www.nature.com/scientificreports OPEN Systematic Genome-wide Screening and Prediction of microRNAs in EBOV During the Received: 26 November 2014 Accepted: 23 March 2015 2014 Ebolavirus Outbreak Published: 26 May 2015 Yue Teng1,*, Yuzhuo Wang1,2,*, Xianglilan Zhang1, Wenli Liu1, Hang Fan1, Hongwu Yao1, Baihan Lin3, Ping Zhu1,2, Wenjun Yuan2, Yigang Tong1 & Wuchun Cao1 Recently, several thousand people have been killed by the Ebolavirus disease (EVD) in West Africa, yet no current antiviral medications and treatments are available. Systematic investigation of ebolavirus whole genomes during the 2014 outbreak may shed light on the underlying mechanisms of EVD development. Here, using the genome-wide screening in ebolavirus genome sequences, we predicted four putative viral microRNA precursors (pre-miRNAs) and seven putative mature microRNAs (miRNAs). Combing bioinformatics analysis and prediction of the potential ebolavirus miRNA target genes, we suggest that two ebolavirus coding possible miRNAs may be silence and down-regulate the target genes NFKBIE and RIPK1, which are the central mediator of the pathways related with host cell defense mechanism. Additionally, the ebolavirus exploits the miRNAs to inhibit the NF-kB and TNF factors to evade the host defense mechanisms that limit replication by killing infected cells, or to conversely trigger apoptosis as a mechanism to increase virus spreading. This is the first study to use the genome-wide scanning to predict microRNAs in the 2014 outbreak EVD and then to apply systematic bioinformatics to analyze their target genes. We revealed a potential mechanism of miRNAs in ebolavirus infection and possible therapeutic targets for Ebola viral infection treatment. The genus Ebolavirus belongs to the family Filoviridae and order Mononegavirales, where the members of this genus are called ebolaviruses1. The Ebolavirus (EBOV, formerly designated “Zaire ebolavirus”) is one of the five known Ebolaviruses. EBOV cause Ebolavirus disease (EVD) in humans and other mam- mals. EVD is a type of hemorrhagic fever having a high case fatality rate2. From March 2014 to October 2014, the Ebola outbreak in West Africa has sickened 8,399 people, killing 4,033 of them3. This is the largest EBOV outbreak in the history4. Each month, several thousand people from affected areas travel to the North America, and even more travellers enter and leave Europe, other parts of Africa, and Asia5. Such air travel situations increase the possibility of EVD transmission. The United States and France have reported that several patients tested positive for Ebola3–6. Moreover, no current antiviral medications are available for EBOV7, and no effective EVD treatment exists at this time8. 1The State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China. 2Department of Physiology and Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan 750004, PR China. 3Computational Neuroscience Program, Department of Psychology, Physics, and Computer Science and Engineering, University of Washington, Seattle, WA 98195, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.T. (email: [email protected]) or Y.T. (email: [email protected]) or W.C. (email: [email protected]) SCIENTIFIC REPORTS | 5:09912 | DOI: 10.1038/srep09912 1 www.nature.com/scientificreports/ MicroRNAs (miRNAs) are small regulatory non-coding RNAs, ranging from 19 to 24 nucleotides, that post-transcriptionally regulate target gene expression by inhibiting the translation of mRNA transcripts or cleaving them9–12. While encoded not only by cellular genomes but also by viral genomes13–15, miRNAs play a vital role in numerous cellular processes, including cell metabolism, viral infection, and antiviral immune response16,17. Viral miRNAs are mostly identified by traditional cloning from virus-infected cells18–20. Computational prediction and hybridization analysis are also applied to viral miRNA identifi- cation21,22. For known viral miRNAs, the majority of them are encoded by DNA viruses while only a few derived from RNA viruses23,24. To date, the RNA virus-encoded miRNAs have been identified in a few retroviruses including bovine leukemia vius (BLV)25,26, human immunodeficiency virus (HIV)25, West Nile virus (WNV)27, Dengue virus28 and hepatitis A virus (HAV)23. Recently, Liang et al.24 have identified two EBOV miRNAs, which means that EBOV can encode functional miRNAs. Unlike the method used by Liang et al.24, based on bioinformatics whole genome-wide scanning and screening, we predicted the potential mature 2014 EBOV miRNAs, their target genes, and related sig- naling pathways. To the best of our knowledge, this is the first paper to systemic genome-wide analyze and predict the potential non-coding RNAs in the 2014 