MERS-CoV (strain HCoV-EMC/2012) was provided by the Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands. The virus was propagated in Vero E6 cells cultured in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum, 2 mmol/L glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin.

Three native-born adult male dromedary camels (Camelus dromedarius) were obtained through private sale; the animals tested negative by neutralization assay for MERS-CoV and for bovine coronavirus by ELISA. Camels 1, 2, and 3 were 2, 3, and 5 years old, respectively. Camels 1 and 2 were intact males, and camel 3 had been castrated. Animals were housed in an Animal Biosafety Level 3 facility for the duration of the experiment and fed ad libitum. Camels were acclimated to the facility for 2 weeks before virus inoculation. We sedated the camels with xylazine, then inoculated them with a total dose of 10⁷ 50% tissue culture infective dose (TCID₅₀) of MERS-CoV (strain HCoV-EMC/2012) in a total volume of 15 mL, by way of intratracheal (8 mL using transcutaneous catheter), intranasal (3.3 mL in each nostril by expulsion from a syringe), and conjunctival (0.2 mL in each conjunctival sac) routes. The routes of inoculation and infectious dose were chosen to reflect a combination of most likely routes of exposure and to increase the potential of infection. The animals were observed at least 1× daily for the duration of the experiment for behavior, food consumption, activity level, and nasal discharge. Rectal temperature was taken daily from 2 to 7 days postinoculation, then 3× weekly until the animals were euthanized. Nasal and oral swab samples and fecal samples were collected into virus transport medium or virus lysis buffer daily from 0 to 7 days postinoculation (dpi), then 3× weekly until the animal was euthanized. Blood was collected into evacuated EDTA and serum-separating tubes daily at 0–7 dpi and 3× weekly thereafter. Urine was collected by convenience and at necropsy. To evaluate whether virus is exhaled from infected camels, a funnel was placed over the muzzle of each camel and connected to a vacuum pump to capture exhaled air in tissue culture media (10 mL Dulbecco modified Eagle medium, 1% fetal bovine serum, 0.013% SE-15 (anti-foam) with an All Glass Impinger (Ace Glass Inc., Vineland, NJ, USA). Exhaled breath was collected for ≈2 minutes and analyzed by quantitative real-time PCR (qPCR) and virus titration. On days 5, 28, and 42, camels 1, 2, and 3, respectively, were euthanized, and samples were collected from nasal turbinates, lungs, trachea, larynx, pharynx, liver, spleen, kidney, bladder, urine, duodenum, jejunum, colon, rectum, abomasum, forestomachs, prescapular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, mesenteric lymph node, medulla, and olfactory cortex.

We extracted RNA from swab samples, fecal samples, and serum samples using the QIAamp Viral RNA kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. For detection of viral RNA, we used 5 μL of RNA in a one-step real-time reverse transcription PCR upE assay (24) using the Rotor-GeneTM probe kit (QIAGEN) according to manufacturer’s instructions. Standard dilutions of a titered virus stock were run in parallel, to calculate TCID₅₀ equivalents in the samples (25).

We titrated swab samples in viral transport medium, whole blood, and homogenized tissues (≈10% wt/vol) for MERS-CoV virus by plaque assay. Briefly, 10-fold serial dilutions of samples were prepared in BA-1 medium (MEM, 1% bovine serum albumin, 350 mg/L sodium bicarbonate, 50 mM Tris, pH 7.6, 5 mg/L phenol red) containing 100 mg gentamicin, 200,000 U penicillin G, 100 mg streptomycin, and 5 mg amphotericin/L; plaque assay was conducted as described for West Nile virus (26). Plaques were counted on days 1 and 3 after the second overlay and virus titers were expressed as PFUs per mL or gram. We determined neutralizing antibody titers by plaque reduction neutralization test as described (26), using a 90% neutralization cutoff.

Tissues were fixed for >7 days in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. To detect MERS-CoV antigen, we completed immunohistochemical testing using a rabbit polyclonal antiserum against HCoV-EMC/2012 (1:1,000) as a primary antibody.

