MicroRNAs (miRNA) is a class of small non-coding RNA, about 20–24 nucleotides in length. miRNA is first transcribed from the genome to form primary miRNA (pri-miRNA), and then cleaved by Drosha enzyme and Dicer enzyme successively to form mature miRNA. One strand of mature miRNA typically degrades mRNA or inhibits translation by binding to the 3′untranslated region (UTR) of the target gene (Bartel, 2004). Not only in diverse biological pathways, cellular miRNAs also play vital roles in virus-host interactions, which are post-transcriptional regulators in directing regulation of virus proliferation (Wong et al., 2020).

Flavivirus genus contains more than 50 arthropod-borne viruses (arboviruses), of which important family members include Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), Yellow fever virus (YFV), Tick- borne encephalitis virus (TBEV) and Tambusu virus (TMUV). Flavivirus is a positive-sense RNA virus with a full length of about 11 kb. The structure of viral genome contains a single open reading frame (ORF) that encodes multiple proteins, cleaved by host and viral proteases into three structural proteins (C, prM, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), flanked by a 5′UTR and a 3′UTR, which is conserved throughout the Flavivirus genus (Heiss et al., 2011; Figure 1).

The expression of host miRNA can be altered by flavivirus infection (Campbell et al., 2014; Kumari et al., 2016; McLean et al., 2017; Cui et al., 2018; Bagasra et al., 2021). Further, differentially expressed miRNAs can directly regulate virus replication by complementing viral genome or indirectly by binding to crucial host genes, which has been reported in various viruses (Smith et al., 2012). For instance, Kumar et al. (2018) confirmed that miR-324-5p suppresses H5N1 highly pathogenic influenza A virus (HPAIV) replication through directly binding within the viral genome, thereby inhibiting viral gene expression. miR-140 inhibits classical swine fever virus (CSFV) genome amplification via interacting with an oncogene Rab25 in swine umbilical vein endothelial cells (Xu et al., 2020). Moreover, it is worth mentioning that miRNAs are usually negatively correlated with target genes. Nevertheless, miR-122 is a special case, as it specifically expresses and is highly abundant in human livers, while promoting the Hepatitis C virus (HCV) life cycle by targeting its 5′UTR (Jopling et al., 2005; Kunden et al., 2020).

Recently, interaction networks composed of non-coding RNAs, including miRNA, long non-coding RNA (lncRNA) and circular RNA (circRNA), have attracted increasing attention for their important roles in various biological processes such as viral infection. lncRNA/circRNA, as competing endogenous RNA (ceRNA), competitively bind to miRNA through their miRNA recognition element (MRE), thereby regulating the expression level of miRNA target mRNA (Salmena et al., 2011). Hence, lncRNA/circRNA–miRNA–mRNA interaction may be an important mechanism of flavivirus pathogenesis. In addition, the virus itself produces a variety of non-coding RNAs, such as subgenomic flaviviral RNAs (sfRNAs) and viral small RNAs (vsRNAs), the latter also include viral small interfering RNAs (siRNAs) and virus-encoded miRNAs. All of them can interact with cellular and viral RNA to regulate flavivirus replication (Schuessler et al., 2012; Hussain and Asgari, 2014; Zeng et al., 2021).

Based on the character that flavivirus completes its life cycle and pathogenicity is highly dependent on the metabolism of living host cells, clarifying the effect of host miRNA as well as other non-coding RNA on host–virus interactions will help us better understand the pathogenesis of flavivirus and novel candidates for the development of therapeutic interventions (Smith et al., 2017). In this review, we elaborate how miRNAs could relate to flavivirus infections and summarize the potential value of miRNAs in combating flavivirus.

Dengue viruses are four closely related but antigenically distinct viral serotypes (DENV1, DENV2, DENV3, and DENV4) that cause very similar disease spectrum in humans. It is the tenth leading cause of death and morbidity in developing countries and the leading cause of death among children under 15 years of age in some South-East Asian countries (Lim, 2019). At present, DENV is also the most widely studied virus in the Flavivirus genus and has been reported to regulate its own replication by using host miRNA (Zhou et al., 2014; Castillo et al., 2016; Diosa-Toro et al., 2017; Kanokudom et al., 2017; Mishra et al., 2020).

Japanese encephalitis virus is a neurotropic virus that infects the Central Nervous System (CNS) and causes fatal encephalitis. Accumulating evidence points to microglia activation and bystander damage as the main pathogenesis of Japanese encephalitis and its complications (Chang et al., 2021). After viral infection, miRNAs expression alters mostly occur in human/mouse microglia and subsequently respond to viral invasion by influencing viral replication and inflammation in the body.

