Fig. 10: Growth kinetics of MERS-CoV isolates in models of the human and camel respiratory tract.

a Replication on primary human airway epithelium (HAE) of a single donor. Two isolates of each phylogenetic lineage were used for infection in biological triplicates. Virus progeny in apical washes was quantified every 24 h by plaque assay. Statistical significance in difference of PFU/ml between lineage 5 and other lineages was determined by one-way ANOVA and two-tailed Student´s t-test for each time point individually. Statistically significant differences in PFU/ml determined by one-way ANOVA corrected for multiple comparisons using the Tukey method were found at 24 hpi (lineage 5 vs. lineage 3: *; p = 0.0274; lineage 5 vs. EMC: *; p = 0.0301); at 48 hpi (lineage 5 vs. lineage 3: *; p = 0.0248; lineage 5 vs. lineage 4: *; p = 0.0398); at 72 hpi (lineage 5 vs. lineage 3: **; p = 0.0095; lineage 5 vs. lineage 4: *; p = 0.0190) and at 96 hpi (lineage 5 vs. lineage 3: ****; p < 0.0001; lineage 5 vs. lineage 4: *; p = 0.0123; lineage 5 vs. EMC: **; p = 0.0050) b Replication of MERS-CoV isolates on ex vivo lung explants, derived from n = 3 different patients that have undergone lung resection. One isolate of each phylogenetic lineage was used for infection in biological triplicates for each explant and virus progeny in the supernatant was quantified by plaque assay. Differences in PFU/ml between lineage 5 and other lineages was tested for significance using the two-sided Kruskal–Wallis test. Statistically significant differences in PFU/ml were found at 16 hpi (lineage 5 vs. EMC: **; p = 0.0024; lineage 5 vs. lineage 4: **; p = 0.0012), at 24 hpi (lineage 5 vs. EMC: ***; p = 0.0006; lineage 5 vs. lineage 3: **; p = 0.0080; lineage 5 vs. lineage 4: ****; p < 0.0001), at 48 hpi (lineage 5 vs. EMC: **; p = 0.0064; lineage 5 vs. lineage 3: **; p = 0.0034; lineage 5 vs. lineage 4: ****; p < 0.0001) and at 72 hpi (lineage 5 vs. lineage 3: **; p = 0.0029; lineage 5 vs. lineage 4: ****; p < 0.0001) c Replication of MERS-CoV isolates on well-differentiated primary Bactrian camel airway epithelium. Two isolates of each phylogenetic lineage were used for infection in biological triplicates. Virus progeny in apical washes was quantified every 24 h by plaque assay. Statistical significance in difference of PFU/ml between lineage 5 and other lineages was determined by one-way ANOVA and two-tailed Student´s t-test for each time point individually. Statistically significant differences in PFU/ml were found at 72 hpi (lineage 5 vs. lineage 3: *; p = 0.0222; lineage 5 vs. lineage 4: *; p = 0.0266 and at 96 hpi (lineage 5 vs. lineage 3: *; p = 0.0144; lineage 5 vs. lineage 4:*; p = 0.0244). d Immunofluorescent analysis of MERS-CoV lineages replication in ex vivo human lung tissue. Explants from n = 3 different patients were infected with one MERS-CoV isolate of each lineage for 24 h. Histological sections were probed with MERS-CoV nucleocapsid antibody (green), nuclei were counterstained with DAPI (blue), and tissue structure was visualized by differential interference contrast. Scale bar represents 20 µM. e MERS-CoV replication quantified by immunofluorescent analysis. Values represent the area (in µm²) of positive-infection, determined by nucleocapsid immunofluorescent signal in n = 3 infected lung explants at 24 hpi, measured in biological duplicates. The complete piece of each lung explant was analyzed for quantification. The positive-infected area of each respective isolate was normalized to the total lung tissue area and the value obtained for the EMC strain-infected lung tissue. Statistical significance in a quantified infected area between lineage 5 and EMC was analyzed by two-tailed, unpaired t-tests. Scale bar = 20 µm. PFU, plaque forming units.