Methods, kits, and applications for evaluating the aerosol transmission characteristics of respiratory pathogens

Abstract

This invention relates to the fields of microbiology and infectious diseases, and discloses a method, kit, and application for evaluating the aerosol transmission characteristics of respiratory pathogens. The method includes: infecting tracheal epithelial cells with a respiratory pathogen and assessing the aerosol transmission potential of the pathogen by detecting its proliferation level within the tracheal epithelial cells; constructing airway epithelial organoids using tracheal epithelial cells, subsequently infecting the organoids with the respiratory pathogen, and assessing the biophysical basis of the aerosol transmission capacity of the pathogen by detecting the release level of respiratory pathogen aerosol particles from the organoids; and infecting animals with the respiratory pathogen and assessing the aerosol transmission capacity of the pathogen by detecting the aerosol transmission efficiency between animals. This method and kit can rapidly, efficiently, and easily evaluate transmission characteristics, providing a rapid response basis for epidemic prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and infectious disease technology, and more specifically, to a method, kit, and application for evaluating the aerosol transmission characteristics of respiratory pathogens. Background Technology

[0002] Aerosol transmission of respiratory pathogens (pathogen particles with a diameter ≤5 μm that remain suspended in the air for extended periods and diffuse through the air) is a core risk source for the spread of epidemics, especially in enclosed spaces, medical procedures, and laboratory environments, where it can easily lead to cluster infections and iatrogenic / laboratory-acquired infections. To address this risk, aerosol transmission risk assessment methods and corresponding reagent kits have become key technologies for epidemic prevention and control. Their core objective is to quantify the survival activity, transmission efficiency, and exposure dose of pathogens in aerosols, providing data support for the formulation of prevention and control strategies and the optimization of protective measures.

[0003] Currently, existing methods for evaluating the aerosol transmission characteristics of respiratory pathogens are mainly divided into animal model transmission experiments, in vitro aerosol sampling and detection methods, and mathematical model prediction methods. Among them, animal model experiments are the most widely accepted classic method. However, animal model methods require multiple steps, including animal infection, incubation period observation, and sample testing. The complete testing cycle usually takes several days to several weeks, making it difficult to quickly respond to the emergency assessment needs of sudden outbreaks. In vitro sampling and detection methods have high requirements for the stability of the sampling environment. In poorly ventilated, turbulent, enclosed spaces (such as underground passages and makeshift hospitals), the sampling representativeness is poor, making it difficult to fully reflect the transmission risk in the area. Mathematical model prediction methods rely on a large number of idealized assumptions and do not adequately consider unknown interference factors in the actual environment (such as airflow disturbances caused by personnel movement and changes in transmission characteristics caused by pathogen mutations), thus limiting the practical reference value of the prediction results.

[0004] Therefore, there is an urgent need to develop a method for evaluating the aerosol transmission characteristics of respiratory pathogens that is highly accurate, widely applicable, has a short detection cycle, and is easy to operate, so as to provide reliable technical support for rapid prevention and control and precise policy implementation in the event of an outbreak. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems in the prior art and to provide a method, kit and application for evaluating the aerosol transmission characteristics of respiratory pathogens.

[0006] To achieve the above objectives, a first aspect of the present invention provides a method for evaluating the aerosol transmission characteristics of respiratory pathogens, the method comprising the following steps:

[0007] (1) Infect tracheal epithelial cells with respiratory pathogens and assess the aerosol transmission potential of the respiratory pathogens by detecting the proliferation level of the respiratory pathogens in the tracheal epithelial cells.

[0008] (2) Airway epithelial organoids were constructed using tracheal epithelial cells, and then the tracheal epithelial organoids were infected with respiratory pathogens. The biophysical basis of the aerosol transmission ability of the respiratory pathogens was evaluated by detecting the release level of respiratory pathogen aerosol particles from the tracheal epithelial organoids.

[0009] (3) Infect animals with respiratory pathogens and assess the aerosol transmission capacity of the respiratory pathogens by detecting the aerosol transmission efficiency between animals;

[0010] The tracheal epithelial cells in steps (1) and (2) may be the same or different.

[0011] A second aspect of the present invention provides a kit for evaluating the aerosol transmission characteristics of respiratory pathogens, the kit comprising:

[0012] 1) Detection reagents for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells;

[0013] 2) Detection reagents for detecting the release levels of respiratory pathogen aerosol particles from tracheal epithelial organoids;

[0014] 3) Detection reagents for assessing the efficiency of aerosol transmission among animals;

[0015] 4) Tracheal epithelial cells;

[0016] 5) Reagents used to construct tracheal epithelial organoids.

[0017] The third aspect of this invention provides the application of the method described in the first aspect or the kit described in the second aspect in the prevention and control of respiratory pathogens.

[0018] The method and kit for evaluating the aerosol transmission characteristics of respiratory pathogens provided by this invention can comprehensively utilize a three-level evaluation model of "cell line, organoid, and laboratory animal" to measure the infection level of pathogens in the respiratory tract and the generation of pathogen aerosol particles in the airway for emerging and sudden respiratory pathogens with different aerosol transmission characteristics. This provides a rapid, efficient, and simple way to evaluate their transmission characteristics and provide a rapid response basis for epidemic prevention and control.

[0019] Biological Preservation

[0020] The cells provided in this invention are immortalized guinea pig tracheal epithelial cells, deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46763. The immortalized guinea pig tracheal epithelial cells provided in this invention are named GTE1. Attached Figure Description

[0021] Figure 1 It refers to the proliferation level of different subtypes of influenza A virus in tracheal epithelial cells;

[0022] Figure 2 It is a microscopic morphological image of airway epithelial organoids;

[0023] Figure 3 It refers to the level of viral aerosol particles released after different subtypes of influenza A virus infect airway epithelial organoids;

[0024] Figure 4 This represents the aerosol transmission efficiency of different subtypes of influenza A virus in a guinea pig transmission model.

[0025] Figure 5 This is a schematic diagram of a device for measuring the aerosol transmission efficiency of emerging and re-emerging influenza viruses among mammals.

