Published online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 1 Articles Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels a comparative serological study Chantal B E M Reusken Bart L Haagmans Marcel A M ller Carlos Gutierrez Gert Jan Godeke Benjamin Meyer Doreen Muth V Stalin Raj Laura Smits De Vries Victor M Corman Jan Felix Drexler Saskia L Smits Yasmin E El Tahir Rita De Sousa Janko van Beek Norbert Nowotny Kees van Maanen Ezequiel Hidalgo Hermoso Berend Jan Bosch Peter Rottier Albert Osterhaus Christian Gort zar Schmidt Christian Drosten Marion P G Koopmans Summary Background A new betacoronavirus Middle East respiratory syndrome coronavirus MERS CoV has been identifi ed in patients with severe acute respiratory infection Although related viruses infect bats molecular clock analyses have been unable to identify direct ancestors of MERS CoV Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats We investigated possible animal reservoirs of MERS CoV by assessing specifi c serum antibodies in livestock Methods We took sera from animals in the Middle East Oman and from elsewhere Spain Netherlands Chile Cattle n 80 sheep n 40 goats n 40 dromedary camels n 155 and various other camelid species n 34 were tested for specifi c serum IgG by protein microarray using the receptor binding S1 subunits of spike proteins of MERS CoV severe acute respiratory syndrome coronavirus and human coronavirus OC43 Results were confi rmed by virus neutralisation tests for MERS CoV and bovine coronavirus Findings 50 of 50 100 sera from Omani camels and 15 of 105 14 from Spanish camels had protein specifi c antibodies against MERS CoV spike Sera from European sheep goats cattle and other camelids had no such antibodies MERS CoV neutralising antibody titres varied between 1 320 and 1 2560 for the Omani camel sera and between 1 20 and 1 320 for the Spanish camel sera There was no evidence for cross neutralisation by bovine coronavirus antibodies Interpretation MERS CoV or a related virus has infected camel populations Both titres and seroprevalences in sera from di erent locations in Oman suggest widespread infection Funding European Union European Centre For Disease Prevention and Control Deutsche Forschungsgemeinschaft Introduction In 2012 a new betacoronavirus Middle East respiratory syndrome coronavirus MERS CoV was identifi ed in patients with severe respiratory disease in the Middle East As of Aug 2 2013 94 laboratory confi rmed cases including 46 deaths have been reported to WHO 1 Illness associated with MERS CoV infection is characterised primarily by mild to severe respiratory complaints most requiring hospital admission for acute respiratory distress syndrome Comorbidities and immuno suppression seem to predispose for infection and severe disease 2 6 and unpublished serological studies suggest that asymptomatic infections occur 7 All cases reported so far have been linked to Jordan Qatar Saudi Arabia and United Arab Emirates Human to human transmission has been reported particularly in health care settings but on the basis of available evidence the basic reproduction number R 0 is thought to be low suggesting that the virus is not transmitted readily 6 8 Therefore the primary reservoir of MERS CoV is probably animals Di erent coronaviruses have various hosts including wildlife livestock poultry pets and human beings Coronaviruses can adapt to new host species as shown by the zoonotic origin of several human coronaviruses 9 Human coronavirus OC43 has recent common ancestry with bovine coronaviruses 10 Rhinolophid bats were identifi ed as a likely reservoir for severe acute respiratory syndrome coronavirus SARS CoV which emerged in people in 2002 03 through intermediate carnivorous hosts 11 Molecular clock analysis 12 showed that bat and civet strains of viruses closely related to SARS CoV only diverged a few years before the outbreak Human coronavirus 229E has a common ancestor with coronaviruses found in Ghanaian Hipposideros spp bats 13 MERS CoV is able to replicate in various bat cell lines 14 and phylogenetic analyses show that it is closely related to betacoronavirus lineage C viruses from Pipistrellus spp bats in Europe and Asia 15 18 Molecular clock dating of epidemiologically unlinked isolates of human MERS CoV estimated their divergence from a common ancestor in mid 2011 4 19 with a cluster of isolates from the eastern Arabian peninsula diverging in late 2012 4 This fi nding could suggest that the diversity of Published Online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 See Online Comment http dx doi org 10 1016 S1473 3099 13 70193 2 Contributed equally Centre for Infectious Disease Research Diagnostics and Screening Division Virology National Institute for Public Health and the Environment Bilthoven Netherlands C B E M Reusken PhD G J Godeke BSc R De Sousa PhD J van Beek MSc Prof M P G Koopmans PhD Department of Viroscience