Construction method and application of respiratory virus infected alveolar organ model

Abstract

The invention relates to the technical field of biological medicine, in particular to a construction method and application of a respiratory virus infected alveolar organ model. Comprising the following steps: infecting alveolar cell source single cells by using a virus inoculum, coating the infected alveolar cell source single cells with matrigel, and inoculating the infected alveolar cell source single cells into a first culture medium for three-dimensional culture to obtain the respiratory virus infected alveolar organ model. According to the invention, alveolar cells are creatively applied to model characterization of respiratory virus infection, and particularly, alveolar cell source single cells are subjected to infection treatment by adopting influenza A H1N1 virus, so that the constructed model can well simulate and characterize the infection process of influenza A virus in human lung organs; the method provides an experimental basis for drug research and development (such as high-throughput screening of anti-influenza virus drugs), viral disease pathogenesis research, research on defense mechanisms of naturally occurring virus infection or virus infection caused by experiments and the like, and has important guiding significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing a respiratory virus-infected alveolar organoid model and its application. Background Technology

[0002] In traditional virus research methods, researchers typically employ a strategy combining cell line inoculation with animal model validation. For example, the virus is inoculated into immortalized cell lines such as African green monkey kidney cell lines (e.g., Vero, AGMK, BGM) or human embryonic lung fibroblasts for in vitro expansion, followed by animal experiments (e.g., mouse, rat, guinea pig, and hamster models) to explore the viral pathogenesis and host defense responses. While these animal models are widely used to analyze virus-host interaction networks, their application has significant limitations:

[0003] First, the inherent individual biological differences in laboratory animals lead to high data dispersion. To ensure the reproducibility of results, it is often necessary to increase the sample size, which not only violates the "3R" principle of animal ethics (replace, reduce, optimize) but is also more likely to cause ethical disputes. Second, the risk of cross-contamination of pathogens in group-housed environments increases significantly, posing a greater challenge to the biosafety management of laboratory animal facilities. Third, there are technical bottlenecks in model construction—the cycle from virus inoculation to symptom onset is long, and the success rate of the model is affected by multiple factors such as operating techniques, animal strains, and environmental factors. Fourth, traditional animal models are difficult to meet the high-throughput screening requirements of modern drug development, and the model capacity of a single experiment is usually limited to a scale of tens to hundreds of cases. Most importantly, the expression profiles of influenza A virus receptors (such as α-2,6 sialic acid receptors) differ significantly across species, leading to a discrepancy between the susceptibility of laboratory animals to human pathogens and clinical reality. This directly restricts the validity of the research conclusions in clinical translation.

[0004] Chinese invention patent application CN118805740A discloses a method for constructing and applying a mouse model of severe pneumonia caused by human respiratory syncytial virus (RSV). This method constructs a severe pneumonia animal model by infecting humanized IGF1R transgenic BALB / c mice with human respiratory syncytial virus via nasal droplets, solving the problem that existing models cannot simulate the human disease course and achieving susceptibility to infection in mice and effectiveness in drug evaluation. However, this method still relies on animal samples and cannot solve the aforementioned technical problems.

[0005] Against this backdrop, providing a model construction method that avoids animal models and can better simulate the viral infection process has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] Traditional virus research often relies on animal models, which are limited by technical issues such as individual animal differences, ethical controversies, and differences in gene expression. This invention innovatively uses human alveolar organoids to construct a respiratory virus infection model, which not only avoids the problems of animal models, but also can better simulate and characterize the infection process of respiratory viruses in human lung organs, and has positive guiding significance for the field of virus research.

[0007] The first aspect of this invention provides a method for constructing a respiratory virus-infected alveolar organoid model, comprising:

[0008] Alveolar organoids were infected with viral inoculum, and the infected alveolar organoids were coated in matrix gel and inoculated into the first culture medium for three-dimensional culture to obtain a respiratory virus-infected alveolar organoid model.

