Te et al. Veterinary Research (2022) 53:67 https://doi.org/10.1186/s13567-022-01084-3 SHORT REPORT Open Access Evaluation of alpaca tracheal explants as an ex vivo model for the study of Middle East respiratory syndrome coronavirus (MERS‑CoV) infection Nigeer Te1,2,4†, Jordi Rodon1,2†, Rhea Creve1,2, Mónica Pérez1,2, Joaquim Segalés1,3, Júlia Vergara‑Alert1,2*    and Albert Bensaid1,2 Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to public health. Here, we estab‑ lished an ex vivo alpaca tracheal explant (ATE) model using an air-liquid interface culture system to gain insights into MERS-CoV infection in the camelid lower respiratory tract. ATE can be infected by MERS-CoV, being ­103 ­TCID50/mL the minimum viral dosage required to establish a productive infection. IFNs and antiviral ISGs were not induced in ATE cultures in response to MERS-CoV infection, strongly suggesting that ISGs expression observed in vivo is rather a consequence of the IFN induction occurring in the nasal mucosa of camelids. Keywords: Air-liquid interface, alpaca, camelid, ex vivo model, MERS-CoV, tracheal explants Introduction, methods, and results hosts of MERS‐CoV [6], since viral neutralizing antibod- Middle East respiratory syndrome coronavirus (MERS- ies have been reported in this species [7–13], and the CoV) is the etiological agent causing a respiratory disease virus does not require mutations to jump from drom- that emerged in The Kingdom of Saudi Arabia in 2012 edaries to humans [14]. Besides, all studied camelids are [1]. Early reports from the Arabian Peninsula indicated susceptible to MERS-CoV infection under both experi- high case-fatality rates associated with the disease [2–4]. mental and natural conditions [15–24]. As of April 2022, the World Health Organization has Innate immunity, the first host defense system against reported 2585 infections and 890 fatalities (∼34.4% case- viral infections, is thought to play a key role in MERS fatality rate) globally in 27 countries across four conti- pathogenesis. Studies of MERS-CoV infections on nents [5]. The primary manifestation of MERS in humans numerous human cell types led to the conclusion that ranges from asymptomatic or mild respiratory symptoms type I and III interferons (IFNs) are largely inhibited or to pneumonia leading to acute respiratory distress syn- delayed [25–27]. Furthermore, high and persistent secre- drome [4]. Dromedary camels are reservoir/intermediate tions of inflammatory cytokines by lung macrophages and the inhibition of innate immune responses at the † mucosal level are likely to contribute to a more severe Nigeer Te and Jordi Rodon contributed equally to this work infection in humans [28–31]. By contrast, camelids only *Correspondence: julia.vergara@irta.cat show subclinical disease in response to MERS-CoV [15– 1 Unitat Mixta d’investigació IRTA‑UAB en Sanitat Animal, Centre de Recerca 24]. Such an outcome is apparently due to the action of en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain type I and III IFNs generated by the infected nasal epi- Full list of author information is available at the end of the article thelia as suggested by recent work performed in alpacas © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Te et al. Veterinary Research (2022) 53:67 Page 2 of 7 [24]. Infected epithelium produced IFNs, unlike distant uninfected cells, while the expression of interferon-stim- ulated genes (ISGs) was elevated in both infected and uninfected epithelial cells, suggesting that IFNs could act in a paracrine fashion to induce ISGs expression in unin- fected cells [24]. Similarly, IFNs produced at the nasal epithelium might also be sensed via an endocrine path- way, as evidenced by the production of ISGs in the lower respiratory tract (LRT) in absence of detectable