Tracheal fibroblasts and uses thereof

Abstract

The application relates to the technical field of cell culture, and discloses a tracheal fibroblast cell and application thereof, the cell being a guinea pig tracheal fibroblast immortal cell, and the preservation number being CGMCC No. 46764. In the application, the cell has good susceptibility to respiratory tract pathogens and excellent transfection efficiency. The cell can be used for isolating or culturing respiratory tract pathogens, preparing respiratory tract pathogen vaccines, screening and / or preparing medicines for preventing and / or treating diseases caused by respiratory tract pathogens, serving as a host cell for researching respiratory tract pathogens, detecting respiratory tract pathogens for non-diagnostic purposes, and constructing an in-vitro model of respiratory tract pathogen infection, and has high scientific research value and practical application potential.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to tracheal fibroblasts and their applications. Background Technology

[0002] Tracheal fibroblasts are the core functional cells of the tracheal mucosal interstitium, playing a fundamental role in maintaining airway structural stability and synthesizing the extracellular matrix. They also participate in the inflammatory response and tissue repair process following respiratory pathogen infection by secreting inflammatory factors and activating innate immune pathways. During the invasion of respiratory pathogens such as viruses and bacteria, these cells are not only target cells for pathogen invasion but also key participants in regulating the degree of pathological damage caused by infection. Their abnormal proliferation and fibrotic transformation are important contributing factors to complications such as airway stenosis and pulmonary fibrosis after infection.

[0003] Guinea pig tracheal fibroblasts, due to their physiological characteristics and immune response patterns similar to human respiratory cells, and their ease of isolation, culture, and establishment of stable cell lines, have become ideal in vitro models for studying respiratory pathogen infection mechanisms, drug screening, and vaccine development. Currently, research using guinea pig tracheal fibroblasts often faces challenges such as demanding cell culture conditions, difficult passage, low viral infection levels, and low transfection efficiency in transgenic experiments, limiting their application in large-scale experiments and long-term studies. Therefore, there is an urgent need to develop novel cell lines that combine culture stability, high viral susceptibility, and transgenic compatibility to specifically address the shortcomings of existing guinea pig tracheal fibroblast applications and provide a more efficient in vitro tool for respiratory-related research. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and to provide tracheal fibroblasts and their applications.

[0005] To achieve the above objectives, the first aspect of the present invention provides a tracheal fibroblast cell line, which is an immortalized guinea pig tracheal fibroblast cell, with the accession number CGMCC No. 46764.

[0006] Secondly, the present invention provides the application of the cells in the isolation of respiratory pathogens.

[0007] Thirdly, the present invention provides the use of the cells in the preparation of respiratory pathogen vaccines.

[0008] Fourthly, the present invention provides the use of said cells in screening and / or preparing medicaments for the prevention and / or treatment of diseases caused by respiratory pathogens.

[0009] Fifthly, the present invention provides the use of said cells in culturing respiratory pathogens.

[0010] In a sixth aspect, the present invention provides the use of said cells as host cells for studying respiratory pathogens.

[0011] In a seventh aspect, the present invention provides the use of said cells in the detection of respiratory pathogens for non-diagnostic purposes.

[0012] Eighthly, the present invention provides the application of the cells in constructing in vitro models of respiratory pathogen infection.

[0013] In this invention, the construction of in vitro models of respiratory pathogen infection mainly includes three mainstream systems: basic single-cell infection models, co-culture infection models, and organoid infection models. The cell type, pathogen type, and culture system must be selected according to the research objectives. The core is to simulate the in vivo respiratory microenvironment to achieve pathogen adhesion, invasion, and proliferation, while simultaneously reproducing the pathophysiological changes after infection. Preferably, the tracheal fibroblasts provided in this invention can serve as both host cells for single-cell infection models and helper cells for constructing co-culture infection models and organoid infection models.

[0014] In this invention, the respiratory pathogen can be at least one of influenza virus, novel coronavirus (SARS-CoV-2), respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus, preferably influenza virus H1 and / or influenza virus H5.