[0009] Optionally, the viral inoculum is an inoculum of at least one of influenza A virus, influenza B virus (B / Yamagata lineage, B / Victoria lineage), novel coronavirus (SARS-CoV-2), and respiratory syncytial virus (RSV).

[0010] The influenza A viruses mentioned can include, among others, the H1N1, H1N2, H2N2, H3N2, H3N2v, H5N1, H7N1, H7N2, H7N3, H7N7, H7H9, H9N2, and H10N8 influenza viruses.

[0011] Optionally, the viral inoculum is at least one of the following: H1N1 / wsn virus strain, A / PR / 8 / 34 virus strain, PR8-WSNNS virus strain, A2 virus strain, Long virus strain, PIV-3 virus strain, Tor2 virus strain, Urbani virus strain, and HCoV-EMC / 2012 virus strain.

[0012] Alternatively, the viral inoculum may be an H1N1 / wsn virus strain.

[0013] Optionally, the MOI of the viral inoculum is 0.01-5; further optionally, it is 0.05-2; further optionally, it is 0.1.

[0014] MOI, also known as the Multiplicity of Infection, describes the average number of viral particles per host cell in a viral infection experiment; for example, MOI = 5 means that each cell is infected with an average of 5 viral particles.

[0015] Optionally, the matrix gel is a mouse sarcoma cell basement membrane (Matrigel); further optionally, it is 5-80% Matrigel.

[0016] Optionally, the components of the first culture medium include: DMEM / F12 medium, 20-40 μM dexamethasone, 0.01-0.2 mM cAMP, 0.01-0.2 mM IBMX, and 0.1-5 μg / mL trypsin.

[0017] The concentrations of dexamethasone, cAMP, IBMX, and trypsin are all calculated based on the volume of DMEM / F12 medium.

[0018] Further optionally, the trypsin is treated with TPCK (L-1-p-toluenesulfonyl-2-phenylalanylchloromethyl ketone).

[0019] Optionally, when preparing a respiratory virus-infected alveolar organoid model, the three-dimensional culture time is 24-96 hours; more preferably, it is 24-48 hours.

[0020] Lung organoids, as a type of three-dimensional structure formed by spontaneous tissue aggregation, mainly originate from adult stem cells, embryonic lung bud progenitor cells, or pluripotent stem cells. These stem cells, under the synergistic effect of matrix gel (such as Matrigel) and specific growth factors, can spontaneously form organoids with alveolar compartment structures. Their cellular composition includes type II alveolar cells (AT2), type I alveolar cells (AT1), and stromal cell subsets, accurately reproducing events such as branching morphogenesis and terminal differentiation during lung tissue development. This invention pioneers the application of alveolar celloids to the model characterization of respiratory viral infections, particularly using H1N1 influenza A virus to infect single cells derived from alveolar celloids. The constructed model can effectively simulate and characterize the infection process of H1N1 influenza A virus in human lung organs, providing an experimental basis for drug development (such as high-throughput screening of anti-H1N1 influenza A drugs), research on the pathogenesis of viral diseases, and the study of defense mechanisms against naturally occurring or experimentally induced viral infections, and has significant guiding significance.

[0021] In some embodiments, the method for preparing the alveolar cell-derived single cells includes:

[0022] Discarded primary lung tissue was digested and dissociated into single cells, and the single cells were purified and sorted to obtain type II alveolar cells.

[0023] The type II alveolar cells were subjected to planar and three-dimensional culture sequentially to obtain lung bud organoids;

[0024] The lung bud organoids were differentiated and cultured to obtain alveolar organoids;

[0025] The alveolar organoids were digested and dissociated to obtain alveolar cell-derived single cells.

[0026] Optionally, the purification and sorting of single cells includes: staining the single cells with EPCAM antibody and HT2-280 antibody, sorting double-positive cells, and obtaining type II alveolar cells.