Erasmus Medical Centre Rotterdam Netherlands B L Haagmans PhD V S Raj PhD S L Smits PhD Prof A Osterhaus PhD Prof M P G Koopmans Institute of Virology University of Bonn Medical Centre Bonn Germany M A M ller PhD B Meyer MSc D Muth PhD V M Corman MD J F Drexler MD Prof C Drosten MD Department of Animal Medicine and Surgery Veterinary Faculty University of Las Palmas de Gran Canaria Las Palmas Canary Islands Spain Prof C Gutierrez PhD Department of Infectious Diseases and Immunology Utrecht University Faculty of Veterinary Medicine Utrecht Netherlands L Smits De Vries MSc B J Bosch PhD Prof P Rottier PhD Department of Animal and Veterinary Sciences College of Agricultural and Marine Sciences Y E El Tahir PhD and Department of Microbiology and Immunology College of Medicine and Health Sciences Prof N Nowotny PhD Sultan Qaboos University Muscat Oman The European Articles 2 Published online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 Programme for Public Health Microbiology Training European Center for Disease Control Stockholm Sweden R De Sousa Viral Zoonoses Emerging and Vector Borne Infections Group Institute of Virology University of Veterinary Medicine Vienna Vienna Austria Prof N Nowotny Animal Health Service Deventer Netherlands K van Maanen PhD Department of Conservation and Research Parque Zoologico Buin Zoo Buin Chile E Hidalgo Hermoso DVM SaBio IREC CSIC UCLM JCCM Ciudad Real Spain C Gort zar Schmidt PhD and Viroclinics Biosciences BV Rotterdam Netherlands S L Smits Prof A Osterhaus Correspondence to Chantal Reusken Centre for Infectious Disease Research Diagnostics and Screening Division Virology National Institute for Public Health and the Environment PO Box 1 3720BA Bilthoven Netherlands Chantal Reusken RIVM nl MERS CoV in people is the result of multiple independent geographically structured zoonotic events in the Middle East 4 19 Possible animal reservoirs need to be identifi ed to determine how circulation of MERS CoV is maintained and to break the chain of transmission 20 MERS CoV can infect cells of several species including human beings and bats 14 The functional receptor is conserved between species suggesting that receptor use is not an important barrier to cross species transmission 21 Data for exposure history of patients are scarce but suggest contact with livestock including dromedary camels and goats 2 4 5 Food and Agriculture Organization data from 2011 show that cows goats sheep and dromedary camels are the main sources of meat and milk in Jordan Saudi Arabia and United Arab Emirates 22 Serological studies are best suited to screen animal populations but have not yet been reported for MERS CoV in animals although several methods have been described for testing antibodies of people 23 24 For specifi city WHO recommends use of a combination of screening assays with recombinant spike protein and confi rmatory testing by neutralisation assays Here we describe antibody profi ling of serum samples from major livestock species that might be relevant to the epidemiology of MERS CoV in the Middle East using samples collected from herds inside and outside the region Methods Serum sample collection We sampled a cohort of 105 dromedary camels Camelus dromedarius from two herds on the Canary Islands 50 were male 55 were female 88 were adults nine were age 3 4 years seven were age 2 years and one was age 3 months Both herds had the same owner with frequent exchange of animals between the herds One herd is from a coastal dune habitat with no other livestock while the other herd is in an inland valley close to a tropical fruit farm in particular mango and papaya which could attract fruit bats and nearby roughly 500 m to horse and goat farms with 25 and 300 animals respectively The camels were born in the Canary Islands except for three adults which were imported from Morocco 25 The camels are used in the tourist industry 80 sera were taken April June 2012 nine in May 2013 and 16 paired sera were taken in these months in 2012 and 2013 all for routine veterinary purposes Samples were obtained by jugular puncture 50 female dromedary camels from Oman were sampled in March 2013 The camels were aged 8 12 years and belonged to di erent owners from separate locations The camels are retired racing camels now used for breeding and blood was taken by jugular puncture for routine screening for brucellosis Figure 1 Reactivity of livestock sera with three coronavirus S1 antigens Fluorescent intensities per antigen at a serum dilution of 1 20 Black lines indicate median Dashed line is cuto of the assay RFU relative fl uorescence units SARS CoV severe acute respiratory syndrome coronavirus HCoV human coronavirus MERS CoV Middle East respiratory syndrome coronavirus 0 10 000 20 000 30 000 40 000 50 000 60 000 70 000 Antigen reactivity RFU Sheep SARS CoV HCoV OC43 MERS CoV Spanish dromedariesOther camelidsGoatsCows Omani dromedaries Articles