[0015] More preferably, the cells exhibit a faster infection response, higher infection efficiency, and more pronounced infection characteristics to the H5 influenza virus.

[0016] In this invention, the tracheal fibroblasts are immortalized cells.

[0017] In this invention, the tracheal fibroblasts are derived from guinea pigs ( Cavia porcellus ).

[0018] The tracheal fibroblasts provided by this invention have a higher infection level against highly pathogenic influenza viruses with low aerosol transmissibility. They are a target cell line for pathogen proliferation and damage in the respiratory tract. Furthermore, this cell line has extremely high transfection efficiency and can be used to carry out molecular biology experiments such as gene knockout and gene overexpression to further explore the molecular mechanisms related to infection and damage by respiratory pathogens such as influenza viruses.

[0019] The tracheal fibroblasts of this invention exhibit significant advantages in pathogen isolation and amplification, effectively supporting the culture and identification of respiratory pathogens such as influenza virus, and providing a reliable cell matrix for vaccine research and production. Furthermore, they have broad application prospects in molecular mechanism research, antiviral drug screening, and pathogen pathogenicity assessment, helping to reveal key regulatory factors and potential therapeutic targets of pathogen infection.

[0020] The establishment of tracheal fibroblasts in this invention not only helps to improve the efficiency and accuracy of respiratory pathogen research, but also provides important technical support for pathogen early warning identification, risk assessment and prevention and control strategy formulation, and has high scientific research value and practical application potential.

[0021] Biological Preservation

[0022] The cells provided in this invention are immortalized guinea pig tracheal fibroblast 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. 46764. The immortalized guinea pig tracheal fibroblast cells provided in this invention are named GTF1. Attached Figure Description

[0023] Figure 1 This is a diagram showing the experimental results of transfection of immortalized tracheal fibroblast cells according to the present invention;

[0024] Figure 2 This is a graph showing the passage test results of the immortalized tracheal fibroblasts of the present invention;

[0025] Figure 3 It is the relative expression level of viral nucleoprotein (NP) in the immortalized tracheal fibroblasts of the present invention after viral infection;

[0026] Figure 4 It is the relative expression level of the viral M gene mRNA in the immortalized tracheal fibroblasts of the present invention after viral infection;

[0027] Figure 5 This is an image showing the immunofluorescence detection results of the immortalized tracheal fibroblasts of the present invention after viral infection. Detailed Implementation

[0028] The present invention will be described in detail below through embodiments.

[0029] In the following examples, the influenza virus H1 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;

[0030] The influenza virus H5 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.

[0031] Example 1

[0032] This example illustrates the culture of primary cells.

[0033] 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 and euthanized by cervical dislocation. Tracheal tissue was aseptically harvested. The tracheal tissue was then minced and the minced tissue pieces were vigorously washed in PBS buffer. The washing was repeated until the supernatant became clear to remove residual epithelial cells. The tissue blocks were then digested at 37°C with 1 mg / mL type I collagenase (Yeasen, 40507ES60). The resulting cell suspension was subjected to differential adhesion culture to selectively enrich fibroblasts. The cells were cultured in DMEM / F12 medium (GIBCO, 21331020) containing 10% fetal bovine serum (FBS, GIBCO, 10099141C), 10 ng / mL bFGF (Invitrogen, 13256-029), 1% Glutamax (GIBCO, 31765035), and 1% penicillin / streptomycin (GIBCO, 15140148). Once the fibroblast purity met the experimental requirements, subsequent functional verification experiments were conducted.

[0034] Example 2

[0035] 1. Cell validation

[0036] Detection for bacteria, fungi, mycoplasma, etc., ensures that cells are free from microbial contamination. The specific steps are as follows:

[0037] (1) Detection of bacterial and fungal contamination

[0038] 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.

[0039] 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.

[0040] (2) Mycoplasma contamination detection

[0041] 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.

[0042] 2. Cell transduction

[0043] (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.