[0026] Figure 6 This is a diagram showing the results of the tracheal epithelial cell transfection experiment used in this invention;

[0027] Figure 7 This is a graph showing the passage test results of the tracheal epithelial cells used in this invention;

[0028] Figure 8 It is the relative expression level of viral nucleoprotein (NP) in the tracheal epithelial cells used in this invention after viral infection;

[0029] Figure 9 It is the relative expression level of the viral M gene mRNA in the tracheal epithelial cells used in this invention after viral infection;

[0030] Figure 10 This is an image showing the immunofluorescence detection results of the tracheal epithelial cells used in this invention after viral infection. Detailed Implementation

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] The first aspect of this invention provides a method for evaluating the aerosol transmission characteristics of respiratory pathogens, the method comprising the following steps:

[0033] (1) Infect tracheal epithelial cells with respiratory pathogens and assess the aerosol transmission potential of the respiratory pathogens by detecting the proliferation level of the respiratory pathogens in the tracheal epithelial cells.

[0034] (2) Airway epithelial organoids were constructed using tracheal epithelial cells, and then the tracheal epithelial organoids were infected with respiratory pathogens. The biophysical basis of the aerosol transmission ability of the respiratory pathogens was evaluated by detecting the release level of respiratory pathogen aerosol particles from the tracheal epithelial organoids.

[0035] (3) Infect animals with respiratory pathogens and assess the aerosol transmission capacity of the respiratory pathogens by detecting the aerosol transmission efficiency between animals;

[0036] The tracheal epithelial cells in steps (1) and (2) may be the same or different.

[0037] In this invention, preferably, the tracheal epithelial cells are susceptible cells to respiratory pathogens. Susceptible cells refer to cells that can be successfully infected by the target pathogen within a short period (e.g., 24 hours), thus more realistically simulating the pathogen infection process.

[0038] In this invention, the tracheal epithelial cells are immortalized cells.

[0039] In this invention, the tracheal epithelial cells are derived from guinea pigs ( Cavia porcellus ).

[0040] More preferably, the tracheal epithelial cells are immortalized guinea pig tracheal epithelial cells, with the preservation number CGMCC No. 46763.

[0041] In this invention, preferably, the respiratory pathogen is selected from at least one of influenza virus, novel coronavirus (SARS-CoV-2), respiratory syncytial virus, rhinovirus, parainfluenza virus and adenovirus, more preferably influenza virus.

[0042] More preferably, the respiratory pathogen is an influenza A virus, more preferably at least one of the H1, H3, H5, H6, H7, H9 and H11 subtype influenza viruses. Even more preferably, it is at least one of the H1N1, H3N2, H3N8, H5N1, H5N6, H5N8, H6N6, H7N9, H9N2 and H11N3 subtype influenza viruses.

[0043] In this invention, preferably, in the method for evaluating the aerosol transmission characteristics of respiratory pathogens, step (2) involves selecting respiratory pathogens that were determined to have aerosol transmission potential in step (1) to infect airway epithelial organoids.

[0044] In this invention, the aerosol transmission potential is determined by quantitatively detecting at least one target gene related to influenza virus aerosol transmission and comparing it with a standardized control system. The target genes include, but are not limited to, the corresponding genes of nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA), RNA polymerase basic proteins (PB1, PB2, and PA), matrix proteins (M), and non-structural proteins (NS).

[0045] In this invention, preferably, the determination of aerosol transmission potential uses a high aerosol transmissibility influenza virus as a positive control group and a low aerosol transmissibility influenza virus as a negative control group; if the relative expression level of the M gene of the novel emerging influenza virus to be tested is higher than the corresponding value of the negative control group, then the novel emerging influenza virus is determined to have potential aerosol transmission ability; if the relative expression level of the M gene of the novel emerging influenza virus to be tested does not exceed the corresponding value of the negative control group, then the novel emerging influenza virus is determined to not have significant aerosol transmission potential.

[0046] More preferably, the influenza virus with high aerosol transmissibility is influenza A H1N1 virus, and the influenza virus with low aerosol transmissibility is influenza A H5N6 virus.

[0047] In this invention, preferably, in the method for evaluating the aerosol transmission characteristics of respiratory pathogens, step (3) involves selecting animals infected with respiratory pathogens that were determined in step (2) to have aerosol transmission capabilities based on biophysical basis.

[0048] In this invention, the biophysical basis of the aerosol transmission capability is determined by measuring the viral titer level of respiratory pathogens infecting tracheal epithelial organoids. This viral titer level is determined using the half-maximal tissue culture infectious dose (TCID50). 50 ) Conduct quantitative evaluation.

[0049] In this invention, preferably, the determination of the biophysical basis of aerosol transmission capability uses a high aerosol transmissibility influenza virus as a positive control group and a low aerosol transmissibility influenza virus as a negative control group. The novel emerging influenza virus to be tested is used to infect the airway epithelial organoids of guinea pigs. After a preset time, mucus samples are collected from the upper part of the guinea pig airway epithelial organoids, and the viral titer in the samples is determined using a viral titer method. If the viral titer value of the novel emerging influenza virus to be tested is higher than the corresponding value of the negative control group, then the novel emerging influenza virus is determined to have a biophysical basis for aerosol transmission capability; if the viral titer value of the novel emerging influenza virus to be tested does not exceed the corresponding value of the negative control group, then the novel emerging influenza virus is determined not to have a biophysical basis for aerosol transmission capability.

[0050] More preferably, the influenza virus with high aerosol transmissibility is influenza A H1N1 virus, and the influenza virus with low aerosol transmissibility is influenza A H5N6 virus.

[0051] In this invention, preferably, the aerosol transmission efficiency between animals is quantitatively assessed by measuring the aerosol exposure infection status of infected animals to uninfected animals.

[0052] In this invention, the quantitative assessment of the aerosol transmission efficiency between animals is achieved through multi-dimensional detection and quantitative calculation of the infection evaluation of recipient animals. The detection indicators include, but are not limited to: viral titer in the nasal wash fluid of the recipient animal, hemagglutination inhibition (HI) antibody titer in serum, and the infection or distribution of tissues within the recipient animal. In some preferred embodiments, if a recipient animal meets at least one of the following criteria (or two or more consistently positive): "positive viral titer detection," "elevated HI titer," or "positive tissue infection," it is determined to be a positive transmission case, and the transmission rate is calculated as the quantitative result of the aerosol transmission efficiency between animals.

