MersV1, Pmc, Corpus, bibRecord, 000A66

Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model

Identifieur interne : 000A66 ( Pmc/Corpus ); précédent : 000A65; suivant : 000A67

Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model

Auteurs : H. S. Li ; Denise I. T. Kuok ; M. C. Cheung ; Mandy M. T. Ng ; K. C. Ng ; Kenrie P. Y. Hui ; J. S. Malik Peiris ; Michael C. W. Chan ; John M. Nicholls

Source :

Antiviral Research [ 0166-3542 ] ; 2018.

RBID : PMC:7113667

Abstract

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has emerged as a coronavirus infection of humans in the past 5 years. Though confined to certain geographical regions of the world, infection has been associated with a case fatality rate of 35%, and this mortality may be higher in ventilated patients. As there are few readily available animal models that accurately mimic human disease, it has been a challenge to ethically determine what optimum treatment strategies can be used for this disease. We used in-vitro and human ex-vivo explant cultures to investigate the effect of two immunomodulatory agents, interferon alpha and cyclosporine, singly and in combination, on MERS-CoV replication. In both culture systems the combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. PCR SuperArray analysis showed that the reduction of virus replication was associated with a greater induction of interferon stimulated genes. As these therapeutic agents are already licensed for clinical use, it may be relevant to investigate their use for therapy of human MERS-CoV infection.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113667

