yellow catfish baculovirus infection.

[0020] The present invention also provides the application of the above-mentioned yellow catfish brain tissue cell line PYCB in the preparation of yellow catfish small RNA virus and / or yellow catfish baculovirus vaccine.

[0021] This invention also provides the application of the above-mentioned yellow catfish brain tissue cell line PYCB in the preparation of genetically engineered host cells.

[0022] The present invention discloses the following technical effects:

[0023] This invention demonstrated primary and passaged culture of yellow catfish brain tissue cells, achieving good culture results and establishing the yellow catfish brain tissue cell line PYCB, which has been passaged 45 times. The proliferation and passage culture medium formulation for this cell line is simple, relying primarily on serum for growth support without the need for expensive purified growth factors. Passaged cells maintain good growth and can be cryopreserved long-term, providing a large number of cells for yellow catfish-related research.

[0024] The PYCB brain tissue cell line constructed in this invention is simultaneously sensitive to both yellow catfish small RNA virus and yellow catfish baculovirus, exhibiting extremely high viral titers and short cytopathic effect duration. Significant cytopathic effect (CPE) can be observed within 36 hours of infection with yellow catfish small RNA virus, and the viral titer can reach 10 at 72 hours. 8.6 TCID 50 ·mL -1 The cytopathic effect (CPE) is significantly higher than previously reported; a clear cytopathic effect can be observed within 24 hours of infection with yellow catfish snare virus, and the viral infection titer can reach 10 at 48 hours. 9.2 TCID 50 ·mL -1 Therefore, this cell line can be well applied to the isolation, proliferation, gene manipulation, and viral vaccine development of yellow catfish viruses, and also provides cell materials for the isolation, culture, and vaccine development of other fish viruses. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the cell morphology of the yellow catfish brain tissue cell line PYCB (40th generation) from Example 1; scale bar is 500 μm.

[0027] Figure 2 The effect of different culture temperatures on the growth of the yellow catfish brain tissue cell line PYCB in Example 2;

[0028] Figure 3 Example 2 illustrates the effect of different FBS concentrations on the growth of the yellow catfish brain tissue cell line PYCB.

[0029] Figure 4 Metaphase chromosome division phase diagram (×1000) of the 30th generation yellow catfish brain tissue cell line PYCB in Example 4.

[0030] Figure 5 This is a CPE observation image of the yellow catfish brain tissue cell line PYCB after 36 hours of infection with yellow catfish small RNA virus in Example 5; scale bar is 500 μm.

[0031] Figure 6 This is a CPE observation image of the yellow catfish brain tissue cell line PYCB after 24 hours of infection with yellow catfish rotavirus in Example 5; scale bar is 500 μm.

[0032] Figure 7 The image shows the fluorescence signal of the yellow catfish brain tissue cell line PYCB transfected with pEGFP-N1 at 24 h in Example 5 of Example 6; where A is the white light image of the transfected cells and B is the fluorescence image of the transfected cells; the scale bar is 200 μm. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0039] Example 1

[0040] This embodiment provides a method for constructing a yellow catfish brain tissue cell line, specifically including the following steps:

[0041] (1) Processing of brain tissue: Take healthy yellow catfish weighing 20g-60g (negative for small RNA virus and rod-shaped virus), disinfect the yellow catfish with 75% alcohol, dissect and take brain tissue under sterile conditions, place it in the rinsing solution, remove the connective tissue in the brain tissue with sterile forceps, and rinse until the rinsing solution is clear. Soak the processed brain tissue in the culture medium, discard the culture medium and cut the brain tissue into pieces for later use.

[0042] The rinsing solution was an HBSS (1×) (Hank's Balanced Salt Solution) solution containing 500 U / mL penicillin and 500 U / mL streptomycin.

[0043] The culture medium was M199 medium.

[0044] (2) Primary culture: The treated yellow catfish brain tissue was added to trypsin digestion solution and digested at room temperature for 30-40 min. Serum-containing culture medium was added to stop the digestion. The cell suspension was collected and filtered through a 100 μm filter. The filtrate was centrifuged at 1200 rpm for 8 min, the supernatant was discarded, and the pellet was resuspended in proliferation culture medium and dispensed into 25 cm aliquots. 2 The cells were placed in a 28°C incubator and cultured. The proliferation medium was replaced every 2-3 days by changing half of the medium.

[0045] The pancreatic enzyme digestion solution was 0.5% Trypsin-EDTA (10×).