[0053] In this invention, preferably, the determination of the aerosol transmission efficiency between animals uses a high aerosol transmissibility influenza virus as a positive control group and a low aerosol transmissibility influenza virus as a negative control group. Guinea pigs (donor guinea pigs) are infected with the novel emerging influenza virus to be tested. After infection, they are brought into contact with healthy recipient guinea pigs, and nasal wash samples are collected from the recipient guinea pigs. The viral titer in the samples is determined using a viral titer method. The aerosol transmission efficiency of the positive control group is recorded as 100%; the aerosol transmission efficiency of the negative control group is recorded as 0%. If the aerosol transmission efficiency of the novel emerging influenza virus to be tested is higher than 0%, it is determined that the novel emerging influenza virus can be transmitted between guinea pigs via aerosols; if the aerosol transmission efficiency of the novel emerging influenza virus to be tested is 0%, it is determined that the novel emerging influenza virus cannot be transmitted between guinea pigs via aerosols.

[0054] A second aspect of the present invention provides a kit for evaluating the aerosol transmission characteristics of respiratory pathogens, the kit comprising:

[0055] 1) Detection reagents for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells;

[0056] 2) Detection reagents for detecting the release levels of respiratory pathogen aerosol particles from tracheal epithelial organoids;

[0057] 3) Detection reagents for assessing the efficiency of aerosol transmission among animals;

[0058] 4) Tracheal epithelial cells;

[0059] 5) Reagents used to construct tracheal epithelial organoids.

[0060] In this invention, preferably, the detection reagent for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells includes a viral nucleic acid quantitative detection reagent or a viral protein immunoassay reagent.

[0061] Specifically, the viral nucleic acid quantitative detection reagent can be a real-time quantitative PCR (qPCR) detection kit, which contains specific primer pairs designed for conserved gene sequences of respiratory pathogens, fluorescent probes, PCR reaction buffer, thermostable DNA polymerase, dNTPs mixture and positive / negative controls; the viral protein immunoassay reagent can be an enzyme-linked immunosorbent assay (ELISA) kit or a Western blotting detection kit, which contains pathogen-specific monoclonal antibodies, enzyme-labeled secondary antibodies, chromogenic substrates, blocking buffer and washing buffer.

[0062] More preferably, the detection reagent for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells is a real-time quantitative PCR (qPCR) detection kit.

[0063] In this invention, preferably, the detection reagent for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells comprises a primer-probe combination targeting the pathogen's nucleic acid.

[0064] More preferably, the primer-probe combination comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer of the target gene (M) is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer of the target gene (M) is shown in SEQ ID NO:2; the nucleotide sequence of the upstream primer of the internal reference gene (GAPDH) is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer of the internal reference gene (GAPDH) is shown in SEQ ID NO:4.

[0065] In this invention, preferably, the detection reagent for detecting the release level of respiratory pathogen aerosol particles from tracheal epithelial organoids includes a virus particle collection aid reagent and a pathogen quantitative analysis reagent.

[0066] Specifically, the virus particle collection aid reagent includes sterile phosphate-buffered saline (PBS) for eluting virus particles attached to the surface of the sampling carrier; the pathogen quantification reagent includes phosphate-buffered saline (PBS) and red blood cell suspension. The red blood cell suspension serves as an indicator component for virus titer determination, achieving quantitative analysis of virus particles through the agglutination reaction of the virus on red blood cells. It is a key reagent for determining virus titers using the hemagglutination (HA) and hemagglutination inhibition (HI) methods.

[0067] More preferably, the red blood cell suspension is selected from at least one of chicken red blood cell suspension, guinea pig red blood cell suspension, and human type O red blood cell suspension. More preferably, it is chicken red blood cell suspension, and the concentration of the red blood cell suspension is preferably 1%-2% (volume fraction).

[0068] In this invention, preferably, the detection reagent for detecting the efficiency of aerosol transmission among animals includes an aerosol particle collection aid reagent and a pathogen quantitative analysis reagent. Specifically, the virus particle collection aid reagent includes sterile phosphate-buffered saline (PBS) for eluting virus particles attached to the surface of the sampling carrier; the pathogen quantitative analysis reagent includes PBS, penicillin / streptomycin reagent, and chicken embryo.

[0069] More preferably, the chicken embryo is a 9-10 day old SPF (specific pathogen-free) chicken embryo. Such chicken embryos do not carry common pathogenic microorganisms and are in good embryonic development; their allantoic cavity, amniotic cavity, and other parts can provide ideal proliferation sites for various respiratory viruses. By inoculating aerosol virus samples into the chicken embryo for culture, the virus can multiply rapidly using the host environment of the chicken embryo. Subsequently, quantitative analysis of the virus titer can be achieved by detecting the viral activity in the chicken embryo culture (e.g., hemagglutination assay).

[0070] In this invention, the tracheal epithelial cells are as described above and will not be repeated here.

[0071] In this invention, preferably, the reagents for constructing tracheal epithelial organoids include organoid culture medium, which comprises epithelial cell organoid culture medium and calcium chloride.

[0072] The third aspect of this invention provides the application of the method described in the first aspect or the kit described in the second aspect in the prevention and control of respiratory pathogens.

[0073] The method of the present invention can be used for public health research rather than clinical diagnosis; therefore, according to one embodiment of the present invention, the method is a method for non-diagnostic purposes.

[0074] The present invention will be described in detail below through embodiments.

[0075] In the following examples, the H1N1 influenza A virus was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / California / 04 / 2009(H1N1); the Genbank accession number of H1N1 is GCA_038510525.1;

[0076] The influenza A virus H3N2 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / Xiamen / 80 / 2004(H3N2); the Genbank accession number for the HA gene of H3N2 is AY963792.1;

[0077] The influenza A virus H3N8 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / Environment / Fujian / 10643 / 2022(H3N8); the GISAID accession number of H3N8 is EPI2211492.

