Dengue virus causes changes of MicroRNA-genes regulatory network revealing potential targets for antiviral drugs
- Mohamed Shahen†1, 2, 3,
- Zihu Guo†1, 2,
- Akhtar Hussain Shar†1, 2,
- Reham Ebaid4,
- Qin Tao1, 2,
- Wenjuan Zhang1, 2,
- Ziyin Wu1, 2,
- Yaofei Bai1, 2,
- Yingxue Fu1, 2,
- Chunli Zheng1, 2,
- He Wang1,
- Piar Ali Shar1, 2,
- Jianling Liu5,
- Zhenzhong Wang6,
- Wei Xiao6Email author and
- Yonghua Wan1, 2Email author
© The Author(s). 2018
Received: 10 May 2017
Accepted: 23 November 2017
Published: 4 January 2018
Dengue virus (DENV) is an increasing global health threat and associated with induction of both a long-lived protective immune response and immune-suppression. So far, the potency of treatment of DENV via antiviral drugs is still under investigation. Recently, increasing evidences suggest the potential role of microRNAs (miRNAs) in regulating DENV. The present study focused on the function of miRNAs in innate insusceptible reactions and organization of various types of immune cells and inflammatory responses for DENV. Three drugs were tested including antiviral herbal medicine ReDuNing (RDN), Loratadine (LRD) and Acetaminophen.
By the microarray expression of miRNAs in 165 Patients. Results showed that 89 active miRNAs interacted with 499 potential target genes, during antiviral treatment throughout the critical stage of DENV. Interestingly, reduction of the illness threats using RDN combined with LRD treatment showed better results than Acetaminophen alone. The inhibitions of DENV was confirmed by decrease concentrations of cytokines and interleukin parameters; like TNF-α, IFN-γ, TGF-β1, IL-4, IL-6, IL-12, and IL-17; after treatment and some coagulants factors increased.
This study showed a preliminary support to suggest that the herbal medicine RDN combined with LRD can reduce both susceptibility and the severity of DENV.
Dengue fever disease (DF)is a mosquito-borne disease caused by Dengue virus (DENV) which has prominence in the past 20 years with an expanded geographic distribution of both the viruses and the mosquitoes . Its spread rate was estimated as 390 million infections annually worldwide, and it is most prevalent in the developing countries . DF is one of the mosquito-borne diseases which causes death all round equatorial and sub equatorial countries. The propagation of several life-threatening pathogens in human is caused by DF disease . DF is caused by Dengue virus (DENV), a mosquito-borne flavivirus. DENV is a single positive-strand RNA virus of the family Flaviviridae, genus Flavivirus. Its innate immune system towards a variety of diseases in humans .
The symptoms of DF patients suffering dengue infection commonly exist during three to 7 days of hard flu-like sickness inclusive rise fever, nausea, puke, headache and body soreness . After that viral infection innate and adaptive immune response are essential for the survival of the host infected by the DENV, the virus infection induce the immune system to generate type I IFN and proinflammatory cytokines parameters . The host cell starts to defend against viral invasion through an appropriate innate immune response. Response to viral infection must be tightly regulated to defend rapidly against it, while minimizing inflammatory damage, that leads to cellular resistance to viral infection .
Several clinical trials were conducted to treat DENV and reduce the impact and symptoms, but until now no any licensed medicine could deal with DF. However, supportive care with a sedative, the replacement of fluids and bed rest can be useful in some cases [5, 8] recommended that a dosage of Acetaminophen 1 g every 8 h for 3 days dealing with DENV might be useful, while Loratadine (LRD) tablets is an orally effective medicine (ethyl4-(8-chloro-5,6-dihydro-11H-benzo, cyclohepta [1,2-b] pyridin-11-ylidene)-1-iperidinecarboxylate) , that could be used to treat antihistamine and relieve other allergy symptoms . Additionnaly, ReDuNing (RDN) medicine a patented traditional antipyretic-detoxicate Chinese injection medicine, has been widely used as an anti-inflammatory and anti-infectious drug in Chinese clinics . Dealing with the severity of dengue demands taking caution toward bodily fluids exchanges, while using proactive therapy for bleeding . RDN medicine is composed of three herbs, Artemisiae annuae L. (genus Artemisia, Asteraceae), Gardenia jasminoides J.Ellis (genus Gardenia, Rubiaceae) and Lonicera japonica Thunb. (genus Lonicera, Caprifoliaceae) . RDN injection medicine exhibits promising effects against enterovirus 71 in Vero monkey cells . From our previous study, some methods were experimentally established to combat inflammation and provide a systems-based strategy to find the therapeutic mechanism of traditional Chinese medicine RDN dealing with the disease .