West Nile virus belongs to the JEV serotype, which maintains in the environment by an avian–mosquito life cycle (Barrows et al., 2018). The neuro-invasive disease of WNV can be fatal, or recovery from the infection may require specialized care, with symptoms lasting more than a year (Petersen et al., 2013). Such neurological symptoms are also reflected in the concentration of targets of differentially expressed miRNAs.

Zika virus was first isolated from the blood of a non-human primate in 1947 and from Aedes africanus mosquitoes 1 year later, which is mainly transmitted by Aedes albopictus and Aedes aegypti (Musso and Gubler, 2016; Lin et al., 2018). As shown in a variety of experimental models and human samples, ZIKV infection induces neuronal apoptosis and disrupts cellular homeostasis, resulting in microcephaly, anencephaly, ventricular enlargement and calcification (Polonio and Peron, 2021).

In addition to the above widely influential zoonoses, other members of the Flavivirus genus whose infection is also closely related to cellular miRNA. TMUV is an emerging and re-emerging zoonotic pathogen that adversely affects poultry industry in recent years, especially the duck breeding industry (Yang et al., 2021). Cui et al. (2018, 2020) and Huang et al. (2020) first employed deep sequencing to investigate miRNA profiles in DEF cells after DTMUV infection and revealed a novel host evasion mechanism adopted by DTMUV via miR-221-3p inhibiting the negative regulator of host immune response, SOCS5. Subsequently, this miRNA hinders JAK-STAT signaling mediated IFN response and promote virus replication, while miR-148a-5p has a similar effect on the virus replication by modulating SOCS1. TBEV causes neurological symptoms that can lead to permanent disability or death. Although there have been no studies on miRNA expression after TBEV infection, several studies have shown that host-specific miRNAs target sequences integrated into viral genomes (such as tick-specific miR-275 and neuro-specific miR-124a) can effectively control the escape of TBEV chimeric viruses or LGTV (a naturally attenuated member of the TBEV serum complex) and reversal of neurotoxic phenotype (Heiss et al., 2011, 2012; Tsetsarkin et al., 2016). Hermance et al. (2019) confirmed that miRNA changes can regulate Powassan virus (POWV) replication in the host through high-throughput sequencing and transfection of specific miRNA inhibitors in vitro.

Since lncRNA/circRNA can competitively bind with miRNA, the expression level of target mRNA can be indirectly regulated through miRNA. ERG-associated lncRNA (lncRNA-ERGAL) was proved to be involved in promoting stability and integrity of vascular endothelial barrier during DENV infection by sponging miR-183-5p (Zheng et al., 2020). Li et al. (2020) established circRNA–miRNA–mRNA interaction network following JEV infection and unraveled cirC_0000220 binds to miR-326-3p to restrict BCL3, MK2 and TRIM25 mediated inflammatory response.

sfRNA is a product of incomplete degradation of viral genomic RNA by host cellular 5′-3′ exonuclease XRN-1 (Slonchak and Khromykh, 2018). However, the production of sfRNA is limited to Flavivirus genus and not found in the other two genera (Schnettler et al., 2012). This non-coding RNA generally promotes flavivirus pathogenesis by impairing the IFN response, as well documented in DENV, WNV (Schuessler et al., 2012; Pompon et al., 2017). But the specific affinity between ZIKV sfRNA and mosquito DEAD-Box helicase ME31B inhibits viral RNA replication and virion production (Göertz et al., 2019). Only when loaded into Argonaute (AGO), siRNAs and miRNAs perform their functions. Hence, Zeng et al. (2021) constructed AGO-associated viral siRNA profiles and completed a network between ZIKV siRNAs and their probable viral RNA (vRNA) targets via AGO-associated RNA sequencing. Twenty-nine siRNAs and 114 potential targets were obtained from the ZIKV genome, and further functional characterization of ZIKV siRNAs need to be verified. Hussain and Asgari (2014) obtained six miRNA-like vsRNAs mapped to the viral 5′ and 3′UTR regions, termed vsRNA1-6, in Ae. aegypti cells after DENV-2 infection. As vsRNA5 has the most significant effect on virus replication, it was selected for further study. The results showed that vsRNA5 with characteristics similar to miRNAs with AGO-2 targets the viral genome NS1 and inhibits viral replication. The same research group also utilized bioinformatics analysis to identify and coin a WNV-encoded small regulatory viral RNA, KUN-miR-1, from viral 3′UTR (Hussain et al., 2012). The transcription factor GATA4 is identified as a target of KUN-miR-1. GATA mRNA expression also increases after KUN-miR-1 RNA mimic is transfected into Ae. aegypti C6/36 cells, which seems to be necessary for efficient WNV replication.

