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Tropism and innate immune responses of the novel human betacoronavirus lineage C 1 virus in human ex vivo respiratory organ cultures 2 3 Renee WY Chan1,2#, Michael CW Chan1#*, Sudhakar Agnihothram3, Louisa LY Chan1, 4 Denise IT Kuok1, Joanne HM Fong1, Y Guan1,4, Leo LM Poon 1,4, Ralph S.Baric3, John M 5 Nicholls2 and JS Malik Peiris1,4*. 6 7 8 Short title: Tropism and pathogenesis of novel betacoronavirus 9 10 1 Centre of Influenza Research and School of Public Health, LKS Faculty of Medicine, The 11 University of Hong Kong, Pokfulam, Hong Kong SAR, China; 12 13 2 Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, 14 Hong Kong SAR, China; 15 16 3 Departments of Epidemiology and Microbiology and Immunology, Gillings School of 17 Global Public Health and School of Medicine, The University of North Carolina, Chapel Hill, 18 NC 27599-7435, USA. 19 20 4 State Key Laboratory of Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, 21 The University of Hong Kong, Hong Kong SAR, China. 22 23 # Equal contributors 24 25 *Corresponding author: Michael CW Chan and JS Malik Peiris, Centre of Influenza 26 Research, School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, 27 Pokfulam, Hong Kong SAR, China. Phone: (852) 2816-8438; Fax: (852) 2872-5782; E-mail: 28 mchan@hku.hk (MCWC) and malik@hku.hk (JSMP) 29 30 Abstract word count: 234 31 Main text word count: 4845 32 33

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00009-13 JVI Accepts, published online ahead of print on 3 April 2013

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Abstract 34 Since April 2012, there have been thirteen laboratory-confirmed human cases of respiratory 35 disease associated with a newly recognized human betacoronavirus lineage C (HCoV-EMC) 36 virus, seven of them fatal. The transmissibility and pathogenesis of HCoV-EMC remains 37 poorly understood and elucidating its cellular tropism in human respiratory tissues will 38 provide mechanistic insights into the key cellular targets for virus propagation and spread. 39 40 We utilized ex vivo organ cultures of human bronchus and lung to investigate the tissue 41 tropism, virus replication kinetics following experimental infection with HCoV-EMC, 42 compared with human coronavirus 229E (HCoV-229E) and SARS coronavirus (SARS-CoV). 43 Innate immune responses elicited by HCoV-EMC were also investigated. 44 45 HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. 46 While SARS-CoV productively replicated in lung tissue, replication in human bronchial 47 tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected non-ciliated 48 bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells and endothelial 49 cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial 50 epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there 51 was minimal HCoV-229E infection in these tissues. 52 53 HCoV-EMC failed to elicit strong type I or III interferon or pro-inflammatory innate immune 54 responses in ex vivo respiratory cultures. Treatment of human lung ex vivo organ cultures 55 with type I interferons (IFN α and β) at 1 hour post-infection reduced the replication of 56 HCoV-EMC suggesting a potential therapeutic use of IFNs for treatment of human infection. 57 58 (Word court: 234) 59

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Introduction 60 Coronavirus infections in humans are generally mild and self-limited. Until the outbreak of 61 SARS caused by SARS-CoV in 2003, there was limited research on the tissue tropism and 62 host response following human infection with coronaviruses. In comparison to HCoV-229E, 63 SARS-CoV was deficient at eliciting interferon (IFN) β innate immune responses in primary 64 human macrophages and dendritic cells (10, 24) since SARS-CoV encodes several 65 antagonists of innate immune sensing and signaling pathways (15, 39). Tropism of SARS-66 CoV in the respiratory tract was primarily restricted to differentiated human airway 67 epithelium (36), alveolar type II pneumocytes (6, 30, 31) with limited tropism for alveolar 68 type I pneumocytes (34). 69 70 In 2012, a novel coronavirus was detected in two patients from Saudi Arabia and Qatar (4, 71 43). Thereafter, more cases were identified both prospectively and retrospectively in Saudi 72 Arabia, Qatar, Jordan and United Kingdom and as of February 2013, a total of thirteen 73 laboratory confirmed cases have been reported with seven deaths (42). The apparent severity 74 of this novel coronavirus contrasts with those seen in other human coronaviruses, with the 75 exception of SARS-CoV (4, 14). The first reported case, which was fatal, occurred in June 76 2012 in a 60-year-old man in Saudi Arabia (43); the second reported case occurred in a 49-77 year-old Qatari man who was treated and discharged in the United Kingdom. From early case 78 descriptions, it appeared that pneumonia leading to acute respiratory distress syndrome is the 79 primary manifestation of the disease, but renal dysfunction was also observed in some cases. 80 The WHO has provided a working case-definition of the disease (42). The disease appears to 81 have an incubation period of up to 10 days and is not easily transmitted between humans. The 82 retrospective investigation of an outbreak of severe respiratory illness in Zarqa, Jordan in 83 April 2012 (1) confirmed that at least some of these cases were also caused by HCoV-EMC. 84 Of the 13 confirmed cases to date, some give a history of contact with animals (e.g. camels 85 and sheep) (14) and a zoonotic origin of the infection is considered likely. More recently, an 86 index case who acquired infection in the Middle-East transmitted infection to two family 87 contacts in the United Kingdom (19), providing evidence for limited human-to-human 88 transmission, thereby raising the level of public health concern. 89

