Human respiratory organoids sustained reproducible propagation of human rhinovirus C and elucidation of virus-host interaction

Abstract

The lack of a robust system to reproducibly propagate HRV-C, a family of viruses refractory to cultivation in standard cell lines, has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C, and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression, whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly, upon HRV-C infection, we observe an innate immune response that is stronger in airway organoids than in nasal organoids, which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally, an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively, we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C, followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has opened an avenue for propagating and studying other uncultivable human and animal viruses.

Fig. 1. Human airway organoids were susceptible to clinical specimens of HRV-C.

a A schematic illustration of the human nasal organoid and airway organoid culture system was created with Biorender.com. b Airway organoids (in 9 transwell inserts) were inoculated with 9 HRV-C+ nasopharyngeal aspirates. At the indicated day post-infection (d.p.i.), culture media were harvested from the infected airway organoids and applied to viral load detection by RT-qPCR. A schematic graph of the experimental procedure was created with Biorender.com. c Airway organoids were inoculated with HRV-A1 and HRV-C3 at 100 viral gene copy/cell (n = 3). Culture media were harvested from apical and basolateral chambers of infected airway organoids at the indicated d.p.i. and applied to the viral load detection. Data represent mean and SD of the indicated number (n) of biological replicates from a representative experiment independently performed three times. Statistical significance (in panel c) was determined using a two-tailed Student’s t-test. **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 2. CYT387 enabled serial propagation of HRV-C in human airway organoids.

a A schematic graph outlines the experimental procedure for panels (b and c), created with Biorender.com. b In the first (P1) passage, after pre-treatment with CYT387 or DMSO overnight, airway organoids were inoculated with HRV-C3 (n = 2). Culture media were harvested from infected airway organoids at indicated h.p.i. to detect viral replication. In the second (P2) passage, airway organoids pretreated with CYT387 or DMSO were inoculated with 50 µl P1 medium collected from CYT387- or DMSO-treated organoids, respectively (n = 2). Culture media were harvested at indicated h.p.i. to detect viral replication. c From P3, airway organoids pretreated with CYT387 or DMSO were inoculated with medium collected from CYT387-treated organoids at 100 viral gene copy/cell (P3, n = 2; P4 and P5, n = 3). The culture media were harvested at 96 h.p.i. to detect viral replication. The viral loads in P1 and P2 media at 96 h.p.i. are incorporated. Data represent mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (P4 and P5) was determined using a two-tailed Student’s t-test. **P < 0.01. ns not significant. Source data are provided as a Source Data file for Fig. 2b, c. d At 24 h.p.i. of HRV-C3, airway organoids were fixed and immune-labeled with an α-VP3 (green) and α-ACCTUB (red). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. The experiment was independently performed three times with similar results. Scale bar, 10 µm.

Fig. 3. Human nasal organoid sustained serial HRV-C propagation.

a A schematic graph outlines the experimental procedure for panels (b, c, and d) created with Biorender.com. b–d Nasal organoids pre-treated with CYT387 or DMSO were inoculated with HRV-C3 (b), C11 (c), and C15 (d) at 100 viral gene copy/cell and incubated in the presence of CYT387 or DMSO respectively (n = 3). CYT387- and DMSO-treated media were brought forward as inoculum to the next round of infection, during which CYT387 and DMSO treatment continued. Culture media were harvested at 96 h.p.i. to detect viral replication. e At 24 h.p.i., nasal organoids inoculated with HRV-C3 were fixed and doubled stained with an α-VP3 (green) and α-ACCTUB (red). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar, 10 µm. f TEM images of HRV-C3 viral particles in the culture media of infected nasal organoids. Arrows indicate viral particles (black) or empty capsids (white). Scale bar, 100 nm. g, h Nasal organoids derived from 3 different donors inoculated with HRV-C3 (g) and HRV-A1 (h) were incubated at 33 °C or 37 °C (n = 3). The culture media were harvested at the indicated h.p.i. to detect viral replication. Data represent the mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (in b, c, d, g, and f) was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01. ns not significant. The experiments in e and f were independently performed three times with similar results. Source data are provided as a Source Data file for Fig. 3b, c, d, g, and h.