[0046] The proliferation medium was M199 medium containing 15% fetal bovine serum, 2% yellow catfish serum, 1% MEM non-essential amino acids, 100 U / mL penicillin and 100 U / mL streptomycin.

[0047] (3) Subculture: When the primary cultured cells reach a confluence of 60%-90%, add 0.25% trypsin to digest them, resuspend the cells in the subculture medium, and then inoculate them in a culture flask in a constant temperature incubator at 28℃. Subculture once every 3-5 days.

[0048] The passage medium was M199 medium containing 15% fetal bovine serum, 1% yellow catfish serum, 1% MEM non-essential Amino acids, 100 U / mL penicillin and 100 U / mL streptomycin.

[0049] The cells have been passaged more than 45 times, resulting in the PYCB brain tissue cell line from yellow catfish. The cell morphology of the 40th generation PYCB yellow catfish brain tissue cell line is shown below. Figure 1 As shown.

[0050] This cell line has been deposited at the China Center for Type Culture Collection (CCTCC) and named PYCB (Pelteobagrus fulvidraco) brain tissue cell line, with accession number CCTCC NO: C2025148, deposit date May 8, 2025, and deposit address Wuhan University, Wuhan, China.

[0051] Example 2

[0052] This example investigated the effects of different culture conditions on cell growth, and the specific process is as follows:

[0053] 1. Effects of different culture temperatures on cell growth

[0054] Take 8×10 from the above Example 1 4 / mL PYCB cells (passage 30) were seeded into T25 culture flasks containing M199 medium with 15% FBS and cultured at 20℃, 24℃, 28℃, and 32℃. Cell counts were performed at 1, 3, and 7 days after culture using a Scepter™ Handheld Automated Cell Counter to plot the cell line growth at different culture temperatures.

[0055] The results are as follows Figure 2 As shown, the growth rate of PYCB cells at 28℃ was significantly higher than that at 20℃, 24℃, and 32℃.

[0056] 2. Effects of different FBS (fetal bovine serum) concentrations on cell growth

[0057] Take 8×10 from the above Example 1 4 PYCB cells (passage 30) were seeded at a density of / mL in T25 culture flasks containing M199 medium with 10% FBS, 15% FBS, and 20% FBS, respectively, and cultured at 28°C. Cell counts were performed using a Scepter™ Handheld Automated Cell Counter from day 1 to 6 post-culture to plot the cell line growth at different FBS concentrations.

[0058] The results are as follows Figure 3 As shown, during the first 3 days, the cell concentration of PYCB cells in 20% FBS culture medium was significantly higher than that in 15% FBS and 10% FBS culture medium. After 3 days, the cell concentration in 20% FBS culture medium was slightly higher than that in 15% FBS culture medium. However, due to cost control considerations for the culture medium, the fetal bovine serum concentration can be appropriately reduced to 15% during passage culture, especially after passages 15-20.

[0059] Example 3

[0060] This embodiment verifies the cryopreservation and resuscitation capabilities of the PYCB cell line obtained in Example 1. The specific process is as follows:

[0061] (1) Take PYCB cells in the logarithmic growth phase of Example 1 (generations 10, 20, and 30, respectively), digest them with trypsin to obtain a single-cell suspension, centrifuge at 1600 g for 10 min, and discard the supernatant. Add an appropriate amount of cell cryopreservation solution (ZENOAQ, JPN) to the cell pellet, resuspend, and transfer to a 1.8 mL sterile cryovial; place the cryovial in a programmed cooling box and freeze overnight at -80°C, and then place it in liquid nitrogen for long-term storage the next day;

[0062] (2) To thaw the frozen cells, take the cryopreservation tubes out of the liquid nitrogen tank, put them in a 37°C water bath and shake them quickly until they thaw. Add them to a cell culture flask containing 6-8 mL of culture medium and incubate them in a 28°C incubator. Change the medium every other day.

[0063] Tests showed that the recovery rate of cells after cryopreservation at different passages (10th, 20th, and 30th generations) was 82%-96%. The recovered cells were able to adhere to the cell wall, grow and divide, and could be passaged normally. The cell morphology and proliferation capacity were not significantly different from those before cryopreservation.

[0064] Example 4

[0065] Chromosome number and karyotype are fundamental to cytogenetics and are relatively accurate indicators for identifying species and sex. In cell culture, chromosomes are frequently used as reliable indicators of cell origin and whether transformation has occurred. Therefore, this example analyzes the chromosomes of the PYCB cell line obtained in Example 1, as detailed below:

[0066] (1) Take the PYCB cell line (30 generations) from Example 1, allow the 30 generations of cells to adhere stably for 36-48 hours, add colchicine at a final concentration of 0.6-1.0 μg / mL and treat for 12-16 hours, digest with trypsin and centrifuge at 1600 g for 10 minutes to recover the cells.