90 The virus isolate from the Saudi patient has been fully sequenced and identifies the virus as a 91 novel human virus within betacoronavirus lineage C. Phylogenetically, it clusters in the same 92 group as viruses isolated from Pipistrellus (P.pipi/VM314/2008/NLD; BtCoV/355A/2005; 93

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BtCoV/A434/2005; HKU4) and Tylonycteris (BtCoV/133/2005; HKU-5) bats in Hong Kong 94 / China and the Netherlands (3, 5). These bat coronaviruses have not been cultured in vitro. 95 The partial sequence of the ORF1 region of the isolate from the patient from Qatar is more 96 than 99% identical to that of the virus reported from Saudi Arabia (HCoV-EMC) (4, 12). 97 Previously known human coronaviruses (HCoV) are alphacoronaviruses HCoV-229E and 98 HCoV-NL63, betacoronavirus lineage A (HCoV-OC43 and HKU-1) and betacoronavirus 99 lineage B (SARS-CoV) (23, 41). 100 101 We have previously used ex vivo cultures of human bronchial and lung tissues to investigate 102 tropism of influenza viruses (7) and showed that productive infection of the upper airways 103 may correlate with pandemic potential in swine influenza viruses (8). We now utilize ex vivo 104 organ cultures of the human bronchus and lung to study the tissue tropism, virus replication 105 kinetics, apoptosis and innate immune responses of HCoV-EMC infection in comparison 106 with the highly pathogenic SARS-CoV and common cold causing human coronavirus 229E 107 (HCoV-229E). 108 109 As there are no specific antivirals for HCoV-EMC infection, we explore the antiviral effects 110 of IFNs. IFN has been previously reported to inhibit SARS-CoV replication both in vitro and 111 in animal models (13, 17, 22, 35, 37, 38). 112 113 Material and Methods 114 Viruses 115 Human betacoronavirus of lineage C (HCoV-EMC) virus was obtained from Dr R Fouchier, 116 Erasmus MC, Rotterdam and the virus seed stock was prepared in Vero cell culture (ATCC) 117 in minimal essential medium (MEM) containing 2% fetal bovine serum (FBS), 100 units/ml 118 penicillin and 100 μg/ml streptomycin (PS). HCoV-EMC virus produced cytopathic effect 119 (CPE) within 1-2 days post-infection of Vero cells. SARS-CoV (strain HK39849) and HCoV-120 229E was propagated in Vero cells and MRC-5 cells respectively, as described previously 121 (10). Highly pathogenic avian influenza (HPAI) viruses cause severe human respiratory 122 disease and virus stocks of A/Hong Kong/483/1997 (H5N1) were prepared in MDCK cells in 123 MEM with no FBS and 1% PS. All the experiments were performed in a BSL-3 bio-124 containment facility at The University of Hong Kong. 125 126 Virus titration by 50% tissue culture infectious dose (TCID50) assay 127

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Confluent 96-well tissue culture plates of the respective cells were used for the virus titration 128 assay for HCoV-EMC and SARS-CoV (Vero cells), HCoV-229E (MRC-5 cells) or influenza 129 A H5N1 (MDCK cells). The cells were washed once with PBS and the Vero and MRC-5 130 cells were replenished with MEM with 2% FBS and 1% PS; while MDCK cells were 131 replenished with MEM with no FBS and 1% PS. Serial half-log10 dilutions (from 0.5 log to 7 132 log) of virus-infected culture supernatants were added onto the wells in quadruplicate. The 133 plates were observed for CPE daily, for five days. The viral dilution leading to CPE in 50% 134 of inoculated wells was estimated by using the Karber method and designated as one TCID50, 135 and used to compute the viral titer in the test sample. 136 137 Ex vivo organ cultures and infection 138 Fresh biopsies of human bronchus and lung parenchyma obtained from patients undergoing 139 surgical resection of lung tissue at Queen Mary Hospital as part of clinical care but surplus 140 for routine diagnostic requirements were used in this study. This study was approved by the 141 Institutional Review Board of the University of Hong Kong/ Hospital Authority Hong Kong 142 West Cluster. Ex vivo cultures of human bronchus and lungs were performed as previously 143 described (7, 8, 32). The bronchial mucosae were placed on surgical sponge with their apical 144 epithelial surface facing upwards while the lung parenchymal tissues were placed into the 24 145 well-plate directly with 1 ml of F12K culture medium and with 1 % PS at 37°C. Bronchial 146 and lung tissues were infected with HCoV-229E, HCoV-EMC and SARS-CoV with a viral 147 titer of 106 TCID50/ml for 1 h at 37°C and washed with 5 ml of warm PBS for three times to 148 remove unbound virus as previously described (7, 8, 32). UV inactivated virus and mock-149 infected cells were used as controls. Culture supernatants from the infected cultures were 150 collected at 1, 24, 48 and 72 hpi and titrated for infectious virus using the TCID50 assay for 151 HCoV-229E, HCoV-EMC and SARS-CoV as described previously (6, 10). Increasing virus 152 titers along over time provided evidence of productive virus replication. Tissues were 153 collected at 1, 24, 48 and 72 hpi for RNA extraction and at 24, 48 and 72 hpi for fixation in 154 10% formalin or 2.5% glutaraldehyde for immunohistochemistry and electron microscopy, 155 respectively. 156 157 In vitro cell culture and infection 158 Human alveolar epithelial cell line (A549) was cultured using DMEM with 10% FBS and 1% 159 PS and were seeded at 1 × 105 cells per well in 24-well tissue culture plates. The cells were 160 infected with HCoV-EMC, SARS-CoV and HPAI H5N1 virus at a multiplicity of infection 161

