Resistance to SARS-CoV-2 infection in camelid nasal organoids is associated with lack of ACE2 expression

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects a variety of animal species. Susceptibility to SARS-CoV-2 is primarily determined by the utilization of the viral receptor, ACE2. SARS-CoV-2 can utilize a broad range of animal ACE2 isoforms in vitro, including the ACE2 from various camelid species. However, experimental infection of these animals does not lead to productive infection or seroconversion. In this study, we investigate the susceptibility of camelids to SARS-CoV-2 using novel well-differentiated camelid nasal organoids. We show that camelid nasal organoids are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) infection, but not to infection with different SARS-CoV-2 variants (614G, BA.1 or EG.5.1.1). All viruses efficiently infected human airway organoids. Immunohistochemistry analysis revealed the absence of ACE2 on camelid nasal organoids and dromedary camel upper respiratory tract. In contrast, DPP4 was expressed in both camelid nasal organoids and the camel upper respiratory tract, which correlates with MERS-CoV infection. This study indicates that the camelid upper respiratory tract lacks expression of ACE2, which is associated with resistance to SARS-CoV-2 infection.

Fig. 1. MERS-CoV targets multiciliated cells and causes deciliation in camelid nasal organoids.

a, b Establishment of camelid nasal organoids (a, created with BioRender.com) and cells were stained (b) with hematoxylin and eosin. c Camelid nasal organoids were stained for cilia (ACTUB, green) and nuclei (Hoechst, blue). (d, e) Camelid nasal organoids were infected with MERS-CoV with a moi of 0.1 and viral replication was determined by d RT-qPCR and e plaque assay titration. Data is depicted as mean with SEM. Dotted line shows detection limit. f, g Infected cells on transwell inserts (f) and paraffin-embedded inserts (g) were stained at 1 dpi for cilia (ACTUB, green) and MERS-CoV spike (S, red). h Full well imaging was performed on MERS-CoV infected camelid nasal organoid inserts at 3 dpi for cilia (ACTUB, green) and MERS-CoV nucleoprotein (NP, red) to show deciliation. Experiments were repeated at least once. Scale bars are 20 µm (b, c, f, g) and 500 µm (h). Inserts (f’, g’, h’ and h”) show digital zoom of the original image.

Fig. 2. SARS-CoV-2 variants 614G, BA.1 and EG.5.1.1 cannot replicate in camelid nasal organoids.

Camelid nasal organoids and human airway organoids were infected with MERS-CoV and SARS-CoV-2 614G, BA.1 and EG.5.1.1 variants with a moi of 0.1 to assess camelid susceptibility. a, b Viral replication of SARS-CoV-2 variants 614G, BA.1 and EG.5.1.1 in camelid nasal organoids was determined by a RT-qPCR and b plaque assay titration. Data is depicted as mean with SEM, and for (b) values of the individual replicates were shown. Dotted line shows detection limit. c, d Infected inserts of c human airway organoids and d camelid nasal organoids were stained at 3 dpi for ciliated cells (ACTUB, green) and viral antigen (Spike for MERS-CoV and Nucleoprotein for SARS-CoV-2, red). Illustrations were created with BioRender.com. Experiments were repeated at least once. Scale bars are 200 µm. Inserts (squares) show digital zoom of the original image.

Fig. 3. DPP4 but not ACE2 is expressed in camelid nasal organoids and tissue.

Immunohistochemistry for DPP4 (top), ACE2 (middle) and isotype (bottom) was performed on camelid nasal organoids and dromedary camel nasal and intestinal tissues from four donors. Representative images at ×20 magnification were shown of one donor. Scale bars are 100 µm. Inserts (squares) show digital zoom of images of the same region at ×40 magnification, scale bars are 20 µm.

Fig. 4. ACE2 is expressed sporadically in the dromedary camel lower respiratory tract.

Immunohistochemistry for DPP4 (top), ACE2 (middle) and isotype (bottom) was performed on dromedary camel bronchus, bronchiole and lung tissue of four donors. Representative images at ×20 magnification were shown of one donor. Scale bars are 100 µm. Inserts (squares) show digital zoom of images of the same region at ×40 magnification, scale bars are 20 µm.