The initial product of RNA Poll II transcriptions is primary miRNA (pri-miRNA) transcripts. These pri-miRNAs have one or more hairpin loop structures, and their length ranges into several hundred kilobases. The microprocessor complex processes these long transcripts (pri-miRNAs) into 70 nt precursor miRNAs (pre-miRNA) in the nucleus (Conrad et al., 2014). Drosha, an RNase III enzyme along with its double-stranded RNA (dsRNA) binding partner, Pasha, constitutes the microprocessor complex (Lee et al., 2003; Denli et al., 2004; Gregory et al., 2004; Han, 2004; Landthaler et al., 2004; Kawai and Amano, 2012; Burke et al., 2014; Nguyen et al., 2015; Kim et al., 2016a). The stem structure of pri-miRNA with 30 bp has a terminal loop, and flanking segments are processed by the microprocessor complex after recognition. The dsRNA binding partner protein Pasha recognizes substrate pri-miRNA, plays an important part in anchoring to the flanking single-stranded RNA (ssRNA) and dsRNA stem junction, and locating the position 11 bp into the stem, where Dorsha is loaded to cleave the pri-mRNA (Lee et al., 2003; Denli et al., 2004). For further processing, the end product of Drosha-Pasha processing, a 70 nt pre-miRNA, is exported into the cytoplasm by Exportin-5 (Exp-5; Kim et al., 2016). The pre-miRNA generated by Drosha’s action on pri-miRNA has a 2 nt 3' overhang, critical for pre-miRNA export. Exp-5, a dsRNA-binding receptor depending on ran guanosine triphosphate (RanGTP), starts the nuclear export of pre-miRNAs by identifying the 2 nt 3' overhang structure of pre-miRNA in the nucleus (Bohnsack, 2004). The Exp-5/pre-miRNA complex migrates through the nuclear pore complexes into the cytoplasm. In the cytoplasm, RanGTP is hydrolyzed to RanGDP, which results in the release of pre-miRNA from the Exp-5/pre-miRNA complex. The pre-miRNAs are protected from digestion by nucleases of Exp-5, which is also the nuclear export factor of pre-miRNAs (Yi, 2003; Lund, 2004).

An RNase III enzyme, Dicer-1 (Dcr-1), slices the terminal loop structure of pre-miRNA, releasing a ~22 nt miRNA-miRNA* duplex in the cytoplasm (Hutvagner, 2001; Koscianska et al., 2011; Feng et al., 2012; Gurtan et al., 2012; Bogerd et al., 2014). Dicer, a core enzyme in the RNAi pathway, was first identified in Drosophila (Bernstein et al., 2001). Two Dicer enzymes, Dcr-1 and Dcr-2, are encoded by the B. mori genome having the specialized function in two crucial pathways, i.e., miRNA and siRNA pathways (Ponnuvel et al., 2007; Kolliopoulou and Swevers, 2013). A pre-miRNA processing complex is formed by the interaction of Dcr-1 with Loquacious (Loqs) (Saito et al., 2005). The pre-miRNA is accumulated, and mature miRNAs are reduced when RNAi depletes the Loqs; depletion of Dcr-1 also results in similar effects (Jiang, 2005).

From mature miRNA-miRNA* duplex resulted from pre-miRNAs processing by Dcr-1, a strand from the duplex is loaded into the RISC. RNA-binding proteins guided by sRNA from the argonaute family are the fundamental components of the RISC. Four discrete ago proteins encoded by the B. mori genome are classified into two sub-clades: Ago and Piwi (Wang et al., 2013). Subgroup ago includes the ago-1 and ago-2 proteins that bind with miRNAs and siRNAs, respectively (Hammond, 2001). The mature miRNA guide strand is loaded into ago-1, while the miRNA* strand is degraded (Kawamata et al., 2009). The miRNA-Ago complex is ready to perform its action on target sequences.

