Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

Abstract

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium.

Figure 1

Establishment and characterization of Bactrian camel and llama AEC cultures. (A) Immunofluorescence analysis showing the development of tight-junctions (ZO-1, white) and ciliogenesis (β-tubulin, red) in Bactrian camel and llama AEC cultures over time from 1-day to 4 weeks post ALI exposure. The cells were counterstained with DAPI (blue) to visualize the nuclei. (B,C) Ciliogenesis quantification of camel and llama AEC cultures overtime, respectively. Ciliation was quantified by measuring the area above a fluorescence intensity threshold of five random images acquired per condition. (D) Transepithelial electrical resistance (TEER) measurement of camel and llama AEC cultures overtime during the differentiation. (E) Epithelial morphology of ex vivo tissues (upper panel) and well-differentiated camel and llama AEC cultures (lower panel). (F) DPP4 expression in well-differentiated camel and llama AEC cultures, with Vero cells as a positive control. Scale bar is 20 µm.

Figure 2

Efficient MERS-CoV replication in camelid AEC cultures. MERS-CoV titer in TCID₅₀/ml released from camel (A) and llama (B) AEC cultures’ apical side from 24 to 96 h post-infection depicted in a log10 scale. Apical virus release in Bactrian camel (C) and llama (D) AEC cultures measured by quantitative reverse transcription PCR. Data is shown as means and standard deviation from three (A,C) or six (B,D) independent biological replicates. (E) Representative immunofluorescence staining of MERS-CoV-infected camel and llama AEC cultures at 48 post-infection. Double-stranded RNA is shown in green, β-tubulin in red, ZO-I in white and DAPI in blue. Scale bar is 20 µm.

Figure 3

IFN treatment efficiently induces innate immune response and reduces MERS-CoV replication in camelid AEC cultures. MX1, Interleukin-6 (IL6), CXCL10, RIG-I and TNF-α expression in log₂ fold induction over mock in camel and llama AEC cultures is displayed 6- and 12-h post-stimulation with type I IFN (A), type III IFN (B), and poly I:C (C) treatment. (D) MERS-CoV titers in TCID₅₀/ml released from camelid AEC cultures’ apical side 48 h post-infection in presence and absence of type I and III IFN pretreatment. Data is shown as means and standard deviation from three independent biological replicates. (E) Representative immunofluorescence staining of type I and III IFN pretreated camelid AEC cultures at 48 h post-infection. Double-stranded RNA is shown in green, ZO-I in white and DAPI in blue. Scale bar is 50 µm.