Nowadays miRNAs (miRNAs) has carried out numerous cautions and applications because several experimental studies have concluded that miRNAs could perform important roles in various biological processes including viral infection, differentiation, apoptosis, metabolism, cell proliferation, development, aging, signal transduction, as well as the development and progression of the human complex illness [15, 16]. Furthermore, miRNAs have close associations with the development, progression, and prognosis of many human illnesses [16, 17], furthermore, miRNAs are widely identified to be correlated with physiological processes: including apoptosis, differentiation, growth and stress responses, also pathological processes: including prognosis, initiation and progression of various illnesses . miRNAs are small (18–25 base long) noncoding RNAs, which were identified as vital ingredients of immune precision and other biological processes, such as development infection and inflammation . Indeed miRNAs have a primary function in the early differentiation of B cells and maintaining the regulatory T-cell lineage, additionally, they regulate the differentiation of dendritic cells and macrophages via toll-like receptors . Recently, microRNAs have caused great excitement as diagnostic and therapeutic signatures of the DF . However, arguments for the possibility of a major function of microRNAs in controlling target genes of DF were increased . So, our studies on joint activation and inhibition of endogenous miRNAs try to fight against dengue fever that includes effects on the initial Immune reaction.
The present study aimed to evaluate the treatment activity of Acetaminophen, RDN and LRD against DF. In addition, the way of treatment using single or mixed therapy was tested.
Study enrollment and investigations
This study was performed a randomized and investigator blinded. We evaluated outpatients referred to Centre for Tropical Diseases, Guangzhou, China during March–December 2015. It was approved by the ethical committee at Guangzhou University hospital of Traditional Chinese Medicine (Guangzhou, China). Patients diagnosed as DF were recruited from first affiliated at hospital. Patients were divided into two phases; primary phase was indicator chosen through a small group of plasma specimen of 20 patients treated with Acetaminophen or RDN injection contaminated LRD tablet and 15 healthy volunteers. The secondary phase was an open evaluation with a big group number of plasma specimen from 165 patients [average (SD) age, 38 (14.11) years] and 45 healthy persons as a control [age, 39.19 (10.16) years].
Tested DENV specific antibodies IgG and IgM in plasma samples were obtained from screened patients by ELISA antigen enzyme-linked immunosorbent assay (ELISA, Focus Technologies, Cypress, Ca, USA) . Positive IgM cases were serologically confirmed by ELISA. Moreover, DV antigen and viral RNA were detected by a dot blot immunoassay and a dengue serotype specific reverse transcription PCR .
Three days post Acetaminophen or RDN contaminated LRD or non-treated infection patients group. Plasma samples were drawn in PAXgene tubes to maintain miRNAs integrity and provide a snapshot of in vivo blood expression while minimizing time and manipulation variations . Total RNA was extracted by mirVana miRNAs isolation kit (Ambion, Austin, TX, USA), and was amplified and assorted according to the Affymetrix GeneChip Total Transcript Sense Target Labeling protocol . Replicate miRNAs in duplicate samples were averaged and miRNAs with intensities of ≥50 in all samples from each group (DF-infected or treated groups) were selected for calculation of the normalization factor. Expressed data were median-normalized. Following normalization, significantly differentially expressed miRNAs were identified through Volcano Plot filtering as described by Cui and Churchill . The detailed information of these differentially expressed miRNAs and correlation coefficient matrix for this data are listed in the Additional file 1 and the detailed result of volcano Plots filtering differentially expressed miRNAs are listed in the Additional file 2.
