Elsevier

Infection, Genetics and Evolution

Volume 63, September 2018, Pages 126-134
Infection, Genetics and Evolution

Research paper
Analysis of the microRNA expression profiles in DEF cells infected with duck Tembusu virus

https://doi.org/10.1016/j.meegid.2018.05.020Get rights and content

Highlights

  • 287 known and 63 novel miRNAs were identified in DTMUV-infected DEF cells.

  • 37 up-regulated miRNAs and 11 down-regulated miRNAs were identified.

  • 9 miRNAs were randomly selected for validating their expression by qRT-PCR.

  • The dysregulated miRNAs were mainly involved in immune response.

  • This is the first study to evaluate miRNA expression profiles in DTMUV-infected DEF cells.

Abstract

Duck Tembusu virus (DTMUV), belonging to the Flaviviridae family, is a single-stranded positive-sense RNA virus. Since April 2010, the outbreak of DTMUV in southeast provinces of China has caused great economic losses. MicroRNAs (miRNAs) play important regulatory roles in viral infection through binding to the host target genes or the viral genomes. To better understanding the molecular mechanisms of virus-host interaction, here we identified the miRNA expression profiles in DTMUV-infected and uninfected DEF cells by high-throughput sequencing. A total of 287 known and 63 novel miRNAs were identified. 48 miRNAs, including 26 known miRNAs and 22 novel miRNAs, were differentially expressed in response to DTMUV infection. Among these miRNAs, 37 miRNAs were up-regulated and 11 miRNAs were down-regulated. 9 miRNAs were randomly selected for validation by qRT-PCR experiment. The results of qRT-PCR experiment were consistent with the sequencing data. GO enrichment showed that the predicted targets of these differentially expressed miRNAs were mainly involved in the regulation of immune system, cellular process and metabolic process. KEGG pathways analysis showed that predicted target genes were involved in several signaling pathways such as Wnt signaling pathway, TGF-beta signaling pathway, mTOR signaling pathway and FoxO signaling pathway. This is the first study to evaluate changes of miRNA expression in DEF cells upon DTMUV infection. Our findings provide important clues for better understanding the DTMUV-host interaction.

Introduction

In the spring of 2010, duck Tembusu virus (DTMUV) was first isolated in Shanghai, China, then rapidly spread to southeast provinces of China, has caused great economic losses in the duck industry (Su et al., 2011b). The typical clinical features of the disease are high fever, loss of appetite, severe egg drop and neurological signs (Yan et al., 2011). DTMUV, a member of the Flaviviridae, is a single-stranded positive RNA virus (Petz et al., 2014). In common with other flaviviruses such as Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV), DTMUV is also a vector-borne virus that can be transmitted through mosquitos (Tang et al., 2015). The DTMUV genome is about 11 kb in length and with an open reading frame (ORF) encoding a polyprotein. The polyprotein was cut into three structural proteins (capsid, PrM/M and envelope protein) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) under the action of proteases of virus and host (Liu et al., 2012).

MicroRNAs (miRNAs) are 20–25 nucleotides noncoding RNAs that play an important role in regulating gene expression at the post-transcriptional level (Bartel, 2004). The mechanism is to inhibit the translation or promote the degradation of target genes by complementarity between the seed sequence of miRNAs and the 3’-UTR of target genes (Bartel, 2009). The research of miRNAs has been extensively carried out. Up to now, the latest version of miRNA database (miRBase 21) has included 38,589 miRNAs. Many recent researches have denoted that the expression profiles of miRNAs were changed upon virus infection. For example, the expression of 17 miRNAs was modulated when ZIKV infected mosquitos Ae. Aegypti (Saldana et al., 2017). 140 and 265 differentially expressed miRNAs were identified in A. albopictus cells and primary sheep testicular (PST) cells infected with Bluetongue virus (BTV), respectively (Du et al., 2017; Xing et al., 2016). Additionally, the differentially expressed miRNAs can regulate replication of virus, including JEV, DENV and WNV (Pareek et al., 2014; Slonchak et al., 2015; Trobaugh and Klimstra, 2017; Wu et al., 2013). It is known that miRNAs can regulate expression of host immune-related genes via targeting the 3′-UTR of target genes, thereby inhibit or activate the downstream signaling pathway and mediate the anti-viral immune response (Dang et al., 2017; Deng et al., 2017; Fu et al., 2018; Hazra et al., 2017; Smith et al., 2017; Wang et al., 2018). Recently, a number of articles proved that miRNAs can modulate viral replication via direct binding to the viral genome. On the one side, miRNAs suppressed viral replication through inhibiting translation of viral genome (Castrillon-Betancur and Urcuqui-Inchima, 2017; Escalera-Cueto et al., 2015; Khongnomnan et al., 2015; Li et al., 2015a; Zheng et al., 2013). On the other side, miRNAs promoted viral replication by stabilizing the virus RNA (Chang et al., 2008; Scheel et al., 2016; Zhou et al., 2014).

