Middle East Respiratory Syndrome Coronavirus Replication Research Articles

Evaluation of Predicted siRNA as an Antiviral against MERS-CoV Targeting the Membrane Gene in the Vero Cell Line

The Middle East respiratory syndrome coronavirus (MERS-CoV) was first isolated from a patient with acute pneumonia and renal failure in Saudi Arabia in 2012. By July 2023, MERS-CoV had resulted in 2605 human cases worldwide, causing a fatality rate of 36%, with 90.2% of cases being located in the Arabian Peninsula. The dromedary camel (Camelus dromedarius) is presumed to be an intermediate host for viral transmission to humans. So far, no prophylactic vaccines or effective antiviral treatments have been approved for MERS-CoV. RNA silencing is a novel approach for treating several diseases. A web-based bioinformatics tool (i-Score Designer) with integrative computational methods was used to predict and evaluate the designed siRNAs. This approach enabled the targeting of a highly conserved region of the MERS-CoV membrane (M) gene to inhibit virus replication. siRNA-M1, -M2, and -M3 were selected as the best of 559 designed siRNA candidates for an in vitro validation based on 2nd generation algorithm scoring, thermodynamic properties, off-target filtration, position-specific nucleotide preferences, and a free immune-stimulatory motifs. siRNAs were evaluated in Vero cells for their cytotoxicity and antiviral efficacy in vitro. Our results showed that the predicted siRNAs had no apparent cytotoxicity observed in Vero cells. The obtained results from the plaque reduction assay and RT-qPCR indicated that siRNA-M3 was the best candidate to inhibit MERS-CoV replication with a defined concentration of 400 picoMolar (pM). The computational methods used, and the in vitro evaluation, may provide an insight for a new antiviral strategy against MERS-CoV, a further in vivo study will nevertheless be required.

Interleukin 13-Induced Inflammation Increases DPP4 Abundance but Does Not Enhance Middle East Respiratory Syndrome Coronavirus Replication in Airway Epithelia.

Chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine interleukin 13 (IL-13), examining how this affected DPP4 protein levels with MERS-CoV entry and replication. IL-13 exposure for 3 days led to greater DPP4 protein abundance, while a 21-day treatment raised DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly affected by IL-13 treatment. Our results suggest that greater DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13-induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.

Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease.

An overview of the recent progress in Middle East Respiratory Syndrome Coronavirus (MERS-CoV) drug discovery

ABSTRACT Introduction The Middle East respiratory syndrome coronavirus (MERS-CoV) has remained a public health concern since it first emerged in 2012. Although many potential treatments for MERS-CoV have been developed and tested, none have had complete success in stopping the spread of this deadly disease. MERS-CoV replication comprises attachment, entry, fusion and replication steps. Targeting these events may lead to the creation of medications that effectively treat MERS-CoV infection. Areas covered This review updates the research on the development of inhibitors of MERS-CoV. The main topics are MERS-CoV‒related proteins and host cell proteins that are involved in viral protein activation and infection. Expert opinion Research on discovering drugs that can inhibit MERS-CoV started at a slow pace, and although efforts have steadily increased, clinical trials for new drugs specifically targeting MERS-CoV have not been extensive enough. The explosion in efforts to find new medications for the SARS-CoV-2 virus indirectly enhanced the volume of data on MERS-CoV inhibition by including MERS-CoV in drug assays. The appearance of COVID-19 completely transformed the data available on MERS-CoV inhibition. Despite the fact that new infected cases are constantly being diagnosed, there are currently no approved vaccines for or inhibitors of MERS-CoV.

Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines

Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the orf1ab region of the virus genome because of its high sequence conservation and vital role in virus replication. The designed siRNAs were used for experimental evaluation in selected cell lines: Vero cells, HEK-293-T, and Huh-7. Virus inhibition was assessed according to the cycle threshold value during a quantitative real-time polymerase chain reaction. Out of 462 potential siRNAs, we filtered out 21 based on specific selection criteria without off-target effect. The selected siRNAs did not show any cellular toxicity in the tested cell lines at various concentrations. Based on our results, it was obvious that the combined use of siRNAs exhibited a reduction in MERS-CoV replication in the Vero, HEK-293-T, and Huh-7 cell lines, with the highest efficacy displayed in the Vero cells.

HnRNP C modulates MERS-CoV and SARS-CoV-2 replication by governing the expression of a subset of circRNAs and cognitive mRNAs

ABSTRACT Host circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA–mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.