Several studies have reported change in cellular miRNAs expression levels in insects in response to pathogen infection based on deep sequencing or microarray analysis (Figure 2). For example, Karamipour investigated the difference in the expression profile of cellular miRNA-184 and let-7 following Autographa californica nucleopolyhedrovirus (AcMNPV) infection of Sf-9 cells. Findings suggested that following AcMNPV infection, there was a difference in the expression of cellular miRNAs at the post-infection times. The expression profile of the core components of miRNA’s biogenesis pathway, Dcr1, Ago1, and Exp5, was upregulated at 16 h post-infection (hpi) following AcMNPV infection. Though, in response to virus infection, Ran’s expression was decreased (Karamipour et al., 2019). An abundant miRNA, Bantam, accounts for more than 5% of the total miRNA of Sf9 cells. Sequenced data suggested that the bantam level was increased late in AcMNPV infection and confirmed with real-time quantitative PCR in infected Sf9 cells and Spodoptera litura larvae (Shi et al., 2016).

At 24 h pi, there was an increase of 2.5-fold in bantam level in uninfected cells, and a high level of bantam was also present late in the infection. The bantam level was also about 1.5-fold more elevated than the control in AcMNPV-infected S. litura larvae, indicating that bantam has a role in the growth and modulates the insect-baculovirus interaction as well (Shi et al., 2016).

Deep sequencing of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infected midgut of silkworm suggests that the expression profile of host miRNAs were altered after 72 and 96 h of viral inoculation. In the RNA libraries constructed from the BmCPV-infected midgut of silkworm and the control midgut, a total of 316 known miRNAs (including miRNA*) and 90 novel miRNAs were identified. A significant difference in the expression of 58 miRNAs was observed between the infected and the normal midgut (Wu et al., 2013). Two small RNA libraries were constructed from Bombyx mori nuclear polyhedrosis virus (BmNPV) infected larvae and the control larvae. Solexa sequencing results revealed significant downregulation of miR-2819 in infected larvae (Wu et al., 2019). Results showed that miR-2819 has a relatively high abundance in several tissues, including Malpighian tubules, fat body, hemolymph, silk gland, and midgut. The abundance of miR-2819 of infected cells was compared with that of control cells at different time points. The results showed that during the 6–12 hpi, the expression level of miR-2819 was increased, while during 24–72 hpi, the expression level of miR-2819 was significantly declined (Wu et al., 2019).

The knowledge about the modulation by miRNAs in the establishment of infection is increasing as more of these studies are emerging. New dimensions are now discovered how the complex phenomena of host-pathogens occurred upon pathogens invasion and how the host responds to these invading pathogens.

Cellular miRNAs may directly target viral genes and impact the propagation and establishment of infection (Table 1). Apart from regulating cellular processes, host miRNAs also have a crucial role in defense against pathogen attack, thus helping in infection propagation (Skalsky and Cullen, 2010; Asgari, 2011; Hussain and Asgari, 2014). miRNA also addresses the biotic stresses like protein-coding genes (Ghosh et al., 2009; Croston et al., 2018). A large number of miRNAs have a differential expression upon pathogen attack (Ghosh et al., 2009; Maudet et al., 2014; Croston et al., 2018). This differential expression of miRNAs indicated the critical role they play in host-pathogen interactions. For instance, with various putative binding sites, B. mori miR-8 was defined as an antiviral miRNA. The putative binding sites were on the BmNPV immediate-early gene (ie-1) mRNA and other vital genes of BmNPV. On blocking of Bmo-miR-8, a 3-fold increase in the ie-1 transcript level and an 8-fold increase in BmNPV accumulation in fat body tissues of infected larvae was observed, indicating a significant increase in the virus load in the infected B. mori larvae (Singh et al., 2012). Similarly, the role of bantam on viral infection was studied. The results indicated that cellular miRNA bantam in Sf9 cells plays an essential role in insect growth and baculovirus-insect interaction. The expression of the most affected viral genes lef8, gp41, and p10 was increased by 8, 10, and 40 times after applying bantam inhibitor or mimic in Sf9 cells. In infected Spodoptera exigua, larval mortality increased from 47% without antago-miR (bantam inhibitor) to 80% with it (Shi et al., 2016). In another study, it was demonstrated that miR-278-3p plays a vital role in BmCPV replication. Insulin-related peptide-binding protein 2 (IBP2) gene, induced by BmCPV infection, having a vital role in B. mori immune response, was identified as one of the targets of miR-278-3p by using a luciferase reporter assay. Over-expression of miR-278-3p negatively regulates the expression of IBP2 in silkworm larvae and positively regulates the mRNA transcript level of BmCPV, and confirms that the miR-278-3p is important for viral infection establishment (Wu et al., 2016). Bombyx mori miR-2,819 effectively downregulates the immediate early gene ie-1 required for viral replication, affecting the viral infection. miR-2,819 also affects other gene’s expression, i.e., polyhedrin and VP39 of BmNPV (Wu et al., 2019).