Drugs and patients treatment
ReDuNing (RDN, Batch No. 100906) produced by Jiangsu Kanion Pharmaceutical Co., Ltd., (Jiangsu, China); Loratadine (LRD, batch LRD/0909180) produced by Vasudna Pharma. Chem. Ltd., China were used in the research for their potential activities against dengue virus inhibition. Experimental patients were randomly divided into 3 groups; model control (without treatment) received saline only, positive control (treated with 1.3 g Acetaminophen tablets) administered orally three times daily for 3 days, and third group treated with 10 mg LRD tablets and 20 ml RDN injection. RDN dose conversion was according to its clinical dose. LRD and RDN were administered to patients the day after stress orally and by intravenous injection, respectively, for 3 days.
Network construction and analysis
To recognize the reaction mechanism of Acetaminophen and LRD with RDN as an herbal drug in the treatment of dengue fever, all active expression miRNAs and their targets were utilized to create interactions of miRNA and a predict gene were linked to each other if the gene was a predicted target of the miRNAs distributed in human. The potential miRNA were connected with those related target which were obtained from the PharmGKB database , and Therapeutic Targets Database (http://bidd.nus.edu.sg/group/bidd.htm).
In pharmaceutical systems, network consists of nodes and edges (connections between nodes), that is a mathematical, computable and quantifiable description of multiple relationships beneath the complicated biological systems . To explore comprehensively the interrelationship between potential targets and miRNAs expressions which will help us to select out those specific targets genes correlated with DENV disease. In the resultant networks, miRNAs predicted targets genes and enrichment functions diseases were illustrated via nodes; while edges indicated the interactions between them.
Mi–T–P network was set up by overlaying the miRNAs-target gene network with related function pathway. Initially functional pathways defined by the KEGG database (Kyoto Encyclopedia of Genes and Genomes) (http://www.genome.jp/kegg/)  and then constructed by combining the predict targets and their related function pathways, the Database for Annotation, Visualization and Integrated Discovery (DAVID knowledgebase) (https://david.ncifcrf.gov/)  were used  to test for enriched DEmiRs targets function and to identify biological themes among them. For each miRNAs expressions, the DAVID web service provided a ranked list of functionally relevant annotation clusters that represent a summary of several annotation categories, including GO , KEGG  and BioCarta pathways . In these networks, degree is used to characterize the connectedness of a node. The degree of a node is the number of edges associated with it. The topological properties of these networks were analyzed using the Network Analysis plugin and CentiScaPe 1.2 of Cytoscape 3.2.0, a popular bioinformatics package for biological network visualization and data integration, was used to generate all displayed networks .
To detect functional pathways or gene ontologies that are processing of differentially expressed miRNAs and putative targets of differentially expressed miRNAs after Dengue disease treated with Acetaminophen, RDN and LRD .
Effect of antiviral inhibition on inflammatory immune response
The supernatants samples were collected from patients before treatment and 3 days post treatment with Acetaminophen, LRD and RDN. Concentrations of TNF-α, IFN-γ, TGF-β1, IL-4, IL-6, IL-12, and IL-17 in culture supernatants were measured by ELISA according to manufacturer’s instructions .
Coagulations factor influences
Quantitative assessment of coagulations factor was performed in the plasma with DENV isolates using the ELISA Enzygnost (Dade Behring, Deerfield, IL, USA) according to the manufacturer’s instructions. Examination, we used plasma from untreated patients. In the case of the negative control, plasma was utilized. In parallel restrained survey in presence of hirudin (special thrombin restrained) was completed.
The analyses of miRNA expression data for microarray and miRNA-Target gene networks were described in each section. Other statistical analysis of Coagulations factor influences and inflammatory immune response data were performed using Student’s t-test and data are expressed as means ± SE, and differences with probability level P ≤ 0.05 were considered statistically significant.
The herbal medicine is widely used in clinical practices for thousands of years with efficacy and safety. However, there were few studies on anti-dengue with herbs. In this study, we compare single therapy of Acetaminophen with a new regimen of intermittent sequential therapy with Acetaminophen, LRD and RDN in the treatment of dengue virus disease. This randomized clinical trial was performed from March to December 2015. Patients diagnosed clinically with DF were randomized in three groups: patients in arm, not received any treatment, in single therapy arm patients received Acetaminophen 1.3 g tablet three times daily and in other arm, patients received sequential regimen of LRD 10 mg tablet each day and RDN 20 ml injection once daily. We carried out the detailed analysis for (RDN single treatment, RDN with Acetaminophen and LRD with Acetaminophen) however, the results were not satisfactory (data not shown).