Inter-individual Variation in Receptor Expression Influences MERS-CoV Infection and Immune Responses in Airway Epithelia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory infection in humans, with symptom severity that ranges from asymptomatic to severe pneumonia. Known risk factors for severe MERS include male sex, older age, and the presence of various comorbidities. MERS-CoV gains entry into cells by binding its receptor, dipeptidyl peptidase 4 (DPP4), on the surface of airway epithelia. We hypothesized that expression of this receptor might be an additional determinant of outcomes in different individuals during MERS-CoV infection. To learn more about the role of DPP4 in facilitating MERS-CoV infection and spread, we used ELISA and immunofluorescent staining to characterize DPP4 expression in well-differentiated primary human airway epithelia (HAE). We noted wide inter-individual variation in DPP4 abundance, varying by as much as 1000-fold between HAE donors. This variability appears to influence multiple aspects of MERS-CoV infection and pathogenesis, with greater DPP4 abundance correlating with early, robust virus replication and increased cell sloughing. We also observed increased induction of interferon and some interferon-stimulated genes in response to MERS-CoV infection in epithelia with the greatest DPP4 abundance. Overall, our results indicate that inter-individual differences in DPP4 abundance are one host factor contributing to MERS-CoV replication and host defense responses, and highlight how HAE may serve as a useful model for identifying risk factors associated with heightened susceptibility to serious respiratory pathogens.

Development of A MERS-CoV Replicon Cell Line for Antiviral Screening.

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00341-z) contains supplementary material, which is available to authorized users.

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus via interferon-λ in vitro and in mice

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.

The SKI complex is a broad-spectrum antiviral drug target

Starting from a yeast suppressor screening platform, we have identified the SKI complex as a potential broad-spectrum antiviral target. We found that the NS1 protein of influenza A virus (IAV) and the ORF4a protein of Middle East respiratory syndrome coronavirus (MERS-CoV), which both function to bind double-strand RNA and inhibit cellular interferon responses, cause a slow growth phenotype when expressed in yeast. Knockout of the components of the yeast SKI complex caused a loss of this slow growth phenotype, suggesting a functional link between the viral proteins and the SKI complex. The SKI complex is a helicase that unwinds double-strand RNA and sends it to the RNA exosome for degradation. We next investigated whether the highly conserved human SKI complex was important for replication of IAV and MERS-CoV. RNAi based experiments showed that both viruses were inhibited when the SKI complex was removed, suggesting the complex has a proviral role in replication. Through in silico modelling using the published crystal structure of the SKI complex, we looked for potential binding pockets for chemical compounds. We screened a selection of these compounds for antiviral activity and have found four different chemicals capable of inhibiting IAV infection. Our most studied of these also inhibits not only MERS-CoV, but also Ebolavirus Makona. Our data suggests the SKI complex may be a target for broad-spectrum antiviral therapy and we have multiple chemical structures from which to work to develop therapeutic approaches.

The influence of interferon-lambda on restricting Middle East Respiratory Syndrome Coronavirus replication in the respiratory epithelium

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory in human with high mortality and it has been a challenge to determine optimum treatment for MERS-CoV-induced respiratory infection. Here, we observed the distribution of MERS-CoV receptors using human respiratory mucosa and also evaluated the contribution of interferon-lambdas (IFN-λs) in response to MERS-CoV infection using in vitro normal human nasal epithelial (NHNE) and bronchial epithelial (NHBE) cells. We found that the gene and protein expression of DPPIV, MERS-CoV receptor, were more dominantly located in nasal and bronchial epithelium although human nasal mucosa exhibited relatively lower DPPIV expression than lung parenchymal tissues. The quantitative mRNA level of the MERS-CoV envelope (upE) gene was significantly induced in MERS-CoV-infected cultured NHNE and NHBE cells until 3 days after infection. The induction of IFNs was identified in NHNE and NHBE cells after MERS-CoV infection and IFN-λs were predominantly increased in MERS-CoV-infected respiratory epithelial cells. Inoculation of IFN-λs to NHNE and NHBE cells suppressed MERS-CoV replication and in particular, IFN-λ4 showed a strong therapeutic effect in reducing MERS-CoV infection with higher induction of IFN-stimulated genes. Thus, IFN-λ has a decisive function in the respiratory epithelium that greatly limits MERS-CoV replication, and may be a key cytokine for better therapeutic outcomes against MERS-CoV infection in respiratory tract.

Compounds with Therapeutic Potential against Novel Respiratory 2019 Coronavirus.