[0027] The basis for constructing the viral infection model in this invention is alveolar cell-derived single cells, which are derived from discarded primary lung tissue. These alveolar cell-derived single cells are obtained by purifying, sorting, and culturing discarded primary lung tissue, and have the advantage of being widely available. The alveolar cell-derived single cells prepared by specific methods can provide a reliable cellular basis for further experimental research, and help to construct a stable and reliable respiratory virus infection alveolar organoid model.

[0028] Optionally, the type II alveolar cells can be cultured in a plane using a reprogrammed culture medium.

[0029] Further optionally, the reprogramming medium comprises: Ham's F-12 medium and a growth regulator; the growth regulator, based on the volume of Ham's F-12 medium, comprises:

[0030] 0.5-2% N2 nutritional supplement;

[0031] 1-4% Vitamin B27 supplement;

[0032] 10-100 ng / mL cell growth factor;

[0033] 1-50 μM ROCK kinase inhibitor;

[0034] 1-10 μM Wnt signaling pathway activator;

[0035] 0.1-3 μM TGF-β signaling inhibitors.

[0036] Optionally, the cell growth factor includes one or more combinations of epithelial cell growth factor, fibroblast growth factor, and hepatocyte growth factor.

[0037] Optionally, the growth regulator may further include: 0.1-5 μg / mL hydrocortisone.

[0038] Optionally, the growth regulator may further include: 1-5 μg / mL triiodothyronine.

[0039] Optionally, the growth-regulating agent further includes: a 0.1-2 nM p38 MAPK (p38 mitogen-activated protein kinase) inhibitor.

[0040] Optionally, the growth regulator may further include: a 20-200 ng / mL BMP inhibitor.

[0041] The second aspect of this invention provides an application of the method for constructing a respiratory virus-infected alveolar organoid model as described above. The method is applied to disease mechanism research, antiviral drug development (e.g., high-throughput screening of anti-H1N1 influenza drugs), clinical translational research, disease prevention and early intervention research, etc.

[0042] Beneficial effects:

[0043] This invention provides a method for constructing a respiratory virus-infected alveolar organoid model and its application, which has the following advantages:

[0044] (1) This invention innovatively applies alveolar cells to the model characterization of respiratory virus infection, especially by using the H1N1 influenza A virus to infect alveolar cell-derived single cells. The constructed model can well simulate and characterize the infection process of influenza A virus in human lung organs.

[0045] (2) The basis for constructing the viral infection model in this invention is alveolar cell-derived single cells, which are derived from discarded primary lung tissue. They are obtained by purifying, sorting and culturing the discarded primary lung tissue. They have the advantages of being widely available and easy to obtain, avoiding the experimental errors and ethical controversies of animal experimental models. They are highly operable and easy to promote.

[0046] (3) The present invention further uses the H1N1 / wsn virus strain with MOI=0.1 to infect alveolar organoids. The experimental results show that as the time after viral infection increases, the viral load on alveolar organoids increases significantly. That is, the organoid model obtained by the present invention has a high viral load after viral infection and exhibits stable functional consistency, which can be used to simulate and characterize the infection process of influenza A virus in human lung organs.

[0047] (4) Immunofluorescence detection results showed that after the alveolar organoid single cells of the present invention were infected by H1N1 virus, there were obvious brown granular deposits in the cells, that is, the alveolar organoid single cells showed a positive signal of H1N1 virus nucleoprotein after infection, which directly proved that H1N1 virus successfully infected alveolar organoid single cells, and further verified the successful construction of the respiratory virus infection alveolar organoid model of the present invention.

[0048] (5) This invention provides guidance for drug development (such as high-throughput screening of anti-H1N1 virus drugs), research on the pathogenesis of viral diseases, and research on the defense mechanisms against naturally occurring or experimentally induced viral infections. It provides key technical support for the construction of a public health security system, can enhance the prevention and control capabilities of major respiratory infectious diseases, and promote the application of precision medicine in the field of viral infections. It has positive and far-reaching social, economic and scientific significance. Attached Figure Description

[0049] Figure 1 Primer information used in RT-qPCR testing;

[0050] Figure 2 Viral load results at different time points in the respiratory virus-infected alveolar organoid model constructed in Example 1; Figure 2 In the table, a represents the viral load in the culture supernatant, and b represents the viral load in the cells; the blue data points represent the viral infection detection results of alveolar organoids with 5 passages, and the red data points represent the viral infection detection results of alveolar organoids with 15 passages.