To understand host–virus interactions is critical to antiviral response. Based on miRNAs regulate a variety of intracellular biological pathways, including virus infection, which has been demonstrated in many viruses (Patil and Karpe, 2020; Song et al., 2020; Zhao et al., 2021). Hence, we emphasized the interaction between flavivirus and host miRNA. Two primary regulatory approaches are adopted by miRNAs in response to viral infection. Due to the high mutation rate of RNA viruses, antiviral effects resulting from direct binding of miRNAs to viral genomes may be rare, unless the viral genome is extremely capable of binding to miRNA (Trobaugh and Klimstra, 2017). This may well explain the lack of coverage, only in DENV (Zhou et al., 2014; Wen et al., 2015; Castillo et al., 2016; Castrillón-Betancur and Urcuqui-Inchima, 2017). Moreover, it has been widely reported that viruses can manipulate the expression levels of specific miRNAs to establish an environment conducive to virus survival and transmission (Hussain et al., 2013; Wu et al., 2014; Chen et al., 2016; Sharma et al., 2016). Meanwhile, the host can also interrupt the virus life cycle by altering miRNA expression levels. In addition to host miRNA, host/virus also produces some other non-coding RNAs during viral infection, which all play different roles in response to viral invasion (Hussain et al., 2012; Ding et al., 2018; Göertz et al., 2019; Zheng et al., 2020; Zeng et al., 2021).

In this view, we collected the differentially expressed miRNAs infected by Flavivirus genus members, mainly DENV, JEV, WNV, and ZIKV. Through the classification of regulatory effects, it is found that these miRNAs principally participate in the process of viral replication, neuroinflammation, neurogenesis and so on (Escalera-Cueto et al., 2015; Liu et al., 2016; Castrillón-Betancur and Urcuqui-Inchima, 2017; Dang et al., 2019; Hazra et al., 2019; Mukherjee et al., 2019; Bhagat et al., 2021). Furthermore, several miRNAs show identical or opposite expression by different flavivirus infection and thus play similar or opposite roles. miR-146a, upregulated after DENV, JEV and ZIKV infections, promotes viral replication by targeting TRAF6 in different cell lines (Wu et al., 2013; Sharma et al., 2015; Shukla et al., 2021). As a member of the antiviral miR-34 family, miR-34c is down-regulated after JEV infection, while up-regulated after WNV and ZIKV infection. But they all work by targeting the NOTCH gene, therefore, it can be speculated that one of the reasons for this differential expression is the difference in cell lines. Although the anti-viral effect of miR-155 has been extensively studied, its expression is reduced during DENV infection to promote viral survival, but with distinct outcomes in JEV and WNV (Pareek et al., 2014; Arboleda et al., 2019; Natekar et al., 2019; Su et al., 2020). Overall, miR-146a, miR-155, and miR-34 family are expected to be potential antiviral targets, the mechanism by which they control the virus should be further investigated in the future. Beyond that, expressing tissue-specific miRNA recognition element has been used as an effective mechanism to exploit attenuated vaccines, helping to reduce the transmission of flavivirus (Lee et al., 2010; Pham et al., 2012; Yen et al., 2018; Buchman et al., 2019). Based on the close association between flavivirus and vector mosquitoes, a large number of mosquito-derived miRNAs have been reported (Skalsky et al., 2010; Hussain et al., 2013; Avila-Bonilla et al., 2020). These host-specific miRNAs also rely on binding to host genes to regulate virus infection. It is worth mentioning that Zhu et al. (2021) discovered a human blood-derived miRNA (miR-150-5p), which interferes with mosquito antiviral system to promote flavivirus infection through cross-species RNAi mechanism, determining the transmission ability of flavivirus in nature.

Other host-derived RNAs, such as lncRNA and circRNA, often regulate target mRNAs by forming a functional grid together with miRNA. During flavivirus infection, lncRNA-ERGAL and cirC_0000220 can regulate the biological processes of infected cells by associating with miR-183-5p and miR-326-3p, respectively (Li et al., 2020; Zheng et al., 2020). But this type of reporting is still limited and a lot of work needs to be done. Increasing evidence points to that viruses themselves can produce non-coding RNAs in addition to their hosts, such as sfRNAs, siRNAs and viral miRNAs mentioned in this review. The interaction between these non-coding RNAs and host/virus has expanded the novel strategy to manage flavivirus (Hussain et al., 2012; Hussain and Asgari, 2014; Slonchak and Khromykh, 2018; Göertz et al., 2019).

In brief, we discussed the regulatory role of host miRNAs after infection with Flavivirus genus members, mainly through miRNA directly binds to the viral genome or vital host genes, although the latter is more widely reported. Understanding host–virus interactions will help us discover promising targets of antiviral drugs and viral therapeutic, which will provide new thinking directions for preventing viral infection.

WC and YP contributed ideas for the review, wrote the manuscript, and produced the figures. AC, MW, ZY, and RJ edited and revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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