[0067] (2) The recovered cells were hypotonic in 75mM KCl at 37℃ for 40 min, and 2 mL of fixative (methanol: glacial acetic acid 3:1) was added for pre-fixation; centrifuged at 1600 g for 10 min, the supernatant was discarded, and fixative was added at room temperature for 30 min; the fixation steps were repeated 2-3 times; for the last fixation, an appropriate amount of fixative was left and gently blown to mix; the fixative suspension was aspirated and dropped from a height of 30 cm onto a glass slide pre-cooled at -20℃, the slide was tilted to allow the droplet to flow down and spread out; the slide was air-dried, and then observed with a phase contrast microscope. If the cells were spread out evenly without contacting each other, more slides were prepared with the same concentration of cells. If the cells appeared to be clumped and overlapping, the suspension was diluted 2-4 times and the droplet slides were prepared according to the above steps.

[0068] (3) Stain with Giemsa stain for 10 min, observe the chromosome morphology of the cells under an oil immersion microscope, and count the number of chromosomes.

[0069] The results are as follows Figure 4 As shown, in the 100 cell division stages of the PYCB cell line, the chromosome number ranged from 48 to 54, but 85% of the division stages had 52 chromosomes, consistent with the chromosome number characteristics of yellow catfish. Although the chromosome number distribution was uneven, diploid chromosomes occurred most frequently, while other aneuploid chromosomes accounted for only a small proportion.

[0070] Example 5

[0071] This embodiment uses the PYCB cell line obtained in Example 1 to perform a virus infection experiment to verify the cell line's sensitivity to viruses. The specific process is as follows:

[0072] (1) Observation of cytopathic effect (CPE)

[0073] Take the PYCB cell line from Example 1 (30 passages) and wait until the cells reach a density of 25 cm. 2 After the cell culture flasks were filled, the filtrate of yellow catfish small RNA virus (YCPrV 22426-8 strain, disclosed in the literature "Establishment and application of qPCR detection method for yellow catfish small RNA virus. Acta Virologica Sinica. 2023(4): 1062-1071", now held by the applicant) and the filtrate of yellow catfish rod-shaped virus (CCTCC NO:V202256, disclosed in patent literature CN116716260A, now held by the applicant) were inoculated into PYCB cell culture flasks respectively, incubated at 24℃ for 1 h, the virus solution was discarded, and replaced with M199 cell culture medium containing 2% fetal bovine serum, and cultured at 24℃ for a further period of time. Cell pathogenesis (CPE) was observed daily using an inverted microscope.

[0074] Cytopathic characteristics: Cell rounding is a description of light microscopic observation after viral infection, which leads to cell rounding and death.

[0075] (2) Titer determination

[0076] After complete cytopathic effect, the virus suspension was collected, and the harvested virus solution was serially diluted 10-fold to 10⁻⁶. -10 Each dilution was replicated in 6 wells and added to 96-well plates inoculated with PYCB cell line. The plates were incubated at 24°C and CPE production was observed daily. Viral titers were calculated according to the Reed & Muench (1938) method.

[0077] like Figure 5As shown, significant cell morphology (CPE) was observed 36 hours after infection with yellow catfish small RNA virus. Cells became rounder, their refractive index increased, the number of shrunken cells gradually increased, and cells eventually detached. Virus infection titer was measured after complete CPE of yellow catfish small RNA virus-infected cells, reaching a titer of 10. 8.6 TCID 50 ·mL -1 .like Figure 6 As shown, obvious cell morphology (CPE) was observed 24 hours after yellow catfish snare virus infection, with cells becoming rounded, shrinking, and detaching. Virus infection titer was measured after complete CPE of yellow catfish snare virus-infected cells, reaching a titer of 10. 9.2 TCID 50 ·mL -1 .

[0078] In the above experiment, the viral titer of PYCB cells infected with yellow catfish small RNA virus reached 10 after 72 h. 8.6 TCID 50 ·mL -1 The viral titer of PYCB cells infected with yellow catfish snare virus reached 10 after 48 hours. 9.2 TCID 50 ·mL -1 This indicates that the cell line constructed in this invention has extremely high sensitivity to yellow catfish small RNA virus and yellow catfish baculovirus. Obvious CPE can be observed 24-36 h after infection, with cells becoming round and shrinking, and their refractive index increasing. The detection speed is faster than that of conventional cell lines.