[0051] Figure 3 Immunofluorescence detection results of the respiratory virus-infected alveolar organoid model constructed in Example 1. Detailed Implementation

[0052] To address the aforementioned technical problems, this invention provides a method for constructing a respiratory virus-infected alveolar organoid model, comprising:

[0053] Alveolar organoids were infected with viral inoculum, and the infected alveolar organoids were coated in matrix gel and inoculated into the first culture medium for three-dimensional culture to obtain a respiratory virus-infected alveolar organoid model.

[0054] In some embodiments, the method for preparing the alveolar cell-derived single cells includes:

[0055] Discarded primary lung tissue was digested and dissociated into single cells, and the single cells were purified and sorted to obtain type II alveolar cells.

[0056] The type II alveolar cells were subjected to planar and three-dimensional culture sequentially to obtain lung bud organoids;

[0057] The lung bud organoids were differentiated and cultured to obtain alveolar organoids;

[0058] The alveolar organoids were digested and dissociated to obtain alveolar cell-derived single cells.

[0059] The following is some information about the raw materials involved in this invention.

[0060]

[0061]

[0062] Note: Unless otherwise specified, the solvents of the solutions involved in this invention are all water; the reagent concentrations are all mass concentrations; the room temperature is 25°C; and the raw materials, consumables and equipment used are all commercially available.

[0063] Example 1

[0064] This embodiment provides a method for constructing a respiratory virus-infected alveolar organoid model, including:

[0065] Using a viral inoculum (H1N1 / wsn virus strain) with an MOI of 0.1, 1×10⁶ alveolar cell-derived single cells (1×10⁶) were inoculated. 5 Infecting the cells with a single cell of alveolar cell type, the infected cells were coated in Matrigel (equivalent to 50% of the culture medium volume), seeded in low-absorption 6-well plates, and cultured in three dimensions with the first culture medium (seedling density of 1×e per well). 5 Cells were used to obtain an alveolar organoid model of respiratory virus infection.

[0066] The first culture medium consists of: DMEM / F12 medium, 300 μM dexamethasone, 0.1 mM cAMP, 0.1 mM IBMX, and 1 μg / mL trypsin (treated with TPCK).

[0067] In preparing the respiratory virus-infected alveolar organoid model, the three-dimensional culture time was 48 hours (the virus inoculum was removed 2 hours after virus infection); during the culture process, the culture supernatant and cells were collected for detection at 2 hours, 24 hours and 48 hours, respectively.

[0068] The method for preparing the alveolar cell-derived single cells includes:

[0069] S1. Primary lung tissue discarded during surgery was cleaned and disinfected using sterile PBS buffer. The tissue was minced (1-2 mm) and transferred to a 15 mL centrifuge tube. 10 mL of compound enzyme digestion solution was added, and the mixture was incubated at 37°C for 1 h to obtain a cell suspension. With the aid of sterile PBS buffer, the cell suspension was sieved using a 70 μm cell filter, and the filtrate was collected. The filtrate was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected. Red blood cell lysis equilibrium solution was added to the precipitate for resuspending. The mixture was centrifuged again and resuspended repeatedly until no red blood cells were observed in the cell pellet, thus completing the lysis and removal of red blood cells to obtain single cells. EPCAM antibody and HT2-280 antibody were added to stain the single cells (EPCAM antibody was derived from APC anti-human CD326 (EpCAM) Antibody (catalog number 324208) direct labeling, and HT2-280 was derived from Goat anti-Mouse IgG, IgM (H+L) Secondary Antibody, Alexa Fluor). TM 488 (catalog number A10680) secondary antibody (volume added according to the instructions) was used to sort EPCAM and HT2-280 double-positive cells to obtain type II alveolar cells.