DOI: 10.1016/j.antiviral.2018.05.007
PubMed: 29772254
PubMed Central: 7113667

Links to Exploration step

PMC:7113667

Le document en format XML

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<front><div type="abstract" xml:lang="en"><p>Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has emerged as a coronavirus infection of humans in the past 5 years. Though confined to certain geographical regions of the world, infection has been associated with a case fatality rate of 35%, and this mortality may be higher in ventilated patients. As there are few readily available animal models that accurately mimic human disease, it has been a challenge to ethically determine what optimum treatment strategies can be used for this disease. We used in-vitro and human ex-vivo explant cultures to investigate the effect of two immunomodulatory agents, interferon alpha and cyclosporine, singly and in combination, on MERS-CoV replication. In both culture systems the combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. PCR SuperArray analysis showed that the reduction of virus replication was associated with a greater induction of interferon stimulated genes. As these therapeutic agents are already licensed for clinical use, it may be relevant to investigate their use for therapy of human MERS-CoV infection.</p>
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  <front><journal-meta><journal-id journal-id-type="nlm-ta">Antiviral Res</journal-id>
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</journal-title-group>
<issn pub-type="ppub">0166-3542</issn>
<issn pub-type="epub">1872-9096</issn>
<publisher><publisher-name>Elsevier B.V.</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">29772254</article-id>
<article-id pub-id-type="pmc">7113667</article-id>
<article-id pub-id-type="publisher-id">S0166-3542(18)30094-9</article-id>
<article-id pub-id-type="doi">10.1016/j.antiviral.2018.05.007</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="au1"><name><surname>Li</surname>
<given-names>H.S.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au2"><name><surname>Kuok</surname>
<given-names>Denise I.T.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au3"><name><surname>Cheung</surname>
<given-names>M.C.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au4"><name><surname>Ng</surname>
<given-names>Mandy M.T.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au5"><name><surname>Ng</surname>
<given-names>K.C.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au6"><name><surname>Hui</surname>
<given-names>Kenrie P.Y.</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au7"><name><surname>Peiris</surname>
<given-names>J.S.Malik</given-names>
</name>
<xref rid="aff1" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="au8"><name><surname>Chan</surname>
<given-names>Michael C.W.</given-names>
</name>
<email>mchan@hku.hk</email>
<xref rid="aff1" ref-type="aff">a</xref>
<xref rid="cor1" ref-type="corresp">∗</xref>
</contrib>
<contrib contrib-type="author" id="au9"><name><surname>Nicholls</surname>
<given-names>John M.</given-names>
</name>
<email>nicholls@pathology.hku.hk</email>
<xref rid="aff2" ref-type="aff">b</xref>
<xref rid="cor2" ref-type="corresp">∗∗</xref>
</contrib>
</contrib-group>
<aff id="aff1"><label>a</label>
School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region</aff>
<aff id="aff2"><label>b</label>
Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region</aff>
<author-notes><corresp id="cor1"><label>∗</label>
Corresponding author. School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, L6-39, Laboratory Block, Pokfulam, Hong Kong Special Administrative Region. <email>mchan@hku.hk</email>
</corresp>
<corresp id="cor2"><label>∗∗</label>
Corresponding author. Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, L6-39, Laboratory Block, Pokfulam, Hong Kong Special Administrative Region. <email>nicholls@pathology.hku.hk</email>
</corresp>
</author-notes>
<pub-date pub-type="pmc-release"><day>17</day>
<month>5</month>
<year>2018</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
      <pub-date pub-type="ppub"><month>7</month>
<year>2018</year>
</pub-date>
<pub-date pub-type="epub"><day>17</day>
<month>5</month>
<year>2018</year>
</pub-date>
<volume>155</volume>
<fpage>89</fpage>
<lpage>96</lpage>
<history><date date-type="received"><day>14</day>
<month>2</month>
<year>2018</year>
</date>
<date date-type="rev-recd"><day>28</day>
<month>3</month>
<year>2018</year>
</date>
<date date-type="accepted"><day>12</day>
<month>5</month>
<year>2018</year>
</date>
</history>
<permissions><copyright-statement>© 2018 Elsevier B.V. All rights reserved.</copyright-statement>
<copyright-year>2018</copyright-year>
<copyright-holder>Elsevier B.V.</copyright-holder>
<license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p>
</license>
</permissions>
<abstract id="abs0010"><p>Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has emerged as a coronavirus infection of humans in the past 5 years. Though confined to certain geographical regions of the world, infection has been associated with a case fatality rate of 35%, and this mortality may be higher in ventilated patients. As there are few readily available animal models that accurately mimic human disease, it has been a challenge to ethically determine what optimum treatment strategies can be used for this disease. We used in-vitro and human ex-vivo explant cultures to investigate the effect of two immunomodulatory agents, interferon alpha and cyclosporine, singly and in combination, on MERS-CoV replication. In both culture systems the combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. PCR SuperArray analysis showed that the reduction of virus replication was associated with a greater induction of interferon stimulated genes. As these therapeutic agents are already licensed for clinical use, it may be relevant to investigate their use for therapy of human MERS-CoV infection.</p>