Published online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 3 Sera were collected for veterinary purposes from two llamas Lama glama six alpacas Vicugna pacos and two Bactrian camels Camelus bactrianus in the Netherlands Sera were collected for veterinary purposes from two Bactrian camels 18 alpacas fi ve llamas and two guanaco Lama guanicoe in Buin Zoo in Chile Sera from cattle n 40 domestic goats n 40 and sheep n 40 were from routine submission to the Dutch Animal Health Service Sera from Spanish domestic goats n 40 were provided by the Instituto de Investigaci n en Recursos Cineg ticos Ciudad Real Spain from submissions for tuberculosis control in 2011 All sera were obtained in agreement with local regulations and Dutch import regulations with regard to animal disease legislation Positive human control sera for the three antigens used on the microarray were taken as described previously 24 All samples were stored at 20 C until testing Laboratory procedures We tested the sera for the presence of IgG antibodies reactive with MERS CoV SARS CoV and human coronavirus OC43 S1 antigens in a protein microarray The receptor binding domains which contain the S1 subunit of spike proteins of MERS CoV residues 1 747 SARS CoV residues 1 676 and human coronavirus OC43 residues 1 760 were expressed purifi ed and spotted on glass slides Slides were incubated with serum and species specifi c conjugates as previously described 24 Goat sera were incubated with Alexa Fluor 647 conjugated rabbit anti goat IgG Fc fragment Jackson Immuno Research West Grove PA USA cow sera with Alexa Fluor 647 conjugated goat anti bovine IgG H L Jackson Immuno Research sheep sera with Alexa Fluor 647 conjugated donkey anti sheep IgG H L Millipore Temecula CA USA and camelid sera with Dylight Figure 2 MERS CoV and human coronavirus OC43 or bovine coronavirus cross reactivity Combinations of the mean fl uorescent intensities of reactions of sera with MERS CoV and human coronavirus OC43 antigens from 105 Spanish dromedary camels A plaque reduction neutralisation tests for bovine coronavirus and MERS CoV B two representative sera are shown numbers 15 and 5 corresponding to camel ID numbers in table 2 in dilutions of 1 40 1 160 and 1 640 as well as the virus input control All samples were tested in duplicates only one well shown and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90 IgG reactivity of both camel sera to MERS CoV antigen and human coronavirus OC43 antigen in a two step dilution series in the microarray C IgG reactivity of two two step serially diluted Omani dromedary camel sera with human coronavirus EMC antigen and human coronavirus OC43 antigen in the microarray D RFU relative fl uorescence units MERS CoV Middle East respiratory coronavirus 0 20 000 40 000 60 000 0 20 000 40 000 60 000 Human coronavirus OC43 reactivity RFU MERS CoV reactivity RFU Input Serum dilution 1 40 1 160 1 640 Bovine coronavirus MERS CoV 15 5 15 5 Serum number Serum number AB 1 20 1 40 1 80 1 160 1 320 1 640 0 20 000 40 000 60 000 Serum dilution Antigen reactivity RFU 1 20 1 40 1 80 1 160 1 320 1 640 1 12801 25601 5120 1 10 2401 20 4801 40 960 1 81 920 1 163 840 1 327 680 Serum dilution CD MERS CoV 15 MERS CoV 5 HCoV OC43 15 HCoV OC43 5 MERS CoV MERS CoV HCoV OC43 HCoV OC43 Articles 4 Published online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 650 conjugated goat anti llama IgG H L Agrisera V nnas Sweden Fluorescence signals were quantifi ed as described previously 24 We tested the sera for IgG reactivity at a dilution of 1 20 and set an arbitrary cuto at an average signal intensity of 20 000 relative fl uorescence units RFU This high cuto was chosen to reduce cross reactive false positives 24 We present results as RFU for each set of quadruplicate spots per antigen Negative fl uorescent intensities caused by background correction were assigned to 0 Analyses were done with GraphPad Prism version 6 02 Sera were heat inactivated before virus neutralisation by incubation for 30 min at 56 C Two fold serial dilutions of sera were prepared using 96 well plates starting dilution 1 10 MERS CoV was diluted in Iscove s modifi ed Dulbecco s medium IMDM supplemented with clemizole penicillin penicillin G streptomycin and 1 fetal bovine serum to a dilution of 2000 tissue culture infective dose 50 per mL 50 L virus suspension was added to the plates and the plates were incubated at 37 C for 1 h The mixtures of virus and serum were then incubated on 96 well plates containing Vero cells for 1 h followed by washing with phosphate bu ered saline and incubation with IMDM and 1 fetal bovine serum for 3 4 days after which endpoint titres were measured All tests were repeated twice independently We tested neutralisation activity of sera against MERS CoV Erasmus MC isolate and bovine coronavirus Nebraska strain by plaque reduction neutralisation test 90 plaque reduction with African green