Ali, MA

Chu, KW

El Rifay, AS

Gomaa, MM

Guan, Y

Kayali, G

Peiris, JSM

Perera, RAPM

Poon, LLM

Shehata, MM

Siu, YL

Webby, RJ

Zeid, DA

Emerging Infectious Diseases

HKU Scholars Hub

	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	20,	No.	6,	June	2014	 1049MERS Coronaviruses in Dromedary Camels, EgyptDaniel K.W. Chu,1 Leo L.M. Poon,1  Mokhtar M. Gomaa, Mahmoud M. Shehata, Ranawaka A.P.M. Perera, Dina Abu Zeid,  Amira S. El Rifay, Lewis Y. Siu, Yi Guan,  Richard J. Webby, Mohamed A. Ali,  Malik Peiris, and Ghazi KayaliWe	 identified	 the	near-full-genome	sequence	 (29,908	nt,	>99%)	of	Middle	East	respiratory	syndrome	coronavirus	(MERS-CoV)	 from	a	nasal	swab	specimen	from	a	drome-dary	camel	in	Egypt.	We	found	that	viruses	genetically	very	similar	 to	 human	 MERS-CoV	 are	 infecting	 dromedaries	beyond	 the	Arabian	Peninsula,	where	human	MERS-CoV	infections	have	not	yet	been	detected.Middle East respiratory syndrome (MERS) is a pneu-monic illness caused by a novel lineage C beta-coronavirus (CoV). During September 2012–January 20, 2014, a total of 178 confirmed cases in humans resulted in 76 deaths (1). Primary infections have originated from countries within the Arabian Peninsula, although travel-associated cases and some secondary transmission have been reported in other countries. Limited human-to-human transmission has resulted in clusters of cases, some associ-ated with multiple rounds of human-to-human transmission (2); the remaining sporadic cases in humans are presumed to be of zoonotic origin.MERS-CoV genomes are phylogenetically classified into 2 clades, clade A and B clusters (3). The viral genomes detected in the earliest cases in humans (clade A cluster; EMC/2012 and Jordan-N3/2012) are genetically distinct from the others (i.e., clade B). The proximate source of hu-man infection remains unclear. MERS-CoV is related to, but not identical to, viruses detected in bats (4,5). A short RNA fragment of the conserved viral polymerase region identical to MERS-CoV has been identified in Taphozous perforates bats, but these findings need to be confirmed (6).Serologic studies of domestic livestock in Jordan, Sau-di Arabia, Qatar, United Arab Emirates, and Egypt have found high seroprevalance to a MERS-like CoV in drom-edary camels but not in other domestic animals (7–11). An investigation of domestic animals in the vicinity of 2 persons with related infections detected fragments of viral RNA in dromedary camels in contact with these persons but whether this represented transmission from a unidenti-fied source to humans and dromedaries, transmission from humans to dromedaries, or vice versa is not clear (12).The StudyWe collected nasal swab specimens from 110 appar-ently healthy dromedaries (Camelus dromedarius) >6 years of age in abattoirs on 12 occasions during June–De-cember 2013 (Table 1). Serum was collected from 52 of these dromedaries. Serum from 179 persons working in the camel abattoirs also was collected. Median age of these workers was 38 years (range 9–67 years), 84% were male, and 25 reported comorbidities (i.e., cardiovascular, renal, diabetes, other). Collection of the human specimens was approved by the ethics committee of the National Research Centre (Giza, Egypt), and Institutional Animal Care and Use Committee approval for collection of animal samples was obtained from St Jude Children’s Research Hospital (Memphis, TN, USA).Real-time reverse transcription PCR (RT-PCR) tar-geting upstream of E gene of MERS-CoV was used for screening. The open reading frame (ORF) 1a gene was used for confirmation as recommended by the World Health Organization(www.who.int/csr/disease/coronavirus_ infections/MERS_Lab_recos_16_Sept_2013.pdf?ua=1). We also used a previously described pan-CoV nested Author	affiliations:	The	University	of	Hong	Kong,	Hong	Kong,	China	(D.K.W.	