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Contents lists available at ScienceDirect Antiviral Research journal homepage E ect 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 H S Li a Denise I T Kuok a M C Cheung a Mandy M T Ng a K C Ng a Kenrie P Y Hui a J S Malik Peiris a Michael C W Chan a John M Nicholls b a School of Public Health Li Ka Shing Faculty of Medicine The University of Hong Kong Hong Kong Special Administrative Region b Department of Pathology Li Ka Shing Faculty of Medicine The University of Hong Kong Queen Mary Hospital Pokfulam Hong Kong Special Administrative Region ARTICLE INFO Keywords Middle East Respiratory Syndrome Coronavirus MERS CoV Type I interferon Cyclosporine Ex vivo explants ABSTRACT Middle East Respiratory Syndrome Coronavirus MERS CoV has emerged as a coronavirus infection of humans in the past 5 years Though con ned 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 e ect 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 e ective than either agent used alone in reducing MERS CoV replication PCR SuperArray analysis showed that the re duction 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 1 Introduction The past 15 years has seen the emergence of two novel cor onaviruses 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 cor onavirus 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 rst identi ed in June 2012 from a 60 year old patient from Middle East who devel oped clinical symptoms and signs similar to SARS and who eventually died from multi organ failure Zaki et al 2012 A novel coronavirus was isolated initially called HCoV EMC but renamed as Middle East Respiratory Syndrome coronavirus MERS CoV de Groot et al 2013 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 Arabi et al 2017 Chan et al 2014 Perlman and McCray 2013 In May 2015 a large outbreak occurred in Korea Korean Society of Infectious Korean Society for Healthcare associated Infection and Prevention 2015 highlighting the threat to global public health security Previously we showed that MERS CoV replicate in human upper and lower respiratory tract where it infected non ciliated bronchial epi thelial cells bronchiolar epithelial cells type I and type II alveolar pneumocytes and endothelial cells using ex vivo explants culture Chan et al 2013 Furthermore we showed that in contrast to SARS CoV infection MERS CoV infection elicited a lower pro in ammatory cy tokine response including the type I and III interferons Chan et al 2013 This evasion of innate immune induction and reduced interferon IFN response suggested that exogenous IFN may be a possible treat ment options for human MERS CoV infection A previous study showed that pegylated IFN exhibited a more potent antiviral e ect to MERS CoV than SARS CoV in cell culture and macaque model de Wilde et al 2013 Falzarano et al 2013 and it was proposed that this was due to the lack of a MERS CoV homolog of SARS CoV ORF6 protein that blocks https doi org 10 1016 j antiviral 2018 05 007 Received 14 February 2018 Received in revised form 28 March 2018 Accepted 12 May 2018 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 Corresponding author Department of Pathology LKS Faculty of Medicine The University of Hong Kong L6 39 Laboratory Block Pokfulam Hong Kong Special Administrative Region E mail addresses mchan hku hk M C W Chan nicholls pathology hku hk J M Nicholls Antiviral Research 155 2018 89 96 0166 3542 2018 Elsevier B V All rights reserved T the IFN induced translocation of STAT1 a factor essential for signaling via the IFN receptor that leads to the induction of IFN associated an tiviral genes In the macaque model IFN was used together with riba virin and this led to improved clinical symptoms following MERS CoV infection with microarray analysis showing lower expression of in ammatory genes Falzarano et al 2013 Nevertheless a number of recent clinical studies reported that the IFN and ribavirin combination did not improve long term survival and was not bene cial to patients who received the treatment late after infection Al Taw q et al 2014 Omrani et al 2014 This indicates that there is a need to consider other therapeutic combination for treating MERS CoV infection Cyclosporine such as cyclosporin A CsA and its derivatives has been shown to inhibit MERS CoV replication in vitro de Wilde et al 2013 It has been demonstrated that CsA could restore type I IFN ex pression upon hepatitis C or rotavirus virus infection Liu et al 2011 Shen et al 2013 Combined use of CsA and type I IFN was shown to inhibit hepatitis C virus replication and trigger greater virological re sponse than IFNtreatment alone Henry et al 2006 Inoue et al 2003 As CsA has known immune suppressive function non im munosuppressive cyclophilin inhibitors have been tried in combination with ribavirin for MERS CoV infection Though these have an in vitro e ect on MERS CoV and SARS CoV this did not translate into a bene t