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(MOI) of 1. After 1 h of virus adsorption at 37°C, the virus inoculum was removed; the cells 162 were washed with PBS and replenished with fresh culture medium without FBS. Evidence of 163 viral infection and virus replication kinetics was determined by a) assaying viral RNA of 164 HCoV-EMC, SARS-CoV and influenza type A virus at 1, 24 and 48 hpi by quantitative RT-165 PCR and b) titrating infectious viral titers in infected culture supernatants to demonstrate 166 productive virus replication in Vero or MDCK cells, as appropriate. 167 168 Quantification of viral and host cytokine and chemokine mRNAs by quantitative RT-PCR 169 Bronchial and lung fragments were homogenized using a Tissueruptor (Qiagen, Hilden, 170 Germany) in 700 μl RLT lysis buffer with beta-mercaptoethanol on ice. A549 cell cultures 171 were lysed in 350 μl RLT lysis buffer with beta-mercaptoethanol. RNA extraction was 172 carried out using RNeasy Mini kit (Qiagen, Hilden, Germany) following manufacturer’s 173 instruction with the addition of DNase-treatment and eluted in 50μl RNase free water. 174 175 One step RT-PCR assay specific for the upstream of E gene of HCoV-EMC was adapted 176 from a recently described protocol (12). In brief, 5 μl of purified RNA was amplified in a 25-177 μl reaction containing 1 μl SSIII/Taq enzyme mix (Superscript III OneStep RT-PCR System 178 with Platinum Taq DNA polymerase (Invitrogen), 12.5 μl of 2× reaction buffer, 0.8 mM 179 MgSO4, 0.4 μM forward primer (upE-Fwd: 5′-GCAACGCGCGATTCAGTT-3′), 0.4 μM 180 reverse primer (upE-Rev: 5′-GCCTCTACACGGGACCCATA-3′), and 0.2 μM probe (upE-181 Prb: 5’-FAM-CTCTTCACATAATCGCCCCGAGCTCG-TAMRA-3’). Reactions were first 182 incubated at 55 °C for 20 min. After a 3 min denaturation at 95 °C, reactions were then 183 thermal-cycled for 40 cycles (94 °C for 15 sec, 58 °C for 30 sec). Total RNA harvested from 184 HCoV-EMC infected Vero cells and water were used as positive and negative controls, 185 respectively. The expression of HCoV-EMC gene was expressed as fold change when 186 compared to its expression at 1 hpi, using 2-ΔΔCT method as described previously (27). The 187 SARS-CoV nucleocapsid protein gene expression (33) and influenza virus matrix protein 188 gene expression were also quantified as previously described and expressed in terms of fold 189 change corresponding to the expression detected at 1 hpi. 190 191 The host gene expression profiles for proinflammatory cytokines (TNFα, IFNβ, IL-29), 192 chemokines (IP-10, MCP-1) and the housekeeping (β-actin) gene were detected in absolute 193 copy numbers, determined from a standard curve generated from a standard plasmid with a 194

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known copy number, which was included in the qPCR simultaneously, as previously 195 described (7, 9). The mRNA levels of selected genes were quantified by real-time qPCR 196 analysis with ABI 7500 Real-Time PCR System (Applied Biosystems). These gene 197 expressions were normalized by using the housekeeping gene product β-actin mRNA. 198 199 Generation of Antisera against BtCoV HKU 5-5 Nucleocapsid protein 200 The gene for BtCoV HKU 5 Nucleocapsid protein (gene bank Accession No: EF065512) was 201 inserted into the Venezuelan Equine Encephalitis (VEE) replicon plasmid pVR21 by overlap 202 polymerase chain reaction, the replicon particles (VRP-HKU5 N) were packaged, and titers 203 were determined on baby hamster kidney cells as previously described (16, 26). Five week 204 old balb/c mice (Harlan laboratories) were immunized with 105 Infectious Units (IU) of VRP- 205 HKU 5 N in 10µl volume, and 21 days later the mice were gain boosted with same dose of 206 antigen. Twenty-one days post boost, serum was collected by tail nick and used in 207 immunohistochemistry analysis. The BtCoV HKU5 N protein shares 68% identity to HCoV-208 EMC (40), and the antisera against the N protein was found to cross react with HCoV-EMC 209 nucleocapsid protein. 210 211 Immunohistochemistry 212 The 10% formalin fixed tissues were embedded with paraffin and stained using a polyclonal 213 mouse antibody raised against coronavirus HKU5 N glycoprotein (1:200) which cross reacts 214 with HCoV-EMC by Western Blot and immunofluorescence assays; mouse monoclonal 215 antibody against SARS-CoV nucleoprotein reactive with SARS-CoV at 1:50 dilution as 216 previously described (31); mouse monoclonal antibody 1E7 reactive with HCoV-229E at 217 1:100 dilution (provided by Dr Lia van der Hoek) and cleaved-caspase 3 staining (Cell 218 signaling, CST-9661S) as a marker of apoptotic cells. The reaction of the primary antibody 219 was revealed by the use of biotinylated goat anti-mouse (Jackson 115-065-146) antibody 220 (1:500) and developed using NovaRed substrate kit (Vector lab SK-4800). Antibodies to 221 CD68 for macrophages, AE1/AE3 for epithelial cells, β-tubulin (Sigma, F-2043) for ciliated 222 bronchial epithelial cells, MUC5AC (Life technology, 18-2261) for goblet cells and 223 podoplanin for type I pneumocytes were used for double labeling using immunofluorescence 224 with secondary antibody conjugated with FITC. HCoV-EMC was detected using the HKU5N 225 antisera and Vector Red substrate in these double-labeling experiments, as this substrate is 226 also fluorescent when using a TRITC filter. The stained preparations were examined using a 227 Nikon Ni immunofluorescence microscope and images captured using a SPOT Slider 2MP 228