monkey kidney cells cell line Vero B4 DSMZ ACC 33 or bovine kidney cells cell line PT CCLV RIE11 in a 24 well plate format Virus 30 60 plaque forming units and heat inactivated sera diluted from 1 40 to 1 640 were pre incubated in 200 L of serum free OPTIpro medium Life Technologies Karlsruhe Germany at 37 C for 1 h Virus adsorption was done at 37 C for 1 h Supernatants were removed and overlaid with Avicel resin FCM BioPolymer Brussels Belgium 5 Assays were stopped after 3 days by fi xation with 8 paraformaldehyde for 30 min All samples were tested in duplicate and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90 Role of the funding source The sponsors had no role in study design data collection data analysis data interpretation or writing of the report The corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication Results Sera were tested for IgG antibodies reactive with MERS CoV SARS CoV and human coronavirus OC43 S1 antigens in a protein microarray fi gure 1 Human coronavirus OC43 is serologically closely related to bovine coronavirus 26 diverging at the end of the 19th century 10 Bovine coronavirus circulates in cows sheep goats and Old and New World camelids 27 30 Because bovine coronavirus S1 was not available human coronavirus OC43 S1 antigen was used as a proxy Sera from three llamas four alpacas one guanaco and two Bactrian camels reacted with human coronavirus OC43 antigen One cow and one goat serum reacted with human coronavirus OC43 antigen as did sera from 16 of 105 15 Spanish dromedary camels All sera from cattle sheep and goats tested negative for MERS CoV antigen but sera from 15 Spanish camels 14 did react with MERS CoV antigen fi gure 1 The reactivity was highly specifi c the same sera did not bind to SARS CoV antigen but a positive control specimen did No correlation existed between the reactivity of sera with MERS CoV antigen and human coronavirus OC43 antigen fi gure 2 All but one serum sample that reacted with MERS CoV antigen were from adult animals One reactive serum was from a 2 year old animal To confi rm the presence of MERS CoV specifi c IgG in the Spanish camel sera we used a MERS CoV neutralisation assay to test a subset of 49 camel sera with di erent degrees of reactivity with MERS CoV and human coronavirus OC43 antigen according to microarray Nine Spanish camels had MERS CoV neutralising antibodies with titres varying between 1 20 Number of serum samples Positive MERS CoV neutralisation titre n Titre range Spanish samples no geographic link MERS CoV antigen array signal RFU 40 000 12 9 75 1 20 to 1 320 Human coronavirus OC43 antigen array signal RFU 40 000 4 0 0 Omani camel samples geographic link MERS CoV antigen array signal RFU 0 40 000 0 0 0 40 000 50 50 100 1 320 to 1 2560 Human coronavirus OC43 antigen array signal RFU 40 000 34 34 100 RFU relative fl uorescence units MERS CoV Middle East respiratory syndrome coronavirus Table 1 Results of neutralising assay for MERS CoV from Spanish and Omani camel serum samples Articles Published online August 9 2013 http dx doi org 10 1016 S1473 3099 13 70164 6 5 and 1 320 table 1 Three of the 12 sera reacted with MERS CoV spike antigen but did not neutralise MERS CoV most likely because of recognition of non neutralising epitopes All MERS CoV neutralising sera had almost saturating reactivity with MERS CoV antigen on the microarray whereas reactivity with human coronavirus OC43 antigen varied from negative to 50 of saturating reactivity table 1 The variable human coronavirus OC43 signals suggest that MERS CoV did not generally cross react with human coro navirus OC43 or bovine coronavirus antigens All nine camels with MERS CoV neutralising antibodies were born and raised on the Canary Islands seven were female two were male Eight camels were adults one was 2 years old To show that the reactivity of the camel sera with human coronavirus OC43 antigen according to the microarray was caused by the presence of bovine coronavirus IgG and to further exclude MERS CoV neutralising activity caused by cross neutralisation by the bovine coronavirus antibodies we tested camels that had su cient serum left n 15 in a comparative MERS CoV and bovine coronavirus plaque reduction neutralisation test fi gure 2 table 2 All camel sera neutralised bovine coronavirus but with varying titres suggesting a lower cuto than 20 000 RFU for OC43 in the microarray fi gure 1 Five camels had high neutralising antibody titres against bovine coronavirus and a mean signal intensity of greater than 50 000 RFU for human coronavirus OC43 antigen on microarray but were negative for MERS CoV neutralisation suggesting that cross neutralisation in this direction did not occur and that the MERS CoV neutralising activity was not caused by the presence of bovine coronavirus neutralising antibodies A serum sample from a patient who had MERS neutralised MERS CoV with a high titre 1 640 but neutralised bo