Human miRNAs expression network and predicted target gene response to dengue virus infection
DDX3X plays an important role in the regulation of the cell proliferation, which indicated that it is involved in antiviral response against DENV infection , moreover DDX3X is able to stimulate the IFN promoter branches in DENV-infected cells and can be used as human immunodeficiency virus to control dengue infection. It has been suggested that the up-regulation of DDX3X may lead to treat dengue infection . The suppression of PTEN gene represents a powerful strategy for ESCC therapy while PTEN has potent antiviral activity against both HIV-1 and dengue virus . RORA has been implicated in several different cellular processes, including cell growth, translational control and nuclear–cytoplasmic shuttling . It was previously proved that RORA played a critical role in the cellular response, such as inhibitor of NF-κB signaling in cell to achieve inhibitory effects on immune response programs [37, 38] Reported that RORA is a candidate transcription factor for suppressing the inflammatory response employed by H1N1 of human monocytes. PPP3R1 was reported to participate in several biological processes such as improve therapeutic strategy to treat autoimmune disorders and neurological disorders such as Alzheimer disease either responses of skeletal muscle and cardiac muscle .
The highest target genes response to dengue virus infection
Phosphatase and Tensin Homolog
DEAD-Box Helicase 3, X-Linked
Adrenoceptor Beta 2
Vascular Endothelial Growth Factor A
Protein Phosphatase 3 Regulatory Subunit B, Alpha
Mitogen-Activated Protein Kinase Kinase Kinase 1
Insulin Like Growth Factor 1 Receptor
RELA Proto-Oncogene, NF-KB Subunit
Neuroblastoma RAS Viral Oncogene Homolog
Conserved Helix-Loop-Helix Ubiquitous Kinase
Mitogen-Activated Protein Kinase 1
B-Cell CLL/Lymphoma 10
ADAM Metallopeptidase Domain 10
RPTOR Independent Companion Of MTOR Complex 2
Mitogen-Activated Protein Kinase 9
Coiled-Coil Domain Containing 88A
Protein Kinase C Epsilon
Gene Ontology (GO) analysis of therapy target genes
regulation of cell proliferation
regulation of programmed cell death and apoptosis
response to cytokine stimulus
protein amino acid phosphorylation
protein kinase cascade
T cell receptor signaling pathway
regulation of lymphocyte activation
Toll-like receptor signaling pathway
myeloid cell differentiation
RIG-I-like receptor signaling pathway
Network construction and analysis for clinical treatment
In general, RDN Injection combined with LRD oral administration can induce the expression of miR-128a, miR-389 and miR-9 to suppress dengue virus replication.  Previously reported that in vivo miR-128a, miR-389 and miR-9 suppresses dengue virus replication by promoting NF-kB–dependent IFN production. After 3 days’ post treatment with Acetaminophen, LRD and RDN, we carried out the GO enrichment and clustering analysis to find out the targets associated with the physiological features of dengue infections related to immune and inflammatory response.
Immunity miRNAs expression profiling
With post-treated Acetaminophen medicine as a single therapy, there is a weak correlation between miRNAs expression and their targets. (Figure 2b) shows miRNAs differential expression in the supernatant of blood samples of patients. Among the up-regulated, M003 (miRNAs, hsa-miR-200c-3p) has the highest number of target candidate (degree = 10), followed by M002 (degree = 6), M005 (degree = 5), M001 (degree = 2) and M004 (degree = 1), indicating the one-target properties of ingredients. Results also showed 23 anticorrelated targets with the number of targets per miRNAs ranging from 2 to 8; and genes being targeted by one miRNA and their corresponding 12 functional annotations. The highest degree miRNAs are differently expressed compared with the patients treated with Acetaminophen therapy (regulation of programmed cell death and apoptosis, regulation of cell proliferation and protein amino acid phosphorylation with degree of 11, 10 and 10, respectively), which may be due to the effect of Acetaminophen on the expression of miRNAs in response to dengue virus. The detailed information of these miRNAs expressions, immune target genes and functional pathway post-treated with Acetaminophen are listed in the Additional file 6: Table S4.
