Method for constructing aedes albopictus a a01 cell line and a decapod rhinovirus 1 susceptible cell model

Abstract

The application discloses a construction method of Aedes albopictus Aa01 cell line and susceptible cell model of Decapod iridovirus 1, and belongs to the technical field of virology and aquaculture disease prevention and control, wherein the Aedes albopictus Aa01 cell line is preserved in the China Center for Type Culture Collection of Wuhan University, and the preservation number is CCTCC NO: C202648. The method for constructing the DIV1 susceptible cell model based on the above cell line comprises the following steps: culturing the Aedes albopictus Aa01 cell line, and subculturing or using for virus inoculation when the density reaches 80%-90%; inoculating the DIV1 into the Aedes albopictus Aa01 cell line, removing the virus liquid after virus adsorption, and adding cell maintenance liquid for continuous culture; and observing the cytopathic effect CPE, collecting the cell culture when the CPE reaches 70%-80%, and obtaining the amplified DIV1 virus liquid. The DIV1 susceptible cell model constructed by the method can solve the problem that there is currently a lack of stable and continuously subcultured DIV1 in vitro culture.

Description

Technical Field

[0001] This invention belongs to the field of virology and aquaculture disease prevention and control technology, specifically involving the construction method of Aedes albopictus Aa01 cell line and Decapoda iridovirus 1 susceptible cell model. Background Technology

[0002] Decapodiform iridovirus 1 (DIV1) is an emerging pathogen that poses a serious threat to the global crustacean aquaculture industry. This virus has a wide host range, infecting many major commercially farmed species, including giant freshwater prawns, Litopenaeus vannamei, and Chinese mitten crabs, and has an extremely high mortality rate, causing enormous economic losses to the aquaculture industry.

[0003] Currently, research on the DIV1 infection mechanism and virus-host interaction remains very limited, with the core bottleneck being the lack of an in vitro cell culture model that can stably support DIV1 proliferation. Existing research largely relies on primary cells obtained from crustaceans (such as hepatopancreas and hematopoietic tissue). However, primary cells have inherent limitations such as short lifespan, inability to be continuously passaged, low standardization, and poor reproducibility, which severely restrict the development of basic virology research (such as viral invasion and replication kinetics) as well as applied research such as antiviral drug screening and vaccine development.

[0004] To address the challenge of lacking permanent crustacean cell lines, researchers have attempted to cultivate shrimp viruses using non-host, established cell lines. For example, insect cell lines (such as Sf9 and C6 / 36) have been used to cultivate white spot syndrome virus (WSSV) and giant freshwater prawn Nodamura virus (MrNV). For instance, patent 202510254461X discloses an insect ovarian tissue cell line, sfo-25, for culturing decapod iridoviruses and its construction method. In this method, the fall armyworm ovarian tissue cell line sfo-25 is constructed through multiple rounds of domestication from sfo-1 to sfo-4. However, the above cell model has the following drawbacks in practical applications: 1. In the above scheme, viral infection is only demonstrated through cytopathic effect (CPE) and electron microscopy (Figures 6-7), but it does not verify whether the amplified virus retains its pathogenicity against the original host, giant freshwater prawn. 2. SFO-25 requires multiple rounds of acclimatization and gradual replacement with four different culture media. The process is complicated and dependent on specific culture media formulations. The stability of the cell line may be affected by operational errors or fluctuations in the composition of the culture media.

[0005] Based on the inventor's professional research, there are currently no reports on successfully culturing DIV1 using insect cell lines and systematically verifying its pathogenicity. Therefore, developing a stable, efficient, and standardized in vitro cell model for amplifying DIV1 is of great significance for elucidating the pathogenic mechanism of DIV1 and developing effective prevention and control technologies. Summary of the Invention

[0006] This invention provides a method for constructing the Aedes albopictus Aa01 cell line and a susceptible cell model of Decapoda iridovirus 1. The susceptible cell model of Decapoda iridovirus 1 constructed by this method can solve the current problem of lacking stable and continuously passaged DIV1 in vitro culture.

[0007] The objective of this invention is achieved through the following technical solution: The Aedes albopictus Aa01 cell line, deposited at the China Center for Type Culture Collection of Wuhan University, China, with accession number CCTCC NO: C202648, was obtained from Aedes albopictus embryonic tissue culture.

