Tropism and replication of Middle East respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study

Abstract

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection causing severe viral pneumonia, with index cases having resided in or recently travelled to the Arabian peninsula, and is a global concern for public health. Limited human-to-human transmission, leading to some case clusters, has been reported. MERS-CoV has been reported in dromedary camels but phenotypic characterisation of such viruses is limited. We aimed to compare MERS-CoV isolates from dromedaries in Saudi Arabia and Egypt with a prototype human MERS-CoV to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung.

Methods: We characterised MERS-CoV viruses from dromedaries in Saudi Arabia and Egypt and compared them with a human MERS-CoV reference strain. We assessed viral replication kinetics and competence in Vero-E6 cells (rhesus monkey), tissue tropism in cultures of ex-vivo human bronchial and lung tissues, and cytokine and chemokine induction, gene expression, and quantification of viral RNA in Calu-3 cells (human respiratory tract). We used mock-infected tissue as negative controls for ex-vivo experiments and influenza A H5N1 as a positive control for cytokine and chemokine induction experiments in Calu-3 cells.

Findings: We isolated three dromedary strains, two from Saudi Arabia (Dromedary/Al-Hasa-KFU-HKU13/2013 [AH13] and Dromedary/Al-Hasa-KFU-HKU19D/2013 [AH19D]), and one from Egypt (Dromedary/Egypt-NRCE-HKU270/2013 [NRCE-HKU270]). The human and dromedary MERS-CoV strains had similar viral replication competence in Vero-E6 cells and respiratory tropism in ex-vivo cultures of the human respiratory tract, and had similar ability to evade interferon responses in the human-respiratory-tract-derived cell line Calu-3.

Interpretation: The similarity of virus tropism and replication competence of human and dromedary MERS-CoV from the Arabian peninsula, and genetically diverse dromedary viruses from Egypt, in ex-vivo cultures of the human respiratory tract suggests that dromedary viruses from Saudi Arabia and Egypt are probably infectious to human beings. Exposure to zoonotic MERS-CoV is probably occurring in a wider geographical region beyond the Arabian peninsula.

Funding: King Faisal University, Egyptian National Research Centre, Hong Kong Food and Health Bureau, National Institute of Allergy and Infectious Diseases, and European Community Seventh Framework Program.

Figure 1

Phylogenetic tree of MERS-CoV full genome sequences Virus isolates used for this study are in bold. The tree was built by the maximum likelihood method in Mega 6.06 with 500 bootstrap replications. Bootstrap values are shown at the major nodes of the tree. The scale bar shows the mutation rate for the tree. Accession numbers of virus genomes retrieved for this analysis are JX869059, KC776174, KC164505, KC667074, KF192507, KJ156881, KJ156949, KJ156944, KJ556336, KJ156952, KF600613, KF186564, KF600627, KF186567, KF600651, KJ156866, KF600634, KF600632, KF600644, KF186565, KF186566, KF600645, KF600647, KF600630, KF600652, KF745068, KJ156869, KJ650297, KJ650296, KJ650295, KF600628, KF961221, KF961222, KJ156874, KJ156910, KJ156934, KF600612, KF600620, and KJ477102. MERS-CoV=Middle East respiratory syndrome coronavirus. EMC=human MERS-CoV EMC. AH13=MERS-CoV Dromedary/Al-Hasa-KFU-HKU13/2013. AH19D=MERS-CoV Dromedary/Al-Hasa-KFU-HKU19D/2013. NRCE-HKU270=MERS-CoV Dromedary/Egypt-NRCE-HKU270/2013.

Figure 2

Replication kinetics of the human and camel MERS-CoV strains in Vero-E6 cells Vero-E6 cells were infected with the indicated viruses at multiplicity of infections of 0·01 and 2 and cultured at 37°C for 72 h. Culture supernatants were harvested at the indicated times, and virus titres were measured by TCID₅₀ assay. Results are presented as individual replicate (mean [SEM] for the three experiments). MOI=multiplicity of infection. TCID₅₀=50% tissue culture infective dose.