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Camera. Double chromogen immunohistochemistry for caspase 3 was performed by first 229 detecting the viral antigen using an alkaline phosphatase conjugated streptavidin technique, 230 followed by ImmPRESS HRP (Vectorlab, MP-7401) for the cleaved caspase 3 (Cell 231 Signalling, 9661S). 232 233 Transmission Electron Microscopy 234 Bronchial and lung tissues were fixed in 2.5% glutaraldehyde, washed three times in PBS and 235 serially dehydrated. The tissues were post-fixed in 1% osmium tetroxide and embedded in 236 Araldite resin (Polysciences, Inc., Washington, PS, USA). Sections were examined with a 237 transmission electron microscope (Philips CM100). 238 Antiviral effect of interferon treatment in ex vivo human lung 239 Recombinant IFN-α (Invitrogen, PHC4014) and IFN-β (Invitrogen, PHC4244) were 240 reconstituted into 1000 U/ml in F12K medium with 1% PS. Pre-infection and post-infection 241 administration regimes were applied. For the pre-infection IFNs treatment, lung fragments 242 were pre-treated with either IFN-α or IFN-β 24 h prior to infection. The IFNs pre-incubated 243 lung tissue fragments were then infected with HCoV-EMC or SARS-CoV as described 244 above, and the infected culture was replenished with culture medium with the corresponding 245 IFNs after infection. For the post-infection treatment, untreated lung tissue fragments were 246 infected and at 1 hpi, the infected culture was replenished with medium containing 1000U/ml 247 of IFNs. Control lung cultures without any IFNs were used for comparison. Culture 248 supernatants from the infected lung explant cultures were collected at 1, 24, 48 and 72 hpi 249 and titrated for infectious virus using the TCID50 assay. 250 251 Statistical analysis 252 Experiments were performed independently with at least three different donors in duplicates. 253 Results shows in figures are the calculated mean and standard error of mean. Mock infected 254 tissues served as negative controls. The differences of log10-transformed viral titers among 255 different viruses at different time points post-infection and the quantitative cytokine and 256 chemokine mRNAs of coronavirus-infected cells were compared by using one-way analysis 257 of variance followed by a Bonferroni multiple-comparison test. Differences were considered 258 significant at a p < 0.05. The statistical analysis was performed using Graph-Pad Prism 5 259 software. 260 261

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Results 262 263 Infection and replication of HCoV-EMC, SARS-CoV and HCoV 229E in ex vivo 264 cultures of human bronchus and lung 265 In ex vivo cultures of human bronchus, HCoV-EMC infected and productively replicated with 266 a 2-log increase in viral titer from 1 hpi to 72 hpi (p < 0.005) (Figure 1B, 1D). SARS-CoV 267 infected bronchial tissue (Figure 1C) and showed less than 1 log increase in viral titer 1 hpi 268 to 24 hpi without reaching statistical significance (Figure 1D). HCoV-229E had no 269 detectable infection or replication in bronchial tissues (Figure 1A, 1D). In ex vivo cultures of 270 human lung, viral antigen was not detected in HCoV-229E inoculated tissues (Figure 1E) 271 and no productive replication was observed (Figure 1H) indicating that HCoV-229E does not 272 replicate in the human lung. Both HCoV-EMC (Figure 1F) and SARS-CoV (Figure 1G) 273 viruses extensively infected and replicated (Figure 1H) in the lung parenchymal tissues as 274 shown by the presence of viral antigen in these tissues along with approximately 2 log 275 increase in HCoV-EMC and SARS-CoV in viral load from 1 hpi to 48 hpi. 276 277 HCoV-EMC cell tropism in the human respiratory tract 278 In order to identify the target cells of HCoV-EMC virus in the human respiratory tract, we 279 performed co-staining of HCoV-EMC viral antigen (stained in red) with specific cell markers 280 (stained in green) in HCoV-EMC infected human bronchial and lung ex vivo culture tissues 281 (Figure 2). In bronchial tissues, we stained the ciliated cells (Figure 2A) and goblet cells 282 (Figure 2B) using β-tubulin and MUC5AC, respectively. The infected cells (in red) were not 283 co-localized with these two major cell types in bronchus and viral antigen was mainly found 284 in non-ciliated bronchial epithelial cells. 285 286 In lung, macrophages, epithelial cells, type I pneumocytes were stained with specific markers 287 CD68 (Figure 2C), AE1/AE3 (Figure 2D) and podoplanin (Figure 2E) respectively. HCoV-288 EMC did not co-stain with macrophages (Figure 2C) but there was overlapping of staining 289 with the AE1/AE3 marker (Figure 2D), which suggested that the principal target cells of 290 HCoV-EMC infection were of epithelial origin and focal co-localization was found in type I 291 pneumocytes (Figure 2E). 292 293 Cellular morphology and immunohistochemistry for viral antigen (nucleoprotein) in the 294 HCoV-EMC infected lung tissues (Figure 1F) gave additional information on cell types 295