[0008] The culture method for the above-mentioned Aedes albopictus Aa01 cell line includes the following steps: (1) Pretreatment steps: Aspirate the original culture medium from the culture flask, add 2 mL of phosphate buffer to the culture flask, gently shake the culture flask to rinse the cell layer, and then aspirate and discard the phosphate buffer. (2) Digestion steps: Add a 0.25% trypsin solution containing EDTA to the pretreated culture flask, shake the culture flask, and digest for 2-3 minutes; (3) Termination and pipetting steps: Add complete culture medium to the culture flask, gently pipet to detach the cells from the bottom of the culture flask, and repeatedly pipet in the liquid to disperse the cells into a suspension mainly composed of single cells. (4) Centrifugation collection steps: Collect the cell suspension into a centrifuge tube and centrifuge at 800 rpm / min for 5 minutes. After centrifugation, aspirate and discard the supernatant, and retain the cell pellet. (5) Resuspension and inoculation steps: Add fresh complete culture medium to the cell pellet, mix well by pipetting, and inoculate into new culture flasks at a mass ratio of 1:2-1:3.

[0009] As a preferred option, during the digestion process in step (2), the cell morphology is observed under a microscope, and digestion is terminated when the cells in the middle of the cell block are observed to be obviously rounded and have gaps.

[0010] Preferably, in step (3), the complete culture medium is made of the following components: MEM medium, non-essential amino acids, fetal bovine serum and penicillin-streptomycin solution in a mass ratio of 88:1:10:1.

[0011] Preferably, in step (3), the non-essential amino acids in equimolar concentrations are L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

[0012] This invention also provides a method for constructing a susceptible cell model of Decapoda iridovirus 1 based on the Aedes albopictus Aa01 cell line, the method comprising the following steps: (1) Cell culture: Culture the Aedes albopictus Aa01 cell line and passage it when the density reaches 80%-90% or use it for virus inoculation; (2) Virus inoculation: DIV1 solution was inoculated into the Aedes albopictus Aa01 cell line cultured in step (1) and incubated at 28°C to allow the virus to come into full contact with the cells; (3) After the virus is adsorbed, remove the virus solution and add cell maintenance medium to continue culturing; (4) Observe the cytopathic effect (CPE). When the CPE reaches 70%-80%, collect the cell culture to obtain the amplified DIV1 virus solution.

[0013] In this method, the Aa01 cell line, as a mosquito-derived cell, may be more adapted to the replication environment of arboviruses (although DIV1 is not an arbovirus, Aa01 cells have better controllability and standardization). Furthermore, the embodiments of this application partially verify its pathogenicity retention in giant freshwater prawns, indicating stronger biological relevance.

[0014] Meanwhile, the Aa01 cell line directly uses the established mosquito-derived cell line, without the need for complex domestication. It only requires optimization of virus inoculation conditions, making the operation steps simpler and more reproducible and standardized.

[0015] Preferably, the optimal incubation time between DIV1 and cells in step (2) is 6 hours.

[0016] Preferably, the cytopathic effects described in step (4) include cell rounding, increased particle size, aggregation, and detachment from the culture surface.

[0017] Preferably, the constructed cell model is highly susceptible to DIV1, and after DIV1 is amplified in the cell model, the viral titer can reach 10. -2.67 TCID 50 / 0.1mL.

[0018] Preferably, the DIV1 susceptible cell model constructed using the above method has the following applications: (1) Used for in vitro amplification of DIV1; (2) Determine the toxicity or titer of DIV1; (3) Used for pathogenicity studies of DIV1; (4) Screening for anti-DIV1 drugs; (5) Prepare a vaccine or diagnostic antigen for DIV1.

[0019] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include: This invention is the first to discover and confirm that the mosquito-derived Aa01 cell line is highly susceptible to DIV1 and can support the complete viral replication cycle, filling the gap in the field of stable in vitro culture systems. Compared with primary crustacean cells that are difficult to obtain, have short lifespans, and cannot be passaged, the cell model of this invention has the advantages of unlimited passage, controllable culture conditions, good reproducibility, and ease of standardization, providing stable and reliable experimental materials for DIV1 research.

[0020] DIV1 titers can reach 10 after amplification in the cell model of this invention. -2.67 TCID 50 / 0.1mL, and animal infection experiments have confirmed that the cell-amplified virus still retains the same pathogenicity as the original virus, and can cause dose-dependent death and typical histopathological changes in giant freshwater prawns.