outbreak EBOVs along with their target genes. The results show several target genes regulated by the possible miRNAs may have important functions in human immune and antiviral response systems. Our research helps to further assess the roles of the 2014 outbreak EBOV miRNAs and their potential targets during viral infection and virus-host interactions, and thus to speed up the process of effective EVD treatment development. Results Alignment of EBOV whole genomes and prediction of mature EBOV miRNAs. As shown in Fig. 1A, we first aligned the complete genome sequences of the 102 human EBOV strains acquired from the 2014 outbreak29. The mutation sites were all highlighted in different colors30. Four real microRNA precursor candidates, with the names of EBOV-pre-miRNA-T1, EBOV-pre-miRNA-T2, EBOV-pre-miRNA-T3 and EBOV-pre-miRNA-T4, were picked out. Their sequences are listed in Table 1. EBOV-pre-miRNA-T1 is 136-base long, EBOV-pre-miRNA-T2 is 80-base long, EBOV-pre-miRNA-T3 is 99-base long, and EBOV-pre-miRNA-T4 is 99-base long. Their locations were marked in Fig. 1B. The start position of EBOV-pre-miRNA-T1 is 653, while its end position is 788. EBOV-pre-miRNA-T2 is located between the position of 4373 and of 4452. EBOV-pre-miRNA-T3 is at the range between 488 and 586. EBOV-pre-miRNA-T4 is located from the position of 479 to 568. Seven different mature EBOV miRNA candidates were predicted using the four EBOV miRNA precursor candidates. The sequences of the eight mature EBOV miRNAs are listed in Table 2, where EBV-miR-T3-5p has the same sequence with EBV-miR-T4-5p. Their lengths are all 22-base long. By aligning and blasting the seven miRNAs, including EBV-miR-T1-5p, EBV-miR-T1-3p, EBV-miR-T2-5p, EBV-miR-T2-3p, EBV-miR-T3-5p, EBV-miR-T3-3p, EBV-miR-T4-5p and EBV-miR-T4-3p, to other species of viral or host miRNAs, only EBV-miR-T3-5p has some similar miRNAs. We analyzed the conservation between EBV-miR-T3-5p and its similar miR- NAs, which is described in the Table 3. A flowchart describing the computational prediction of the putative miRNAs is shown in Fig. 2. Structures of potential EBOV pre-miRNAs and mature miRNAs. In order to achieve more accurate prediction, we predicted the primary, secondary, and tertiary structure of the four poten- tial EBOV pre-miRNAs31,32. RNAfold was used to predict consensus secondary structures of the pre-miRNAs. Attempts to fold these pre-miRNAs, along with their flanking sequences, into the expected hairpin structures were successful in candidates EBOV-pre-miRNAs-T1, EBOV-pre-miRNAs-T2, EBOV-pre-miRNAs-T3 and EBOV-pre-miRNAs-T4, while other pre-miRNAs failed to fold into any form of stable RNA structures because that they could only give rise to stem-loops that were too short to be authentic pre-miRNA stem-loops. These findings led us to believe that EBOV-pre-miRNAs-T1, EBOV-pre-miRNAs-T2, EBOV-pre-miRNAs-T3 and EBOV-pre-miRNAs-T4 are indeed authentic EBOV pre-miRNAs. The optimal secondary structures in dot-bracket notation with a minimum free energy of − 39.4 kcal/mol of EBOV-pre-miRNA-T1, − 18.8 kcal/mol of EBOV-pre-miRNA-T2, − 33.51 kcal/mol of EBOV-pre-miRNA-T3 and − 31.91 kcal/mol of EBOV-pre-miRNA-T4 are given in Fig. 3A,B shows the predicted hairpin structures of confirmed EBOV pre-miRNAs. Hairpins longer than 100 nt were trun- cated; these hairpins are indicated by a double slash preceding the stem. Furthermore, the 3D structures of the potential EBOV pre-miRNAs were computationally predicted. Even though EBOV-pre-miRNA-T3 and EBOV-pre-miRNA-T4 have similar 2D structures31, they own different 3D structures (Fig. 3C). The positions of possible mature miRNAs were colored in second and tertiary structures (Figs. 3B,C)32. For EBOV-pre-miRNAs-T1, EBOV-pre-miRNAs-T2, EBOV-pre-miRNAs-T3 and EBOV-pre-miRNAs-T4 alternative predictions, which are likely to represent the authentic stem-loop structures recognized and cleaved by Drosha are shown underneath the minimal energy structures. Bioinformatics analysis of mature EBOV miRNAs and prediction of their target genes. Based on the sequences of the seven EBOV miRNAs, EBV-miR-T1-5p, EBV-miR-T1-3p, EBV-miR-T2-5p, EBV-miR-T2-3p, EBV-miR-T3-5p, EBV-miR-T3-3p, EBV-miR-T4-5p and EBV-miR-T4-3p, their target genes were searched by TargetScan33 and the other miRNA regulation database (the integration data- base of miRecords, TarBase, starbase, and miR2Disease)34–37. All these potential mature miRNAs have SCIENTIFIC REPORTS | 5:09912 | DOI: 10.1038/srep09912 2 www.nature.com/scientificreports/ Figure 1. The 102 complete genome sequences alignment of the 2014 outbreak EBOV. (A) Full alignment map of all 102 EBOV complete genomes. (B) Locations and putative target sites of the four real miRNA precursor candidates.