in a mouse model de Wilde et al 2017 Here we report the individual and combined e ects of CsA and IFN 1 on inhibiting MERS CoV re plication in an in vitro and human lung and bronchus ex vivo explant culture model We found the combined use of CsA and IFN 1 had inhibitory e ects 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 2 Material and methods Ex vivo explants culture of human respiratory tract was obtained from patients undergoing surgery at Queen Mary Hospital according to previously established criteria Hui et al 2017 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 2 1 Ex vivo organ culture and infection 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 Ex vivo infections of human bronchus and lungs was performed as previously published Chan et al 2013 2014 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 1ml of culture medium at 37 C Human betacoronavirus of lineage C virus HCoV EMC was provided by R Fouchier Erasmus MC Fig 1 Evaluation of the changes in MERS CoV re plication after the addition of IFN 1 CsA and a combination of the two agents in Vero cells 9 M CsA and or 2 4 10 4 U ml of IFN 1 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 post hoc test P 0 05 P 0 01 P 0 001 Fig 2 Evaluation of the changes in MERS CoV replication after the addition of IFN 1 CsA and a combination of the two agents in A human bronchus and B human lung explant cultures 9 M CsA and or 2 4 10 4 U ml of IFN 1 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 post hoc test P 0 05 P 0 01 P 2 folds The red dots lie above the upper boundary line are the up regulated genes in EMC infected tissues with di erent 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 gure legend the reader is referred to the Web version of this article H S Li et al Antiviral Research 155 2018 89 96 92 Fig 6 Replication of MERS CoV with di erent treatments in human microvascular endothelial cells A 9 M CsA and or 2 4 10 4 U ml of IFN 1 were used E ect of IFN 1 and or CsA on protein expression level ofMERS 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 quanti ed by densitometry using ImageJ software normalized expression activation levels were indicated on the histogram 9 M CsA and or 2 4 10 4 U ml of IFN 1 were used H S Li et al Antiviral Research 155 2018 89 96 93 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 de signated as one TCID 50 ml 2 4 Quanti cation of viral and host cytokine and chemokine mRNAs by quantitative RT PCR Bronchial and lung fragments were homogenized using a TissueRuptor Qiagen Hilden Germany in 700 l RLT lysis bu er 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 l RNase free water 25ng of total RNA was used for the rst strand cDNA synthesis with PrimeScript RT reagent Kit System TaKaRa 2 5 Evaluation of interferon pathway pro les by superarray The expression of 84 key genes involved in cell signaling mediated by interferon receptors and ligands was pro led by RT PCR based RT 2 Pro ler Interferon and receptors Arrays Qiagen RT PCR reactions were performed in 96 well plate format using the ViiA7 Real Time PCR System Thermo Fisher Scienti c Fold changes of IFN gene expression in experimental samples relative to the control samples e g mock in fected were calculated using the Ct method The Ct value of each sample was normalized by up to a total of 5 housekeeping genes 2 microglobulin B2M hypoxanthine phosphoribosyltransferase 1 HPRT1 ribosomal protein L13a RPL13A glyceraldehyde 3 phos phate dehydrogenase GAPDH and actin ACTB All data was analyzed by the RT 2 Pro ler PCR Array Data Analysis Template v3 5 and all gene expression changes greater than 5 fold was considered signi cant and signi cant gene changes were con rmed by individual qPCR 2 6 Western blotting Cell lysate were prepared using RIPA bu er which were heated for 10min 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 di erent protein of in terest were detected by enhanced chemiluminescence method 2 7 Statistical analysis Experiments were performed independently with three di erent donors Results shown in gures 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 post hoc test between groups Statistical signi cance is de ned when p 0 05 3 Results 3 1 Interferon 1 and or cyclosporine A inhibit the replication of HCoV EMC Viral replication in Vero cells culture was determined with regimens of 9 M cyclosporine A and 2 4 10 4 U ml of IFN alfacon 1 separately or in combination Although there was a signi cant reduction of viral titres with IFN alfacon or CsA used separately cultures treated with both agents in combination had signi cantly lower viral titres at 24 48 and 72h when compared with cultures treated with a single drug as well as untreated cells Fig 1 While HCoV EMC replicated in untreated ex vivo 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 56h post infection Fig 2A and B In ex vivo cultures of bronchus Fig 2A combination of CsA and IFN alfacon 1 treatment