</abstract>
<abstract abstract-type="author-highlights" id="abs0015"><title>Highlights</title>
<p><list list-type="simple" id="ulist0010"><list-item id="u0010"><label>•</label>
<p id="p0010">The effect of interferon-α and/or cyclosporine on MERS-CoV replication was evaluated with a human ex-vivo culture model.</p>
</list-item>
<list-item id="u0015"><label>•</label>
<p id="p0015">All treatments were able to reduce MERS-CoV replication.</p>
</list-item>
<list-item id="u0020"><label>•</label>
<p id="p0020">The combined treatment was more effective than either agent used alone in reducing MERS-CoV replication.</p>
</list-item>
<list-item id="u0025"><label>•</label>
<p id="p0025">The effect of the combined treatment group was associated with a greater induction of interferon stimulated genes.</p>
</list-item>
</list>
</p>
</abstract>
<kwd-group id="kwrds0010"><title>Keywords</title>
<kwd>Middle East Respiratory Syndrome Coronavirus (MERS-CoV)</kwd>
<kwd>Type I interferon</kwd>
<kwd>Cyclosporine</kwd>
<kwd>Ex vivo explants</kwd>
</kwd-group>
</article-meta>
</front>
<body><sec id="sec1"><label>1</label>
<title>Introduction</title>
<p id="p0030">The past 15 years has seen the emergence of two novel coronaviruses that have infected humans resulting in a high morbidity and mortality. Until 2003 it was thought that coronavirus infections, such as OC43 and 229E were associated with mild respiratory disease and there was little reason to develop novel therapeutic options for these coronavirus infections. The global outbreak of SARS in 2003 with a 10% fatality rate, and the recent emergence of MERS-CoV infection, as well as other recently recognized coronavirus infections such as NL63 and HKU1 have demonstrated the need for investigation into treatment options of severe coronavirus infections. MERS-CoV was first identified in June 2012 from a 60 year old patient from Middle East who developed clinical symptoms and signs similar to SARS, and who eventually died from multi organ failure (<xref rid="bib24" ref-type="bibr">Zaki et al., 2012</xref>
). A novel coronavirus was isolated, initially called HCoV-EMC but renamed as Middle East Respiratory Syndrome coronavirus (MERS-CoV) (<xref rid="bib6" ref-type="bibr">de Groot et al., 2013</xref>
). Since 2012 the virus has continued to cause severe zoonotic human disease in the Middle East, sometimes associated with outbreaks of human-to-human transmission within health care facilities (<xref rid="bib3" ref-type="bibr">Arabi et al., 2017</xref>
; <xref rid="bib5" ref-type="bibr">Chan et al., 2014</xref>
; <xref rid="bib20" ref-type="bibr">Perlman and McCray, 2013</xref>
). In May 2015 a large outbreak occurred in Korea (<xref rid="bib15" ref-type="bibr">Korean Society of Infectious, Korean Society for Healthcare-associated Infection and Prevention, 2015</xref>
), highlighting the threat to global public health security.</p>
<p id="p0035">Previously we showed that MERS-CoV replicate in human upper and lower respiratory tract where it infected non-ciliated bronchial epithelial cells, bronchiolar epithelial cells, type I and type II alveolar pneumocytes and endothelial cells using <italic>ex vivo</italic>
 explants culture (<xref rid="bib4" ref-type="bibr">Chan et al., 2013</xref>
). Furthermore, we showed that in contrast to SARS-CoV infection, MERS-CoV infection elicited a lower pro-inflammatory cytokine response including the type I and III interferons (<xref rid="bib4" ref-type="bibr">Chan et al., 2013</xref>
). This evasion of innate immune induction and reduced interferon (IFN) response suggested that exogenous IFN may be a possible treatment options for human MERS-CoV infection. A previous study showed that pegylated IFN exhibited a more potent antiviral effect to MERS-CoV than SARS-CoV in cell culture and macaque model (<xref rid="bib8" ref-type="bibr">de Wilde et al., 2013</xref>
; <xref rid="bib9" ref-type="bibr">Falzarano et al., 2013</xref>
) and it was proposed that this was due to the lack of a MERS-CoV homolog of SARS-CoV ORF6 protein that blocks the IFN induced translocation of STAT1 - a factor essential for signaling via the IFN receptor that leads to the induction of IFN associated antiviral genes. In the macaque model, IFN was used together with ribavirin and this led to improved clinical symptoms following MERS-CoV infection, with microarray analysis showing lower expression of inflammatory genes (<xref rid="bib9" ref-type="bibr">Falzarano et al., 2013</xref>
). Nevertheless, a number of recent clinical studies reported that the IFN and ribavirin combination did not improve long-term survival, and was not beneficial to patients who received the treatment late after infection (<xref rid="bib2" ref-type="bibr">Al-Tawfiq et al., 2014</xref>
; <xref rid="bib19" ref-type="bibr">Omrani et al., 2014</xref>
). This indicates that there is a need to consider other therapeutic combination for treating MERS-CoV infection.</p>
<p id="p0040">Cyclosporine, such as cyclosporin A (CsA) and its derivatives has been shown to inhibit MERS-CoV replication <italic>in vitro</italic>
 (<xref rid="bib8" ref-type="bibr">de Wilde et al., 2013</xref>
). It has been demonstrated that CsA could restore type I IFN expression upon hepatitis C or rotavirus virus infection (<xref rid="bib18" ref-type="bibr">Liu et al., 2011</xref>
; <xref rid="bib23" ref-type="bibr">Shen et al., 2013</xref>