expres- sion of IFNs in these tissues [22, 24]. Besides the effective innate immune response of camelid hosts, another factor that may contribute to the mild pathogenesis of MERS-CoV in these species Figure 1 Schematic diagram of the experimental time course is the ability of the virus to moderately replicate within and settings of the air-liquid (ALI) device for the alpaca cells from the LRT. Previous research showed that very tracheal explant (ATE) model. (A) A schematic overview of the few infected cells were found in trachea upon intrana- ALI interface set-up and (B) a photo of an ATE (arrow) resting onto sal MERS-CoV inoculation [15, 19, 20, 22, 24], although an inox gauze within a 6-well culture plate. (C) General overview of the experiment indicating the time points in which explants were dipeptidyl peptidase 4 (DPP4) expression levels are com- collected, cultured, infected, and sampled. Upon necropsy, ATE were parable to that of nasal mucosa in camelids [32, 33]. It is isolated from alpacas (n = 3) and cultured in an ALI interface system. unclear how MERS-CoV replication towards the LRT is ATE were apically infected after 24 h of culture with MERS-CoV restrained. Thus, in the present study, we established an Qatar15/2015 strain at three different doses: ­102, ­103, and ­104 ­TCID50/ ex vivo tracheal organ culture using an ALI system to ful- mL. Explant replicates from each animal were collected for virological, pathological, and immunological assessments at -24, 0, 24, 48 and fill these knowledge gaps. 72 h respectively. Three 10 to 12-month-old alpacas (Vicugna pacos) were purchased by private sale, housed at IRTA farm facilities at Alcarràs (Catalonia, Spain) during the accli- mation period and transferred to the Autonomous Uni- 5% ­CO2. The ciliary beating was checked daily (each 24 h) versity of Barcelona, in Barcelona (Spain), for necropsy under a light microscope. procedures. Animals were euthanized with an overdose A passage-3 of the clade B MERS-CoV Qatar15/2015 of pentobarbital injected into the jugular vein. The mid- strain (GenBank Accession MK280984) stock was propa- dle portion of tracheas (n = 3) was aseptically collected gated in Vero E6 cells and titrated by calculating the dilu- and transported to the laboratory in pre-heated transport tion that caused cytopathic effect in 50% of the inoculated medium consisting of a 1:1 mixture of Dulbecco’s modi- cell cultures (50% tissue culture infectious dose endpoint fied Eagle’s medium (DMEM, Lonza) and Roswell Park ­[TCID50]), as previously described [19]. The MERS-CoV Memorial Institute (RPMI) 1640 medium supplemented Qatar 15/2015 strain was selected on the basis that only with penicillin (100 U/mL), streptomycin (100 μg/mL), clade B strains are currently circulating in the Arabian L-glutamine (10 mM); all of them from ThermoFisher Peninsula [35] and it has enhanced in vivo replicative Scientific (Life Technologies, Waltham, USA) and capabilities in alpacas compared to the prototypical clade amphotericin (2 μg/mL; Sigma-Aldrich). Obtained tra- A MERS-CoV EMC/2012 strain [22]. ATE were inocu- cheal tissues were washed by immersion into fresh warm lated with the MERS-CoV Qatar15/2015 strain at three transport medium three times, followed by the isolation different doses: ­102, ­103, and ­104 ­TCID50/mL in CM (Fig- of ATE according to a previously published protocol [34] ure 1C). Briefly, tracheal explants were prepared in trip- with some modifications. Briefly, the tracheal mucosa licates for each time point per animal (n = 3) onto sterile was stripped from the tracheal rings. The cartilage was stainless-steel gauzes placed on 6-well culture plates carefully discarded, and each segment was then cut with the epithelial surface upwards (Figures 1A, B). After into squares of 25 m ­ m2, prior to transfer to fine meshed 24 h of culture, explants were then transferred to a new inox steel gauzes placed in 6-well culture plates with the 6-well culture plate prior to infection with each MERS- epithelium facing up. Each well contained 2 mL of cul- CoV dose apically. CM alone was used for mock-infected ture media (CM) consisting of transport media without controls. After 1 h of viral exposure in a humidified incu- amphotericin. ATE were cultivated in ALI interface, so bator at 37 °C and 5% C ­ O2, explants were washed three the epithelium was slightly immersed in fluid with cilia times with warm PBS and placed back to the gauze at exposed to the air (Figures 1A, B). Explants were cultured the original 6-well plates containing CM. Explants were in a humidified incubator for up to 4 days at 37 °C and collected at 24, 48 and 72 h post-inoculation (hpi) and Te et al. Veterinary Research (2022) 53:67 Page 3 of 7 introduced in tubes containing 1) 10% formalin for mor- values obtained from infected explants collected at 24, 48 phometric, histological and immunohistochemical anal- and 72 hpi were compared to those obtained from non- ysis (two of the triplicates); or 2) TRI-Reagent (Zymo infected ATE at 0 hpi. The up-or down-regulated expres- Research, California, USA) for RNA virus quantification sion of each cytokine gene was expressed in fold-change as well as innate immune response analysis. values (Sheet B in Additional file 1). Explants were fixed by immersion in 10% neutral‐buff- MERS-CoV infection did not significantly alter the ered formalin for 5 days, embedded in paraffin blocks average thickness of the epithelium layer (ranging and processed for histological analysis (hematoxylin and between 63.3 to 86.7 µm) (Figure 2) nor caused obvious eosin stain, H&E). Besides, the thickness of the tracheal histopathological changes in ATE for the duration of the epithelium was assessed by measuring five randomly entire ex vivo experiment (Figures 3A, B). All cultured selected fields across each trachea section. A monoclonal ATE maintained nearly intact cellular and tissular mor- mouse anti-MERS-CoV N protein antibody (Sino Biolog- phology despite vacuolation of very few cells within the ical Inc., Beijing, China) was used to detect the presence epithelium and the lamina propria from 48 hpi onwards of MERS-CoV antigen, following a previously established (Figure 3B). Figure 4A shows the kinetics of MERS-CoV protocol [33]. A grading system for immunohistochemis- yields after infection with three different viral doses (­ 102, try (IHC) was established by a board-certified veterinary ­103 and 1 ­04 ­TCID50/mL, respectively). Viral genomic pathologist (−, no positive cells detected; ± , less than 10 RNA loads in ATE were dose- and time-dependent (Fig- positive cells per tissue section; + , 10–50 positive cells ure 4A left). An infection dose of ­104 ­TCID50/mL led to per tissue section; + + , 50 to 150 positive cells per tissue the highest viral RNA detection in ATE and a plateau section). was reached at 48 hpi onwards. To ascertain active viral Total RNA from ATE samples collected at different gene transcription within ATE, the presence of MERS- time points was extracted and converted into cDNA fol- CoV subgenomic RNA (M gene) was also assessed (Fig- lowing a previous standard protocol [24]. A microflu- ure 4A right). M mRNA loads were only detected in ATE idic RT-qPCR assay was utilized to relatively quantify infected with 1 ­ 03 or 1 ­ 04 ­TCID50/mL MERS-CoV, while the expression of immune related genes. The selection no M mRNA transcription occurred upon inoculation of innate immune genes and specific pairs of primers to with ­102 ­TCID50/mL doses. In agreement with the results amplify their transcriptional products were described obtained by viral