[0021] This cell model can be used not only for basic research on DIV1, such as replication mechanism and pathogenesis, but also for high-throughput screening of antiviral drugs and preparation of diagnostic reagents. It is of great significance for controlling the spread of DIV1 and ensuring the healthy development of crustacean aquaculture. Attached Figure Description

[0022] Figure 1 This is a diagram illustrating the cytopathic effect of DIV1 infection on Aa01 cells in Example 1 of this invention. A represents uninfected control cells with normal morphology; B represents DIV1-infected cells, exhibiting irregular morphology and cell shedding.

[0023] Figure 2 This is a graph showing the effect of different incubation times between the virus and cells on viral load in Example 2 of the present invention. The results show that the viral load gradually increases within 6 hours after infection, reaches its peak at 6 hours, and then tends to stabilize.

[0024] Figure 3 These are histopathological and transmission electron micrographs of the artificial infection experiment in Example 4 of this invention. A is a section of hepatopancreas tissue from a healthy giant freshwater prawn (H&E staining); B is a section of hepatopancreas tissue from a giant freshwater prawn infected with DIV1, showing obvious structural disorder; C is viral particles in the cytoplasm of the infected tissue observed under a transmission electron microscope. Detailed Implementation

[0025] The following detailed description of the embodiments of this application, in conjunction with the accompanying drawings, will provide a thorough understanding of how this application uses technical means to solve technical problems and achieve corresponding technical effects, enabling its implementation. The embodiments of this application and the various features within them can be combined with each other without conflict, and all resulting technical solutions are within the protection scope of this application.

[0026] It should be clearly stated that the embodiments described below are merely some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0027] Example 1: Establishment of a DIV1 susceptible cell model and observation of cytopathic effects 1. Material preparation Cell line: Aedes albopictus Aa01 cell line, deposited by the Microbiology Laboratory of Zhejiang Freshwater Fisheries Research Institute at the China Center for Type Culture Collection, Wuhan University, China, accession number CCTCC NO: C202648. Virus strain: DIV1 strain, isolated from the hepatopancreatic tissue of naturally infected giant freshwater prawns.

[0028] Main reagents: MEM medium (containing non-essential amino acids), fetal bovine serum (FBS), penicillin-streptomycin antibiotics, 0.25% trypsin (containing EDTA), PBS buffer.

[0029] 2. Cell Culture Aa01 cells were seeded into T25 culture flasks containing 5 mL of complete culture medium and cultured in a 28°C, 5% CO2 incubator. When the cells reached approximately 90% confluence, they were passaged. During passage, the old culture medium was discarded, the cells were rinsed once with PBS, and then digested with 1 mL of 0.25% trypsin for 2-4 minutes. When the cells became rounded and detached under a microscope, 1 mL of complete culture medium was added to stop the digestion. The cells were gently pipetted to form a single-cell suspension, collected in centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium. The cells were then aliquoted into new culture flasks at a 1:2 or 1:3 ratio for continued culture.

[0030] 3. Virus vaccination Collect Aa01 cells in logarithmic growth phase, with good growth and approximately 80% confluence, and discard the old culture medium. Perform a 10-fold serial dilution of the DIV1 virus stock solution in MEM medium, and inoculate 500 μL of the diluted virus solution into the cells. Incubate the cells at 28°C for 1 hour to allow virus adsorption. Then discard the virus solution, add 5 mL of cell maintenance medium (97% MEM + 2% FBS + 1% penicillin-streptomycin), and transfer to a 28°C, 5% CO2 incubator for further culture.

[0031] 4. Observation of cytopathic effects Cell status was observed daily under an inverted microscope. Results showed that uninoculated control cells exhibited normal morphology throughout the culture period, showing semi-adherent growth, round shape, and strong refractive index. However, cells inoculated with DIV1 began to show significant cytopathic effect (CPE) 48 hours post-infection, characterized by rounded cells, increased refractive index, and increased intracellular granules; by 96 hours, CPE had further progressed, with many cells becoming irregular in shape, some elongated spindle-shaped, and beginning to detach from the culture flask surface, aggregating into floating cell clumps (e.g., ...). Figure 1 (As shown). This indicates that Aa01 cells are highly susceptible to DIV1, which is able to replicate in these cells and cause typical cytopathic effects.

[0032] Example 2: Determination of the optimal virus-cell incubation time DIV1 cells were seeded into Aa01 cells according to the method in Example 1, and cell samples were collected at 0, 2, 4, 6, 8, 10, and 12 hours post-seedling. Total DNA was extracted from the cells using the reported method. Taq The Man probe-based real-time quantitative PCR method for detecting viral load.