Currently, there are many studies about changes of miRNAs expression caused by flavivirus infection, such as DENV (Avila-Bonilla et al., 2017; Campbell et al., 2014; Liu et al., 2015; Su et al., 2017), WNV (Mukesh and Nerurkar, 2014), JEV (Cai et al., 2015; Zhang et al., 2015) and ZIKV (Kozak et al., 2017; Saldana et al., 2017). There are no reports about the miRNA expression changes induced by DTMUV infection. In order to solve this problem, we used deep sequencing approach to analyze the miRNA profiles in DEF cells upon DTMUV infection. Our study identified the differentially expressed miRNAs in DTMUV-infected DEF cells, and demonstrated these differentially expressed miRNAs play an important role in physiological and pathological processes by bioinformatics analysis. The finding provides new insights for us to understand the pathogen-host interaction and to search therapeutic measures for DTMUV infection.

Section snippets

Ethics approval and consent to participate

The usage of duck embryos in this paper was approved by the Animal Ethics Committee of Sichuan Agricultural University (approval No. 2015-016) and followed the National Institutes of Health guidelines for the performance of animal experiments.

Cell and virus

Primary DEF cells were prepared from 9-day-old duck embryos. DEF cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO2. The DTMUV CQW strain (GenBank: KM233707.1)

DTMUV replication in DEF cells

DTMUV infection in DEF cells was confirmed by CPE and western blot. As shown in Fig. 1A, we observed that DTMUV caused minimal CPE in DEF cells at 36hpi. CPE were obviously observed in DTMUV-infected DEF cells compared with the uninfected cells. As shown in Fig. 1B, the viral E protein expression was detected at 36 hpi, and widely expression was observed at 48 hpi, 60 hpi and 72 hpi by western blot. Because of the optimal time for miRNA sequencing analysis is during high virus yield without

Discussion

As a member of Flaviviridae family, DTMUV is a potential zoonotic pathogen and has caused huge economic loss in poultry industry in China (Su et al., 2011b; Wang et al., 2016). However, the molecular mechanisms of DTMUV-host interaction have not been completely elucidated and there is no effective way to control DTMUV infection. Increasing studies showed that miRNAs play important regulatory roles in viral pathogenesis through targeting host genes or viral genome (Asgari, 2014; Chen et al.,

Conclusions

In summary, this is the first time to detect the miRNA profiles in DEF cells upon DTMUV infection through deep sequencing. We identified 48 differentially expressed miRNAs in DTMUV-infected DEF cells, including 37 up-regulated miRNAs and 11 down-regulated miRNAs. 9 differentially expressed miRNAs were randomly selected for validating their expression by qRT-PCR. GO enrichment and KEGG analysis showed that these target genes of 48 differentially expressed miRNAs were mainly involved in immune

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by National Key Research and Development Program of China (2017YFD0500800), National Key R & D Program (2016YFD0500800), China Agricultural Research System (CARS-43-8) and Sichuan Province Research Programs (2017JY0014/2017HH0026). We would like to thank our funding sources and the assistance on bioinformatics analysis provided by Chengdu Basebiotech Co., Ltd.

Availability of data and materials

The data generated or analyzed during this study are available from the corresponding author upon reasonable request.

References (85)

  • N. Fan et al.

    MicroRNA 34a contributes to virus-mediated apoptosis through binding to its target gene Bax in influenza A virus infection

    Biomed Pharmacother

    (2016)
  • Y. Fu et al.

    Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication

    Antivir. Res.

    (2015)
  • Z. Li et al.