Chu,	L.L.M.	Poon,	R.A.P.M.	Perera,	Y.	Guan,	M.	Peiris);	 National	 Research	 Centre,	 Giza,	 Egypt	 (M.M.	 Gomaa,	 M.M.	 Shehata,	D.A.	Zeid,	A.S.	El	Rifay,	M.A.	Ali);	HKU-Pasteur	Research	Pole,	Hong	Kong	(L.Y.	Siu);	St.	Jude	Children’s	Research	Hospital,	Memphis,	Tennessee,	USA	(R.J.	Webby,	G.	Kayali)DOI:	http://dx.doi.org/10.3201/eid2006.140299 1These authors contributed equally to this article. Table	1.	Results	of	testing	nasal	swab	specimens	from	dromedary	camels	by	RT-PCR	for	MERS-CoV	and	for	other	CoVs,	Egypt,	2013* Location	of	animals	sampled No.	samples tested No.	MERS-CoV	positive† No.	other	CoV	positive.	(virus	identified) Alexandria‡ 17 0 0 Cairo     Abattoir 1§ 46 0 3	(BCoV)  Abattoir 2§ 10 3 0 Nile	Delta	region,	abattoir§ 37 1 5	(BCoV) *RT-PCR,	reverse	transcription	PCR;	MERS,	Middle	East	respiratory	syndrome;	CoV,	coronavirus;	BCoV,	bovine	coronavirus. Nasal	swabs	from	dromedary	camels	were	placed	in	phosphate-buffered saline/glycerol	transport	medium,	kept	on	ice	during	the	field	trip	and	stored at 80°C	on	arriving	at	the	laboratory.	Specimens	were	shipped	in	dry	ice	to	the	University	of	Hong	Kong	laboratory, where they were stored at 80°C until testing. †Taken as positive only when both upstream of E gene and open reading frame	1a	RT-PCR	were	positive. ‡Locally reared animals. §Imported from Sudan or Ethiopia.  DISPATCHES1050	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	20,	No.	6,	June	2014PCR targeting the viral RNA-dependent RNA polymerase (RdRp) region (13), and PCR products were analyzed by sequencing (online Technical Appendix, wwwnc.cdc.gov/EID/article/20/6/14-0299-Techapp1.pdf).We detected MERS-CoV RNA in 4 (3.6%) of 110 nasal swab specimens from dromedary camels with the upstream of E gene assay (cycle threshold [Ct] 23.2–36.8), confirmed by the ORF1a assay (Ct 23.2–39.1), fulfilling the World Health Organization criteria for diagnosis of MERS-CoV infection (Table 1). PCR was repeated from a fresh RNA extract to confirm positive results. One positive sample was obtained from a camel in an abattoir in the Nile Delta region in November 2013 and 3 other samples from an ab-attoir in Cairo in December 2013. Virus culture attempts in Vero E6 cells (ATCC CRL-1586) so far have been un-successful. The pan-CoV nested PCR detected CoV in an additional 8 specimens from dromedary camels. Sequence analyses of these additional positive samples showed that these amplicon sequences were genetically similar to those of bovine coronavirus (BCoV) (>99% nucleotide simi-larity). No animal was co-infected with MERS-CoV and BCoV-like viruses. The animals positive for either MERS-CoV or BCoV-like virus were all imported from Sudan or Ethiopia for slaughter.Figure	1.	Phylogenetic	analyses	of	a	partial	RNA-dependent	RNA	polymerase	(RdRp)	sequence	determined	from	samples	from	dromedary	camels	(Camelus dromedarius)	NRCE-HKU205	and	NRCE-HKU270	that	were	positive	for	Middle	East	respiratory	syndrome	coronavirus	(MERS-CoV).	The	viral	RdRp	region	analyzed	is	a	highly	conserved	region	of	the	genome	(covering	motif	B	of	RdRp)	in	nonstructural	protein	12,	at	position	15202–15582	of	MERS-CoV	genome.	The	partial	RdRp	sequence	of	NRCE-HKU205	(GenBank	accession	no.	KJ477102)	and	 NRCE-HKU270	 (GenBank	 accession	 no.	 KJ477103)	 was	 aligned	 with	 human	MERS-CoVs	 (GenBank	 accession	 nos.	 