[0078] The H5N1 influenza A virus was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / Anhui / 1 / 2005(H5N1); the Genbank accession number for the HA gene of H5N1 is DQ371928.1.

[0079] The influenza A virus H5N6 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / chicken / Hebei / 3399 / 2017(H5N6); the HA and NA gene sequences of H5N6 correspond to Genbank accession numbers PX884831 and PX884830, respectively.

[0080] The influenza A virus H5N8 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / goose / Hebei / HG12 / 2021(H5N8). The HA, NA, M, PB1, PB2, PA, NP, and NS gene sequences of H5N8 correspond to GenBank accession numbers OM868039.1, OM868040.1, ON878716.1, ON878720.1, ON878721.1, ON878719.1, ON878717.1, and ON878718.1, respectively.

[0081] The H6N6 influenza A virus was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / chicken / Jilin / 3.08_CCNQM002-O / 2017(H6N6); the GISAID accession number for H6N6 is EPI1820944.

[0082] The H7N9 influenza A virus was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / quail / Hebei / 0607 / 2018(H7N9). The HA, NA, M, PB1, PB2, PA, NP, and NS gene sequences of H7N9 correspond to GenBank accession numbers MT498676.1, MT498678.1, MT498679.1, MT498674.1, MT498673.1, MT498675.1, MT498677.1, and MT498680.1, respectively.

[0083] The influenza A virus H9N2 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with strain numbers A / Fujiansiming / 19 / 2021(H9N2) and A / Environment / Xiamen / 03 / 2021(H9N2), abbreviated as H9N2-H19 and H9N2-E03, respectively. The HA gene sequence of H9N2-H19 corresponds to GenBank accession number ON856627.1, and the NA gene sequence corresponds to GenBank accession number ON856631.1. The HA gene sequence of H9N2-E03 corresponds to GenBank accession number ON856630.1, and the NA gene sequence corresponds to GenBank accession number ON856634.1.

[0084] The influenza A virus H11N3 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / Environment / Fujian / EV01 / 2020(H11N3). The HA, NA, M, PB1, PB2, PA, NP and NS gene sequences of H11N3 correspond to GenBank accession numbers ON968457.1, ON968459.1, ON968458.1, ON968463.1, ON968464.1, ON968462.1, ON968460.1 and ON968461.1, respectively.

[0085] The tracheal epithelial cells were immortalized guinea pig tracheal epithelial cells, provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with accession number CGMCC No. 46763;

[0086] Complete culture medium: DMEM / F12 medium (GIBCO, 21331020), 10% fetal bovine serum (GIBCO, 10099141C), 1% epithelial cell culture additive (Zhejiang Meisen Cell Technology Co., Ltd., CTCC-009-613-2), 1% penicillin / streptomycin reagent (GIBCO, 15140148).

[0087] Differentiation medium: Epithelial cell organoid culture medium (CELLnTEC Advanced Cell Systems, CnT-PR-AD), 1 mM CaCl2.

[0088] Example 1

[0089] 1. To detect the proliferation ability of influenza virus in guinea pig tracheal epithelial cells and to preliminarily assess the potential for viral aerosol transmission.

[0090] Diluted H1-H11 subtype novel influenza virus samples were diluted with Opti-MEM medium and inoculated into monolayer guinea pig tracheal epithelial cell lines at an MOI of 0.01. Cells were incubated at 37°C for 1 hour, while control cells were incubated in Opti-MEM medium. After incubation, the viral supernatant was discarded, and 2 mL of maintenance medium (0.5% fetal bovine serum + DMEM / F12 + 1% epithelial cell culture additive + 1% penicillin / streptomycin) was added to each well. Control cells underwent maintenance medium replacement at the same time, and cells were returned to the incubator for another 24 hours. Infected cells were collected 24 hours post-infection, and RNA was extracted using an RNA extraction kit (TransGen, ER111) following the kit's instructions. RNA concentration was measured using NanoDrop, and the extracted RNA was reverse transcribed using a reverse transcription kit (TAKARA, RR047) following the kit's instructions to obtain cDNA. Subsequently, a qPCR detection kit (TransGen, AQ621) was used to determine the relative expression level of the viral M gene mRNA, following the kit's instructions. The primer sequences used are shown in Table 1, and the qPCR reaction program is shown in Table 2.

[0091] Table 1

[0092]

[0093] Table 2

[0094]

[0095] Note: Fluorescence signal collection was performed in the second program segment (56℃ annealing and extension).

[0096] pass Figure 1 As shown in Table 3, when influenza A (H1N1) virus was selected as the control group for highly aerosol-transmittable influenza viruses, the relative expression level of the virus in cells was increased by 10 compared to the uninfected control group. 7.48 The H5N6 influenza A virus was selected as a low aerosol transmissibility influenza control group, and its relative intracellular expression level was found to be 10 times higher than that of the uninfected control group. 3.89 The relative expression level of viral proteins in the H1N1 group was 10 times that of the H5N6 group.3.59 The H1N1 group was significantly higher than the H5N6 group (p<0.0001). According to 10 3.89 As the lower limit of detection for determining whether a virus has the potential for aerosol transmission, a value 10 times or more above this limit and significantly higher than that of the low aerosol transmissibility influenza control group is considered positive. After testing (Table 3), it was found that the relative expression level of the viral M gene of H1N1, H3N2, H7N9, H9N2-H19, and H11N3 subtype influenza viruses in guinea pig tracheal epithelial cells was greater than 10. 3.89 The number of cases was several times higher than that of the influenza control group with low aerosol transmissibility ( ). p<0.05, (p<0.0001), suggesting potential for aerosol transmission. Meanwhile, the relative expression levels of the viral M gene in guinea pig tracheal epithelial cell lines for H3N8, H5N1, H5N6, H5N8, H6N6, and H9N2-E03 subtype influenza viruses did not exceed 10. 3.89 If the value is less than 10 times (including 10 times) and there is no significant difference, it may not have the potential for aerosol transmission. Influenza virus samples with positive test results will be used for further experiments.

[0097] Table 3

[0098]

[0099] 2. To detect the ability of influenza virus to produce viral aerosol particles in airway epithelial organoids, and to determine the biophysical basis of the virus's aerosol transmission capability.