Qi, et al.  Reported that cytokines and epigenetic regulators may be putative target genes of these miRNAs, and the pathway of biological regulation may be modulated by these miRNAs, miRNAs were classified as important ingredients of immune regulation . Distinct miRNAs are capable of targeting genes that inhibit epigenetic pathways, while several miRNAs were confirmed to control down-regulation inflammatory responses by directly targeting genes encoding molecules [43, 44]. Additionally, during DENV2 infection miRNAs were differentially expressed, among these upregulated are (R-4290, −4279, −625*, −let-7e, −1290, −33a, −378, −1246, −767-5p, −320c, −720, −491-3p, −3647, −451 and −4286) but downregulated miRNA were (miR-106b,-20a, −30b and −3653) .
Inflammatory miRNAs expression profiling
In order to clarify the effect of Acetaminophen as anti-inflammatory therapy and to find the relationships between the active ingredients of miRNAs and their targets, the network was generated by connecting the potential miRNAs 14 and the potential targets 24 associated with the DENV. The mean number of potential targets per potential compound was 1.045. Among the 14 potential miRNAs, 2 have high degree distributions and each of them hits more than 10 and 6 potential targets (hsa-miR-107, hsa-miR-320d). All of the 24 targets showing down-regulations were linked by 2 until 10 miRNAs and some of them are verified in previous research (Fig. 3b). The information of these parameters has been listed in the Additional file 8: Table S6.
Effect of antiviral inhibition on inflammatory immune response
Influence of coagulation factor
Treatment of DENV is presently a global infection and it is a viral disease for which there is no licensed therapeutic agent . Treatment and diagnosis via new molecular-based techniques have become a new hope for early control, but are still exclusive because of their costs and normalization. DENV is the most important factors transmitted by mosquitoes worldwide and causing viral disease, almost 100 million cases of dangerous DENV each year, resulting in severe and life-threatening . Direct anti-virus treatment that reduces the risk of dengue fever can be helpful although this may need to keep all serotypes effectively . Predicting potential miRNA-disease associations created valuable contributions to promoting new drugs, explaining the pathogenesis concerning disorders and medication for distinct individual complex illnesses . However, we have not completed a comprehensive outlook of all miRNAs expression regulation target genes through virus-human interactions in patients of DENV. A few human miRNAs might show antiviral miRNAs. When some delusive antiviral miRNAs were blocked by locked nucleic acid-modified antisense oligoribonucleotides, the hosts miss carriage to suppress viral replication . Otherwise, in few cases, human miRNAs expression might be corroborated by viruses to reshape the human intracellular medium to benefit viral replication .
In the present study, results showed that about 89 human miRNAs are important in the DENV interactions before medication, which are of interest in human immune and inflammatory responses. Therefore, our study provides the most human miRNAs and their predicted functions pathways mediated by them in response to virus infection. In contrast, similar studies in mammals, revealed that the miRNAs profiles were reshaped by hepatitis C virus and miRNAs of human might have been correlated with the regulation of human immune systems . Accordingly, virus contagion has a capability of destructing processions of cellular functions of the host on several levels, like the effects of the cell cycle or apoptosis of cells infected with viruses, including miRNAs . Through cellular, miRNAs of virus-host interactions play a key regulators of gene expression . Further experiments concluded that the regulation of Ad6 by miR-122 could significantly improve the safety profile of the entire body rearward systemic administration. Consequently, also recognizing disease-related miRNAs could adequately improve disease biomarker detection for the medication, it is necessary to develop robust computational models to predict innovative human disease-miRNA associations [15, 61].