Accumulation of 20-hydroxyecdysone (20E) in the late phase of larval instar results in the initiation of molting and metamorphosis (Riddiford et al., 2003). MiRNA bantam affects baculovirus-host interaction by regulating 20E. Reduction in bantam level results in an increased level of 20E, resulting in high mortality in virus-infected S. exigua (Ran et al., 2018). Bmo-miR-390 effectively downregulates the expression of BmNPV-cg30 in BmNPV-infected BmN cells. Cg30 gene of BmNPV is required to replicate BmNPV as a deletion or mutation in this gene results in decreased occlusion bodies (OB) production and reduction in toxicity to the silkworm larvae (Ishihara et al., 2013; Kang et al., 2018).

The impact of miR-274-3p on the BmCPV replication in the silkworm larvae infected with the BmCPV was investigated. The experimental techniques, including bioinformatics analysis, identified BmCPV Nonstructural protein 5 (NS5) as the potential target of miR-274-3p. qRT-PCR and Western blotting results revealed that the level of NS5 was reduced significantly by miR-274-3p inhibitors, while the polyhedrin gene expression was increased after the application of miR-274-3p inhibitors. The inhibition of miR-274-3p facilitates BmCPV replication by upregulating BmCPV NS5 gene expression (Wu et al., 2017). The authors demonstrated that mature artificial miRNAs (amiRNAs) expressed successfully target the viral lef-11 gene. Mature amiRNAs efficiently inhibited the BmNPV proliferation by silencing the target gene. The overexpression of mature amiRNAs may induce acute cellular toxicity (Zhang et al., 2014). Next-generation sequencing (NGS) showed that 167 genes were upregulated and 141 genes were downregulated in larval instars of B. mori following pathogenic infection. Several genes with a role in B. mori immune response against BmCPV were identified. The 2-fold upregulation of the core RNAi genes Ago-2 and Dcr-2 was observed during pathogenic infection (Kolliopoulou et al., 2015). Reports revealed that exo-RNAi is operative in the silkworm B. mori against pathogenic infection of BmCPV, which is characterized by a segmented dsRNA genome (Zografidis et al., 2015).

MicroRNAs carry out their functions through their targets, and for viral miRNAs, their targets can be the host genes or the viral genes. MiRNAs can bind to the 3' or 5' UTR of the target mRNA, and they also can bind to coding sequences to manipulate target gene expression (Table 2). For instance, bmnpv-miR-3 miRNA encoded by BmNPV modulates the expression of DNA binding protein (P6.9), vital for the late stage of viral infection in the host, B. mori. The downregulation of BmNPV late genes helps BmNPV escape the host’s early immune response (Singh et al., 2014). The viral miRNAs are mainly involved in the active regulation of viral genes. It may reduce the viral DNA load and the number of infectious budded virions (BVs) to avoid host surveillance and prolong residence duration inside the host cell.

AcMNPV-miR-1 is an AcMNPV encoded miRNA. The ac-94 gene involved in producing infectious BVs was downregulated by AcMNPV-miR-1, resulting in the decreased production of BVs and enhancing the formation of occlusion-derived virions (ODVs). The overexpression of AcMNPV-miR-1 reduces the budded virus’s infectivity, affecting viral DNA replication and accelerating ODVs formation. AcMNPV-miR-1 moderately downregulated ac95 and upregulated ac18. These findings suggest that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae (Zhu et al., 2016; Wang et al., 2020a). The most distinct downregulation for AcMNPV-miR-2 was observed for lef-6, with lef-11, orf-49, and orf-63 also showing apparent downregulation, while orf-38 did not exhibit significant regulation (Wang et al., 2020a). AcMNPV-miR-3 plays a regulatory role in BV and ODV production. AcMNPV-miR-3 is located on the opposite strand of the viral gene ac101 coding sequence in the AcMNPV genome and detected at 6 hpi. and reaches a maximum level of around 12 hpi. in AcMNPV-infected Sf9 cells. AcMNPV-miR-3 downregulates ac101 through a siRNA-like cleavage mode. Ac101 is a core gene required for BV and ODV production, viral infectivity, and virus-induced nuclear actin polymerization. Dual-luciferase reporter assay revealed a slight downregulation with ac23, ac25, ac86, and ac98 target sites (Jiao et al., 2019; Wang et al., 2020a).