RNA quantification, IHC positive labe- in previous works [22, 24]. In addition, specific primers ling was detected in ATE infected with either ­103 or ­104 for the detection of genomic (UpE) and subgenomic (M) ­TCID50/mL sampled at 48 or 72 hpi (Figure 4A and Addi- viral RNA were added to the assay (Sheet A in Additional tional file 2). Specific staining was mostly located in the file 1) [24]. Data were analyzed with the Real-Time PCR cytoplasm of tracheal epithelial cells (Figure 4B). Analysis 4.1.3 software (Fluidigm Corporation, South To explore antiviral and inflammatory pathways San Francisco, USA) and the DAG expression software activated upon MERS-CoV infection in ATE, the rela- 1.0.5. 6, as previously described [24]. HPRT1, GAPDH tive mRNA expression levels of 39 innate immune and UBC genes were used as normalizer genes, and response genes were monitored in mock-treated Figure 2 Evaluation of alpaca tracheal explants (ATE) thickness after ex vivo culture by means of light photomicrographs. The trachea was harvested upon necropsy, followed by infection at different doses of MERS-CoV (right of the figure). ATE were collected at different time points and were fixed in 10% neutral-buffered formalin. Sections of each formalin-fixed paraffin embedded ATE were stained by hematoxylin-eosin to evaluate the thickness of the epithelium by measuring five randomly selected fields across each section (indicated with a black arrow at the left) at –24, 0, 24, 48 and 72 hpi, respectively. Te et al. Veterinary Research (2022) 53:67 Page 4 of 7 Figure 4 MERS-CoV loads in alpaca tracheal explants (ATE). (A) MERS-CoV genomic (left) and subgenomic (right) RNA was determined by microfluidic RT-qPCR assays (Sheet A in Additional file 1). Each line represents the mean Cq value and error bars display standard deviation intervals of ATE from 3 animals sampled at 0, 24, 48 and 72 hpi, respectively. Blue, purple, and red dots indicate ATE infected with 1­ 02, ­103 and ­104 ­TCID50/mL MERS-CoV, respectively. (B) Tissue tropism of MERS-CoV in ATE. At 72 hpi, MERS-CoV N protein (brown staining) was detected by immunohistochemistry in the epithelium of ATE that were infected with ­103 and ­104 ­TCID50/mL doses respectively; original magnification: × 200 for both tissues. ATE, Figure 3 Histopathological study of alpaca tracheal explants alpaca tracheal explants; 1/3 and 2/3 indicate the detection of viral (ATE). Hematoxylin-eosin staining was applied to assess histological RNA in one out of three or two out of three explants, respectively. alterations of ATE (A) prior and (B) after MERS-CoV infection. No significant lesions were observed in any of the studied explants, inoculated or not with MERS-CoV. Original magnification: × 200 for all tissues. Discussion The pathogenesis of MERS-CoV in the intermediate host is yet to be fully understood. Although camelids and viral-infected ATE. Three highly stable normal- can be experimentally infected, such an approach repre- izer genes were included in the assay. All genes were sents a significant financial cost, ethical concern, safety detected at basal levels in non-infected ATE controls risk and requires special household facilities under a collected at 0 hpi, thus, allowing normalization of the biosafety level-3 (BSL-3) environment. Thus, we devel- whole data sets against this reference time point. Tran- oped an ex vivo ATE model to address these hurdles. scription of pro-inflammatory cytokines, chemokines, ATE retained the ciliary integrity during the course of and transcription factors (except for IRF5 that the experiment, indicating that the coordination and remained unaltered) showed a slight down-regulation modulation of the ciliary beating of tracheal epithelium trend upon infection of ATE with any of the MERS- remained