Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential

Abstract

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Keywords: Africa; MERS-CoV; characterization; coronaviruses; phenotype.

Fig. 1.

Phylogenetic relationships of the clade A, B, and C MERS-CoVs used in the investigation. Full genome sequences of human MERS-CoV strain (EMC) and camel MERS-CoV strains (AH13, C270, Mor213, BF785, and Nig1657) used in the investigation marked with asterisks are shown to illustrate clade designations and phylogenetic relationships. The tree was constructed by the maximum likelihood method using PhyML. Scale bar indicates the pairwise nucleotide substitutions per site. Taxa labeled with blue and red represent MERS-CoV sequences from human and camels, respectively. ORF3 and ORF4b deletions in the virus genomes are indicated as green and purple boxes, respectively, and more details of the individual deletions are shown in SI Appendix, Table S2. The virus clade designations are denoted.

Fig. 2.

Comparison of virus replication kinetics in ex vivo cultures of human bronchial and lung tissues. Viral replication kinetics of African viruses BF785 (clade C1.1), CAC9690 (clade C2), and CAC10200 (clade C2) are compared with EMC (clade A) and AH13 (clade B). The bar chart shows the virus titers (TCID₅₀) of culture supernatants at 1, 24, 48, and 72 h postinfection in ex vivo cultures of human bronchus and lung tissues infected with each virus with 10⁶ TCID₅₀. Data are shown as mean and SD of viral titers from three independent tissue donors. The horizontal dotted line denotes the limit of detection of virus TCID₅₀ assay. Statistical significance was determined by two-way ANOVA. Analysis of the 24- to 72-h data are shown on the AUC chart for the bronchus and lung tissues of each virus, and statistical analysis was done using one-way ANOVA. Bonferroni’s comparisons were performed. Statistical significance is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Comparison of virus replication kinetics in the lung of hDPP4 knockin mice infected by different MERS-CoVs from Saudi Arabia, Burkina Faso, Ethiopia, Kenya, Nigeria, Morocco, and Egypt. Five (A and B) or three (C and D) mice per group (6 to 10 wk old, female) were infected intranasally with 10⁴ PFU of the respective MERS-CoV strains. Virus titers in the lungs were measured at day 3 (A and C) and 5 (B and D) postinfection. Titers are expressed as TCID₅₀ per milliliter of tissue homogenate. MERS-CoV titers in the lung of mice infected with viruses from Burkina Faso BF785 (clade C1.1), Kenya CAC10200 (clade C2), Ethiopia CAC9600 (clade C2), or Saudi Arabia EMC (clade A) and AH13 (clade B) were compared at day 3 (A) or day 5 (B) postinfection. MERS-CoV strains from Burkina Faso BF785, Nigeria Nig1657 (clade C1.1), Egypt C270 (clade C1.2), and two viruses from Saudi Arabia EMC (clade A) and AH13 (clade B) were compared at day 3 (C) or 5 (D) postinfection. Means and SE of means are shown. Statistical significance was determined by comparing the results of EMC with each of the other viruses using Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

MERS-CoV spike gene contributes to lower replication competence in clade C MERS-CoVs. (A) Effect of S protein substitutions of BF785 and Nig1657 on cell entry in Calu-3 cells. Calu-3 cells were infected with 2.5 and 5 ng p24 of pseudoparticles carrying S of EMC/2012, BF785, Nig1657, or empty vector. At 66 h postinfection, cells were lysed and cell entry was measured by luciferase activity. Means and SD of representative experiments were presented. Each experiment was repeated six times. (B) Calu-3 cells were infected at a MOI of 0.01 of rgEMC/2012 and rgBF785-S. Culture supernatants were collected at 1, 24, 48, and 72 h postinfection. Virus titers of the supernatants were determined by the TCID₅₀ assay. Means and SEM of three independent experiments (n = 3) are shown. (C) Human DPP4 knockin mice were infected intranasally with 10⁴ PFU of the recombinant viruses. Virus titers in the lungs of mice at 1, 3, and 5 d postinfection were measured by the TCID₅₀ assay. The virus titers at each time point represent the mean (SE) from four mice. (D) Calu-3 cells were infected with the rgEMC/2012 and rgBF785-S at a MOI of 5 for 16 h. At 16 h postinfection, the cells were fixed and immunolabeled for nucleoprotein in green and stained for nuclei (DAPI) in blue. Percentages of infected cells were counted. Results are shown as percentages of relative infection (means ± SD) compared with rgEMC/2012 from three independent experiments (n = 3). (E) Huh-7 cells were transfected with plasmids to express S proteins of MERS-CoV BF785 or EMC/2012. After 20 h postinfection, the cells were fixed and immunolabeled for S proteins in green, and nuclei were stained with DAPI (blue). Nine syncytia were randomly selected for examination from three independent experiments (n = 3) and syncytial sizes quantified using Metamorph software. The size of the syncytia are shown as average values of the nuclei/syncytium ratio. Error bars indicate SD. Statistical significance was determined by Student’s t test and virus titers were log10 transformed in analysis. **P < 0.01; ***P < 0.001.