[0070] S2. The type II alveolar cells were then subjected to 10 4 pcs / cm 2 The cells were seeded at the specified density in 6-well plates, and 2 mL of the second culture medium (reprogramming medium) was added to each well for amplification culture. When the cell confluence reached ≥80%, 25% trypsin digestion solution (solvent is water) was added for digestion for 6 min. Then, the cells were passaged in reprogramming medium and cultured in a plane for 3-4 days to obtain lung bud epithelial-like cells.

[0071] The second culture medium comprises: Ham's F-12 medium and growth regulators; the growth regulators, based on the volume of Ham's F-12 medium, comprise:

[0072] 1% N2 nutritional supplement;

[0073] 2% B27 nutritional supplement;

[0074] 20 ng / mL EGF;

[0075] 50 ng / mL bFGF;

[0076] 0.5 μg / mL hydrocortisone;

[0077] 10μM ROCK kinase inhibitor;

[0078] 3μM Wnt signaling pathway activator CHIR-99021;

[0079] 1 μM TGF-β signaling inhibitor SB431542 (Taoshu Company, T1726).

[0080] S3. When the lung bud epithelial-like cells grow to a cell confluence of ≥80%, add 0.25% trypsin digestion solution (solvent is water) for digestion for 6 min; then place them in a reprogramming medium containing 5% basement membrane matrix and culture for 3 days to obtain lung bud organoids.

[0081] S4. The lung bud organoids were cultured in the third culture medium (differentiation medium) for 7 days to obtain alveolar organoids. The alveolar organoids with 5 passages and 15 passages (when the cells grew to a degree of cell confluence ≥ 80%, 0.25% trypsin digestion solution was added for digestion and recorded as one passage) were used as the basis for subsequent virus infection treatment and testing.

[0082] The third culture medium comprises: DMEM / F12 medium, 30 μM dexamethasone, 0.1 mM cAMP, and 0.1 mM IBMX; wherein the concentrations of dexamethasone, cAMP, and IBMX are calculated based on the volume of DMEM / F12 medium.

[0083] S5. The alveolar organoids are digested and dissociated (specifically, TrypL E is added for digestion and agitation for 5 minutes) to obtain alveolar cell-derived single cells.

[0084] Example 2

[0085] This embodiment provides a method for constructing a respiratory virus-infected alveolar organoid model, with the specific implementation method being the same as in Embodiment 1; the difference being that the reprogramming culture medium comprises: Ham's F-12 medium and growth regulators; based on the volume of Ham's F-12 medium, the growth regulators comprise:

[0086] 1% N2 nutritional supplement;

[0087] 2% B27 nutritional supplement;

[0088] 20 ng / mL EGF;

[0089] 20 ng / mL HGF

[0090] 10μM ROCK kinase inhibitor;

[0091] 200 ng / mL R-spondin-1 (MCE, HY-P7114), an activator of the Wnt signaling pathway;

[0092] 1 μM TGF-β signaling inhibitor (A8301);

[0093] 100 ng / mL BMP inhibitor Noggin (Taoshu Company TMPY-05202).

[0094] Example 3

[0095] This embodiment provides a method for constructing a respiratory virus-infected alveolar organoid model, with the specific implementation method being the same as in Embodiment 1; the difference being that the reprogramming culture medium comprises: Ham's F-12 medium and growth regulators; based on the volume of Ham's F-12 medium, the growth regulators comprise:

[0096] 1% N2 nutritional supplement;

[0097] 2% B27 nutritional supplement;

[0098] 20 ng / mL EGF;

[0099] 2 μg / mL triiodothyronine;

[0100] 10μM ROCK kinase inhibitor;

[0101] 3μM Wnt signaling pathway activator CHIR-99021;

[0102] 1 μM TGF-β signaling inhibitor SB431542 (Tao Shu T1726);

[0103] 1 nM p38 MAPK inhibitor (Taoshu Company T36010).