). Combined use of CsA and type I IFN was shown to inhibit hepatitis C virus replication and trigger greater virological response than IFN treatment alone (<xref rid="bib10" ref-type="bibr">Henry et al., 2006</xref>
; <xref rid="bib12" ref-type="bibr">Inoue et al., 2003</xref>
). As CsA has known immune suppressive function, non-immunosuppressive cyclophilin inhibitors have been tried in combination with ribavirin for MERS-CoV infection. Though these have an <italic>in vitro</italic>
 effect on MERS-CoV and SARS-CoV, this did not translate into a benefit in a mouse model (<xref rid="bib7" ref-type="bibr">de Wilde et al., 2017</xref>
). Here, we report the individual and combined effects of CsA and IFN-α1 on inhibiting MERS-CoV replication in an <italic>in vitro</italic>
 and human lung and bronchus <italic>ex vivo</italic>
 explant culture model. We found the combined use of CsA and IFN-α1 had inhibitory effects on MERS-CoV infection and replication, as well as on the induction of interferon stimulated genes (ISG), which sheds light on the potential use of this combination in curing MERS-CoV infection.</p>
</sec>
<sec id="sec2"><label>2</label>
<title>Material and methods</title>
<p id="p0045"><italic>Ex vivo</italic>
 explants culture of human respiratory tract was obtained from patients undergoing surgery at Queen Mary Hospital, according to previously established criteria (<xref rid="bib11" ref-type="bibr">Hui et al., 2017</xref>
). We selected areas of morphologically normal lung, and histology was performed on a control sample. The samples were subjected to virus culture and bacterial culture. If there was intrinsic disease or infection in the resected lung specimens, they were not used for research. The project was approved by the local institutional review board (UW 14-549).</p>
<sec id="sec2.1"><label>2.1</label>
<title>Ex vivo organ culture and infection</title>
<p id="p0050">Fresh biopsies of human bronchi and lung were sampled from human lungs that were removed at surgery as part of clinical care, but surplus for routine diagnostic requirements. <italic>Ex vivo</italic>
 infections of human bronchus and lungs was performed as previously published (<xref rid="bib4" ref-type="bibr">Chan et al., 2013</xref>
, <xref rid="bib5" ref-type="bibr">2014</xref>
). In brief, the bronchial mucosae were placed on a surgical sponge with their apical epithelial surface facing upwards while the lung parenchymal tissues were placed into 24 well-plates directly with 1 ml of culture medium at 37 °C. Human betacoronavirus of lineage C virus (HCoV-EMC) was provided by R. Fouchier, Erasmus MC, Rotterdam, the Netherlands, and used as the prototype MERS-CoV for examining the efficacy of different treatments. Fresh bronchial and lung tissues were infected with HCoV-EMC with a viral titer of 10<sup>6</sup>
 TCID<sub>50</sub>
/ml for 1 h at 37 °C and then washed with 5 ml of PBS at room temperature for three times to remove unbound virus. Fresh culture medium with a regimen of 9 μM cyclosporine A (Novartis Pharmaceuticals Corporation) and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN alfacon-1 (Kadmon pharmaceuticals) was then added to the cultures, and the treatments were replenished in a 16-h interval. Supernatants from the infected cultures were collected at 1, 8, 24, 40 56 hpi and titrated for infectious virus using the TCID<sub>50</sub>
 assay for HCoV-EMC. Increasing virus titers over time provided evidence of productive virus replication. Tissues were collected at 24, and 56 hpi for RNA extraction and fixation in 10% formalin for immunohistochemical staining of MERS-CoV nucleocapsid protein (NP) using an antibody provided by Dr. R. Baric, and cleaved caspase 3 (Cell Signaling Technology). Vero cells were incubated with alpha-interferon and/or cyclosporine, for determination of optimal inhibition before application to <italic>ex vivo</italic>
 samples (<xref rid="appsec1" ref-type="sec">Supplementary Fig. 1</xref>
).</p>
</sec>
<sec id="sec2.2"><label>2.2</label>
<title>Cell cultures</title>
<p id="p0055">Vero (ATCC CCL-81) cell line was cultured in Minimum Essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 units/mL of penicillin and 100 μg/mL of streptomycin. Human lung microvascular endothelial cells were prepared from fresh human lung by selection of CD31<sup>+</sup>
 cells using anti-human CD31 antibody (Abcam) and Dynabeads cell separation system (ThermoFisher). The isolated cells were maintained in EGM-2 medium supplemented with 5% FBS (Lonza). All cells used in this study were maintained at 37 °C with 5% CO<sub>2</sub>
 humidified atmosphere.</p>
</sec>
<sec id="sec2.3"><label>2.3</label>
<title>Viral replication kinetics by 50% tissue culture infectious dose (TCID<sub>50</sub>
/ml) titration</title>
<p id="p0060">Confluent 96-well tissue culture plates of Vero E6 cells were prepared 1 days before the virus titration assay. Cells were washed once with PBS and replenished with DMEM with 2% FBS, 100 units/mL of penicillin and 100 μg/mL of streptomycin. Serial half-log10 dilutions (from 0.5 log to 7 log) of virus-infected culture supernatants was added onto the wells in quadruplicate. The plates were observed for CPE daily, for seven days. The endpoint of viral dilution leading to CPE in 50% of inoculated wells was estimated by using the Karber method and designated as one TCID<sub>50</sub>
/ml.</p>
</sec>
<sec id="sec2.4"><label>2.4</label>
<title>Quantification of viral and host cytokine and chemokine mRNAs by quantitative RT-PCR</title>
