Research paperAnalysis of the microRNA expression profiles in DEF cells infected with duck Tembusu virus
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.
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