KF600652,	KF600630,	KF600651,	KF186567,	KF600627,	KF186564,	KF600634,	KF600632,	KF600644,	KF600647,	KF600645,	KF186565,	KF186566,	KF745068,	 KF600620,	 KF600612,	 KC667074,	 KC164505,	 KF192507,	 KF600613,	 KF600628,	 KF961222,	 KF961221,	 KC776174,	 and	JX869059)	 and	other	 representative	animal	 betacoroanviruses	 (GenBank	accession	nos.	HKU5–1,	EF065509;	BtCoV/PML/Neo	 cf.	 zul/RSA/2011,	KC869678).	Bat	CoV	HKU5–1	and	bat	CoV/PML/Neo	cf.	zul/RSA/2011	were	 included	 in	 the	analysis	as	outgroups	because	they	are	phylogenetically	closest	to	MERS-CoV.	Phylogenetic	trees	were	constructed	by	using	MEGA5	(14)	with	neighbor-joining	method.	Numbers	at	nodes	 indicate	bootstrap	values	determined	by	500	 replicates.	Only	bootstrap	values	>70	are	denoted.	Bold	 type	 indicates	MERS-CoV	identified	in	the	current	study.	Scale	bars	indicate	the	estimated	genetic	distance	of	these	viruses.	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	20,	No.	6,	June	2014	 1051MERS-CoV	in	Camels,	EgyptOn phylogenetic analysis, the partial RdRp sequences from MERS-CoV–positive samples NRCE-HKU205 and NRCE-HKU270 grouped within the cluster of MERS-CoV (Figure 1). The viral load in the other 2 specimens was too low to provide amplicons suitable for genetic sequencing. Viral RNA from NRCE-HKU205, the first positive specimen detected, was selected for more de-tailed genomic sequencing. We amplified viral RNA by PCR, using primers specific for overlapping regions of human MERS-CoV genome. PCR products were se-quenced and assembled to produce a near full-length genome, lacking only the 3′ untranslated region (29908 nt, >99% of the MERS-CoV genome) (online Technical Appendix). The camel MERS-CoV genome has an over-all nucleotide similarity of 99.2%−99.5% and deduced amino acid similarity of 98.0%–100% to ORFs of human MERS-CoV EMC/2012 (Table 2). The 5′ untranslated re-gion and internal transcriptional regulatory sequences of NRCE-HKU205 were identical to published human MERS-CoV sequences.Using MERS-CoV EMC/2012 as a reference sequence, we found 12 aa differences (residues 23, 26, 194, 434, 666, 696, 756, 886, 888, 918, 1158, and 1333 of EMC/2012) in the spike protein of dromedary MERS-CoV NRCE-HKU205 and 1 aa deletion (residue 1293 of EMC/2012) in the N terminal of the transmembrane domain. NRCE-HKU270 virus spike does not have a deletion of residue 1293. Of these, only residue 434 falls within the proposed receptor binding domain of spike protein, but it is located at the core (stem) subdomain of the receptor binding do-main, suggesting that the camel MERS-CoV is still likely to bind human CD26 (online Technical Appendix Figure). The biological impact of this difference and other amino acid differences needs to be fully explored.We used phylogenetic analyses of the full genome, the spike gene, and nucleocapsid gene of MERS-CoV NRCE-HKU205 to compare with the same genes of other human MERS-CoVs (Figure 2). NRCE-HKU205 is within the clade A group but distinct from MERS-CoV EMC/2012, the only other MERS-CoV isolate available in our laboratory, excluding laboratory cross-contamina-tion as an explanation for the detection of MERS-CoV from the dromedary specimens.Using a previously described pseudoparticle neutral-ization assay (8), we detected antibodies against MERS-CoV (titer >20) in 48 (92.3%) of 52 dromedary serum samples with titers ranging from 20 to >640. Dromedary NRCE-HKU205 had a serum antibody titer of 640, possi-bly indicating a developing serologic response, as was not-ed previously (7), or the possibility of re-infection. Serum was