[0100] Airway epithelial organoids with an air-liquid interface were constructed by uniformly seeding guinea pig tracheal epithelial cells above the membrane of a Transwell (Corning, 3470) culture chamber. After 24 hours, once the cells had fully adhered, the culture medium in the upper chamber was removed, and the complete culture medium in the lower chamber was replaced with differentiation medium. Culture was monitored continuously for 2-6 weeks to establish airway epithelial organoids for future use. Results are as follows: Figure 2 As shown. Diluted H1-H11 subtype samples of newly emerging influenza virus were added to the airway epithelial organoids in a Transwell culture chamber at an MOI of 1 and incubated at 37°C for 2 hours. The virus solution and differentiation medium in the lower chamber were discarded, and the sample was washed three times with PBS. Differentiation medium was then added back to the lower chamber. After 36 hours, mucus samples were collected from the airway epithelial organoids with PBS, and TCID was measured. 50 The mucus sample to be tested was serially diluted 10-fold (10... -1 Up to 10 -8The virus was inoculated into a monolayer of guinea pig tracheal epithelial cells in a 96-well plate at different dilutions. 100 µL of virus solution was inoculated into three wells per well. After incubation at 37°C and 5% CO2 for 36 hours, the hemagglutination titer of the cell culture medium in each well was determined. The specific steps were as follows: 50 µL of the sample to be tested was taken from each well of the 96-well plate and transferred to a corresponding well in a 96-well V-shaped microplate. Then, 50 µL of 1% (v / v) chicken blood erythrocyte suspension was added to each well. The 96-well V-shaped microplate was placed in a 37°C incubator for 25 minutes, and the results were observed. When the hemagglutination reaction plate was held upright, a trail of chicken blood flowing from the bottom of the well was considered negative; if the chicken blood agglutinated and remained stationary, it was considered positive. The hemagglutination titer of the virus was read as the highest dilution that could cause chicken blood to agglutinate. The results are as follows: Figure 3 As shown in Table 4.

[0101] Table 4

[0102]

[0103] pass Figure 3 It can be seen that, by selecting the H1N1 influenza A virus as a control for highly aerosol-transmittable influenza viruses, its viral titer can reach 10. 3.50 TCID 50 / mL; meanwhile, influenza A H5N6 virus was selected as a control for influenza with low aerosol transmissibility, and its viral titer was determined to be 10. 1.33 TCID 50 / mL. According to 10 1.33 TCID 50 / mL is used as the detection limit for determining whether a virus has the potential for aerosol transmission; values ​​higher than this are considered positive. Testing revealed viral titers greater than 10 for H1N1, H3N2, H7N9, H9N2-H19, and H11N3 influenza subtypes. 1.33 TCID 50 / mL, which can release a large number of viral aerosol particles from airway epithelial organoids. Meanwhile, the viral titers of H3N8, H5N1, H5N6, H5N8, H6N6, and H9N2-E03 subtype influenza viruses are less than 10. 1.33 TCID 50 / mL, the release capacity of viral aerosol particles after infecting airway epithelial organoids is relatively weak. Influenza virus samples with positive test results were used for further experiments.

[0104] 3. To detect the aerosol transmission efficiency of influenza virus in a guinea pig transmission model and determine the aerosol transmission capability of the virus.

[0105] Diluted samples of emerging and re-emerging influenza virus were administered at a rate of 100 μL virus droplets per nasal cavity (10 μL / 100 μL). 6 EID 50(200 μL) was nasally inoculated into both nostrils of anesthetized guinea pigs. Three donor guinea pigs were inoculated for each sample. After inoculation, they were placed in six IVC cages and housed on one side. Figure 5 Twenty-four hours after inoculation, six healthy recipient guinea pigs were placed in an adjacent IVC cage on the opposite side, 5 cm apart, to avoid direct contact with the infected donor guinea pigs. Nasal wash fluid from the recipient guinea pigs was collected three days later, and the EID of the nasal wash samples was measured. 50 To detect the efficiency of viral aerosol transmission.

[0106] EID 50 The experimental method is as follows: Dilute the nasal wash sample to be tested with PBS buffer containing 1% penicillin and bismuth subsalicylate at a concentration of 10 μL. 1 -10 8 Diluted samples were inoculated into 9-10 day old SPF-grade chicken embryos at a rate of 200 μL / embryo, with 3 embryos inoculated for each dilution. Embryos were incubated at 37°C. Embryos that died nonspecifically within 24 hours were discarded. The remaining embryos were incubated for another 48 hours before being collected. The hemagglutination status of each embryo was determined, and the median infectious dose (EID) was calculated using the internationally recognized Reed-Muench method. 50 .

[0107] The calculation formula is as follows:

[0108] EID 50 =10 X+Y / 200 μL;

[0109] X = the base-10 logarithm of the viral dilution factor when the positivity rate is higher than 50%;

[0110] Y = (Percentage of positive rate above 50% - 50%) / (Percentage of positive rate above 50% - Percentage of positive rate below 50%)

[0111] Viral titer in nasal wash >10 0.7 EID 50 A positive result is indicated by a test line of / mL, meaning the recipient guinea pig has been infected with the influenza virus.

[0112] Aerosol transmission efficiency = number of infected recipient guinea pigs / total number of recipient guinea pigs × 100%.

[0113] pass Figure 4As shown in Table 5, the H1N1 influenza virus was selected as the control group for influenza viruses with high aerosol transmissibility, and its aerosol transmission efficiency reached 100%. Meanwhile, the H5N6 influenza virus was selected as the control for influenza viruses with low aerosol transmissibility, and its aerosol transmission efficiency was 0%. 0% was used as the lower limit of detection for whether a virus has the potential for aerosol transmission; values ​​higher than this were considered positive. The tests revealed that the aerosol transmission efficiencies of H1N1, H3N2, H7N9, H9N2-H19, and H11N3 subtype influenza viruses were greater than 33%, indicating they could be transmitted aerosolally among guinea pigs. Conversely, the aerosol transmission efficiency of H3N8, H5N1, H5N6, H6N6, H5N8, and H9N2-E03 subtype influenza viruses was 0%, indicating they could not be transmitted aerosolally among guinea pigs.