[0079] Example 6

[0080] This example demonstrates the application of the PYCB cell line in gene manipulation, as detailed below:

[0081] To evaluate the transfection efficiency and gene expression of the PYCB cell line, 30th generation PYCB cells were seeded into 24-well plates and transfected using the green fluorescent protein (GFP) expression vector pEGFP-N1 (Clontech, Mountain View, California, USA). Cells were transfected only after reaching at least 90% confluence. Lipofectamine 2000 (Invitrogen, USA) was used for transfection, with a ratio of 2 μL Lipofectamine 2000 per 1 μg plasmid DNA. Specifically, 1 μg plasmid DNA and 2 μL Lipofectamine 2000 per well were diluted in 100 μL of Opti-MEM low-serum medium, gently mixed, and incubated at room temperature for 15–20 min to form a complex. The transfection mixture was then added to the cells and incubated at 28°C for 6 h; subsequently, the medium containing the transfection reagent was replaced with fresh M199 medium. GFP expression was observed using a fluorescence microscope (Leica) at 24 h and 48 h post-transfection (hpt).

[0082] The results are as follows Figure 7 As shown, pEGFP-N1 was successfully transfected into PYCB cells, and a green fluorescent signal was detected at 24 hpt. No significant morphological changes were observed in the transfected cells during this period.

[0083] Example 7

[0084] This embodiment provides a method for rapid detection of small RNA viruses in yellow catfish using the PYCB cell line, as detailed below:

[0085] Total RNA was extracted from PYCB cells infected with yellow catfish small RNA virus as described in Example 5, and RT-qPCR amplification was performed using specific primers. The results showed that, compared with uninfected cells, the infected cells exhibited good specificity and sensitivity, with a viral load reaching 10-1. 2 copies / μL.

[0086] The specific primer sequences are as follows:

[0087] F: CCTGAGTAYAGCTGGTGC, SEQ ID NO.1;

[0088] R: TGCTGTAACATTCATGGC, SEQ ID NO.2;

[0089] P: FAM-TAGCAACAACAAGACAAGCCACATC-BHQ1, SEQ ID NO.3.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A yellow catfish brain tissue cell line PYCB (Pelteobagrus fulvidraco), characterized in that, The yellow catfish brain tissue cell line PYCB is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2025148 and deposit date of May 8, 2025.

2. A method for constructing the yellow catfish brain tissue cell line PYCB as described in claim 1, characterized in that, Includes the following steps: (1) Brain tissue from healthy yellow catfish was obtained, and single cells were obtained through washing and digestion; (2) The single cells are cultured in a proliferation medium for primary culture; (3) Primary culture until the cell confluence reaches 60%-90%, digest and then passage culture in subculture medium to obtain the yellow catfish brain tissue cell line PYCB; The proliferation medium was M199 medium containing 15% fetal bovine serum, 2% yellow catfish serum, 1% MEM non-essential amino acids, 100 U / mL penicillin and 100 U / mL streptomycin; The passage medium was M199 medium containing 15% fetal bovine serum, 1% yellow catfish serum, 1% MEM non-essential amino acids, 100 U / mL penicillin and 100 U / mL streptomycin.

3. The construction method according to claim 2, characterized in that, The rinsing solution used is an HBSS solution containing 500 U / mL penicillin and 500 U / mL streptomycin.

4. The construction method according to claim 2, characterized in that, The digestive fluid used during digestion is pancreatic enzyme digestive fluid.

5. The construction method according to claim 2, characterized in that, The primary culture temperature is 28℃; the subculture temperature is 28℃, and subculture is performed every 3-5 days.

6. The application of the yellow catfish brain tissue cell line PYCB as described in claim 1 in the culture of yellow catfish small RNA virus and / or yellow catfish baculovirus.

7. The application of the yellow catfish brain tissue cell line PYCB as described in claim 1 in constructing a yellow catfish small RNA virus and / or yellow catfish baculovirus infection cell model.

8. The use of the yellow catfish brain tissue cell line PYCB as described in claim 1 in screening drugs for preventing and treating yellow catfish small RNA virus and / or yellow catfish baculovirus infection.

9. The use of the yellow catfish brain tissue cell line PYCB as described in claim 1 in the preparation of yellow catfish small RNA virus and / or yellow catfish baculovirus vaccine.

10. The application of the yellow catfish brain tissue cell line PYCB as described in claim 1 in the preparation of genetically engineered host cells.

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