functional. Morphometric and histological CoV dosages (Figure 5 and Sheet B in Additional assessments demonstrated that ATE remained integral file 1). The rest of the genes, including IFNs, ISGs, with good cellular and tissular morphology for at least 72 pattern recognition receptors, downstream signaling hpi, showing great resemblance to the freshly collected enzymes, adaptors, and receptors, fluctuated around trachea. Of note, a massive ciliary loss occurs in the nasal basal levels (Figure 5 and Sheet B in Additional file 1), epithelia of dromedary camels experimentally infected suggesting inhibition of antiviral responses in ATE with MERS-CoV [36] but not in llamas [33] or alpacas upon MERS-CoV infection. [24]. Te et al. Veterinary Research (2022) 53:67 Page 5 of 7 Figure 5 Kinetics of innate immune gene profiles of ATE in response to different MERS-CoV infectious doses. Total RNA was extracted from ATE, followed by conversion to cDNA. The Fluidigm Biomark microfluidic assay was used to quantify transcripts from innate immune genes at different hpi. After normalization, fold change values between controls (freshly prepared explants) and mock or infected ATE were calculated. The resulting heatmap shows color variations corresponding to ­log2 fold change values, blue for increased and brown for decreased gene expression, respectively. The khaki rectangles indicate no expression of the corresponding gene. TFs, transcription factors; CKs, chemokines; ADs, adaptors; RT, receptor. In the present study, ATE were unable to express IFNs and MERS-CoV infection in camelids. In that respect, in agreement with previous observations on the trachea explant models might better reflect an in vivo scenario of alpacas infected with MERS-CoV [22, 24]. In addi- since mucosa and submucosa are both co-cultured tion, ISGs were not induced in ATE upon MERS-CoV maintaining the tissue structure. Nonetheless, AEC and infection, supporting the fact that upregulation of ISGs tracheal explant models overcome natural physiological in tracheal mucosa observed in vivo is due likely to the drawbacks inherent to epithelial cell lines including dif- paracrine action of IFNs produced by the nasal epithe- ferences in cell polarization, coordinated ciliary activ- lia [24]. Recently, pseudostratified airway epithelial cell ity and apical contact with air [39]. Furthermore, tissue (AEC) culture models for llamas and Bactrian camels pieces obtained from a single animal can be used in have been developed. These AEC were generated from numerous replicates, thereby reducing the experimen- the tracheobronchial tract, supported the growth of sev- tal variability and the number of animals used, in line eral MERS-CoV lineages and were sensitive to the antivi- with the 3Rs principle [40]. ral effect of IFNs [37, 38]. However, in these experiments, In conclusion, a novel ex vivo culture model was the production of IFNs and cytokines upon MERS-CoV developed using ATE, which is suitable to study MERS- infection were not checked. Nevertheless, when treated CoV infection and replication as it occurs in natural with IFNs, camelid tracheal cells can produce ISGs, reservoir hosts. We demonstrated as a proof of prin- denoting a functional innate immune system [38]. Both, ciple that ATE can be infected by MERS-CoV without ATE and AEC could be exploited to test agonists and triggering innate antiviral responses. Besides, ATE antagonists of several innate immune pathways leading could also be suitable to study other respiratory path- to MERS-CoV clearance. ogens of camelids by reducing the burden of animals The exclusive use of camelid tracheal cells to study used for experimentation. host-MERS-CoV interactions might not be sufficient since, at least in vivo, infected nasal epithelial cells are Abbreviations able