<p id="p0065">Bronchial and lung fragments were homogenized using a TissueRuptor (Qiagen, Hilden, Germany) in 700 μl RLT lysis buffer with beta-mercaptoethanol on ice. RNA extraction was carried out using RNeasy Mini kit (Qiagen, Hilden, Germany) following manufacturer's instruction with the addition of DNase-treatment and eluted in 50 μl RNase free water. 25 ng of total RNA was used for the first-strand cDNA synthesis with PrimeScript RT reagent Kit System (TaKaRa).</p>
</sec>
<sec id="sec2.5"><label>2.5</label>
<title>Evaluation of interferon pathway profiles by superarray</title>
<p id="p0070">The expression of 84 key genes involved in cell signaling mediated by interferon receptors and ligands was profiled by RT-PCR-based RT<sup>2</sup>
 Profiler Interferon and receptors Arrays (Qiagen). RT-PCR reactions were performed in 96-well plate format using the ViiA7 Real-Time PCR System (Thermo Fisher Scientific). Fold changes of IFN gene expression in experimental samples relative to the control samples (e.g. mock-infected) were calculated using the ΔΔCt method. The ΔΔCt value of each sample was normalized by up to a total of 5 housekeeping genes (β2-microglobulin [B2M], hypoxanthine phosphoribosyltransferase 1 [HPRT1], ribosomal protein L13a [RPL13A], glyceraldehyde-3-phosphate dehydrogenase [GAPDH] and β-actin [ACTB]). All data was analyzed by the RT<sup>2</sup>
 Profiler PCR Array Data Analysis Template v3.5 and all gene expression changes greater than 5 fold was considered significant, and significant gene changes were confirmed by individual qPCR.</p>
</sec>
<sec id="sec2.6"><label>2.6</label>
<title>Western blotting</title>
<p id="p0075">Cell lysate were prepared using RIPA buffer, which were heated for 10 min at 95 °C. The protein were then resolved in SDS-PAGE and transferred to PVDF membrane. Mouse anti-actin (Millipore) and rabbit anti-MERS-CoV nucleocapsid protein (kindly provided by Dr. R. Baric) were used as loading and infection controls. Other proteins of interest, including phosphor-STAT1 (Tyr701), STAT1, phosphor-AKT (Ser473), AKT, phosphor-mTOR (Ser2448), mTOR, phosphor-p38 (Thr180/Tyr182), and p38, were also detected (all from Cell Signaling Technology). The membranes were incubated with the respective HRP conjugated secondary antibody, and signals of different protein of interest were detected by enhanced chemiluminescence method.</p>
</sec>
<sec id="sec2.7"><label>2.7</label>
<title>Statistical analysis</title>
<p id="p0080">Experiments were performed independently with three different donors. Results shown in figures included the calculated mean and standard error of mean. Comparison among three or more groups was analyzed using two-way analysis of variance (ANOVA) with Bonferroni's multiple-comparisons as <italic>post-hoc</italic>
 test between groups. Statistical significance is defined when <italic>p</italic>
 < 0.05.</p>
</sec>
</sec>
<sec id="sec3"><label>3</label>
<title>Results</title>
<sec id="sec3.1"><label>3.1</label>
<title>Interferon-α1 and/or cyclosporine A inhibit the replication of HCoV-EMC</title>
<p id="p0085">Viral replication in Vero cells culture was determined with regimens of 9 μM cyclosporine A and 2.4 × 10<sup>4</sup>
 U/ml of IFN alfacon-1 separately or in combination. Although there was a significant reduction of viral titres with IFN-alfacon or CsA used separately, cultures treated with both agents in combination had significantly lower viral titres at 24, 48 and 72 h when compared with cultures treated with a single drug as well as untreated cells (<xref rid="fig1" ref-type="fig">Fig. 1</xref>
).<fig id="fig1"><label>Fig. 1</label>
<caption><p>Evaluation of the changes in MERS-CoV replication after the addition of IFN-α1, CsA and a combination of the two agents in Vero cells. 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni's <italic>post hoc</italic>
 test (*/#<italic>P</italic>
 < 0.05, **/##<italic>P</italic>
 < 0.01, ***/###<italic>P</italic>
 < 0.001).</p>
</caption>
<alt-text id="alttext0030">Fig. 1</alt-text>
<graphic xlink:href="gr1_lrg"></graphic>
</fig>
</p>
<p id="p0090">While HCoV-EMC replicated in untreated <italic>ex vivo</italic>
 lung and bronchial tissues, titres in the bronchus were higher than those in the lung, especially at 40 and 56 hpi. Similar to the replication in cell lines, viral replication was reduced by all treatments at 40 and 56 h post-infection (<xref rid="fig2" ref-type="fig">Fig. 2</xref>
A and B). In <italic>ex vivo</italic>
 cultures of bronchus (<xref rid="fig2" ref-type="fig">Fig. 2</xref>
A), combination of CsA and IFN alfacon-1 treatment had significantly lower viral titres compared to CsA or IFN treatment alone. In the <italic>ex vivo</italic>
 lung cultures, combination therapy or CsA therapy resulted in significantly lower viral titres than untreated cultures or IFN treatment alone (<xref rid="fig2" ref-type="fig">Fig. 2</xref>
B). However, the effect of CsA alone was comparable with the combination therapy. These data suggested additive or synergistic effects of IFN and CsA in limiting HCoV-EMC replication in the bronchus.<fig id="fig2"><label>Fig. 2</label>
<caption><p>Evaluation of the changes in MERS-CoV replication after the addition of IFN-α1, CsA and a combination of the two agents in (A) human bronchus and (B) human lung explant cultures. 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. The data shown are the mean ± standard error of the mean in at least three independent experiments, which are analyzed by two-way ANOVA followed by Bonferroni's <italic>post hoc</italic>
 test (*/#<italic>P</italic>
 < 0.05, **/##<italic>P</italic>
 < 0.01, ***/###<italic>P</italic>
 < 0.001).</p>
</caption>
<alt-text id="alttext0035">Fig. 2</alt-text>
<graphic xlink:href="gr2_lrg"></graphic>
</fig>
</p>
</sec>
<sec id="sec3.2"><label>3.2</label>
