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DISPATCHES

1012

Human Infection with MERS

Coronavirus after Exposure to

Infected Camels, Saudi Arabia, 2013

Ziad A. Memish, Matthew Cotten, Benjamin Meyer, Simon J. Watson,

Victor Max Corman, Andrea Sieberg, Hatem Q. Makhdoom, Abdullah Assiri, Malaki Al Masri, Souhaib Aldabbagh,

Berend-Jan Bosch, Martin Beer, Marcel A. Müller, Paul Kellam, and Christian Drosten

cross-species transmission. Camels may act as a direct

Middle East respiratory syndrome coronavirus (MERS- from a patient who died of pneumonia in Saudi Arabia (1).

of MERS-CoV infection, most resulting in respiratory tract illness, have been reported to the World Health Organiza- tion; 97 of these cases were fatal. Known cases have been directly or indirectly linked to countries in the Arabian Pen- insula (2). Dromedary camels across and beyond the region

show high rates of antibodies against MERS-CoV (3–7), and viral RNA has been detected in camels in different countries (8,9). In 1 instance, a camel and 2 humans caring for the camel were found to be infected with viruses that were highly similar but distinct within 4,395 nt of the cam- el-derived virus sequence, including several phylogeneti- cally informative nucleotide changes (10). To investigate possible camel–human virus transmission, we analyzed an infection with MERS-CoV in a man after he had contact with an infected camel.

The Study On November 3, 2013, the Ministry of Health of Saudi

infection in a 43-year-old male patient at King Abdulaziz University Hospital in Jeddah. The patient had cared for ill camels in his herd of 9 animals starting in early October, when the patient noted respiratory signs of illness with na- sal discharge in several animals; he continued caring for the sick animals until October 27, the day of onset of his

per day 3 days per week, applying herbal remedies to the animals’ snouts and nostrils. He did not clean the stables or milk the animals, but he routinely consumed raw, unpas- teurized camel milk from the herd.

Presence of MERS-CoV RNA in the patient was con-

transcription PCR (RT-PCR) targeting the upE and orfA gene fragments (11,12). Respiratory swab specimens yielded detectable signal after 28 RT-PCR cycles, indica- tive of an approximate viral load of 350,000 RNA copies per sample. A nearly complete viral genome was obtained (Jeddah_1_2013; GenBank accession no. KJ556336), con-

- 2)

(phylogeny shown in online Technical Appendix Figure 1, wwwnc.cdc.gov/EID/article/20/6/14-0402-Techapp1.pdf).

To identify potential sources of infection, on Novem- ber 9, the Ministry of Health investigated 5 close house- hold contacts and the animal attendant on a farm owned by the patient. Nasopharyngeal swab samples were taken and tested at Jeddah Regional Laboratory by using RT-PCR. Deep nasal swab specimens were taken on the same day from 3 of the 9 camels at the farm. Testing of all samples by RT-PCR using the upE assay (11,12) did not detect MERS- CoV RNA in any of the human patients, but 1 of the 3 cam- els (camel G) tested positive (cycle threshold [Ct] = 33). On November 13, nasal swab samples were obtained from all 9 animals. upE RT-PCR results were positive for camel G (Ct = 38) and a second camel (camel B; Ct = 39).

Samples from November 13 and a small remaining amount of RNA extract from camel G from November 9 were sent to the Sanger Institute in Cambridge, UK, and

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t samples with the upE assay from camel G but not for camel

of Virology in Bonn, Germany, for the same samples by using real-time RT-PCRs targeting the upE and 1A diag- nostic target regions.

determined from 8 RT-PCR fragments, 2 of them partially overlapping (4,608 nt total) (13). Phylogenetic analyses supported the conclusion that transmission occurred be- tween camel and patient, but no direction was implied (e.g, camel to human vs. human to camel; online Technical Ap- pendix Figure 1). The human- and camel-derived sequenc- es shared a signature of single nucleotide polymorphisms that occurred in no other known MERS-CoV sequences (Figure). Single nucleotide exchanges occurred at nt posi- tions 21945 and 29662; these exchanges might have come

arisen during virus transmission, as described (10). How- ever, because of the low RNA concentration in the samples,