to notably up regulate the transcriptional expres- AEC: pseudostratified airway epithelial cell; ATE: alpaca tracheal explant; sion of IFN genes [22, 24]. Therefore, the development BSL-3: biosafety level-3; CM: culture media; DPP4: dipeptidyl peptidase 4; of nasal explants and well differentiated nasal epithelial hpi: hours post-inoculation; IFN: interferon; IHC: immunohistochemistry; ISG: interferon-stimulated genes; LRT: lower respiratory tract; MERS-CoV: Middle cell models will be necessary for future studies to fully East respiratory syndrome coronavirus; TCID50: 50% tissue culture infectious appreciate the interplay between the epithelial barrier dose endpoint. Te et al. Veterinary Research (2022) 53:67 Page 6 of 7 Supplementary Information 2. Arabi YM, Arifi AA, Balkhy HH, Najm H, Aldawood AS, Ghabashi A, Hawa H, Alothman A, Khaldi A, Al Raiy B (2014) Clinical course and outcomes The online version contains supplementary material available at https://​doi.​ of critically ill patients with Middle East respiratory syndrome corona‑ org/​10.​1186/​s13567-​022-​01084-3. virus infection. Ann Intern Med 160:389–397. https://​doi.​org/​10.​7326/​ M13-​2486 Additional file 1: Viral loads (UpE and M mRNA) and Fc of innate 3. Assiri A, Al-Tawfiq JA, Al-Rabeeah AA, Al-Rabiah FA, Al-Hajjar S, Al-Barrak immune response genes in ATE. A, Flemban H, Al-Nassir WN, Balkhy HH, Al-Hakeem RF, Makhdoom HQ, Additional file 2: MERS-CoV N protein detection by IHC in ATE. Zumla AI, Memish ZA (2013) Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome corona‑ virus disease from Saudi Arabia: a descriptive study. Lancet Infect Dis Acknowledgements 13:752–761. https://​doi.​org/​10.​1016/​S1473-​3099(13)​70204-4 We thank Dr Bart L. Haagmans from Erasmus Medical Center (EMC, Rotterdam) 4. Zumla A, Hui DS, Perlman S (2015) Middle East respiratory syndrome. for providing the MERS-CoV Qatar-15/2015 strain. We also thank Dr Hans Lancet 386:995–1007. https://​doi.​org/​10.​1016/​S0140-​6736(15)​60454-8 Nauwynck and staff from his lab (Laboratory of Virology, Faculty of Veteri‑ 5. WHO | Middle East respiratory syndrome coronavirus (MERS-CoV). nary Medicine, Ghent University) for providing advanced training on ex vivo https://​www.​who.​int/​emerg​encies/​mers-​cov/​en/. Accessed 18 May 2022 cultures. We are particularly indebted to the staff of the BSL-3 biocontainment 6. Sabir JSM, Lam TT-Y, Ahmed MMM, Li L, Shen Y, Abo-Aba SEM, Qureshi animal facility at IRTA-CReSA (Cerdanyola del Vallès, Barcelona). MI, Abu-Zeid M, Zhang Y, Khiyami MA, Alharbi NS, Hajrah NH, Sabir MJ, Mutwakil MHZ, Kabli SA, Alsulaimany FAS, Obaid AY, Zhou B, Smith DK, Authors’ contributions Holmes EC, Zhu H, Guan Y (2016) Co-circulation of three camel corona‑ NT, JR, JS, JV-A, and AB designed research; NT, JR, MP, JS, and JV-A performed virus species and recombination of MERS-CoVs in Saudi Arabia. Science research; NT, JR, RC, MP, JS, JV-A, and AB analyzed data; and NT, and AB wrote 351:81–84. https://​doi.​org/​10.​1126/​scien​ce.​aac86​08 the paper. All authors read and approved the final manuscript. 7. Corman VM, Jores J, Meyer B, Younan M, Liljander A, Said MY, Gluecks I, Lattwein E, Bosch BJ, Drexler JF, Bornstein S, Drosten C, Müller MA (2014) Funding Antibodies against MERS coronavirus in dromedary camels, Kenya, This research was supported by European Commission: [Call: H2020- 1992–2013. Emerg Infect Dis 20:1319–1322. https://​doi.​org/​10.​3201/​ INFRAIA-2016–2017 grant N°: 731014, Innovative Medicines initiative (IMI) eid20​08.