[0114] Table 5

[0115]

[0116] Example 2

[0117] This embodiment illustrates the preparation of tracheal epithelial cells used in this invention.

[0118] (I) Two healthy SPF (specific pathogen-free) female Hartley guinea pigs (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing approximately 300-350 g, were selected. Tracheal tissue was aseptically harvested after cervical dislocation. The tracheal tissue was minced and seeded into cell culture dishes, then cultured in a medium containing DMEM / F12 (GIBCO, 21331020), 1% epithelial cell culture additive (Zhejiang Meisen Cell Technology Co., Ltd., CTCC-009-613-2), and 1% penicillin / streptomycin (GIBCO, 15140148). To screen for and remove contaminating fibroblast populations in the primary cell preparation, the primary cells were passaged and purified for two consecutive generations using serum-free medium. After the epithelial cell purity met the experimental requirements, subsequent functional verification experiments were conducted.

[0119] (II) Preparation and Validation of Tracheal Epithelial Cells

[0120] 1. Cell validation

[0121] Detection for bacteria, fungi, mycoplasma, etc., ensures that cells are free from microbial contamination. The specific steps are as follows:

[0122] (1) Detection of bacterial and fungal contamination

[0123] Culture method: 100 μL of cell culture medium was spread onto LB agar plates (Solepro, L1015) and PDA agar plates (Solepro, P8931); a blank control was also set up by spreading 100 μL of sterile PBS onto the same agar plates. The plates were inverted after spreading. The LB plates were incubated at 37°C for 24 h, and the PDA plates were incubated at 28°C for 48 h. Colonies were observed on the plates.

[0124] Microscopic observation: Observe whether there are granular, filamentous, or moving small dots; and observe whether the culture medium is turbid, yellow, or contains flocculent or membrane-like substances.

[0125] (2) Mycoplasma contamination detection

[0126] qPCR method: Take 2 μL of cell culture medium and prepare the reaction system according to the instructions of the Mycoplasma qPCR Detection Kit (Beyotime, C0303S). Set the qPCR reaction program as follows: pre-denaturation 95℃ for 2 min; denaturation 95℃ for 15 s; annealing / extension (60℃) for 20 s, for a total of 40 cycles. If the test sample shows no typical S-type amplification curve in the FAM channel or the Ct value is >35, and the positive control is positive and the negative control is negative, the result is judged to be negative for mycoplasma.

[0127] 2. Cell transduction

[0128] (1) The day before the experiment, target cells were seeded into a six-well plate. The number of wells was seeded according to the number of experimental groups, and at least one well was reserved as a negative control. The cell content per well was 2-3 × 10⁶ cells. 5 Each cell.

[0129] (2) When the cell count reaches 60-70%, digest the cells in one well and count them. Using MOI=100, substitute the virus titer and cell count into the formula to calculate the volume (μL) of virus solution to be used. The formula is as follows:

[0130] MOI = Titration × Sample Volume × 10 -3 / cell number

[0131] (3) Except for the negative control wells, remove the culture medium from the wells, add 1 mL of fresh culture medium, and place them in an incubator for later use.

[0132] (4) 1×10 lentivirus 8 TU / mL (Suzhou Haixing Biotechnology Co., Ltd., HX-SV40T-LV1) and polybrene (Suzhou Haixing Biotechnology Co., Ltd., GUOR-R001) were dissolved at 4℃, and polybrene was added to each experimental group to make a final concentration of 5 μg / mL.

[0133] (5) Prepare the necessary consumables and reagents in the biosafety cabinet, add the required virus solution to each well, shake gently to mix, and place in a 37°C, 5% CO2 incubator for incubation.

[0134] (6) Observe cell morphology 6 h after transfection. If there is a toxic reaction, change the medium. If there is no abnormality, the time can be extended to a maximum of 24 h for medium change.

[0135] (7) Observe cell fluorescence 24 h after transfection. Take one bright-field image and one fluorescence image of each group of cells in the same field of view. The results of the cell transfection experiment are as follows: Figure 6 As shown, the cells of this invention have extremely high transfection efficiency.

[0136] (8) When the cell count reached 90% or higher, the six-well cells were passaged and seeded in 6 cm culture dishes. By comparing the proliferation of the primary cells with that of the primary cells, when the primary cells stopped proliferating while the immortalized cells could continue to proliferate, the expression level of SV40T was detected by qPCR.

[0137] (9) The expression levels of the target gene (SV40-T) were analyzed by qPCR in primary tracheal epithelial cells and recombinant cell lines transfected with lentivirus (CGMCC No. 46763). A qPCR detection kit (Novizan, Q712-02 / 03) was used, and the qPCR reaction was performed according to the kit instructions. The primer sequences used are shown in Table 6; the qPCR reaction program is shown in Table 7; and the detection results are shown in Table 8.

[0138] Table 6

[0139]

[0140] Table 7

[0141]

[0142] Note: Fluorescence signal collection was performed in the second program segment (60℃ annealing and extension).

[0143] Table 8

[0144]

[0145] As shown in Table 8, the cells of this invention can successfully express SV40-T, that is, the immortalized guinea pig tracheal epithelial cell line has been successfully constructed.

[0146] 3. Continuous passage culture, amplification, and quality control

[0147] (1) Primary cell passage: The cells were passaged continuously at a ratio of 1:3 until P3 cells showed obvious signs of aging and their proliferation almost stopped, at which point the experiment was terminated; the primary cells were derived from guinea pig tracheal epithelial tissue.

[0148] (2) Cell passage (CGMCC NO. 46763): Cells were cultured in a medium containing DMEM / F12 + 10% FBS (fetal bovine serum, GIBCO, 10099141C) + 1% epithelial cell culture additive + 1% penicillin / streptomycin. Cells were passaged continuously at a 1:3 ratio to P5. Cell morphology was normal and proliferation was rapid. One plate of cells was selected for further passage, and the remaining four plates were cryopreserved for future use. Cells were passaged continuously at a 1:3 ratio to P15. Cell morphology was normal and proliferation was stable. These cells were then cryopreserved for seed culture.