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targeted by HCoV-EMC at 48 hpi. Endothelial cells within the medium size interstitial 296 vessels of the lung (Figure 2F) and the bronchiolar epithelial cells (Figure 2G) were found 297 to stain positive with the nucleoprotein of HCoV-EMC. The tropism of HCoV-EMC in lung 298 endothelial cells may suggest the possibility of extrapulmonary dissemination. 299 300 Electron microscopy shows budding HCoV-EMC virions in bronchial and alveolar 301 epithelial cells 302 To further confirm the sites of HCoV-EMC viral replication within the human lung, 303 transmission electron microscopy of the ex vivo infected bronchus and lung parenchymal 304 tissues was performed. Virion containing cells in the bronchus were flattened non-ciliated 305 bronchiolar-type epithelial cells (Figure 3A) and in the lung were type II pneumocytes with 306 visible lamellar bodies (Figure 3C). While virions were identified intracellularly, large 307 aggregates of virions in intracytoplasmic secretory vesicles were not conspicuous. 308 Ultrastructural examination of the bronchus and lung showed enveloped viral particles from 309 75 – 85 nm in diameter (Figure 3B and 3D). These were often associated with a rim of 310 spikes or corona measuring approximately 8 nm in length. 311 312 Extensive apoptosis in HCoV-EMC infected human lung ex vivo cultures 313 By immunohistochemistry, we found extensive expression of cleaved caspase 3, an apoptosis 314 marker, in ex vivo lung tissue infected with HCoV-EMC (Figure 4B) and SARS-CoV 315 (Figure 4C) but not in mock (Figure 4A) infected human lung tissue. In order to investigate 316 if the apoptosis was induced directly by coronavirus virus infection in the human lung, we 317 performed co-staining by immunohistochemistry of HCoV-EMC viral antigen (stained in 318 pink) with cleaved caspase 3 (stained in reddish brown) using HCoV-EMC (Figure 4D) and 319 SARS-CoV (Figure 4E) infected human lung ex vivo culture tissues. As showed in both 320 HCoV-EMC and SARS-CoV infected lung tissue, both revealed that the apoptotic cells were 321 not the viral protein expressing cells (Figure 4D and 4E) suggesting that paracrine 322 mechanisms may contribute to induction of apoptosis. 323 324 Viral and host gene expression upon HCoV-EMC infection in ex vivo cultures of 325 bronchus and lung and in vitro culture of A549 cells 326 Ex vivo bronchial and lung tissues from three donors were infected with HCoV-EMC and 327 RNA was extracted from infected cells at 1, 24, 48 and 72 hpi. Viral RNA was quantitated by 328 RT-PCR. Host mRNA expression of type I (IFN-β) and type III (IL-29) interferons and pro-329

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inflammatory cytokines and chemokines TNF-α and IP-10 was quantitated in HCoV-EMC or 330 mock infected bronchial and lung tissues. Viral gene expression increased by more than 331 2000-fold in bronchial cultures and more than 180-folds in lung cultures, with one donor 332 showing an exceptional 6000-fold increase in a lung culture (Figure 5A). However, 333 compared to mock infected cultures, HCoV-EMC infection in bronchus and lung failed to 334 induce IFN-β or TNF-α (Figure 5B and 5D). There was marginal induction of IL-29 in virus 335 infected lung cultures at 48 hpi (p < 0.05) when compared to mock infected tissue (Figure 336 5C) and higher IP-10 mRNA expression at 24 hpi compared with mock infected bronchial 337 tissue (p < 0.05) (Figure 5E). IL-1β, MCP-1 and RANTES mRNA were also similarly 338 quantified with no upregulation of these genes detected in bronchial or lung tissues infected 339 with HCoV-EMC (data not shown). Inactivation of the HCoV-EMC by ultraviolet irradiation 340 prior to infection of ex vivo bronchus and lung cultures completely abolished the viral 341 replication and any cytokine induction (data not shown). 342 343 In order to confirm the apparent lack of host IFN responses elicited by HCoV-EMC in the ex 344 vivo bronchus and lung cultures, we carried out further experiments quantitating viral RNA 345 and host type I and III IFNs, TNF-α and IP-10 mRNA in the alveolar epithelial cell line A549 346 infected with HCoV-EMC, SARS-CoV (non replicating negative control) and HPAI H5N1 347 (high cytokine inducing positive control) viruses or uninfected A549 cells (control). We 348 showed that HCoV-EMC and HPAI H5N1 virus replicated in A549 cells as demonstrated by 349 TCID50 assay and viral gene expression whilst SARS-CoV showed no evidence of replication 350 (Figure 6A and 6B). While influenza A H5N1 virus strongly induced IFN-β (Figure 6C), 351 TNF-α (Figure 6D) and IP-10 (Figure 6E) gene expression, none of these cytokines were 352 induced by HCoV-EMC infection of A549 cells. 353 354 IFNs inhibit HCoV-EMC replication in ex vivo lung cultures 355 HCoV-EMC and SARS-CoV productively replicated in ex vivo lung cultures (Figure 1H). 356 Using this ex vivo lung culture model, we investigated the effect of IFN-α or IFN-β treatment 357 commencing 1 hour after infection on viral replication. A significant decrease in virus 358 replication kinetics of HCoV-EMC was observed with approximately of 2-log decrease in 359 infectious viral titers at 48 hpi and 72 hpi (Figure 7A) which paralleled with a reduction in 360 viral gene-copy load assayed by quantitative RT-PCR assays (data not shown). IFN-α or IFN-361 β treatment also inhibited SARS-CoV replication in ex vivo lung organ cultures, but the effect 362 was less pronounced compared with HCoV-EMC infection (Figure 7B). In addition, the 363