Currently, the expansion of the novel human respiratory coronavirus (known as SARS-CoV-2 [severe acute respiratory syndrome coronavirus 2], COVID-2019 [coronavirus disease 2019], or 2019-nCoV [2019 novel coronavirus]) has stressed the need for therapeutic alternatives to alleviate and stop this new epidemic. The previous epidemics of infections by high-morbidity human coronaviruses, such as SARS-CoV in 2003 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, prompted the characterization of compounds that could be potentially active against the currently emerging novel coronavirus, SARS-CoV-2. The most promising compound is remdesivir (GS-5734), a nucleotide analog prodrug currently in clinical trials for treating Ebola virus infections. Remdesivir inhibited the replication of SARS-CoV and MERS-CoV in tissue cultures, and it displayed efficacy in nonhuman animal models. In addition, a combination of the human immunodeficiency virus type 1 (HIV-1) protease inhibitors lopinavir/ritonavir and interferon beta (LPV/RTV-IFN-β) was shown to be effective in patients infected with SARS-CoV. LPV/RTV-IFN-β also improved clinical parameters in marmosets and mice infected with MERS-CoV. Remarkably, the therapeutic efficacy of remdesivir appeared to be superior to that of LPV/RTV-IFN-β against MERS-CoV in a transgenic humanized mouse model. The relatively high mortality rates associated with these three novel human coronavirus infections, SARS-CoV, MERS-CoV, and SARS-CoV-2, have suggested that proinflammatory responses might play a role in the pathogenesis. It remains unknown whether the generated inflammatory state should be targeted. Therapeutics that target the coronavirus alone might not be able to reverse highly pathogenic infections. This minireview aims to provide a summary of therapeutic compounds that have shown potential in fighting SARS-CoV-2 infections.

Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection

The continued emergence of Middle East Respiratory Syndrome (MERS) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. Remdesivir (GS-5734) effectively inhibited MERS coronavirus (MERS-CoV) replication in vitro, and showed efficacy against Severe Acute Respiratory Syndrome (SARS)-CoV in a mouse model. Here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of MERS-CoV infection, the rhesus macaque. Prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented MERS-CoV-induced clinical disease, strongly inhibited MERS-CoV replication in respiratory tissues, and prevented the formation of lung lesions. Therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. The data presented here support testing of the efficacy of remdesivir treatment in the context of a MERS clinical trial. It may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-nCoV.

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

A Yeast Suppressor Screen Used To Identify Mammalian SIRT1 as a Proviral Factor for Middle East Respiratory Syndrome Coronavirus Replication.

Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we used the yeast Saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a accessory gene is expressed in yeast it causes a slow-growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow-growth phenotype as a reporter of ORF4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the YDL042C/SIR2 yeast gene as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation, there was reduced MERS-CoV replication, suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-κB signaling, demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of Saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV ORF4a proteins and the YDL042C/SIR2 yeast gene. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.

Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication.

Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro.

The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

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

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.

MERS-CoV pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) presents an emerging threat to public health worldwide by causing severe respiratory disease in humans with high virulence and case fatality rate (about 35%) since 2012. Little is known about the pathogenesis and innate antiviral response in primary human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) upon MERS-CoV infection. In this study, we assessed MERS-CoV replication as well as induction of inflammatory cytokines and chemokines in MDMs and immature and mature MDDCs. Immature MDDCs and MDMs were permissive for MERS-CoV infection, while mature MDDCs were not, with stimulation of proinflammatory cytokine and chemokine upregulation in MDMs, but not in MDDCs. To further evaluate the antiviral activity of well-defined drugs in primary antigen presenting cells (APCs), three compounds (chloroquine, chlorpromazine and toremifine), each with broad-spectrum antiviral activity in immortalized cell lines, were evaluated in MDMs and MDDCs to determine their antiviral effect on MERS-CoV infection. While chloroquine was not active in these primary cells, chlorpromazine showed strong anti-MERS-CoV activity, but it was associated with high cytotoxicity narrowing the potential window for drug utilization. Unlike in established cells, toremifene had marginal activity when tested in antigen presenting cells, with high apparent cytotoxicity, also limiting its potential as a therapeutic option. These results demonstrate the value of testing drugs in primary cells, in addition to established cell lines, before initiating preclinical or clinical studies for MERS treatment and the importance of carefully assessing cytotoxicity in drug screen assays. Furthermore, these studies also highlight the role of APCs in stimulating a robust protective immune response to MERS-CoV infection.

Pathogenicity and Viral Shedding of MERS-CoV in Immunocompromised Rhesus Macaques.

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged in the Middle East. Since 2012, there have been approximately 2,100 confirmed cases, with a 35% case fatality rate. Disease severity has been linked to patient health status, as people with chronic diseases or an immunocompromised status fare worse, although the mechanisms of disease have yet to be elucidated. We used the rhesus macaque model of mild MERS to investigate whether the immune response plays a role in the pathogenicity in relation to MERS-CoV shedding. Immunosuppressed macaques were inoculated with MERS-CoV and sampled daily for 6 days to assess their immune statues and to measure viral shedding and replication. Immunosuppressed macaques supported significantly higher levels of MERS-CoV replication in respiratory tissues and shed more virus, and virus disseminated to tissues outside of the respiratory tract, whereas viral RNA was confined to respiratory tissues in non-immunosuppressed animals. Despite increased viral replication, pathology in the lungs was significantly lower in immunosuppressed animals. The observation that the virus was less pathogenic in these animals suggests that disease has an immunopathogenic component and shows that inflammatory responses elicited by the virus contribute to disease.