Microarray analysis in the present study displayed that hsa-miR-320a; hsa-miR-107 and hsa-miR-361-5p were extremely preserved in human. In addition, analyses predicted that miR-320a; one of the miRNAs highly preserved could target the Mitogen-Activated Phosphatase and Tensin Homolog (PTE), and DEAD-Box Helicase 3, X-Linked (DDX3X). Recent studies revealed that PTEN and DDX3X were activated by invading DENV, suggesting that host might inhibit virus infection by targeting viral transcripts with host miRNAs . To reduce the dengue fever impact using a potential anti-dengue medicine, we administered several known antiviral drugs (Acetaminophen, LRD and RDN), oral treatment or injection targeting DENV were found to significantly reduce viremia. Additionally, LRD combination with RDN showed a significant reduction of viremia and indicated that RDN inhibitors may be interesting drug candidates for DENV. They also have a potential for antiviral efficacy in vivo, similar to their proposed used in anti–hepatitis C , whereas Acetaminophen treatment may have little effect during dengue fever treatment.
Furthermore the potential of RDN and LRD against DENV in patient are bitten by carrier mosquitoes. The results showed that RDN could reduce the immune parameters in blood of patients if mixed with LRD tablet. The suggested mechanism was that RDN down-regulated NF-B activation reducing the expression of pro-inflammatory cytokines and increasing IFITM3 expression via MAVS, giving a sign that the anti-virus effects of RDN were related to adjusting the unbalanced homeostasis of the body caused by stress response, but unrelated with direct anti-virus activity [64, 65] Reported that RDN could inhibit the susceptibility of influenza in restraint-stressed mice, through RDN down-regulating NF-kB activation to constrict the expression of pro-inflammatory cytokines and enhance IFITM3 mRNA expression via MAVS. This outline suggested that the anti-influenza effects of RDN were regarding homeostasis of the body caused by stress response, however not concerning with direct anti-virus activity. RDN injection has various functions; such as absolution heat, eliminate flatus, and detoxification, clinically used in the curative of hyperpyrexia, influenza and body pain, cough, yellow sputum, and other symptoms raised by respiratory tract infection. RDN injection has been openly applied with good clinical performance .
Until now, it has been cloudy how dengue viremia levels contribute to the severity of the disease, after that reducing viremia by antiviral drugs to reduce the symptoms of DF . Found that higher level of viremia was linked with increased DENV intensity and proposed that lowering viremia may reduce morbidity and the risk of dengue hemorrhagic fever or dengue shock syndrome. Infected mice by dengue showing increased TNF-a, IFN-g and IL-6 levels, which were recognized as triggers for DENV were also reported. We have found that reducing blood some immune parameters through the use of antiviral treatment significantly decreases proinflammatory cytokine levels and increases the concentrations of coagulants factors in the blood of treated patients. These results indicated that lowering proinflammatory cytokine parameters through timely antiviral drug treatment may improve the severity of DF symptoms and perhaps decrease the risk of progression.
Finally, this work suggests ways to prohibit the gaining of DENV which is to avoid being bitten by mosquitoes that carries the disease. Although this can be done by avoiding areas where dengue is endemic, but may be that is not the ideal strategy because it would require a person to avoid almost of the tropical and subtropical areas of the world, many of which are popular purpose to travel, work or at least using natural repellent for mosquitoes.
Based on the results of this study, it was concluded that RDN could be used as anti-dengue in vivo, to reduce the susceptibility and severity of dengue virus. This study also warrants the future in vivo anti-viral as part of the developmental process for development RDN as potential anti-dengue therapeutic. We therefore, need more investigations and specific diagnostic criteria. However, this study with a few number of sample size, gives a promising opportunity in the treatment of DENV using RDN in combination with LRD.
We are grateful to the director and the staff of the Centre for Tropical Diseases in Guangzhou University hospital in particular, the doctors and nurses of the Intensive Care Unit—for their help and support during the study.
This work was supported by Grants from Northwest A & F University, National Natural Science Foundation of China (31170796 and 81373892). It also was supported in part by Grants from, National Natural Science Foundation of China (31540008).
Availability of data and materials
All data generated or analyzed during this study are included in this published article (and its Additional files).
MS, ZG and YW conceived the project. MS, QT, WZ, AHS, ZW, YB, YF, CZ, HW and PAS performed experiments. MS and QT analyzed most data. MS, ZG, JL, ZW, WX and YW provided critical intellectual input to the study and the manuscript preparation. MS, ZG, AHS and RE wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
We evaluated outpatients referred to Centre for Tropical Diseases. This study was approved by the ethics committee of Guangzhou University hospital of Traditional Chinese Medicine (Guang-zhou, China).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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