<title>Immunohistochemistry for viral antigen and apoptotic cell death in interferon-α1 and/or cyclosporine A treated <italic>ex vivo</italic>
 cultures of human lung and bronchus</title>
<p id="p0095">Lung and bronchus explants tissues were collected and fixed with 10% formalin after infection. MERS-CoV nucleocapsid protein (NP) was used as an indicator of HCoV-EMC infectivity (<xref rid="fig3" ref-type="fig">Fig. 3</xref>
). Extensive HCoV-EMC infection was found in both bronchial ciliated and non-ciliated cells and alveolar pneumocytes without any treatment. In bronchial explants, it has been found that IFN-α1 treatment could inhibit HCoV-EMC infection, and the level of inhibitory effect in infection was greatest in the combined treatment group; CsA alone inhibited the least HCoV-EMC infection. Furthermore, in <italic>ex vivo</italic>
 lung explants infection, HCoV-EMC infection was significantly inhibited in all treatment groups when compared to the control treatment.<fig id="fig3"><label>Fig. 3</label>
<caption><p>IFN-α1 and/or CsA inhibited MERS-CoV infection in bronchus & lung explant culture. 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. Sections of infected human bronchus and lung were stained with a polyclonal antibody against the MERS-CoV nucleocapsid protein. Positive cells were identified with a reddish brown colour. Magnification: x200. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p>
</caption>
<alt-text id="alttext0040">Fig. 3</alt-text>
<graphic xlink:href="gr3_lrg"></graphic>
</fig>
</p>
<p id="p0100">Next, we determined the ability of different treatments to reduce cellular apoptosis induced by HCoV-EMC infection by staining for cleaved caspase 3 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>
). Extensive staining of cleaved caspase 3 were found in the <italic>ex vivo</italic>
 bronchial and lung explants without treatment, while the level of staining was reduced in all treatments groups in bronchus tissues. IFN-α1 and CsA combined treatment group had the greatest impact on reducing cleaved caspase 3 staining, and the effect by CsA alone was the lowest in bronchus tissues. For lung tissues, the level of apoptosis was minimal in both IFN-α1 and CsA combined treated and CsA treated groups.<fig id="fig4"><label>Fig. 4</label>
<caption><p>IFN-α1 and/or CsA reduced apoptosis caused by MERS-CoV infection in bronchus & lung explant culture. 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. Sections of infected human bronchus and lung were stained with a monoclonal anti-cleaved caspase 3 antibody. Positive cells were identified with a Vector Red staining (pink colour). Magnification: x200. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p>
</caption>
<alt-text id="alttext0045">Fig. 4</alt-text>
<graphic xlink:href="gr4_lrg"></graphic>
</fig>
</p>
</sec>
<sec id="sec3.3"><label>3.3</label>
<title>Induction of interferon stimulated genes but not interferon receptors by the treatment of Interferon-α1 and cyclosporine A</title>
<p id="p0105">After showing the ability of IFN-α1 and CsA combined treatment in inhibiting HCoV-EMC replication, we investigated the potential antiviral mechanisms underlying such inhibition using an interferon and receptor PCR array. The data showed that the combined treatment of IFN-α1 and CsA had the most potent effect on inducing interferon-stimulated genes (ISGs) in both lung (24 hpi) and bronchial (56 hpi) tissues (<xref rid="fig5" ref-type="fig">Fig. 5</xref>
A). The combined treatment group also induced the expression of IFN beta-1 but not IFNAR1, 2 and IFN gamma receptors in both lung and bronchus (<xref rid="fig5" ref-type="fig">Fig. 5</xref>
B). ISGs such as ISG15, MX1, IRF7, IFI44, IFI44L, OAS1, SP110, IFIT1, IFIT2, IFIT3, and IFI27 were highly induced and these data were verified in independent qPCR using lung and bronchus tissues (<xref rid="appsec1" ref-type="sec">Supplementary Figs. 2–3</xref>
), and in Vero E6 cell line (Data not shown), the induction of ISGs were significant compared with the no treatment group.<fig id="fig5"><label>Fig. 5</label>
<caption><p>Combined treatment of IFN-α1 and CsA induced highest level of interferon stimulated genes (ISGs). 9 μM CsA and 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. Distribution of gene expression was shown in the clustergram (A), the X axis indicates the normalized expression level of EMC infected lung or bronchus tissue without treatment, and the Y axis indicates the normalized expression level of EMC infected lung or bronchus tissue with different treatments; each dot on the plot represents the corresponding expression of a gene, the central line indicates no differences in gene level between two groups, while the boundary lines indicates the fold-change threshold (>=2 folds). The red dots lie above the upper boundary line are the up-regulated genes in EMC infected tissues with different treatments as compared to EMC infected tissues without treatment; and the green dots in lower section of the plot are the down-regulated genes. Gene expression of the combined treatment group in human bronchus (B) and lung (C) explant culture relative to the no treatment group (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p>
</caption>
<alt-text id="alttext0050">Fig. 5</alt-text>
<graphic xlink:href="gr5_lrg"></graphic>
</fig>
</p>
</sec>
<sec id="sec3.4"><label>3.4</label>
<title>Activation of pathways related to the increase in ISGs</title>