not available from the other 3 animals with MERS-CoV RNA-positive nasal swab specimens.Serum from 179 persons working in the dromedary ab-attoirs was negative for antibody to MERS-CoV. This find-ing includes 114 persons working in the 2 abattoirs from which the MERS-CoV–positive animal swab specimens were obtained.ConclusionsOur findings confirm that MERS-CoV infects drom-edary camels and that this virus is genetically very similar to a MERS-CoV that is infecting humans. The detection of MERS-CoV in nasal swab specimens of camels in 2 of 12 sampling occasions in abattoirs, taken together with the high seropositivity to MERS-CoV in dromedaries previ-ously reported, supports the contention that MERS-CoV infection is common in dromedaries. Studies of drom-edaries within camel herds and through the animal mar-keting system supplying abattoirs are needed to define the epidemiology of the infection. Our findings strengthen the plausibility that dromedaries may be a potential source of human infection and emphasize the need for detailed epide-miologic investigation of the exposure histories of humans with MERS. However, the lack of serologic evidence of in-fection of humans working in these abattoirs suggests that transmission of this virus to humans is uncommon. The de-tection of MERS-CoV in dromedaries in Egypt, in animals imported from Sudan and Ethiopia, suggests that cases may occur in humans beyond the Arabian Peninsula. MERS CoV diagnostic tests should be considered for all patients with unexplained severe pneumonia in Egypt, northeastern Africa, and beyond.This research was supported by an internal research grant from the National Research Centre, Giza, Egypt; by a research contract from the National Institute of Allergy and Infectious Diseases con-tract HHSN266200700005C; and a grant from the European Com-munity Seventh Framework Program (FP7/2007-2013) under proj-ect European management Platform for Emerging and Re-emerging Disease entities (grant agreement no. 223498) (EMPERIE). Table	2.	Percentage	identity	between	ORFs	of	dromedary	camel	MERS-CoV	(NRCE-HKU205)	and	human	MERS-CoV	(EMC/2012)	at	the	nucleotide	and	amino	acid	levels* ORF % Identity	to	HCoV-EMC/2012 Nucleotide Amino acid ORF1a 99.5 99.2 ORF1b 99.5 99.7 S 99.2 98.9 ORF3 99.0 98.0 ORF4a 99.0 100 ORF4b 99.4 99.1 ORF5 99.4 98.6 E 100 100 M 100 100 N 99.4 99.2 ORF8b 99.1 98.2 *ORF,	open	reading	frame;	MERS,	Middle	East	respiratory	syndrome;	CoV,	coronavirus;	H,	human; E,	envelope;	M,	membrane;	N,	nucleocapsid.  DISPATCHES1052	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	20,	No.	6,	June	2014Dr Chu is a postdoctoral fellow at the Centre of Influenza Research at the University of Hong Kong. His primary research interests are virus ecology. evolution of coronavirus, astroviruses, and zoonotic infections.References  1. World Health Organization. Middle East respiratory syndrome coronavirus (MERS-CoV) summary and literature update—as of 20 January 2014 [cited 2014 Feb 20]. http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_20_Jan_2014.pdf  2. Assiri A, McGeer A, Perl TM, Price CS, Al-Rabeeah AA, Cummings DAT, et al. Hospital outbreak of Middle East respira-tory syndrome coronavirus. N Engl J Med. 2013;369:407–16. http://dx.doi.org/10.1056/NEJMoa1306742  3. Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, et al. Transmission and evolution of the Middle East respi-ratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study. Lancet. 