Field Investigations

Five days after the patient was admitted to the hospital, a veterinarian examined the nine camels, a procedure that was repeated for the following 27 days. During these examinations, the camels were found to be completely healthy, with no nasal discharge.

Sample Collection

Nasal swabs were obtained from the patient on hospital days 1, 4, 14, and 16. Blood samples were also collected from the patient on days 1 and 14. In addition, a nasal swab was obtained from the patient's daughter 1 day after the onset of her illness. Six days after the patient's admission, blood samples were collected from his three friends, who were asymptomatic. Nasal swabs, blood, milk, urine, and rectal swabs were collected from the nine camels 5 days after the patient's admission. Blood and nasal samples were collected again from the camels 28 days later (i.e., 33 days after the patient's hospital admission). All nasal swabs, which were immersed in viral transport medium, and other samples were transported in a cold container to the Special Infectious Agents Unit, a biosafety level 3 laboratory at King Fahd Medical Research Center, King Abdulaziz University, Jeddah, for analysis. Blood samples were centrifuged and serum samples were collected for analysis.

Cell Culture

Vero cells (ATCC CCL-81) were inoculated with 100 μl of the nasal swab medium and maintained in complete Dulbecco's Modified Eagle's Medium, as described previously.4 The cells were incubated in a humidified atmosphere at 37°C in 5% carbon dioxide and examined daily for a cytopathic effect. Cell-culture supernatants were collected when a cytopathic effect was observed and were analyzed by means of real-time RT-PCR. The MERS-CoV isolate that was generated from the first culture passage was used for whole viral genome sequencing.

Molecular Detection

RNA was extracted from the nasal swabs or culture supernatants with the use of the QIAamp Viral RNA Mini Kit (Qiagen), according to the manufacturer's instructions. Eluted RNA was screened for the MERS-CoV upstream region of the E gene (upE region) and confirmed by targeting the open reading frame region ORF1a and ORF1b with the use of real-time RT-PCR, as described previously.2 Further confirmation was performed by partially sequencing the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) regions of the viral genome, as recommended by the World Health Organization.5 Both assays were conducted on the original samples collected from the patient and from Camel B. (Details regarding the RT-PCR assay and sequencing are provided in the Supplementary Appendix.)

Viral Genome Sequencing

Viral RNA extracted from culture supernatants that had been inoculated with samples from the patient and from Camel B were subjected to RT-PCR amplification with an ABI Veriti thermal cycler (Applied Biosystems) with the use of primer pairs covering the whole length of the viral genome. The RT-PCR fragments were then sequenced, as described in the Supplementary Appendix. Sequences were deposited in GenBank and given accession numbers (KF958702 MERS-CoV-Jeddah-human-1 for the patient's isolate and KF917527 MERS-CoV-Jeddah-camel-1 for the camel's isolate). Genomic regions containing unique mutations were also partially resequenced from the original samples obtained from both the patient and the camel.

Phylogenetic Analysis

Sequences were aligned with all MERS-CoV reference strains retrieved from GenBank. Phylogenetic analysis and distance calculations were performed with the use of Molecular Evolutionary Genetics Analysis (MEGA) software, version 5, by means of the neighbor-joining method with 1000 bootstrap replicates.

Conventional Immunofluorescence Assay

Serum samples that were collected from the patient and the camels were serially diluted up to 1:51,200. A conventional immunofluorescence assay to detect anti–MERS-CoV antibodies was performed in Vero cells infected with MERS-CoV, with the use of a commercially available test kit (Anti-MERS-CoV IFT, EUROIMMUN), as described previously.2