Fig. 4. Nasal organoids were more permissive to HRV-C than airway organoids.

a–c Airway (AwO) and nasal (NsO) organoids derived from different donors were inoculated in parallel with HRV-C3 at 1000, 100, 10, and 1 viral gene copy/cell (n = 3). Culture media were harvested from the infected organoids at the indicated h.p.i. to detect viral replication. d After co-staining with α-VP3 and α-ACCTUB, HRV-C3- and mock-infected airway and nasal organoids were applied to flow cytometry analysis (n = 2). Representative histograms are shown on the left. Data on the right represent the mean and SD from a representative experiment. e SEM images of HRV-C3- and mock-infected airway organoids. The experiment was independently performed three times with similar results. Scale bar, 200 nm. Data represent mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (in a, b, and c) was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. ns not significant. Source data are provided as a Source Data file for Fig. 4a–d.

Fig. 5. Host transcriptional response in human airway and nasal organoids.

HRV-C3 infected airway organoids (AwO) and nasal organoids (NsO) treated with CYT387 (HRV-C^+CYT387) or DMSO (HRV-C), together with mock-infected organoids (Mock) were applied to RNA sequencing analysis. a Volcano plot shows DEGs in the infected airway and nasal organoids compared with mock-infected organoids. DEGs with a log2(fold change) > 1 and < −1 are shown in red and blue, respectively. b Venn diagram shows the numbers of unique and common upregulated DEGs in the infected airway and nasal organoids. c Heatmap depicts DEGs in the indicated organoids and the assigned GO biological processes (GO:0009615, GO:0045088, GO:0060337, GO:0034341, GO:0019221, GO:0032602, GO:0050727). d The heatmap demonstrates the enriched GO terms in the indicated airway and nasal organoids. The color of the dots represents the normalized enrichment score (NES) value for each enriched GO term. e GAPDH normalized expression levels of innate immune molecules in HRV-C3-infected airway and nasal organoids at 24 and 48 h.p.i (n = 3). f Data show the percentage of virus-aligned reads over total reads in the indicated organoids (n = 2). g Three lines of airway and nasal organoids were treated with 10 μg/ml Poly(I:C) or mock-treated (n = 3). At 6 h post-treatment, organoids were harvested for RT-qPCR. Results show the GAPDH normalized expression level of innate immune molecules in the airway and nasal organoids. Data represent mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (in e, g) was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. ns not significant. Source data are provided as a Source Data file for Fig. 5e, g.

Fig. 6. Receptor blocking, antiviral inhibition, and virus titration in airway and nasal organoids.

a Airway (AwO) and nasal organoids (NsO) and their un-differentiated counterparts were immune-stained with an α-CDHR3 and an isotype IgG control and applied to flow cytometry analysis (n = 2). The left panel shows the representative histograms. b Nasal organoids were fixed and double-stained with an α-CDHR3 (green), α-ACCTUB (red). Confocal images of en face (top) and cross-section (bottom) are shown. Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar, 10 µm. c Nasal organoids were treated with two α-CDHR3 of 20 μg/ml and autologous IgG for 2 h (n = 3), followed by HRV-C3 inoculation and further incubation with the same antibodies. Culture media were harvested at the indicated h.p.i. to detect viral replication. Results show the relative viral load in the α-CDHR3 treated organoids versus those in IgG-treated organoids. d Airway organoids infected with HRV-A1 or HRV-C3 were treated with 1 μM Rupintrivir or 3 μM Itraconazole or DMSO (n = 3). Culture media were harvested from the infected organoids at the indicated h.p.i. and applied to the detection of viral replication by RT-qPCR. Results show the relative viral load in the drug-treated organoids versus those in DMSO-treated organoids. e, f Nasal organoids were infected with HRV-C3 after 10-fold serial dilutions (10⁻¹ ~ 10⁻³). At 24 h.p.i., the organoid monolayers were fixed and immune-stained with an α-VP3, followed by imaging analysis. e Representative high-content confocal images show the VP3+ cells (green) in the monolayers infected by serially-diluted viruses. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. f Three different areas in each organoid monolayer were randomly selected for calculating VP3+ cells. The results show the average number of VP3+ cells at different dilutions. Data represent mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (in c, d) was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. ns not significant. The experiments in b and e were performed three times independently with similar results. Source data are provided as a Source Data file for Fig. 6a, c, d, and f.