[0033] The results are as follows Figure 2 As shown, the intracellular viral load gradually increased within 0 to 6 hours after infection, reaching a peak at 6 hours; the viral load tended to stabilize between 6 and 12 hours. Therefore, 6 hours was determined to be the optimal incubation time for virus-cell interaction in the cell model described in this invention.

[0034] Example 3: DIV1 viral titer (TCID) 50 Determination of ) After digesting Aa01 cells in the logarithmic growth phase, the cell concentration was adjusted to approximately 2 × 10⁻⁶ cells using complete culture medium. 5 Cells were seeded at a density of 100 μL per well in a 96-well cell culture plate and cultured at 28°C in a 5% CO2 incubator until the cells formed a monolayer.

[0035] The DIV1 virus solution collected in Example 1 was serially diluted 10-fold using MEM medium (10... -1 Up to 10 -9 ).

[0036] Remove the old culture medium from the 96-well plate, and add the virus solution of each dilution to the corresponding well. Inoculate 8 replicates per dilution, 100 μL per well. Also, set up negative control wells (add 100 μL of MEM culture medium).

[0037] After incubating the culture plates in a 28°C, 5% CO2 incubator for 6 hours, add 100 μL of cell maintenance medium (97% MEM + 2% FBS + 1% penicillin-streptomycin) to each well.

[0038] CPE was observed under a microscope daily, and the number of wells with CPE was counted on day 4 post-infection (4 dpi). The results are shown in Table 1.

[0039] Viral titers were calculated using the Reed-Muench method. The results showed that the TCID of the DIV1 viral fluid amplified in the cell model of this invention was [data missing]. 50 10 -2.67 / 0.1mL, meaning that each 0.1mL of virus solution contains half the tissue culture infection dose of 10. 2.67 TCID 50 .

[0040] Table 1 TCID 50 Measurement results

[0041] Example 4: Pathogenicity verification of cell-cultured DIV1 against giant freshwater prawns To verify whether DIV1 cells cultured and passaged in Aa01 cells still retain pathogenicity against the original host, Macrobrachium rosenbergii, an artificial infection experiment was conducted.

[0042] Virus preparation: The cell culture supernatant that produced obvious CPE in Example 1 was collected, filtered through a 0.45 μm filter membrane to remove cell debris, and DIV1 virus solution in cell culture was obtained. The virus solution was diluted 10-fold, 100-fold, and 1000-fold with sterile PBS.

[0043] Animal grouping and challenge: 125 healthy giant freshwater prawns (average weight approximately 12g) were randomly divided into 5 groups of 25 prawns each. These groups were: original virus solution group, 10-fold dilution group, 100-fold dilution group, 1000-fold dilution group, and PBS control group. Challenge was performed via intramuscular injection, with each prawn injected with 0.1mL of the corresponding virus dilution or sterile PBS.

[0044] Results Observation and Recording: Shrimp were observed continuously for 7 days after challenge, and daily mortality was recorded. The results are shown in Table 2. The cumulative mortality rate in the original virus group and the 10-fold dilution group reached 100% within 7 days; the cumulative mortality rate in the 100-fold dilution group was 72%; the cumulative mortality rate in the 1000-fold dilution group was 38%; while all shrimp in the PBS control group survived. All dead shrimp tested positive for DIV1 by PCR. The experimental results indicate that DIV1 cultured in Aa01 cells retains complete pathogenicity and can induce dose-dependent mortality in giant freshwater prawns.

[0045] Table 2 Results of artificial infection experiments

[0046] Example 5: Histopathological and Ultrastructural Observation of Infected Tissues The giant freshwater prawns infected with DIV1 in Example 4 and the healthy control group were dissected, and their hepatopancreas tissues were collected for histopathological and transmission electron microscopy observation.

[0047] Histopathology: Tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned (6 μm), stained with hematoxylin and eosin (H&E), and observed under a light microscope. Results showed that the hepatic tubules of healthy shrimp were tightly and orderly arranged, and oval in shape. Figure 3 A); while the hepatic tubules of shrimp infected with DIV1 showed severe disorder, loose arrangement, and ring-like structure ( Figure 3 B).