[0104] Performance testing

[0105] 1. RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) Detection of Specific Genes in a Respiratory Virus Infected Alveolar Organoid Model

[0106] Taking Example 1 as an example, during the construction of the respiratory virus-infected alveolar organoid model, culture supernatant and cells were collected at 2h, 24h, and 48h for detection. The collected cells were partially fixed in 4% PFA (paraformaldehyde) for immunofluorescence detection. The remaining cells were centrifuged, the supernatant was discarded, and 1mL of Trizol (total RNA extraction reagent) was added for lysis. Simultaneously, 1mL of Trizol (total RNA extraction reagent) was added to the culture supernatant for lysis and qPCR detection. RNA was extracted from the cells and supernatant after Trizol treatment and detected using primers for H1N1 NP detection (primer information is available in [link to primer information]). Figure 1qPCR was performed to determine the viral load of H1N1 in alveolar organoids at different time points. Test results are shown below. Figure 2 .

[0107] The qPCR testing conditions are as follows:

[0108] Reverse transcription reaction stages (Stage 1, 2)

[0109] Cycle number: 1

[0110] Temperature and time conditions: react at 42℃ for 5 minutes first, then react at 95℃ for 10 seconds.

[0111] PCR reaction stage (Stage 3)

[0112] Number of cycles: 40

[0113] Temperature and time conditions: 95℃ for 3 seconds, 60℃ for 30 seconds.

[0114] Annealing stage (Stage 4):

[0115] Temperature and time conditions: 95℃ reaction time for 3 seconds.

[0116] 2. Immunofluorescence detection

[0117] Cells infected for 48 hours were fixed in 4% PFA (paraformaldehyde) for immunofluorescence detection. The detection steps included:

[0118] 1) Prepare paraffin sections from the fixed samples and bake them at 62°C for 1 hour.

[0119] 2) Dewaxing the sections: Immerse the sections in xylene I for 20 minutes, xylene II for 20 minutes, and xylene III for 20 minutes in sequence (do not immerse in xylene for 20 minutes continuously);

[0120] 3) Hydration: Soak the slices in anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, 95% ethanol for 1 min, 95% ethanol for 1 min, and 70% ethanol for 1 min in sequence, then rinse with tap water for 5 min, and then rinse once with distilled water.

[0121] 4) Add 100 μL of hydrogen peroxide solution (3% concentration) to each slice and incubate at room temperature for 10 min to block the activity of endogenous peroxidase.

[0122] 5) Wash three times with PBS (phosphate buffer), 5 min each time;

[0123] 6) Heat antigen retrieval: Place the PBS-washed slides on a plastic sample holder and immerse them in boiling antigen retrieval solution (1 mmol Tris-EDTA, pH=9.0). Boil for 15 min, then keep warm for 15 min. After turning off the power, allow them to cool naturally.

[0124] 7) Wash three times with PBS, 5 minutes each time;

[0125] 8) Remove PBS, add 100 μL of 5% BSA blocking solution (from Sigma) to each slice, and incubate at room temperature for 20 min;

[0126] 9) Remove serum, add 100 μL of primary antibody dilution buffer (Influenza Avirus Nucleoprotein, catalog number HL1089) to each slide, and incubate overnight at 4°C;

[0127] 10) Remove the humidifier box the next day and place it at 37℃ for 30 minutes;

[0128] 11) Wash three times with PBS, 5 minutes each time;

[0129] 12) Remove PBS, add 50 μL of labeled secondary antibody (Goat anti-rabbit IgG H&L(HRP)abcam) to each slide, and incubate at 37°C for 30 min;

[0130] 13) Wash three times with PBS, 5 minutes each time;

[0131] 14) Remove PBS, add 100 μL of try-594 tyramine conversion reagent to each slice, and incubate at room temperature for 20 min;

[0132] 15) Wash three times with PBS, 5 minutes each time;

[0133] 16) Remove PBS and add 100 μL of anti-fluorescence quenching mounting medium (containing DAPI) to each slide.