2013;382:1993–2002. http://dx.doi.org/10.1016/S0140-6736(13)61887-5  4. Annan A, Baldwin HJ, Corman VM, Klose SM, Owusu M, Nkrumah EE, et al. Human betacoronavirus 2c EMC/2012–related viruses in bats, Ghana and Europe. Emerg Infect Dis. 2013;19:456–9. http://dx.doi.org/10.3201/eid1903.121503Figure	 2.	 Phylogenetic	 analyses	of	 Middle	 East	 respiratory	syndrome	 coronavirus	 (MERS-CoV)	 from	 dromedary	 camels.	Genomic	 (A),	 spike	 (B),	 and	nucleocapsid	 (C)	 sequences	 of	the	 dromedary	 camel	 MERS-CoV	 NRCE-HKU205	 (GenBank	accession	 no.	 KJ477102)	 were	aligned with the corresponding human	 MERS-CoV	 (N	 =	 25)	sequences	 retrieved	 from	GenBank	 (accession	 nos.	 as	 in	Figure	 1	 legend).	 Phylogenetic	trees were constructed by using MEGA5	 (14)	 with	 neighbor-joining	 method.	 Numbers	 at	nodes	 indicate	 bootstrap	 values	determined	 by	 500	 replicates.	Only	 bootstrap	 values	 >70	 are	denoted. Bold type indicates MERS-CoV	 identified	 in	 the	current study. Scale bars indicate the estimated genetic distance of these	viruses.	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	20,	No.	6,	June	2014	 1053MERS-CoV	in	Camels,	Egypt  5. Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards LR, Schoeman MC, et al. Close relative of human Middle East respira-tory syndrome coronavirus in bat, South Africa. Emerg Infect Dis. 2013;19:1697–9. http://dx.doi.org/10.3201/eid1910.130946  6. Memish ZA, Mishra N, Olival KJ, Fagbo SF, Kapoor V, Epstein JH, et al. Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg Infect Dis. 2013;19:1819–23. http://dx.doi.org/10.3201/eid1911.131172  7. Reusken CBEM, Haagmans BL, Müller MA, Gutierrez C, Godeke G-J, Meyer B, et al. Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect Dis. 2013;13:859–66. http://dx.doi.org/10.1016/S1473-3099(13)70164-6  8. Perera RA, Wang P, Gomaa MR, El-Shesheny R, Kandeil A, Bagato O, et al. Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013. Euro Surveill. 2013;18: 20574.  9. Reusken CB, Ababneh M, Raj VS, Meyer B, Eljarah A, Abutarbush S, et al. Middle East respiratory syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, June to September 2013. Euro Surveill. 2013;18:20662.10. Hemida MG, Perera RA, Wang P, Alhammadi MA, Siu LY, Li M, et al. Middle East respiratory syndrome (MERS) coronavirus serop-revalence in domestic livestock in Saudi Arabia, 2010 to 2013. Euro Surveill. 2013;18:20659.11. Meyer B, Müller MA, Corman VM, Reusken CBEM, Ritz D, Godeke G-D, et al. Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013. Emerg Infect Dis [Internet]. 2014 Apr [cited 2014 Feb 22]. http://dx.doi.org/10.3201/eid2004.131746 12. Haagmans BL, Al Dhahiry SH, Reusken CB, Raj VS, Galiano M, Myers R, et al. Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. Lancet Infect Dis. 2014;14:140–5. http://dx.doi.org/10.1016/S1473-3099(13)70690-X13. Chu DK, Leung CY, Gilbert M, Joyner PH, Ng EM, Tse TM, et al. Avian coronavirus in wild aquatic birds. J Virol. 2011;85:12815–20. http://dx.doi.org/10.1128/JVI.05838-1114. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using maxi-mum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9. http://dx.doi.org/10.1093/molbev/msr121Addresses for correspondence: Malik Peiris, School of Public Health, The University of Hong Kong, 21 Sassoon Rd, Pokfulam, Hong Kong Special Administrative Region, China; email: malik@hku.hk; Ghazi Kayali, Division of Virology, Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA; email: ghazi.kayali@stjude.org

MERS coronaviruses in dromedary camels, Egypt

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MERS coronaviruses in dromedary camels, Egypt

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