[0048] Transmission electron microscopy: After fixation with glutaraldehyde, osmium tetroxide, resin embedding, ultrathin sectioning, and staining with uranium acetate and lead citrate, the tissue was observed under a transmission electron microscope. The results showed that a large number of hexagonal, high-electron-density viral particles were visible in the cytoplasm of infected shrimp hepatopancreatic cells. Figure 3 C).

[0049] The above results further confirm that DIV1 amplified using the cell model of the present invention can induce pathological changes in giant freshwater prawns consistent with natural infection, and this cell model has extremely high biological relevance.

[0050] In summary, this invention successfully constructed a stable and efficient DIV1 susceptible cell model. Based on the mosquito-derived Aa01 cell line, this model supports the complete DIV1 replication cycle, maintaining stable viral titers and strong pathogenicity after viral amplification. The establishment of this model provides a crucial experimental platform for studying the pathogenic mechanism of DIV1, screening antiviral drugs, and developing diagnostic reagents, and has significant application value for controlling the spread of DIV1.

Claims

1. The Aedes albopictus Aa01 cell line, characterized in that, The Aedes albopictus Aa01 cell line is deposited at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: C202648, and was obtained from Aedes albopictus embryonic tissue culture.

2. The Aedes albopictus Aa01 cell line according to claim 1, characterized in that, The cultivation method includes the following steps: (1) Pretreatment steps: Aspirate the original culture medium from the culture flask, add 2 mL of phosphate buffer to the culture flask, gently shake the culture flask to rinse the cell layer, and then aspirate and discard the phosphate buffer. (2) Digestion steps: Add a 0.25% trypsin solution containing EDTA to the pretreated culture flask, shake the culture flask, and digest for 2-3 minutes; (3) Termination and pipetting steps: Add complete culture medium to the culture flask, gently pipet to detach the cells from the bottom of the culture flask, and repeatedly pipet in the liquid to disperse the cells into a suspension mainly composed of single cells. (4) Centrifugation collection steps: Collect the cell suspension into a centrifuge tube and centrifuge at 800 rpm / min for 5 minutes. After centrifugation, aspirate and discard the supernatant, and retain the cell pellet. (5) Resuspension and inoculation steps: Add fresh complete culture medium to the cell pellet, mix well by pipetting, and inoculate into new culture flasks at a mass ratio of 1:2-1:

3.

3. The Aedes albopictus Aa01 cell line according to claim 2, characterized in that, During the digestion process in step (2), cell morphology is observed under a microscope. Digestion is terminated when the cells in the middle of the cell block are observed to be obviously rounded and have gaps.

4. The Aedes albopictus Aa01 cell line according to claim 2, characterized in that, In step (3), the complete culture medium is made from the following components: MEM medium, non-essential amino acids, fetal bovine serum and penicillin-streptomycin solution in a mass ratio of 88:1:10:

1.

5. The Aedes albopictus Aa01 cell line according to claim 4, characterized in that, In step (3), the non-essential amino acids are specifically L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine in equimolar concentrations.

6. A method for constructing a susceptible cell model of Decapoda iridovirus 1 based on the Aedes albopictus Aa01 cell line according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Cell culture: Culture the Aedes albopictus Aa01 cell line and passage it when the density reaches 80%-90% or use it for virus inoculation; (2) Virus inoculation: DIV1 was inoculated into the Aedes albopictus Aa01 cell line cultured in step (1) and incubated at 28°C to allow the virus to come into full contact with the cells; (3) After the virus is adsorbed, remove the virus solution and add cell maintenance medium to continue culturing; (4) Observe the cytopathic effect (CPE). When the CPE reaches 70%-80%, collect the cell culture to obtain the amplified DIV1 virus solution.

7. The method according to claim 6, characterized in that, In step (2), the optimal incubation time between DIV1 and cells is 6 hours.

8. The method according to claim 6, characterized in that, The cytopathic effects described in step (4) include cell rounding, increased granulation, aggregation, and detachment from the culture surface.

9. The method according to claim 6, characterized in that, The constructed cell model was highly susceptible to DIV1, and after DIV1 was amplified in the cell model, the viral titer could reach 10. -2.67 TCID 50 / 0.1mL.

10. The method according to any one of claims 6-9, characterized in that, The constructed DIV1 susceptible cell model has the following applications: (1) Used for in vitro amplification of DIV1; (2) Determine the toxicity or titer of DIV1; (3) Used for pathogenicity studies of DIV1; (4) Screening for anti-DIV1 drugs; (5) Preparation of diagnostic antigens for DIV1.

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