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effect of IFN-α or IFN-β treatment 24 h prior to infection continued into the post-infection 364 period was also examined. This did not further enhance the antiviral effect on HCoV-EMC 365 replication (data not shown). 366 367 Discussion 368 We compared the tropism of the novel human coronavirus HCoV-EMC, SARS-CoV and 369 HCoV-229E on ex vivo cultures of the human lung and bronchus using 370 immunohistochemistry to indicate infection and by quantitating infectious viral titers from 1 371 to 72 hpi to indicate productive viral replication. HCoV-EMC and SARS-CoV infect and 372 productively replicate in ex vivo cultures of human lung. While both viruses also infect 373 bronchial epithelial cells as assessed by immunohistochemistry, there is no significant 374 increase in infectious viral titers of SARS-CoV in the bronchus ex vivo cultures while HCoV-375 EMC demonstrates evidence of productive replication. 376 377 Human infections associated with HCoV-EMC have so far clinically appeared to be relatively 378 severe, but it is unclear whether this reflects an ascertainment bias where milder disease goes 379 unrecognized. Future studies, including sero-epidemiological investigations are needed to 380 establish the true severity of this infection in human populations. The tropism and replication 381 competence of HCoV-EMC in human lung and bronchus demonstrated here suggests that 382 HCoV-EMC replicates at least as well, or even better than, SARS-CoV in the human lung 383 and bronchial tissues and targets type I and type II alveolar epithelial cells, highlighting the 384 potential threat posed by this novel virus. Type II alveolar epithelial cells are crucial in the 385 regeneration of the alveolar epithelium following injury by infection and a virus that targets 386 this cell type is likely to lead to significant lung pathology. Previous studies have shown that 387 HPAI H5N1 virus, one that is also known to cause severe primary viral pneumonia and acute 388 respiratory distress syndrome, also replicates efficiently in alveolar epithelium of ex vivo lung 389 cultures (32). In agreement with the milder clinical disease caused by HCoV-229E, HCoV-390 229E failed to infect or replicate in either bronchial or lung tissues. However, it is relevant to 391 note that not all viruses that can replicate in alveolar epithelium of the ex vivo cultures of lung 392 are consistently associated with severe respiratory disease (e.g. 2009 pandemic H1N1) (7). 393 Thus capacity for replication in alveolar epithelium in ex vivo cultures of lung appears to be a 394 necessary, but not sufficient correlate of disease severity. 395 396

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In previous studies with swine influenza viruses, we found that lack of tropism for ex vivo 397 cultures of the bronchus and nasopharynx correlate with lack of transmission in humans (8). 398 HCoV-229E, which is known to transmit efficiently in humans also failed to replicate 399 efficiently in ex vivo bronchial cultures, although infection and replication of it was 400 demonstrated in in vitro cultures of pseudostratified human airway epithelial model (20) and 401 well-differentiated normal human bronchial epithelial cells (6). “Common cold” 402 coronaviruses have been shown to infect the nasal mucosa of humans (2) and presumably 403 would replicate in ex vivo cultures of human nasopharynx or tonsil. Since ex vivo tissue of 404 human nasopharynx is less readily available, we have not so far obtained data from HCoV-405 229E, SARS-CoV or HCoV-EMC infection in such cultures. More systematic investigations 406 of HCoV-EMC in ex vivo nasopharyngeal cultures would provide further information on the 407 tropism for this virus in the upper respiratory tract providing insights into potential human 408 transmissibility or lack thereof. 409 410 Immunohistochemical analysis of virus infected ex vivo lung cultures also demonstrated that 411 endothelial cells within medium sized interstitial blood vessels of the lung were also targets 412 for HCoV-EMC infection. This may imply that the virus, as with SARS-CoV, may spread 413 systemically to affect distant organs. Thus the renal dysfunction that was repeatedly seen in 414 patients with HCoV-EMC may possibly be due to virus dissemination to the kidney although 415 there is no direct evidence of this at present. Further clinical studies are needed to address 416 whether viral dissemination does in fact occur and whether the renal dysfunction is due to 417 viral invasion of the kidneys. 418 419 Immunofluorescence study and transmission electron microscopy of infected ex vivo cultures 420 of lung and bronchus allowed us to further define the cell types targeted by HCoV-EMC 421 virus. Non-ciliated bronchial epithelial cells, bronchiolar epithelial cells and type I and type II 422 pneumocytes appear to be the major target for HCoV-EMC infection. We did not observe 423 virus infected alveolar macrophages in ex vivo lung cultures under these experimental 424 conditions. A preliminary study of human peripheral blood monocyte derived macrophages 425 also showed that these cells did not support the replication of the HCoV-EMC (unpublished 426 data). 427 428 Mechanisms that contribute to the pathogenesis of respiratory viruses such as SARS-CoV and 429 avian influenza A H5N1 include direct virus replication induced cell apoptosis, necrosis or 430