Each camel showed minor clinical signs of disease, consisting of rhinorrhea (Figure 1, panel A) and a mild elevation in body temperature at 2 dpi and 5–6 dpi (Figure 1, panel B); no other clinical signs were observed. Rhinorrhea developed in all 3 camels beginning at 2 (camels 1 and 3) and 5 (camel 2) dpi, and persisted <2 weeks. The nasal discharge drained from both nares and varied in character from serous to purulent; minor hemorrhage was observed on some occasions, but may have been caused by trauma that occurred during collection of samples.

MERS-CoV shedding started during 1–2 dpi, as detected by the presence of infectious virus and viral RNA by qPCR in nasal swab samples. Infectious virus shedding was detected <7 dpi, and shedding of viral RNA was detected <35 dpi in nasal swab samples (Figure 2). Low concentrations of infectious virus and viral RNA were detected in oral samples, likely originating in drainage from the nasal cavity (Figure 3). No viral RNA was detected in fecal samples or in urine samples collected by convenience or at necropsy at 0, 1, 5, 14, 21, 28, and 42 dpi from the 3 camels. No infectious virus or viral RNA was detected in any of the serum or whole blood samples. Small quantities of MERS-CoV RNA were detected in exhaled breath by qPCR (10^1.2 and 10^1.4 TCID₅₀ equivalent/mL) at 3 and 5 dpi, but infectious virus was not detected.

Infectious virus was detected in tissues from camel 1, which was euthanized on 5 dpi, but not in tissues obtained from camels 2 and 3, which were euthanized at28 and 42 dpi, respectively. Infectious virus was detected in tissues of the upper respiratory tract (URT), including nasal turbinates, olfactory epithelium, pharynx, and larynx. In the lower respiratory tract, infectious virus was detected in the trachea and in 1 of 4 lung lobes tested. Infectious virus was also detected in the retropharyngeal, mediastinal, mesenteric, and tracheobronchial lymph nodes (Figure 4). On necropsy of camel 1 at 5 dpi, histologic lesions were found in the pseudostratified epithelial cells in the URT and the lower respiratory tract (trachea, bronchi, and bronchioles) but not in the alveoli (Figure 5). The lesions were characterized as mild to moderate acute intraepithelial and submucosal inflammation with multifocal necrosis and loss of pseudostratified epithelial cells, comparable to the common cold among humans. Multifocal loss of epithelial polarity and cilia with squamous metaplasia were observed. The epithelium was infiltrated by small-to-moderate numbers of neutrophils with fewer macrophages; similar inflammatory cells also permeated the submucosa. The submucosal glands of the trachea were multifocally necrotic and infiltrated by small numbers of neutrophils. Viral antigen was detected within the epithelial cells of the nasal turbinates, larynx, trachea, bronchi, and bronchioles, but not the alveoli. In addition, viral antigen was present at the follicular mantle zone of the tonsils and mediastinal and retropharyncheal lymph nodes (Figure 5). The nasal turbinates, larynx, and trachea of camel 2 (necropsied at 28 dpi) had similar but milder lesions when compared with those of camel 1. The nasal turbinate, larynx, and bronchus showed small numbers of infiltrating neutrophils; however, in contrast with the condition of camel 1, the cilia and goblet cells were intact. The remainder of the respiratory tract of camel 2 was unaffected. Immunohistochemical testing revealed the presence of limited viral antigen in the nasal turbinate but not in any of the other tissues at that time. No lesions or viral antigens were detected in camel 3 at 42 dpi.

Serum samples were collected weekly from the camels to monitor the generation of neutralizing antibodies specific to MERS CoV. Each of the 3 camels was seronegative before inoculation. Robust MERS-CoV specific antibody responses developed in camels 2 and 3 (euthanized on 28 and 42 dpi, respectively), detected first on 14 dpi with a plaque-reduction neutralization test titer from 20 to 40 that increased to 640 at 35 dpi (Table). Camel 1 was euthanized at 5 dpi and was not tested for development of antibodies against the virus.