key: cord-0822999-9hll754i authors: Te, Nigeer; Vergara‐Alert, Júlia; Lehmbecker, Annika; Pérez, Mónica; Haagmans, Bart L.; Baumgärtner, Wolfgang; Bensaid, Albert; Segalés, Joaquim title: Co‐localization of Middle East respiratory syndrome coronavirus (MERS‐CoV) and dipeptidyl peptidase‐4 in the respiratory tract and lymphoid tissues of pigs and llamas date: 2018-12-28 journal: Transbound Emerg Dis DOI: 10.1111/tbed.13092 sha: 298a64fc7158cb78592bd96d53b4e58fbf562276 doc_id: 822999 cord_uid: 9hll754i This study investigated the co‐localization of the Middle East respiratory syndrome coronavirus (MERS‐CoV) and its receptor dipeptidyl peptidase‐4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS‐CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post‐inoculation (p.i.), DPP4‐MERS‐CoV co‐localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co‐localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS‐CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS‐CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS‐CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection. September 8th, 2018, South Korea reported the first MERS-CoV case since the end of an outbreak in 2015, suggesting that MERS-CoV is still a worldwide threat (WHO, 2018) . Dromedary camels have been considered as the main reservoir hosts for MERS-CoV, as viral neutralizing antibodies have been reported in this species (Corman et al., 2014; Reusken et al., 2013) . Moreover, animal-to-human transmissions have been described (Azhar et al., 2014) . More recently, a surveillance study showed that a MERS-CoV strain responsible for human outbreaks was isolated from the upper respiratory tract of dromedaries, demonstrating that the virus does not require mutations to jump between species (Sabir et al., 2016) . Besides dromedaries, several animal species, including common marmosets, rhesus macaques, llamas, pigs and alpacas, are experimentally susceptible to MERS-CoV infection (Adney, Bielefeldt-Ohmann, Hartwig, & Bowen, 2016; Falzarano et al., 2014; Vergara-Alert et al., 2017; de Wit et al., 2013) . Middle East respiratory syndrome coronavirus is a positivestranded RNA virus that belongs to the betacoronavirus genus (Zaki et al., 2012) . It has a genome of approximately 30 Kb nucleotides that encodes four structural proteins: spike (S), nucleocapsid (N), membrane (M) and envelope (E) proteins, and a RNA polymerase (Cotten, Lam, et al., 2013; Cotten et al., 2014; Zhang, Shen, & Gu, 2016) . The receptor binding domain (RBD) of the S-protein mediates viral entrance through binding dipeptidyl peptidase-4 (DPP4, also known as CD26), a serine protease expressed on the surface of many cell types (Raj et al., 2013) . The tissue distribution of DPP4 has been described in some animal species, including dromedary camels, bats, pigs, llamas, sheep and horses (Vergara-Alert et al., 2017; Widagdo et al., 2016 Widagdo et al., , 2017 . However, although DPP4 is decisive for MERS-CoV entry in vitro (Raj et al., 2013; Chan et al., 2016) , the role of the protein in determining tissue tropism with regards to MERS-CoV pathogenesis in vivo has not been fully elucidated. MERS-CoV antigen has been demonstrated in nasal epithelial cells expressing DPP4 Vergara-Alert et al., 2017) . However, no double staining studies to detect potential co-localization of both antigens were reported until very recently. Haverkamp et al. (2018) demonstrated that nasal epithelial cells infected with MERS-CoV in dromedaries seemed to lose DPP4 expression, while adjacent non-infected cells retained positivity for DPP4. Moreover, ciliary damage was also a feature of dromedary camels infected with MERS-CoV (Haverkamp et al., 2018) . These authors postulated that the mild and transient disease in dromedaries is, at least in part, potentially attributable to the down-regulation of its own cell entry receptor, thus self-limiting the viral infection. Taking into account that a number of animal species may potentially act as reservoirs for MERS-CoV (Vergara-Alert et al., 2017) , it is important to establish if DPP4 determines or not tissue tropism with regards to viral pathogenesis in vivo. Moreover, if the ciliary loss is a particular finding of dromedary camels infected with MERS-CoV or may also affect other susceptible species is not known. Therefore, the objective of this study was to assess the co-localization of MERS-CoV and DPP4 by means of double immunostaining in two susceptible species: llama and pig. In addition, the degree of ciliation of the upper respiratory tract in both species was studied by scanning electron microscopy (SEM). All paraffin blocks and fresh tissue samples used