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autophagy; by-stander apoptosis or dysregulation of host innate inflammatory responses. We 431 found extensive apoptosis in SARS CoV and HCoV-EMC infected ex vivo lung tissues which 432 did not co-localize with viral antigen suggesting that both viruses can induce apoptosis via 433 paracrine mechanisms. Unlike influenza A H5N1 which induces a prominent pro-434 inflammatory cytokine response and SARS-CoV which induces a dysregulated cytokine 435 responses (i.e. poor IFNs response but potent proinflammatory chemokine responses), HCoV-436 EMC infection elicited poor pro-inflammatory chemokine and cytokine responses, including 437 poor type I and III IFN responses both in ex vivo lung cultures and in the alveolar epithelial 438 cell line A549. Thus, immuno-modulatory therapies that have been investigated in 439 experimental models for H5N1 disease (25, 29, 44) may have no clinical utility in HCoV-440 EMC infection. On the other hand, IFN therapy may be of potential benefit. SARS-CoV also 441 appeared to evade induction of IFNs responses although other pro-inflammatory cytokines 442 were potently induced (10) and it was found that a number of SARS-CoV proteins functioned 443 as IFN antagonists in vitro (21). IFNs therapy has been shown to have therapeutic potential in 444 SARS-CoV infections using in vitro (11) and non-human primate models (17) and some trend 445 towards clinical benefit was observed in human studies that were based on retrospective 446 controls rather than in randomized controlled clinical trials (28). Ex vivo cultures of human 447 lung have been used to demonstrate the therapeutic benefits of IFNs in HPAI H5N1 virus 448 infections (18). Therefore, we explored the antiviral effects of IFNs on HCoV-EMC infection 449 in human ex vivo lung culture model. 450 451 Both IFN alpha and beta significantly suppressed viral replication when added to ex vivo 452 cultures of human lung one hour post HCoV-EMC infection. IFNs treatment of the cells 24 h 453 prior to infection and continued into the post-infection period did not enhance the antiviral 454 effect on HCoV-EMC replication. As there are currently no antivirals for treatment of HCoV-455 EMC, these findings provide a therapeutic option for this serious disease. Further studies to 456 confirm the effect of IFNs using relevant animal models would be a priority to confirm the 457 utility of IFNs as therapeutic and/or prophylactic interventions for HCoV-EMC infection. 458 The biological basis for innate immune evasion by HCoV-EMC deserves investigation. 459 460 A recent study (published on-line, after the submission of our manuscript) has reported that 461 HCoV-EMC replicates more efficiently than SARS-CoV in a pseudostratified culture of 462 human airway epithelium (HAE) that morphologically and functionally resembles human 463 upper conducting airways in vitro. Their data pertained only to the bronchial region of the 464

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respiratory tract, and furthermore, the extent of HCoV-EMC replication in that in vitro HAE 465 model was more limited than we observe in the ex vivo bronchus or lung cultures. As with 466 our results, they showed that HCoV-EMC is a weak inducer of interferon of IFNs and that 467 IFNs can inhibit replication of HCoV-EMC in HAE cultures (20). Our results support and 468 extend those observations using human bronchial tissue cultured ex vivo, add information on 469 the tropism of HCoV-EMC in ex vivo lung and bronchial infection and demonstrate the effect 470 of interferon on human tissues infected with this virus. 471 472 In conclusion, this study illustrates the clinical utility of using ex vivo cultures of the human 473 respiratory system to investigate newly emerging respiratory viruses. There have so far been 474 no autopsy reports describing the virus induced pathology in the lung and such studies will 475 complement and confirm the data we report here. It must be noted that autopsy data even 476 when available often reflects the late-stage disease in patients who may have been kept alive 477 on mechanical ventilation for long periods of time. Thus studies with ex vivo experimental 478 infection of the human respiratory tract are invaluable to understand virus tropism and 479 pathogenesis as well as to provide evaluation of potential therapeutic options. 480 481

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Acknowledgement 482 Dr Lia van der Hoek in University of Amsterdam in Netherlands for sharing the mouse anti-483 HCoV-229E antibody to carry out the immunohistochemistry of this experiment. Mr Kevin 484 Fung in the Department of Pathology, The University of Hong Kong, Ms Sara SR Kang, SF 485 Sia, Icarus WW Chan, Christine BH Trang, Iris HY Ng, Chloe KS Wong, Drs. Kenrie PY 486 Hui and Francois Kien in the Centre of Influenza Research, School of Public Health, The 487 University of Hong Kong and Mr. WS Lee in the HKU Electron Microscope Unit for the 488 technical assistant of the experiment. This study was supported in part by research grants to 489 JSMP from the European Community Seventh Framework Program (FP7/2007-2013) under 490 project European management Platform for Emerging and Re-emerging Disease entities 491 (Grant agreement No. 223498) (EMPERIE), National Institute of Allergy and Infectious 492 Diseases (NIAID) contract HHSN266200700005C and the Research Fund for Control of 493 Infectious Diseases of the Health & Welfare Bureau of the Government of the Hong Kong 494 Special Administrative Region. RSB is supported by a Southeastern Regional Center of 495 Excellence Grant in Biodefense and grants from the National Institutes of Health (U54-496 AI057157; R01AI085524; RO1AI075297). 497 498 499 on A