[0149] (3) Frozen cell thawing and passage: P15 cells were thawed and seeded in T25 culture flasks, and passaged continuously to P20 at a ratio of 1:3. The cells had normal morphology and stable proliferation.

[0150] (4) Cell expansion and cryopreservation: Cells were seeded in 10 cm culture dishes until 90% of the cells were cryopreserved. Three tubes were cryopreserved per dish and stored in liquid nitrogen.

[0151] (5) Quality Inspection:

[0152] Culture method detection and microscopic observation: negative for bacteria and fungi;

[0153] qPCR test: Mycoplasma negative.

[0154] The results are as follows Figure 7 As shown in Table 9.

[0155] Table 9

[0156]

[0157] As can be seen from Table 9, the cells of the present invention can be stably and continuously passaged.

[0158] 4. Cell proliferation detection

[0159] (1) P20 cells were administered at a dose of 1×10 5 10 cells / well were seeded in 10 wells of a 6-well plate;

[0160] (2) After 24 h, digest the cells in the two wells and count the number of cells in each well (take 3 samples from each well).

[0161] (3) For five consecutive days, at the same time each day, digest the cells in two wells and count the number of cells (take three samples from each well).

[0162] The results are shown in Table 10.

[0163] Table 10

[0164]

[0165] As can be seen from Table 10, the cells of the present invention still exhibit good proliferation characteristics after 20 passages.

[0166] 5. Measurement of viral infection and viral replication levels

[0167] Experimental methods:

[0168] (1) Virus incubation

[0169] Immortalized guinea pig tracheal epithelial cells (CGMCC NO. 46763) were collected, and the cell supernatant was discarded. Influenza virus H1 (H1N1) or H5 (H5N6) was diluted to a final MOI of 0.01 using serum-free OPTIMEM medium (GIBCO, 31985070). The diluted virus solution was added to the wells of a six-well plate at a volume of 500 μL per well and incubated at 37°C for 1 h. After incubation, the viral supernatant was discarded, and then 2 mL of maintenance medium (0.5% FBS + DMEM / F12 + 1% epithelial cell culture additive + 1% penicillin / streptomycin) was added to each well. The plate was then returned to the incubator and cultured for another 60 h.

[0170] (2) WB sampling and testing methods

[0171] a. Discard the supernatant from each well of the virus-infected cells from step (1), add 200 μL of the prepared cell protein lysis buffer (Beyotime, P0013C and P1065), incubate on ice for 10 min, then collect into a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 10 min at 4℃, and add the supernatant to a new pre-chilled 1.5 mL centrifuge tube; add 5×SDS-PAGE loading buffer (Yisheng, 20315ES05) to the supernatant, boil at 100℃ for 10 min to denature, ensuring that the virus particles rupture and release proteins. Centrifuge at 12000 r / min for 1 min, and use the supernatant for Western blotting.

[0172] b. Place a 10% high-resolution precast gel (Yaxin, LK403) in an electrophoresis tank and add an appropriate amount of protein electrophoresis buffer. Add markers and protein samples to the gel wells, adjust the voltage to 80 V, and start electrophoresis. After 20 min, observe the lanes (running under the stacking gel). Once the bands have separated, adjust the voltage to 120 V and stop electrophoresis after 70 min. Cut a PVDF membrane (Millipore, IPVH00010) according to the gel size. Immerse the PVDF membrane in 100% methanol (Shanghai Test, 10010018) for 5 min and in 20% methanol for 2 min. Start the PVDF membrane transfer at a constant current of 400 mA for 30 min. Place the PVDF membrane in a container and block it with TBST (Yaxin, PS103S) containing 5% skim milk powder (Solepro, D8340) at room temperature for 1 h. After blocking, wash the membrane three times with TBST for 10 min each time.

[0173] c. Primary antibody: Place the PVDF membrane containing the target protein in a solution of influenza A virus nucleoprotein antibody (Xinbosheng, GTX636282-S) diluted 1:5000 using universal antibody dilution buffer. Place the PVDF membrane containing the internal control in a solution of actin antibody (MBL, M177-3) diluted 1:8000 using universal antibody dilution buffer. Incubate at 4°C on a shaker for 16-18 h. Wash the membrane three times with TBST for 10 min each time.

[0174] d. Secondary antibody: The PVDF membrane containing the target protein was placed in a solution of goat anti-rabbit antibody (Xinbosheng, GTX213110-01) diluted 1:5000 using a universal antibody diluent. The PVDF membrane containing the internal control was placed in a solution of goat anti-mouse antibody (Immunoway, RS0001) diluted 1:14000 using a universal antibody diluent. The membranes were incubated at room temperature for 1.5 h. The membranes were washed three times with TBST for 10 min each time. A developing solution (Pulley, P1010) was prepared to develop the PVDF membranes.

[0175] (3) qPCR sampling and testing methods

[0176] a. After discarding the supernatant from each well of the virus-infected cells from step (1), extract RNA using an RNA extraction kit (TransGold, ER111) following the kit instructions. Measure the RNA concentration using NanoDrop and store at -80°C.

[0177] b. Using a reverse transcription kit (TAKARA, RR047), follow the kit instructions to reverse transcribe the RNA extracted in step a to obtain cDNA.

[0178] c. qPCR reaction: A qPCR detection kit (TransGold, AQ621) was used, and the qPCR reaction was performed according to the kit instructions. The upstream and downstream primer sequences for the M gene, and the upstream and downstream primer sequences for the internal reference gene (GAPDH) are shown in Table 1. The qPCR reaction was performed according to the reaction conditions in Table 2.