[0134] 17) Microscopic examination: Observe under a microscope and record the results. Figure 3 .

[0135] like Figure 2 As shown, the viral load on alveolar organoids increased significantly with increasing time after viral infection, reaching 10 in the culture supernatant. 6 Around 10, the viral load in the cells can reach 10. 8The viral load changes of alveolar organoids with passage numbers of 5 and 15 after viral infection showed a high degree of consistency. These results demonstrate that the present invention has successfully constructed an alveolar organoid model of respiratory virus infection. The obtained organoid model has both a high viral load and stable functional consistency after viral infection, and can be used to simulate and characterize the infection process of influenza A virus in human lung organs.

[0136] like Figure 3 As shown, immunofluorescence detection results revealed distinct red markers within alveolar organoid single cells infected with H1N1 virus. This is precisely the location of the H1N1 virus nucleoprotein as marked by the DAB staining method. In other words, the presence of a positive signal for H1N1 virus nucleoprotein after alveolar organoid single-cell infection directly demonstrates that H1N1 virus successfully infected alveolar organoid single cells, further validating the successful construction of the respiratory virus-infected alveolar organoid model of this invention. The test results of Examples 2 and 3 are similar to those of Example 1, both successfully constructing a respiratory virus-infected alveolar organoid model, which can be used to simulate and characterize the H1N1 virus infection process in human lung organs.

Claims

1. A method for constructing a respiratory virus-infected alveolar organoid model, characterized in that, include: Alveolar organoids were infected with viral inoculum, and the infected alveolar organoids were coated in matrix gel and inoculated into the first culture medium for three-dimensional culture to obtain a respiratory virus-infected alveolar organoid model.

2. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 1, characterized in that, The viral inoculum is an inoculum of at least one of influenza A virus, influenza B virus, novel coronavirus, and respiratory syncytial virus.

3. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 1, characterized in that, The viral inoculum is at least one of the following: H1N1 / wsn virus strain, A / PR / 8 / 34 virus strain, PR8-WSNNS virus strain, A2 virus strain, Long virus strain, PIV-3 virus strain, Tor2 virus strain, Urbani virus strain, and HCoV-EMC / 2012 virus strain.

4. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 3, characterized in that, The viral inoculum was the H1N1 / wsn virus strain.

5. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 1, characterized in that, The MOI of the viral inoculum is 0.01-5.

6. The method for constructing a respiratory virus-infected alveolar organoid model according to any one of claims 1-5, characterized in that, The method for preparing the alveolar cell-derived single cells includes: Discarded primary lung tissue was digested and dissociated into single cells, and the single cells were purified and sorted to obtain type II alveolar cells. The type II alveolar cells were subjected to planar and three-dimensional culture sequentially to obtain lung bud organoids; The lung bud organoids were differentiated and cultured to obtain alveolar organoids; The alveolar organoids were digested and dissociated to obtain alveolar cell-derived single cells.

7. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 6, characterized in that, The purification and sorting of single cells includes: staining the single cells with EPCAM antibody and HT2-280 antibody, sorting double-positive cells, and obtaining type II alveolar cells.

8. The method for constructing a respiratory virus-infected alveolar organoid model according to any one of claims 1-5, characterized in that, The first culture medium consists of: DMEM / F12 medium, 20-40 μM dexamethasone, 0.01-0.2 mM cAMP, 0.01-0.2 mM IBMX, and 0.1-5 μg / mL trypsin. The concentrations of dexamethasone, cAMP, IBMX, and trypsin are all calculated based on the volume of DMEM / F12 medium.

9. The method for constructing a respiratory virus-infected alveolar organoid model according to claim 8, characterized in that, The trypsin was treated with TPCK.

10. An application of the method for constructing a respiratory virus-infected alveolar organoid model according to any one of claims 1-9, characterized in that, The proposed method can be applied to disease mechanism research, antiviral drug development, clinical translational research, and disease prevention and early intervention research.

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