PCR-based mutations could not be done. A serum sample taken from camel G during the initial

investigation on November 9 was tested by recombinant 6,14) and

showed reactivity to MERS-CoV (titer 320). To investigate signs of recent MERS-CoV infection in the group of cam- els, we obtained blood samples at short intervals from all 9 animals during November 14–December 9, 2013. All sam- ples were tested by ELISA against recombinant MERS- CoV spike antigen domain S1 fused to human Fc fragment, using a formulation as described (15). Serum samples from all 9 animals showed reactivity to the MERS-CoV antigen; serum samples from control animals showed no reactivity (online Technical Appendix Figure 2, panel A).

DISPATCHES

ELISA signals were constant over time in most animals, but a small, yet visible, change of signal over the observa- tion period was noted for camels B and G. To clarify the

serum samples (obtained on November 14 and December 9) from all animals were re-tested by IFA. As summarized in the Table, camels B and G showed 4-fold increases of

tests that rely on 2-fold endpoint titrations, a titer increase >

sequential samples from camels B and G were compared with sequential samples from camels E and I by using IFA

of titers for camels B and G (online Technical Appendix Figure 2, panel B).

Because bovine CoV occurs in camels, we tested for antibodies against bovine CoV in camels B and G to ex- clude potential cross-reactions. Using IFA (6), we found no bovine CoV antibodies in camel G, but camel B showed rising bovine CoV antibody titers (Table). To obtain further differentiation, we performed neutralization assays against MERS-CoV and bovine CoV (7). Titers in serum samples from November 14 and December 9, respectively, were 160 and 320 for camel B and 160 and 160 for camel G. None of the animals showed serum neutralization against bovine CoV.

Conclusions

similarity of MERS-CoVs carried by humans and cam- els (8,10), supporting the hypothesis that human MERS- CoV infection may be acquired directly from camels. In addition, both animals that showed signs of recent in- fection were juvenile, which provides further support to

by MERS-CoV (7,8). Given the synchronized parturi- tion pattern of dromedary camels, with birthing in the

winter months, an increase of epizootic activity might

each year. Our data provide particular insight into the timing of

infections and transmission. Antibody titers rose and vi- ral RNA concentrations were already on the decline in the camels while the patient was hospitalized with acute symptoms. Assuming a time before appearance of anti- bodies of 10–21 days, at least some of the camels would have been actively infected during middle to late October, when some animals showed signs of respiratory illness and the patient acquired his infection. Nevertheless, we cannot rule out other infectious causes of the animals’ up- per respiratory signs. Also, because of the retrospective nature of this investigation, we cannot rule out the possi- bility of a third source of MERS-CoV infection for camels and humans.

The work was funded by the European Community’s Sev- enth Framework Programme (FP7/2007–2013) under the proj- ect EMPERIE, European Community grant agreement number 223498 and ANTIGONE, contract number 278976. C.D. has re- ceived infrastructural support from the German Centre for Infec- tion Research.

Dr Memish is Deputy Minister for Public Health, Ministry of Health; director of the WHO Collaborating Center for Mass Gathering Medicine; and professor at Alfaisal University College of Medicine, Riyadh, Kingdom of Saudi Arabia. His research in- terest is MERS coronavirus.

References

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Saudi Arabia, 2013*

Camel Age Anti– titers

Anti– titers

A 13 y B 3 mo 320 1,280–

C 12 y D E 13 y

8 y G† 8 mo <10 <10 H 8 mo I 2 y

†For camel G, a sample taken on November 9 was also available and yielded an immunofluorescence titer of 320.

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Addresses for correspondence: Ziad A. Memish, Ministry of Health, Riyadh 11176, Saudi Arabia; email: [email protected]; or Christian Drosten, Institute of Virology, University of Bonn Medical Centre, Sigmund Freud Str 25, 53105 Bonn, Germany; email: [email protected]