[0179] (4) Immunofluorescence experiment

[0180] After removing the supernatant from each well of the virus-infected cells from step (1), add 500 μL of pre-chilled 4% paraformaldehyde fixative (Solepro, P1110) and let stand at room temperature for 30 min. After fixation, discard the 4% paraformaldehyde and wash three times with PBS for 5 min each time. Add 1 mL of 1% Triton X-100 solution to the washed cells and permeabilize at room temperature for 30 min. After permeabilization, wash three times with PBS for 5 min each time. After washing, add 1 mL of 3% BSA and incubate at 37℃ for 30 min. After incubation, wash three times with PBS for 5 min each time. Add 200 µL of primary antibody (Xinbosheng, GTX636675 and GTX631830, diluted 1:200 and 1:300 respectively) and incubate overnight at 4℃. Wash three times with PBST on a shaker for 5 min each time. Then, add 200 µL of secondary antibody (positive energy, AF594, 1:200 dilution; Jackson ImmunoResearch, 115-545-003, 1:200) and incubate at 37°C in the dark for 2 h. Wash the cells 5 times with PBST on a shaker for 5 min each time. After washing, add 20 µL of DAPI staining solution (Solepro, S2110) and incubate at room temperature for 10 min.

[0181] Experimental results:

[0182] (1) Cellular lesions: direct observation under a microscope.

[0183] Evaluation criteria: Using the uninfected control as a reference, the evaluation is based on changes in cell morphology and adherence. When typical changes such as cell shrinkage, increased refractivity, loose intercellular connections, cell detachment / floating, monolayer destruction, and focal plaques or fusions are observed, it is judged as a positive cytopathic effect (CPE), and the same criteria are used to interpret different treatment groups consistently.

[0184] (2) Western blot (WB) determination of viral protein (NP) expression level: After viral infection of cells, cell proteins were extracted, and the expression level of the protein was determined using viral protein-specific antibodies and Western blot (WB) detection technology. The relative protein expression level is a quantitative value obtained by correcting the gray value of the target protein (NP) band with the gray value of the internal reference protein (β-actin) band in the same sample.

[0185] (3) qPCR determination of viral M gene mRNA expression level: After viral infection of cells, cellular RNA was extracted, and qPCR was performed using specific primers targeting viral M gene mRNA. The relative expression level of viral M gene mRNA was calculated using a relative quantification method. Specifically, the target gene and internal reference gene were detected in each sample. ① The ΔCt value of the target gene and internal reference gene was calculated: ΔCt = Ct(target gene) - Ct(internal reference gene). ② The ΔΔCt value between the treatment group and the control group was calculated: ΔΔCt = ΔCt(treatment group) - ΔCt(control group). ③ The relative expression level was calculated: 2^(-ΔΔCt).

[0186] (4) Immunofluorescence: After the virus infects the cells, the cells are fixed with formaldehyde, and the viral proliferation level is determined using a virus matrix protein (M) specific antibody and immunofluorescence technology. The relative fluorescence intensity is calculated by reading the fluorescence value using ImageJ.

[0187] The results are shown in Table 11.

[0188] Table 11

[0189]

[0190] As shown in Table 11, 12 h after infection, cytopathic effects were observed in tracheal epithelial cells after H1 infection, but not after H5 infection. The infection rate of H1 in tracheal epithelial cells was significantly higher than that of H5. Western blot results showed that the relative expression levels of NP protein in both groups increased significantly after 24 h of infection, and the relative expression levels of NP protein in the H1 group were significantly higher than those in the H5 group from 24 to 60 h after infection. p<0.0001) Figure 8 qPCR results showed that, 6 h after infection, the relative expression levels of M gene mRNA in groups H1 and H5 increased significantly, and, 12-60 h after infection, the relative expression levels of M gene mRNA in group H1 were significantly higher than those in group H5. p<0.0001) Figure 9 Immunofluorescence results showed that H1 could proliferate and infect tracheal epithelial cells in large quantities, significantly higher than the infection level of H5. Figure 10In summary, the results from cytopathic effects, qPCR, Western blotting, and immunofluorescence are consistent, indicating that H1 has a faster, more efficient, and more pronounced infectivity on tracheal epithelial cells, suggesting that these cells are more susceptible to respiratory pathogens with high aerosol transmission capacity.

[0191] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method of evaluating the aerosol transmission characteristics of a respiratory pathogen, characterized by, The method includes the following steps: (1) Infect tracheal epithelial cells with respiratory pathogens and assess the aerosol transmission potential of the respiratory pathogens by detecting the proliferation level of the respiratory pathogens in the tracheal epithelial cells; the tracheal epithelial cells are immortalized guinea pig tracheal epithelial cells, and their preservation number is CGMCC No. 46763. (2) Select respiratory pathogens that are identified as having aerosol transmission potential in step (1) to infect airway epithelial organoids; construct airway epithelial organoids using tracheal epithelial cells, and then infect tracheal epithelial organoids with respiratory pathogens. By detecting the release level of respiratory pathogen aerosol particles from tracheal epithelial organoids, assess the biophysical basis of the aerosol transmission capability of the respiratory pathogen. (3) Select animals infected with respiratory pathogens that are determined to have aerosol transmission capability in step (2) and infect them with respiratory pathogens. By detecting the aerosol transmission efficiency between animals, the aerosol transmission capability of the respiratory pathogen is evaluated. The tracheal epithelial cells in steps (1) and (2) are the same; the method is for non-disease diagnosis purposes.

2. The method of claim 1, wherein, The respiratory pathogen is selected from at least one of influenza virus, novel coronavirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus.

3. The method according to claim 2, wherein, The respiratory pathogen is the influenza virus.

4. The method according to claim 1, wherein, The efficiency of aerosol transmission between animals is quantitatively assessed by measuring the infection status of infected animals through aerosol exposure to uninfected animals.

5. A kit for evaluating the aerosol transmission characteristics of respiratory pathogens, characterized in that, The kit includes: 1) Detection reagents for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells; 2) Detection reagents for detecting the release levels of respiratory pathogen aerosol particles from tracheal epithelial organoids; 3) Detection reagents for assessing the efficiency of aerosol transmission among animals; 4) Tracheal epithelial cells; the tracheal epithelial cells are immortalized guinea pig tracheal epithelial cells, with the preservation number CGMCC No. 46763; 5) Reagents used to construct tracheal epithelial organoids.

6. The kit according to claim 5, wherein, Detection reagents for detecting the proliferation level of respiratory pathogens in tracheal epithelial cells include a primer-probe combination targeting the pathogen's nucleic acid.

7. The kit according to claim 5 or 6, wherein, The reagents used to construct tracheal epithelial organoids include organoid culture media containing epithelial cell organoid culture media and calcium chloride.

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