​140596 grant N°115760]. IRTA is supported by CERCA Programme / Generalitat de 8. Drosten C, Meyer B, Müller MA, Corman VM, Al-Masri M, Hossain R, Catalunya. N.T. is a recipient of a China Scholarship Council grant (CSC NO. Madani H, Sieberg A, Bosch BJ, Lattwein E, Alhakeem RF, Assiri AM, 201608150108). The funders had no role in study design, data collection and Hajomar W, Albarrak AM, Al-Tawfiq JA, Zumla AI, Memish ZA (2014) analysis, decision to publish, or preparation of the manuscript. Transmission of MERS-coronavirus in household contacts. N Engl J Med 371:828–835. https://​doi.​org/​10.​1056/​NEJMo​a1405​858 Availability of data and materials 9. Farag EABA, Reusken CBEM, Haagmans BL, Mohran KA, Stalin Raj V, Pas The data that support the findings of this study are available from the authors SD, Voermans J, Smits SL, Godeke G-J, Al-Hajri MM, Alhajri FH, Al-Romaihi upon reasonable request. HE, Ghobashy H, El-Maghraby MM, El-Sayed AM, Al Thani MHJ, Al-Marri S, Koopmans MPG (2015) High proportion of MERS-CoV shedding drom‑ edaries at slaughterhouse with a potential epidemiological link to human Declarations cases, Qatar 2014. Infect Ecol Epidemiol 5:28305. https://​doi.​org/​10.​3402/​ iee.​v5.​28305 Ethics approval and consent to participate 10. Reusken CB, Ababneh M, Raj VS, Meyer B, Eljarah A, Abutarbush S, Godeke The authors confirm that the ethical policies of the journal, as noted on the GJ, Bestebroer TM, Zutt I, Muller MA, Bosch BJ, Rottier PJ, Osterhaus AD, journal’s author guidelines page, have been adhered to. All animal experi‑ Drosten C, Haagmans BL, Koopmans MP (2013) Middle East Respiratory ments were approved by the Ethical and Animal Welfare Committee of IRTA Syndrome coronavirus (MERS-CoV) serology in major livestock species (CEEA-IRTA) and by the Ethical Commission of Animal Experimentation of the in an affected region in Jordan, June to September 2013. Euro Surveill Autonomous Government of Catalonia (file No. CEA-OH/10943/1). The work 18:20662. https://​doi.​org/​10.​2807/​1560-​7917.​es2013.​18.​50.​20662 involving MERS-CoV was performed at the BSL-3 facilities of the Biocontain‑ 11. Reusken CB, Farag EA, Jonges M, Godeke GJ, El-Sayed AM, Pas SD, Raj ment Unit of IRTA-CReSA in Barcelona, Spain. VS, Mohran KA, Moussa HA, Ghobashy H, Alhajri F, Ibrahim AK, Bosch BJ, Pasha SK, Al-Romaihi HE, Al-Thani M, Al-Marri SA, AlHajri MM, Haagmans Competing interests BL, Koopmans MP (2014) Middle East respiratory syndrome coronavirus The authors declare that they have no competing interests. (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014. Euro Surveill Author details 19:20829. https://​doi.​org/​10.​2807/​1560-​7917.​es2014.​19.​23.​20829 1 Unitat Mixta d’investigació IRTA‑UAB en Sanitat Animal, Centre de Recerca 12. Raj VS, Osterhaus ADME, Fouchier RAM, Haagmans BL (2014) MERS: emer‑ en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona gence of a novel human coronavirus. Curr Opin Virol 5:58–62. https://​doi.​ (UAB), 08193 Bellaterra, Catalonia, Spain. 2 IRTA, Programa de Sanitat Animal, org/​10.​1016/j.​coviro.​2014.​01.​010 Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat 13. Reusken CBEM, Haagmans BL, Müller MA, Gutierrez C, Godeke G-J, Meyer Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain. 3 Departa‑ B, Muth D, Raj VS, Smits-De Vries L, Corman VM, Drexler J-F, Smits SL, El ment de Sanitat i Anatomia Animals, Facultat de Veterinàriaia, Universitat Tahir YE, De Sousa R, van Beek J, Nowotny N, van Maanen K, Hidalgo- Autònoma de Barcelona (UAB), Campus de la UAB, 08193 Bellaterra, Catalonia, Hermoso E, Bosch B-J, Rottier P, Osterhaus A, Gortázar-Schmidt C, Drosten Spain. 4 School of Public Health, Li Ka Shing, Faculty of Medicine, The University C, Koopmans MP (2013) Middle East respiratory syndrome coronavirus of Hong Kong, Hong Kong, China. neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect Dis 13:859–866. https://​doi.​org/​10.​1016/​ Received: 22 April 2022 Accepted: 12 July 2022 S1473-​3099(13)​70164-6 14. 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