The cellular state from susceptible to resistant or vice versa may be remodeled upon abnormal-expression of cellular miRNAs due to the abnormal expression of their target genes (Ghosh et al., 2009). A huge amount of literature is available describing miRNAs’ identification from insect hosts, but the exact role in host-pathogen interactions is still limited to a few reports (Ylla et al., 2016; Kozomara et al., 2019). Moreover, these studies mainly focus on miRNA alterations due to the viral attack in insects (Tang et al., 2019).

Recent investigations have revealed that viral miRNAs might target and regulate host genes to establish infection and successfully replicate. However, the information is minimal concerning insect viruses. The miRNAs’ impact might be via regulation of a specific gene leading to modifying a particular pathway or might cause a global reduction in the host miRNA depending on the target genes (Table 3). For instance, a BmNPV encoded miRNA (bmnpv-miR-1) has been shown to downregulate the host GTP-binding nuclear protein Ran’s expression, thereby inhibiting small RNA transport from the nucleus into the cytoplasm to ensure their active proliferation (Singh et al., 2012). Reports indicated that an insect double-stranded RNA virus (BmCPV) might generate miRNAs, and these miRNAs play an essential role in host viral interaction. BmCPV-miR-3 and BmCPV-miR-5 regulate host target genes and manipulate viral replication and proliferation (Pan et al., 2017) AcMNPV encoded miRNA AcMNPV-miR-4 interfere with the host cell cycle, cytokine secretion, exocytosis, and membrane fusion as it targets the ALG2 and ROP genes. ALG-2 is involved in apoptosis initiation. Rop is highly expressed in the nervous system and in specialized tissues where intensive exocytic/endocytic cycles occur, providing evidence that it impacts viral infection and propagation (Jiao et al., 2019). A BmCPV-derived miRNA (BmCPV-miR-1) effectively up-regulates the expression of the B. mori inhibitor of apoptosis protein (BmIAP) gene. It inhibits cell apoptosis mechanisms, hence favoring better replication of the virus and helps in viral infection establishment (Guo et al., 2020).

E66 a structural protein of the ODV encoded by ORF37 of the BmNPV genome. It also encodes a miRNA BmNPV-miR-415, which produces bmo-miR-5738 when introduced into the host cells. Bmo-miR-5738 upregulates the expression of TOR2 through its 3' UTR. TOR2, as a member of the phosphatidylinositol kinase-related kinase family, plays a critical role in metabolism, development, growth, and survival at the cellular and organism levels. Recently it has been revealed that TORE2 also upregulates the molting hormone 20E (Cao et al., 2017). Apoptosis in Sf9 cells due to the inhibition of miR-14 indicates that it is essential for constitutive cell survival. When mimics of miR-14 precursor molecules were applied, they partially inhibited the cell death induced by actinomycin-D (Act-D). MiR-14 might have inhibitory interactions with caspases, as it functions downstream of mitochondrial cytochrome and hence averting Act-D-induced apoptosis (Kumarswamy and Chandna, 2010). Similar exploitation of host-pathogen interaction by miRNAs was also observed in other diamondback moths. Certain miRNAs are also produced by Cotesia vestalis, (a parasitoid of Plutella xylostella) and bracovirus (CvBV). The parasitized host revealed that most of the miRNAs that arrest the host’s growth were of C. vestalis origin. In contrast, the expression profile of the miRNAs encoded by CvBV was 100-fold increased in parasitized hosts than non-parasitized ones. miRNAs arrest the growth of the host by modulation of the host ecdysone receptor (EcR; Wang et al., 2018).

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.