had signi cantly lower viral titres compared to CsA or IFN treatment alone In the ex vivo lung cultures combination therapy or CsA therapy resulted in signi cantly lower viral titres than untreated cultures or IFN treatment alone Fig 2B How ever the e ect of CsA alone was comparable with the combination therapy These data suggested additive or synergistic e ects of IFN and CsA in limiting HCoV EMC replication in the bronchus 3 2 Immunohistochemistry for viral antigen and apoptotic cell death in interferon 1 and or cyclosporine A treated ex vivo cultures of human lung and bronchus Lung and bronchus explants tissues were collected and xed with 10 formalin after infection MERS CoV nucleocapsid protein NP was used as an indicator of HCoV EMC infectivity Fig 3 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 1 treatment could inhibit HCoV EMC infection and the level of inhibitory e ect in infection was greatest in the combined treatment group CsA alone inhibited the least HCoV EMC infection Furthermore in ex vivo lung explants infection HCoV EMC infection was signi cantly inhibited in all treatment groups when compared to the control treatment Next we determined the ability of di erent treatments to reduce cellular apoptosis induced by HCoV EMC infection by staining for cleaved caspase 3 Fig 4 Extensive staining of cleaved caspase 3 were found in the ex vivo bronchial and lung explants without treatment while the level of staining was reduced in all treatments groups in bronchus tissues IFN 1 and CsA combined treatment group had the greatest impact on reducing cleaved caspase 3 staining and the e ect by CsA alone was the lowest in bronchus tissues For lung tissues the level of apoptosis was minimal in both IFN 1 and CsA combined treated and CsA treated groups 3 3 Induction of interferon stimulated genes but not interferon receptors by the treatment of Interferon 1 and cyclosporine A After showing the ability of IFN 1 and CsA combined treatment in inhibiting HCoV EMC replication we investigated the potential anti viral mechanisms underlying such inhibition using an interferon and receptor PCR array The data showed that the combined treatment of IFN 1 and CsA had the most potent e ect on inducing interferon sti mulated genes ISGs in both lung 24 hpi and bronchial 56 hpi tis sues Fig 5A The combined treatment group also induced the ex pression of IFN beta 1 but not IFNAR1 2 and IFN gamma receptors in both lung and bronchus Fig 5B ISGs such as ISG15 MX1 IRF7 IFI44 IFI44L OAS1 SP110 IFIT1 IFIT2 IFIT3 and IFI27 were highly in duced and these data were veri ed in independent qPCR using lung and bronchus tissues Supplementary Figs 2 3 and in Vero E6 cell line Data not shown the induction of ISGs were signi cant compared with the no treatment group 3 4 Activation of pathways related to the increase in ISGs To evaluate the possible mechanisms related to the augmented ISG levels associated with IFN and CsA we further examined the phos phorylation level of STAT1 AKT mTOR and p38 MAPK which are the H S Li et al Antiviral Research 155 2018 89 96 94 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 in vitro and we have demonstrated that HCoV EMC could infect lung endothelial cells in our previous study Chan et al 2013 Our data showed that HMVEC L was highly susceptible to HCoV EMC in fection and the IFN 1 and CsA combined treatment group had the greatest impact on reducing HCoV EMC replication Fig 6A The treatment dosage was not cytotoxic to the cell we used Supplementary Fig 1B In line with the virus replication data we con rmed that the IFN 1 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 1 and CsA treated groups Fig 6B This suggested that the e ect of the combined treatment was independent of the JAK STAT pathway We next examined the p38 MAPK and AKT mTOR pathways Fig 6C 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 Fig 6C This indicated that the bene cial e ects of combination therapy may not be linked to the AKT mTOR translational control of ISGs 4 Discussion MERS continues to cause disease in the Arabian Peninsula with high case fatality in hospitalized patients There are still no proven speci c antiviral therapies for this disease Despite the apparent bene t of in terferon therapy in rhesus macaques and marmosets interferon therapy has not translated into clinical bene t in clinical cases of MERS re viewed in Al Taw q and Memish 2017 Arabi et al 2017 This may be in part related to the late presentation of patients compared to la boratory settings The lack of a good experimental animal model has further hampered progress on antiviral therapies for MERS Our ex vivo 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 ex vivo tissues In this study we nd that a combination of interferon with short term cy closporine Fig 2 may be worthy of pursuit in a clinical trial setting Previously these drugs have been used singly but not in combination de Wilde et al 20