in the present work were obtained from a previous experimental study demonstrating that llamas and pigs were susceptible to MERS-CoV infection (Vergara-Alert et al., 2017) . Briefly, 2-month-old pigs and 6-8-month-old llamas were intranasally inoculated with 10 7 50% tissue culture infective dose (TCID50) MERS-CoV in 3 ml saline solution. Four pigs were euthanized on day 2 post-inoculation (p.i.) with an intravenous overdose of pentobarbital followed by exsanguination, and four animals of each species were killed on day 4 p.i. The remaining animals (six pigs and four llamas) were euthanized on day 24 p.i. following the same protocol. Complete necropsies were performed and respiratory and lymphoid tissues (nasal turbinate, trachea, bronchus, lung, cervical lymph node, mediastinal lymph node, tonsil and thymus) were collected for IHC and RT-qPCR examination. Formalin-fixed samples of nasal turbinate, trachea and lung were used for SEM studies, including those from negative control pigs euthanized to prior to the start of the experiment. Tissues collected from pigs on day 2, 4 and 24 p.i. and from llamas on day 4 and 24 p.i. were fixed by immersion in 10% neutral-buffered formalin for 1 week and embedded in paraffin blocks. The tissues were sectioned (3 μm) onto coated glass slides (DAKO; Agilent Technologies Company, Santa Clara, CA, USA), deparaffinized in xylene and hydrated in decreasing grades of ethanol (100%, 96% and 70%). Endogenous peroxidase was blocked with 3% H 2 O 2 solution in methanol for 30 min. Scanning electron microscopy was done following a previously published protocol (Haverkamp et al., 2018) . For each necropsy day, formalin fixed samples of nasal mucosa, trachea and bronchus of two pigs and two llamas infected with MERS-CoV were post-fixed in 5% glutaraldehyde. Two additional negative control samples were obtained for non-infected pigs. Afterwards the samples were dehydrated in a series of graded ethanol, dried and coated in a sputtercoater (SCD 040; Oerlikon Balzers, Balzers, Liechtenstein) with gold. For visualization, a digital scanning microscope (DSM 940, Carl Zeiss Jena GmbH) was used. Per localization and time point post-infection, eight fields at a magnification of 1,000 were evaluated; the percentage of ciliated area was analysed using GraphPad Prism 5.0 (GraphPad Software, Inc). Mann-Whitney Test was applied and results were considered statistically significant at pvalue < 0.05. A previously published RT-qPCR protocol was used to detect (Figures 4c and d) , it was observed in the cytoplasm of type I pneumocytes (Figure 5b ). In the BALT, notable cytoplasmic DPP4 was located in the plasma cells (Figure 6b ). In lymphoid tissues, DPP4 was rarely observable by IHC (Figure 7b ). No differences on DPP4 distribution were observed when comparing tissues at different days p.i. In pigs, on day 2 p.i., MERS-CoV antigen was detected in the cytoplasm of scattered epithelial cells in the medial part of the nasal Table 1 summarizes the semi-quantitative scores of MERS-CoV antigen found in the different studied tissues. Pigs infected with MERS-CoV showed severe ciliary loss in the nasal mucosa ( Figure 8a , Table 1 ) on days 2 and 4 p.i. (p = 0.0082). In contrast, ciliation of the trachea was unaffected on day 2 p.i., but ciliary loss was significant on days 4 ( Figure 8b , Table 1 ) and 24 p.i. (p = 0.008 and p = 0.0078, respectively). Bronchial ciliation was unaffected during the whole experiment ( Figure 8 , Table 1 ). On the other hand, llamas infected with MERS-CoV showed almost no significant ciliary loss in nasal mucosa, trachea and bronchi at any time; only one animal exhibited moderate ciliary loss in the nasal mucosa ( Figure 9a , Table 1 ) on day 4 p.i. In pigs, cervical and mediastinal lymph nodes and tonsil contained low viral RNA loads (Cq values ranged from 34.5 to 39) on day 4 p.i., but they were negative on days 2 and 24 p.i (Supporting information Figure S1a ). In llamas, comparatively higher viral loads were observed in cervical lymph nodes on day 4 p.i., and to a lower extent, in mediastinal lymph nodes, tonsils and thymus (Supporting information Figure S1b ). On day 24 p.i., a low amount of viral RNA T A B L E 1 Summary of MERS-CoV antigen distribution in respiratory and lymphoid tissues, RT-qPCR Cq values and ciliary coverage (in percentage) of airways of pigs and llamas 2 dpi ID of animal IHC, immunohistochemical score: −, negative; +, low (less than 10 cells/tissue); ++, moderate (10-50 cells/tissue); +++, high (more than 50 cells/tissues). Cq, quantification cycle obtained by RT-qPCR. ND, non-detectable. Cq value of turbinate, trachea and bronchus of both pigs and