[0044] (2) When the cell count reaches 60-70%, digest the cells in one well and count them. Using MOI (Multiple of Infection) = 200, 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:

[0045] MOI = Titration × Sample Volume × 10 -3 / cell number

[0046] (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.

[0047] (4) Lentiviral virus (1×10 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 the final concentration 5 μg / mL.

[0048] (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.

[0049] (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.

[0050] (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 1 As shown, the cells of this invention have extremely high transfection efficiency.

[0051] (8) When the cell confluence reaches 90% or more, the six-well cells are passaged and seeded in 6 cm culture dishes. By comparing the proliferation of primary cells with that of primary cells, when the primary cells no longer proliferate but the immortalized cells can continue to proliferate, the expression level of SV40T is detected by qPCR.

[0052] (9) The expression levels of the target gene (SV40-T) were analyzed by qPCR in primary fibroblasts and recombinant cell lines transfected with lentivirus (CGMCC No. 46764). 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 1; the qPCR reaction program is shown in Table 2; and the detection results are shown in Table 3.

[0053] Table 1

[0054]

[0055] Table 2

[0056]

[0057] Note: Fluorescence signal collection was performed in the second program segment (60℃ annealing and extension).

[0058] Table 3

[0059]

[0060] As shown in Table 3, the cells of this invention can successfully express SV40-T, that is, the immortalized guinea pig tracheal fibroblast cell line has been successfully constructed.

[0061] 3. Continuous passage culture, amplification, and quality control

[0062] (1) Primary cell passage: The cells were passaged continuously at a ratio of 1:3 until P3 cells showed obvious aging and almost stopped proliferation, at which point the experiment was terminated; the primary cells were derived from guinea pig tracheal fibroblast tissue.

[0063] (2) Cell passage (CGMCC No. 46764): Cells were cultured in DMEM / F12 medium containing 10% FBS, 10 ng / mL bFGF, 1% Glutamax, and 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, proliferation was stable, and the cells were cryopreserved for seed culture.

[0064] (3) Frozen cell thawing and passage: P15 cells were thawed and seeded in T25 culture flasks, and passaged continuously to P60 at a ratio of 1:3. The cells had normal morphology and stable proliferation.

[0065] (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.

[0066] (5) Quality Inspection:

[0067] Culture method detection and microscopic observation: negative for bacteria and fungi;

[0068] qPCR test: Mycoplasma negative.

[0069] The results are as follows Figure 2 As shown in Table 4.

[0070] Table 4

[0071]

[0072]

[0073] As can be seen from Table 4, the cells of the present invention can be stably and continuously passaged.

[0074] 4. Cell proliferation detection

[0075] (1) P20 and P60 cells were administered at a rate of 1×10 5 10 cells / well were seeded into 10 wells of a 6-well plate;

[0076] (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 and take the average value).

[0077] (3) For five consecutive days, at the same time each day, digest the cells in two wells and count the number of cells in each well (take three samples from each well and take the average value).

[0078] The results are shown in Table 5.

[0079] Table 5

[0080]

[0081] As can be seen from Table 5, the cells of the present invention still exhibit good proliferation characteristics after 60 passages.

[0082] 5. Measurement of viral infection and viral replication levels

[0083] Experimental methods:

[0084] (1) Virus incubation

[0085] Immortalized guinea pig tracheal fibroblast cells (CGMCC No. 46764) were collected, and the cell supernatant was discarded. 500 μL of virus H1 or H5 (MOI=0.01) diluted in serum-free OPTI MEM medium (GIBCO, 31985070) was added to each well of a six-well plate, and the plates were incubated at 37°C for 1 h. After virus incubation, the viral supernatant was discarded, and 2 mL of maintenance medium (0.5% FBS, 10 ng / mL bFGF, 1% Glutamax, 1% penicillin / streptomycin in DMEM / F12) was added to each well. The plates were then returned to the incubator and cultured for another 60 h.

[0086] (2) WB sampling and testing methods

[0087] 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 rpm for 10 min at 4 °C, and transfer 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 °C for 10 min to denature, ensuring that the virus particles rupture and release proteins. Centrifuge at 12000 rpm for 1 min, and use the supernatant for Western blotting.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] (3) qPCR sampling and testing methods

[0092] 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.