    MicroRNAs in the immune organs of chickens and ducks indicate divergence of immunity against H5N1 avian influenza

    FEBS Lett.

    (2015)
  • Y. Liu et al.

    The expression profile of Aedes albopictus miRNAs is altered by dengue virus serotype-2 infection

    Cell Biosci.

    (2015)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method

    Methods

    (2001)
  • T.K. Scheel et al.

    A broad RNA virus survey reveals both miRNA dependence and functional sequestration

    Cell Host Microbe

    (2016)
  • D.W. Trobaugh et al.

    MicroRNA regulation of RNA virus replication and pathogenesis

    Trends Mol. Med.

    (2017)
  • S. Urcuqui-Inchima et al.

    Interplay between dengue virus and toll-like receptors, RIG-I/MDA5 and microRNAs: implications for pathogenesis

    Antivir. Res.

    (2017)
  • X.S. Wang et al.

    MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia

    Blood

    (2012)
  • T. Wu et al.

    miR-155 modulates TNF-α-inhibited osteogenic differentiation by targeting SOCS1 expression

    Bone

    (2012)
  • S. Wu et al.

    miR-146a facilitates replication of dengue virus by dampening interferon induction by targeting TRAF6

    J. Infect.

    (2013)
  • S. Xing et al.

    Analysis of the miRNA Expression Profile in an Aedes albopictus Cell Line in Response to Bluetongue Virus Infection. Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases

    (2016)
  • P. Yan et al.

    An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China

    Virology

    (2011)
  • D.-b. Yu et al.

    Identification of novel and differentially expressed MicroRNAs in the ovaries of laying and non-laying ducks

    J. Integr. Agric.

    (2013)
  • L. Zhang et al.

    Characterization of MicroRNA* species in Peking duck skin

    J. Integr. Agric.

    (2013)
  • Y. Zhang et al.

    Integration Analysis of miRNA and mRNA Expression Profiles in Swine Testis Cells Infected with Japanese Encephalitis Virus. Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases

    (2015)
  • W. Zhang et al.

    Molecular identification and immunological characteristics of goose suppressor of cytokine signaling 1 (SOCS-1) in vitro and vivo following DTMUV challenge

    Cytokine

    (2017)
  • S. Anders et al.

    Differential expression analysis for sequence count data

    Genome Biol.

    (2010)
  • S. Asgari

    Role of microRNAs in arbovirus/vector interactions

    Viruses

    (2014)
  • P.C. Boutros et al.

    VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R

    BMC Bioinformatics

    (2011)
  • Y. Cai et al.

    Identification and analysis of differentially-expressed microRNAs in Japanese encephalitis virus-infected PK-15 cells with deep sequencing

    Int. J. Mol. Sci.

    (2015)
  • G.A. Calin et al.

    MicroRNA signatures in human cancers

    Nat. Rev. Cancer

    (2006)
  • C.L. Campbell et al.

    MicroRNA levels are modulated in Aedes aegypti after exposure to Dengue-2

    Insect Mol. Biol.

    (2014)
  • A.L. Cardoso et al.

    miR-155 modulates microglia-mediated immune response by down-regulating SOCS-1 and promoting cytokine and nitric oxide production

    Immunology

    (2012)
  • J.C. Castrillon-Betancur et al.

    Overexpression of miR-484 and miR-744 in Vero cells alters dengue virus replication

    Mem. Inst. Oswaldo Cruz

    (2017)
  • J. Chang et al.

    Liver-specific MicroRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells

    J. Virol.

    (2008)
  • S. Chen et al.

    Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways

    Vet. Res.

    (2016)
  • Z. Chen et al.

    MicroRNA-33a-5p modulates Japanese encephalitis virus replication by targeting eukaryotic translation elongation factor 1A1

    J. Virol.

    (2016)
  • A.M. Cheng et al.

    Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

    Nucleic Acids Res.

    (2005)
  • T.G.O. Consortium et al.

    Gene ontology: tool for the unification of biology

    Nat. Genet.

    (2000)
  • L.B. Frankel et al.

    MicroRNA regulation of autophagy

    Carcinogenesis

    (2012)
  • M.R. Friedländer et al.

    Discovering microRNAs from deep sequencing data using miRDeep

    Nat. Biotechnol.

    (2008)
  • Cited by (0)

    View full text