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Figure legends 676 677 Figure 1. Tissue tropism of HCoV-EMC in human bronchus and lung. (A-C) Bronchi, 678 and (E-G) lung tissues were infected with HCoV-229E, HCoV-EMC and SARS-CoV. At 24 679 hpi, the tissues were fixed and tissue sections were stained for human coronaviruses N protein 680 (reddish brown) as described in materials and methods. Viral replication kinetics in ex vivo 681 cultures of (D) bronchi and (H) lung biopsies infected with 106 TCID50/ml of coronaviruses 682 by virus titration at 37°C. The bar chart showed the mean and the SE of mean of the virus 683 titer pooled from at least three independent experiments. Asterisks indicate a statistically 684 significant increase in viral yield when compare to 1 hpi. * : p < 0.05, ***p < 0.0005. 685 686 Figure 2. Cellular localization of HCoV-EMC in lung. HCoV-EMC stained in Vector Red 687 (Red) in bronchus tissue with cell marker conjugated with FITC (green) (A) B-tubulin 688 (ciliated cell marker), (B) MUC5AC (goblet cell marker) and in lung with (C) CD68 689 (macrophage marker) and (D) AE1/3 (epithelial cell marker), (E) Podoplanin (type I 690 pneumocyte marker) at 24hpi. (F) Cellular tropism of HCoV-EMC in lung. Human 691 coronavirus N protein (stained in reddish brown with red arrows) identified in endothelial 692 cells at 24 hpi and (G) bronchiolar epithelial cells at 48hpi. White arrows indicated cells with 693 co-staining. 694 695 Figure 3. Transmission electron microscopy locating the budding site of HCoV-EMC in 696 the human bronchus and lung. (A, B) Bronchial epithelial cells showed at 10500× and 697 92000× respectively and (C, D) alveolar epithelial cells showed at 21000× and 145000× 698 respectively, showing the budding of virions at 48 hpi. Black arrows indicate the type II 699 alveolar epithelial cells with lamellar bodies. 700 701 Figure 4. Apoptotic cell identified in the human lung ex vivo organ culture upon HCoV-702 EMC and SARS-CoV infection. Ex vivo culture of lung infected with (A) Mock, (B) HCoV-703 EMC and (C) SARS-CoV at 48 hpi, reddish-brown stain identifies the presence of cleaved-704 caspase 3. Co-staining of (D) HCoV-EMC and (E) SARS-CoV antigen (pink stain) with 705 cleaved-caspase 3 (reddish-brown stain). 706 707 Figure 5. Human HCoV-EMC viral gene expression and the major cytokine and 708 chemokine expression in bronchus and lung tissues in response to infection. The viral 709

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gene expression of (A) HCoV-EMC was expressed in fold change corresponding to the first 710 hour expression respectively, with grey bars representing the data from ex vivo bronchus and 711 black bars representing the data from ex vivo lung at 1, 24, 48 and 72 hour post infection (hpi) 712 from three donors. Gene expression of (B) IFN-β, (C) IL-29, (D) TNF-α and (E) IP-10 from 713 mock and infected ex vivo cultures at 1, 24, 48 and 72 hpi were plotted. The graph shows the 714 mean and standard error of mean (SEM) of mean mRNA copies expressed per 105 β-actin 715 copies from three representative experiments. * : p < 0.05. 716 717 Figure 6. Viral replication and cytokine and chemokine expression of HCoV-EMC in 718 A549 cells (A) A549 cells infected with HCoV-EMC, SARS-CoV and influenza H5N1 virus 719 (MOI of 1), Viral replication was determined by TCID50 assay. The bar chart showed the 720 mean and the SE of mean of the virus titer pooled from three independent experiments. 721 Asterisks indicate a statistically significant increase in viral yield when compare to 1 hpi. (B) 722 The viral gene expression of HCoV-EMC, SARS-CoV and influenza H5N1 as expressed in 723 fold change corresponding to the first hour expression respectively. (C) IFN-β, (D) TNF-α 724 and (E) IP-10 gene expression from mock and HCoV-EMC, SARS-CoV and influenza H5N1 725 infected A549 cells at 1, 24 and 48 hpi were plotted. The graph shows the mean and standard 726 error of mean (SEM) of mean mRNA copies expressed per 105 β-actin copies from three 727 representative experiments. ** : p < 0.05, ***p < 0.0005. 728 729 Figure 7. Interferon treatments suppressed HCoV-EMC and SARS CoV replication in 730 human lung ex vivo culture. The human lung ex vivo cultures were infected with 106 731 TCID50/ml of HCoV-EMC or SARS CoV for 1 h at 37°C. Control culture medium F12K + 732 1% PS (black bars), IFNα (white bars) and IFNβ (grey bars) at a concentration of 1000U/ml 733 in F12K + 1% PS were used as replenished medium 1hpi. Infected culture supernatants were 734 collected at 1, 24, 48 and 72 hpi and titrated in Vero cells. Virus replication kinetics of (A) 735 HCoV-EMC and (B) SARS-CoV were plotted to show the inhibitory effect on virus 736 replication by IFNs post treatment. Asterisks indicate a statistically significant at the same 737 time point between control and IFNs treatment, * : p < 0.05 738 739 740

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