<p id="p0110">To evaluate the possible mechanisms related to the augmented ISG levels associated with IFN and CsA, we further examined the phosphorylation level of STAT1, AKT, mTOR, and p38 MAPK, which are the molecules linked to the transcriptional activation of interferon-sensitive response element (ISRE). We then performed these experiments in primary human cells in addition to continuous cell-lines. Therefore, we used human lung microvascular endothelial cell (HMVEC-L) which has been associated with extra-pulmonary dissemination of HCoV-EMC, as HCoV-EMC does not replicate well in primary human alveolar epithelial cells <italic>in vitro</italic>
 and we have demonstrated that HCoV-EMC could infect lung endothelial cells in our previous study (<xref rid="bib4" ref-type="bibr">Chan et al., 2013</xref>
). Our data showed that HMVEC-L was highly susceptible to HCoV-EMC infection and the IFN-α1 and CsA combined treatment group had the greatest impact on reducing HCoV-EMC replication (<xref rid="fig6" ref-type="fig">Fig. 6</xref>
A). The treatment dosage was not cytotoxic to the cell we used (<xref rid="appsec1" ref-type="sec">Supplementary Fig. 1B</xref>
). In line with the virus replication data, we confirmed that the IFN-α1 and CsA treated group was more potent at reducing HCoV-EMC NP expression than either IFN or CsA alone. STAT1 expression and phosphorylation were comparably increased in the IFN treated and combine IFN-α1 and CsA treated groups (<xref rid="fig6" ref-type="fig">Fig. 6</xref>
B). This suggested that the effect of the combined treatment was independent of the JAK-STAT pathway. We next examined the p38 MAPK and AKT/mTOR pathways (<xref rid="fig6" ref-type="fig">Fig. 6</xref>
C). The phosphorylation level of p38 in the combined treatment group was the lowest, which was similar to the CsA treated group. On the contrary, phosphorylation level of AKT at the s473 was the lowest in the combined treatment group, and the phosphorylation level of mTOR were comparable among all groups (<xref rid="fig6" ref-type="fig">Fig. 6</xref>
C). This indicated that the beneficial effects of combination therapy may not be linked to the AKT/mTOR translational control of ISGs.<fig id="fig6"><label>Fig. 6</label>
<caption><p>Replication of MERS-CoV with different treatments in human microvascular endothelial cells (A), 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used. Effect of IFN-α1 and/or CsA on protein expression level of MERS-CoV nucleocapsid and phosphorylation/activation of STAT1 protein (B); phosphorylation/activation level of protein involve in PI3K/Akt/mTOR and p38 MAPK pathways (C) were shown. Bands from three independent experiments were quantiﬁed by densitometry using ImageJ software, normalized expression/activation levels were indicated on the histogram. 9 μM CsA and/or 2.4 × 10<sup>4</sup>
 U/ml of IFN-α1 were used.</p>
</caption>
<alt-text id="alttext0055">Fig. 6</alt-text>
<graphic xlink:href="gr6_lrg"></graphic>
</fig>
</p>
</sec>
</sec>
<sec id="sec4"><label>4</label>
<title>Discussion</title>
<p id="p0115">MERS continues to cause disease in the Arabian Peninsula with high case fatality in hospitalized patients. There are still no proven specific antiviral therapies for this disease. Despite the apparent benefit of interferon therapy in rhesus macaques and marmosets, interferon therapy has not translated into clinical benefit in clinical cases of MERS (reviewed in (<xref rid="bib1" ref-type="bibr">Al-Tawfiq and Memish, 2017</xref>
; <xref rid="bib3" ref-type="bibr">Arabi et al., 2017</xref>
). This may be in part related to the late presentation of patients compared to laboratory settings. The lack of a good experimental animal model has further hampered progress on antiviral therapies for MERS. Our <italic>ex vivo</italic>
 cultures of the human bronchus and lung provides an alternative and complementary option for investigating therapeutic agents but still have a limitation in severe CoV infection studies, because patients may present to a health care setting 5 or more days after exposure, which is currently beyond our ability to maintain the <italic>ex vivo</italic>
 tissues. In this study, we find that a combination of interferon with short term cyclosporine (<xref rid="fig2" ref-type="fig">Fig. 2</xref>
) may be worthy of pursuit in a clinical trial setting. Previously, these drugs have been used singly but not in combination (<xref rid="bib8" ref-type="bibr">de Wilde et al., 2013</xref>
). The <italic>ex vivo</italic>
 cultures showed a significant decrease in virus replication when this combination treatment was used compared to single treatment and this was also reflected in the greater number of ISG upregulated, compared to mock or single treatments. This increase in ISG may thus dampen the potential immune suppressive effect of CsA if used as a single agent. The combined treatment of type I interferon with cyclosporine also mitigated the extent of apoptosis caused by HCoV-EMC in our study (<xref rid="fig4" ref-type="fig">Fig. 4</xref>
). As different therapeutic modalities are considered for evaluation in clinical trials, we suggest that IFN and CsA combination therapy should be considered. A recent review has also noted the need to evaluate combination therapies (<xref rid="bib1" ref-type="bibr">Al-Tawfiq and Memish, 2017</xref>
).</p>
<p id="p0120">CsA is a small molecule immunosuppressant while IFN-α1 is an immuno-stimulating protein favoring cell conversion to an antiviral state. Since the mechanism underlying the increment of ISG by this combination is unclear, we tried to identify the possible mechanisms behind the higher induction levels of ISG in the combined treatment of type I IFN and CsA over the other treatment groups. In canonical type I IFN signaling, IFN-α/β activates the JAK-STAT pathway in which STAT1 is an essential member to form the ISGF3 complex, which binds to the ISRE and controls the ISG expression (<xref rid="bib13" ref-type="bibr">Ivashkiv and Donlin, 2014</xref>