[0093] b. Using a reverse transcription kit (TAKARA, RR047), follow the kit instructions to reverse transcribe the RNA extracted in step a to obtain cDNA.

[0094] c. qPCR reaction: A qPCR detection kit (TransGen, AQ621) was used, and the qPCR reaction was performed according to the kit instructions. The upstream primer sequence for the M gene was 5'-CTTCTAACCGAGGTCGAAACG-3' (SEQ ID NO: 5), and the downstream primer sequence for the M gene was 5'-CTTTAGCCACTCCATGAGAGC-3' (SEQ ID NO: 6). The upstream primer sequence for the internal reference gene (GAPDH) was 5'-AACTTCGGCATTGTGGAGGG-3' (SEQ ID NO: 7), and the downstream primer sequence for the internal reference gene was 5'-GGATGCGGGGATGATGTTCT-3' (SEQ ID NO: 8). The qPCR reaction was performed according to the reaction conditions in Table 6.

[0095] Table 6

[0096]

[0097] Note: Fluorescence signal collection was performed in the second program segment (56℃ annealing and extension).

[0098] (4) Immunofluorescence experiment

[0099] 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 (positively potent, AF594, 1:200 dilution) 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.

[0100] Experimental results:

[0101] (1) Cellular lesions: direct observation under a microscope.

[0102] Evaluation criteria: Using the uninfected control group as a reference, the evaluation was 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 were observed, it was judged as a positive cytopathic effect (CPE). The same criteria were used to interpret different treatment groups consistently.

[0103] (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.

[0104] (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).

[0105] (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.

[0106] The results are shown in Table 7.

[0107] Table 7

[0108]

[0109] As shown in Table 7, 12 h after viral infection, H5 infection resulted in significant cytopathic effects in tracheal fibroblasts, while H1 infection did not. The infection rate of H5 in tracheal fibroblasts was significantly higher than that of H1. Western blot results showed that 24 h after infection, the relative expression levels of NP protein in both H1 and H5 groups significantly increased, and from 24 to 60 h after infection, the relative expression levels of NP protein in the H5 group were significantly higher than those in the H1 group. p<0.001, p<0.0001) Figure 3 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 level of M gene mRNA in group H5 was significantly higher than that in group H1. p<0.01, p<0.0001) Figure 4 Immunofluorescence results showed that H5 could proliferate and infect tracheal fibroblasts in large quantities, significantly higher than the infection level of H1. Figure 5 In summary, the results from cytopathic effects, qPCR, Western blotting, and immunofluorescence were consistent, indicating that H5 cell line has a faster, more efficient, and more pronounced infectivity on tracheal fibroblasts, suggesting that this cell line is more susceptible to highly pathogenic respiratory pathogens with low aerosol transmission capacity.

[0110] 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 tracheal fibroblast strain, characterized in that, These cells are immortalized guinea pig tracheal fibroblasts, with the cell preservation number CGMCC No. 46764.

2. The use of the cells described in claim 1 in the isolation of respiratory pathogens.

3. The use of the cells according to claim 1 in the preparation of respiratory pathogen vaccines.

4. The use of the cells of claim 1 in screening and / or preparing medicaments for the prevention and / or treatment of diseases caused by respiratory pathogens.

5. The use of the cells according to claim 1 in culturing respiratory pathogens.

6. The use of the cell according to claim 1 as a host cell for studying respiratory pathogens.

7. The use of the cells of claim 1 in the detection of respiratory pathogens for non-diagnostic purposes.

8. The use of the cells described in claim 1 in constructing an in vitro model of respiratory pathogen infection.

9. The application according to any one of claims 2-8, wherein, The respiratory pathogen is at least one of influenza virus, novel coronavirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus.

10. The application according to any one of claims 2-8, wherein, The respiratory pathogens are influenza virus H1 and / or influenza virus H5.

Images