Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids

Abstract

Background: Human seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development.

Methods: The infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested.

Findings: HAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs.

Interpretation: HAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections.

Funding: This research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Keywords: Airway organoids; Antiviral therapy; Seasonal coronaviruses; Thermal sensitivity; Virus-host interactions.

Figure 1

Characterizing the cell types of undifferentiated hAOs and the permission of hAOs to 229E and OC43 infections. (A) to (D) Characterizing cell types by immunostaining Kertain 5 (basal cells), MUC5AC (goblet cells), β-tubulin (ciliated cells) and CC10 (club cells). (E) schematic diagram for seasonal coronaviruses inoculation in hAOs. (F) Dynamics of intracellular virus RNA copies in hAOs cultured at 33 °C from 1 to 24 h post-inoculation (n=3). (G) Dynamics of infectious viral titers in hAOs cultured at 33 °C from 1 to 24 h post-inoculation (n=3). (H) Immunofluorescence staining for viral double-stranded RNA (dsRNA) in hAOs at 33 °C. Scale bar = 25 μm. (I) Transmission electron microscopy analysis of 229E and OC43 infected hAOs cultured with expansion medium at 33 °C.

Figure 2

Recapitulating 229E and OC43 infections in differentiated hAOs. (A) Transcriptional levels of specific cell-type markers upon differentiation at 37 °C. (B) to (E) Characterizing cell types by immunostaining β-tubulin (ciliated cells), MUC5AC (goblet cells), Kertain 5 (basal cells) and CC10 (club cells) (Scale bar = 25 μm). (F) Dynamics of intracellular virus RNA copies in differentiated hAOs at 33 °C from 1 to 24 h post-inoculation (n=3). (G) Dynamics of infectious viral titers in differentiated hAOs at 33 °C from 1 to 24 h post-inoculation (n=3). (H) Immunofluorescence staining for viral double-stranded RNA (dsRNA) in differentiated organoids at 33 °C (Scale bar = 10 μm).

Figure 3

(A) TEER value of monolayers in transwell membrane tested at 3, 5, 10, 13, 16, 20, 23 and 25 days after seeding. (B) Schematic representation of virus inoculation in organoid cells in transwell system. (C) Quantification of released viruses from monolayers inoculated with 229E particles. (D) Quantification of released viruses from monolayers inoculated with OC43 particles. (E) to (G) Immunofluorescence staining for viral dsRNA and β-tubulin (ciliated cell marker) in transwell monolayers at 72 h post-inoculation (Scale bar = 25 μm).

Figure 4

Kinetics of 229E and OC43 virus replication and production at different temperatures. (A) 229E replication kinetics in undifferentiated hAOs at 33 °C and 37 °C at 1 h, 1 day, 3 day, 5 day, 7 day post-inoculation (n=4). (B) The kinetics of 229E virus production (n=4). (C) The kinetics of OC43 viral replication (n=3–4). (D) The kinetics of OC43 virus production (n=3–4). (E) Immunofluorescence staining of 229E viral dsRNA and EpCAM (epithelial membrane marker) in undifferentiated hAOs at different temperatures at 3 days post-inoculation. (F) Immunofluorescence staining of OC43 viral dsRNA and EpCAM in undifferentiated hAOs. The kinetics of 229E viral replication (G) and production (H) in differentiated hAOs at 33 °C and 37 °C at 1 h, 1 day, 3 days, 5 days, 7 days post-inoculation (n=2–3). The kinetics of OC43 replication (I) and production (J) in differentiated hAOs (n=2–3). (K) and (L) Immunofluorescence staining of viral dsRNA and EpCAM in differentiated hAOs at 33 °C and 37 °C at 3 days post-inoculation. Scale bar = 25 μm.

Figure 5

Genome-wide transcriptomic analysis in human airway organoids upon seasonal coronavirus infection. (A) Principal component analysis (PCA) of different organoid samples (U: Undifferentiated hAOs; D: Differentiated hAOs). (B) Top 40 significantly enriched pathways by gene ontology (GO) analysis of hAOs cultured at 37 °C. (C to E) Venn diagram of overlapped differentially expressed genes (C) in differentiated and undifferentiated airway organoids cultured at 33 °C and 37 °C, (D) 229E virus infection in undifferentiated hAOs or differentiated hAOs cultured at 33 °C and 37 °C, (E) OC43 virus infection in undifferentiated hAOs or differentiated hAOs cultured at 33 °C and 37 °C. (F to I) Differential gene expression analysis by coronavirus infections in undifferentiated hAOs, (F) 229E infection at 33 °C, (G) 229E infection at 37 °C, (H) OC43 infection at 33 °C, (I) OC43 infection at 37 °C.

Figure 6

Antiviral activity of molnupiravir against 229E and OC43 in hAOs cultured at 33 °C. (A) The inhibitory effect of molnupiravir on 229E replication in hAOs cultured at 33 °C (n = 4–10). (B) IC50 and CC50 of molnupiravir in hAOs infected with 229E and cultured at 33 °C (n = 6–8). (C) The inhibitory effect of molnupiravir on OC43 replication in hAOs cultured at 33 °C (n = 4–9). (D) IC50 and CC50 of molnupiravir in hAOs infected with OC43 and cultured at 33 °C (n = 6–8). (E) and (F) The inhibitory effect of low concentration (2.5 μM) and high concentration (50 μM) of molnupiravir by immunostaining dsRNA in hAOs cultured at 33 °C. Scale bar = 25 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Antiviral activity of molnupiravir against 229E and OC43 in hAOs cultured at 37 °C. (A) The inhibitory effect of molnupiravir against 229E in hAOs cultured at 37 °C (n= 4–9). (B) IC50 and CC50 of molnupiravir in hAOs infected with 229E and cultured at 37 °C (n = 6–8). (C) The inhibitory effect of molnupiravir against OC43 in hAOs cultured at 37 °C (n = 4–9). (D) IC50 and CC50 of molnupiravir in hAOs infected with OC43 and cultured at 37 °C (n = 6–8). (E) and (F) The inhibitory effect of low concentration (2.5 μM) and high concentration (50 μM) of molnupiravir by immunostaining dsRNA in hAOs cultured at 37 °C. Scale bar = 25 μm. *P < 0.05; **P < 0.01; ***P < 0.001.