). Since the activation level of STAT1 in the combined treatment was similar or even slightly lower than the IFN-α treatment alone (<xref rid="fig6" ref-type="fig">Fig. 6</xref>
A), we believe that the extra ISG induction could be the result of the other signaling cascades independent of the JAK-STAT signaling. Therefore, we tried to determine whether the combined treatment would have effects on the AKT/mTOR pathway, which in turn has translational controls on the ISGs (<xref rid="bib14" ref-type="bibr">Kaur et al., 2008</xref>
; <xref rid="bib16" ref-type="bibr">Kroczynska et al., 2009</xref>
; <xref rid="bib22" ref-type="bibr">Saleiro et al., 2015</xref>
). Our results showed that mTOR activation was not affected by the combined treatment; and the decreased phosphorylation level of AKT in the combined treatment group may be related to the other signaling cascades that linked to AKT, thereby it was not likely that AKT/mTOR was involved in the enhanced ISGs levels by the double treatment. Furthermore, we also examined the p38 MAPK activation level, which also linked to the ISRE separately (<xref rid="bib21" ref-type="bibr">Platanias, 2005</xref>
; <xref rid="bib22" ref-type="bibr">Saleiro et al., 2015</xref>
). From our results, HCoV-EMC induced p38 phosphorylation (<xref rid="bib17" ref-type="bibr">Lim et al., 2016</xref>
) was reduced due to the effect from CsA, which may contribute to the inhibition of MER-CoV replication levels, while this result did not match with the elevated ISG level. Therefore, it is likely that CsA and type I IFN combination therapy uses alternative pathways to enhance antiviral effect and in enhancing ISG induction by acting directly or indirectly on the ISRE (<xref rid="appsec1" ref-type="sec">Supplementary Fig. 4</xref>
).</p>
<p id="p0125">In conclusion, we have demonstrated that CsA and IFN-α1 is a potent therapeutic combination for inhibiting HCoV-EMC replication <italic>in vitro</italic>
 and <italic>ex vivo</italic>
, which can result in higher ISG expression. This combination may be worth considering in future clinical trials.</p>
</sec>
<sec id="sec5"><title>Declarations of interest</title>
<p id="p0130">None.</p>
</sec>
</body>
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<sec id="appsec1" sec-type="supplementary-material"><label>Appendix A</label>
<title>Supplementary data</title>
<p id="p0140">The following are the supplementary data related to this article:<fig id="dfig1" position="anchor"><label>figs1</label>
<caption><p>The trend of cell survival by MTT assay. Regimens of 9 μM CsA and/or 2.4 x 10<sup>4</sup>
 U/ml of IFN-α was applied on Vero cells (A); and human lung microvascular endothelial cells (B).</p>
</caption>
<alt-text id="alttext0010">figs1</alt-text>
<graphic xlink:href="mmcfigs1_lrg"></graphic>
</fig>
<fig id="dfig2" position="anchor"><label>figs2</label>
<caption><p>Verification of ISG expression using qPCR in human bronchus tissue (A). The corresponding MERS-CoV UpE level is also shown (B). The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni’s <italic>post hoc</italic>
 test (*<italic>P</italic>
<0.05, **<italic>P</italic>
<0.01, ***<italic>P</italic>
<0.001 ).</p>
</caption>
<alt-text id="alttext0015">figs2</alt-text>
<graphic xlink:href="mmcfigs2_lrg"></graphic>
</fig>
<fig id="dfig3" position="anchor"><label>figs3</label>
<caption><p>Verification of ISG expression using qPCR in human lung tissue (A). The corresponding MERS-CoV UpE level is also shown (B).The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni’s <italic>post hoc</italic>
 test (*<italic>P</italic>
<0.05, **<italic>P</italic>
<0.01, ***<italic>P</italic>
<0.001 ).</p>
</caption>
<alt-text id="alttext0020">figs3</alt-text>
<graphic xlink:href="mmcfigs3_lrg"></graphic>
</fig>
<fig id="dfig4" position="anchor"><label>figs4</label>
<caption><p>Schematic diagram of the proposed pathways that may be involved in the IFN-α and/or CsA control of ISGs transcription and translation.</p>
</caption>
<alt-text id="alttext0025">figs4</alt-text>
<graphic xlink:href="mmcfigs4_lrg"></graphic>
</fig>
</p>
</sec>
<ack id="ack0010"><title>Acknowledgements</title>
<p>We thank Kevin Fung of the Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong for the technical assistance on the immunohistochemical staining; we also acknowledge Louisa LY Chan and Christine HT Bui at the School of Public Health, The University of Hong Kong for their technical contributions. Research funding was provided by the <funding-source id="gs1">Health and Medical Research Fund</funding-source>
 (Ref: 14131052) from the Research Fund Secretariat, Food and Health Bureau, Hong Kong Special Administrative Region; US National Institute of Allergy and Infectious Diseases (NIAID) under <funding-source id="gs2">CEIRS</funding-source>
 contract HHSN27220140006C, and the <funding-source id="gs3">Theme Based Research Scheme</funding-source>
 (T11-705/14-N) by the Research Grants Council of the Hong Kong Special Administrative Region.</p>
</ack>
<fn-group><fn id="appsec2" fn-type="supplementary-material"><label>Appendix A</label>
<p id="p0145">Supplementary data related to this article can be found at <ext-link ext-link-type="doi" xlink:href="10.1016/j.antiviral.2018.05.007" id="intref0010">https://doi.org/10.1016/j.antiviral.2018.05.007</ext-link>
.</p>
</fn>
</fn-group>
</back>
</pmc>
</record>

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Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model

Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model

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