MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity

Chu, Daniel K. W.; Hui, Kenrie P. Y.; Perera, Ranawaka A. P. M.; Miguel, Eve; Niemeyer, Daniela; Zhao, Jincun; Channappanavar, Rudragouda; Dudas, Gytis; Oladipo, Jamiu O.; Traoré, Amadou; Fassi-Fihri, Ouafaa; Ali, Abraham; Demissié, Getnet F.; Muth, Doreen; Chan, Michael C. W.; Nicholls, John M.; Meyerholz, David K.; Kuranga, Sulyman A.; Mamo, Gezahegne; Zhou, Ziqi; So, Ray T. Y.; Hemida, Maged G.; Webby, Richard J.; Roger, Francois; Rambaut, Andrew; Poon, Leo L. M.; Perlman, Stanley; Drosten, Christian; Chevalier, Veronique; Peiris, Malik

A genetic feature observed in BF785 and Nig1657 was the deletion in ORF4b. ORF4b gives rise to a gene product that is reportedly associated with evasion of host cell IFN defense mechanisms (10, 11) , and these deletions would be expected to impact ORF4b function. We therefore compared innate immune responses elicited in Calu-3 cells by infection with EMC, AH13, BF785 (which has most of its ORF4b gene deleted), and Nig1657 (which has ORF4b partially deleted as well as an ORF3 deletion) viruses after infection at an MOI of 2 (Fig. 3) . A highly pathogenic avian influenza virus, A/HK/483/1995(H5N1), was used as a positive control of cytokine and IFN-stimulated gene (ISG) expression. UpE gene expression in MERS-CoV-infected cells confirmed that BF785 and Nig1657 had lower replication than EMC and AH13 (Fig. 3A) . IFN-β, IFN-λ1/IL-29, IP-10 (CXCL10), ISG15, and Mx1 mRNA expression was strongly up-regulated as early as 24 h post infection in H5N1-infected cells, at which time there was minimal induction of these genes in MERS-CoV-infected cells (Fig. 3 B-G) . By 48 h after infection IFN-β, IFN-λ1/IL-29, IP10 (CXCL-10), ISG15, MX1, and TNF-α mRNA was detectable in cells infected with all (A) Calu-3 cells were infected at an MOI of 0.01, and virus titers in culture supernatants were determined by the 50% tissue culture infective dose per milliliter (TCID 50 ) assay. The horizontal dotted line denotes the limit of detection in the TCID 50 assay. Statistical significance was determined by two-way ANOVA with Bonferroni's correction: *P < 0.05; **P < 0.01; ***P < 0.001 compared with EMC; ###, P < 0.001 compared with AH13; +, P < 0.05; +++, P < 0.001 compared with BF785. Each experiment was repeated once, and one representative experiment is shown. (B) Ad5-hDPP4-transduced C57BL/ 6 mice (7 wk old, female) were infected intranasally with 1 × 10 4 pfu of MERS-CoV strains EMC, AH13, or BF785. Virus titers in the lungs were measured. The horizontal dotted line denotes the limit of detection. Titers are expressed as pfu per gram of tissue. n = 3 mice per group at each time point. Statistical significance was determined by two-way ANOVA with Bonferroni's comparisons: ***P < 0.001 compared with EMC; ###, P < 0.001 compared with AH13. (C and D) Comparison of virus replication and tropism of MERS-CoV EMC, BF785, and Nig1657 in ex vivo cultures of human bronchus (C) and lung (D). Virus titers in culture supernatants were determined by TCID 50 assay. Data are the means and SEM of three individual tissue donors. The horizontal dotted line denotes the limit of detection in the TCID 50 assay. Statistical significance was determined by two-way ANOVA with Bonferroni's comparisons: *P < 0.05; **P < 0.01 compared with EMC.