Figure 3

Tissue tropism of human and camel MERS-CoV in human respiratory tract tissues Figure shows ex-vivo cultures of formalin-fixed paraffin-embedded sections of bronchus and lung. Immunohistochemical staining of MERS-CoV N protein (stained by vector red in pink) in the human bronchus and lung cultures were determined at 24 and 72 h post infection. (A) Mock virus. (B) Human MERS-CoV EMC. (C) MERS-CoV Dromedary/Al-Hasa-KFU-HKU13/2013 (AH13). (D) MERS-CoV Dromedary/Al-Hasa-KFU-HKU19D/2013 (AH19D). (E) MERS-CoV Dromedary/Egypt-NRCE-HKU270/2013 (NRCE-HKU270). MERS-CoV=Middle East respiratory syndrome coronavirus. hpi=hours post infection.

Figure 4

Replication kinetics of human and camel MERS-CoV in ex-vivo cultures of human bronchus and lung (A) Thermal inactivation of MERS-CoV at 37°C. MERS-CoV strains were all inactivated at 24 h post incubation, irrespective of initial concentration; with the exception of the highest virus input of NRCE-HKU270. Replication curves of MERS-CoV infection of human bronchial (B) and lung (C) tissues, by individual tissue donor. The horizontal dotted line denotes the limit of detection in the TCID₅₀ assay. MERS-CoV=Middle East respiratory syndrome coronavirus. EMC=human MERS-CoV EMC. AH13=MERS-CoV Dromedary/Al-Hasa-KFU-HKU13/2013. AH19D=MERS-CoV Dromedary/Al-Hasa-KFU-HKU19D/2013. NRCE-HKU270=MERS-CoV Dromedary/Egypt-NRCE-HKU270/2013. TCID₅₀=50% tissue culture infective dose.

Figure 5

Cellular localisation of human and camel MERS-CoV in bronchus and lung tissue MERS-CoV was stained with vector red (red) and cell markers conjugated with FITC (green) for detection of MUC5AC (goblet cell marker; A) and β-tubulin (ciliated cell marker; B) in bronchial tissue and human cytokeratin clone AE1/AE3 (epithelial cell marker; C), prosurfactant protein C (type II pneumocyte marker; D) and CD68 (macrophage marker; E), in lung tissue at 72 h post infection. Arrows show cells co-stained with both viral nucleoprotein and prosurfactant C. MERS-CoV=Middle East respiratory syndrome coronavirus. EMC=human MERS-CoV EMC. AH13=MERS-CoV Dromedary/Al-Hasa-KFU-HKU13/2013. AH19D=MERS-CoV Dromedary/Al-Hasa-KFU-HKU19D/2013. NRCE-HKU270=MERS-CoV Dromedary/Egypt-NRCE-HKU270/2013.

Figure 6

MERS-CoV cytokine responses in Calu-3 cells Calu-3 cells were infected with MERS-CoV strains and H5N1 at multiplicity of infection of 2 for 1 h at 37°C. Figure shows gene expression of TNFα (A), CXCL10 (B), interferon β (C), and interleukin 6 (D) at 6, 24, and 30 h post infection. Means and SEMs of the mRNA copies expressed per 10⁵ β-actin are shown. The concentration of the cytokine and chemokine in the culture supernatant collected at 30 h post infection were further examined using (E) ELISA and (F) cytometric bead array. The bar chart represents the means and SEMs of the detected protein concentrations. UD represents samples with undetectable concentration, or concentration of the target protein below the detection limit of the ELISA or cytometric bead array kit. p values for significant differences between human and dromedary MERS-CoV are shown. MERS-CoV=Middle East respiratory syndrome coronavirus. EMC=human MERS-CoV EMC. AH13=MERS-CoV Dromedary/Al-Hasa-KFU-HKU13/2013. NRCE-HKU270=MERS-CoV Dromedary/Egypt-NRCE-HKU270/2013. TNFα=tumour necrosis factor α. IL-6=interleukin 6. IFNβ=interferon β. SEM=standard error of the mean.