llamas is cited from a previously published work (Vergara-Alert et al., 2017) . SEM, percentage of ciliary coverage calculated through scanning electron microscopy. NS, no sample available. NP, not processed, NA, non-applicable. | 837 (Cq = 32) was found in cervical lymph nodes, being even less (Cq = 36) in mediastinal lymph nodes and tonsils (Supporting information Figure S1b ). MERS-CoV RNA was not detected in the thymus of llamas on 24 day p.i (Supporting information Figure S1b ). Middle East respiratory syndrome coronavirus entry into target cells through interaction between the RBD and the DPP4 protein has been well documented (Raj et al., 2013) . However, this is the first study in which co-localization between the virus and DPP4 from respiratory and lymphoid organs of different MERS-CoV susceptible species (pigs and llamas) has been studied. In (Nicholls et al., 2003) , respiratory syncytial virus in humans (Jumat et al., 2015) , influenza virus in humans and ferrets (Zeng et al., 2013) and canine respiratory coronavirus in dogs (Mitchell et al., 2013) . A number of mechanisms have been proposed to explain such ciliary loss, including direct destruction of the ciliary apparatus, ionic variations during respiratory viral attachment and replication stages, damage through regulating the production of oxidizing metabolites, and apoptosis (Jumat et al., 2015; Mall, 2008; Vareille, Kieninger, Edwards, & Regamey, 2011) . Therefore, the severe ciliary loss observed in nasal turbinates of pigs from this study was probably due to a bystander effect, since a very low amount of MERS-CoV antigen and RNA was found in these affected turbinates. More recent evidence suggests that the speed and efficiency of viral antigen to attach the host cell is accelerated by an entry complex that includes DPP4, a CoV-activating transmembrane protease serine 2 (TMPRSS2) and the tetraspanin Cd9 (Earnest et al., 2017) . In their study, mice transfected with the human DPP4 gene became significantly less susceptible to MERS-CoV infection after F I G U R E 9 SEM micrographs of ciliary damage in the respiratory tract of MERS-CoV inoculated llamas. On day 4 p.i., only one animal showed severe ciliary loss in nasal turbinate (a), while ciliary bundles were unaffected in trachea. On day 24 p.i., complete ciliation was seen in in apical surface of nasal turbinate and trachea (scale bar, 10 μm) silencing Cd9 or TMPRSS2 with small RNAs. Furthermore, MERS-CoV was reported to bind to sialic acid (Sia) and depletion of this molecule led to inhibition of MERS-CoV entry in Calu-3 human airway cells . In consequence, it is possible that DPP4 is not the single host determinant for the virus entry, at least for some animal species. Another study has demonstrated that horses express DPP4 in the respiratory tract, but no viral RNA was detected (Vergara-Alert et al., 2017) upon MERS-CoV inoculation, suggesting the presence of host factors such as glycosyls (Peck et al., 2015) that block the entry of MERS-CoV. Nevertheless, whether or not DPP4 is the only functional receptor for MERS-CoV remains to be investigated in susceptible animals. Our observations and analyses will need to be further validated as some questions still remain. For instance, it is not known why ciliary loss did not occur in llamas despite the much higher amount of virus than in the pig. Moreover, Haverkamp et al. (2018) demonstrated down-regulation of surface DPP4 upon MERs-CoV infection, but this was not observed in llamas, since most infected cells still retained DPP4 on its cell surface. In conclusion, the present work provides evidence that MERS-CoV preferably infects respiratory epithelial cells expressing DPP4 in llamas, supporting that DPP4 is necessary for virus entry in these organs. However, based on our observations, DPP4 may not be the single host determinant in regulating virus entry in respiratory cells of pigs and lymphoid tissues of both species. Although pigs showed a significant expression of DPP4 (mostly in the cell cytoplasm), the number of cells permissive for MERS-CoV in this species was lower than that of llamas. ZAPI project; IMI Grant Agreement no. 115760), with the assistance and financial support of IMI and the European Commission, and in-kind contributions from EFPIA partners. The funding from CERCA Programme/Generalitat de Catalunya to IRTA is also acknowledged. Nigeer Te is a recipient of a Chinese Scholarship Council grant Formal analysis: N. Te, J. Segalés. 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The authors also thank Ker- Additional supporting information may be found online in the Supporting Information section at the end of the article.