Mosquito’s innate immune system can recognize various microorganisms and mount a strong immune response against them (26). They recognize foreign particles and then initiate phagocytosis, produce antimicrobial peptides, produce melanin to trap pathogens, form nodules, and promote wound healing (27). RNA interference is a defense strategy used by mosquitoes against viruses (28). miRNAs are involved in the posttranscriptional level gene regulation and might also be involved in the regulation of insect immune response (29). Previous studies have suggested that miRNAs may play an important role in regulating and establishing infection of arboviruses and Wolbachia (an endosymbiotic bacteria) in mosquitoes (30). However, there is limited knowledge regarding the specific targets of these miRNAs.

In a study by Shrinet et al., 2014, it was observed that following the infection of CHIKV, there were significant changes in signaling and metabolic pathways, including protein synthesis in the endoplasmic reticulum and the immunity pathways. KEGG and KOBAS analysis also indicated that altered miRNA expression may regulate the cellular pathways. The authors further evaluated the role of vector miRNA in CHIKV pathogenesis. Different miRNAs, namely aae-miR-1000, aae-miR-190–5p, aae-miR-2b, and aae-miR-2c were downregulated in CHIKV-infected Ae. albopictus Singh’s cell line. Among these miRNAs, aae-miR-1000, aae-miR-2b, and aae-miR-2c have been suggested to be involved in ribosome biogenesis. Similarly, aae-miR-927–5p, aae-miR-305–3p, aae-miR-283, and aae-miR-100 were upregulated in Ae. albopictus Singh’s cell line upon CHIKV infection. The miRNA target prediction analysis revealed that aae-miR-305–3p, aae-miR-100, and aae-miR-283 target protein processing pathways and cell-mediated cytotoxicity. The aae-miR-305–3p and aae-miR-927 play a role in the vesicular transport of the SNARE (Soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein interaction. ( Supplementary Table 1 ) Also, the aae-miR-305–3p plays a role in virus ECM (extracellular matrix) receptor-mediated interaction and endocytosis pathways (31). Similar to CHIKV, differential expression of aae-miR-927 was observed in Dengue viruses (DENV) -infected C6/36 cells (32). Azlan et al., 2022, showed that aae-miR-927 acts as a proviral factor in DENV1-infected C6/36. Further, it was found that the aae-miR-927 targets filamin (FLN), which is part of the Toll signaling pathway (33). ( Supplementary Table 1 ) Certain miRNAs, such as aae-miR-927, may be involved in the pathogenesis of multiple arboviruses.

In contrast, Dubey et al., 2017 reported that in CHIKV-infected Aag-2 (Ae. aegypti cell line), the expression of aae-miR-2b was upregulated. aae-miR-2b has two target genes: URMs (ubiquitin-like modifier) and ubiquitine. ( Supplementary Table 1 ) The expression of these URM was significantly controlled by aae-miR-2b in Aag 2 (Ae. aegypti cell line), affecting CHIKV replication (34). Studies have demonstrated URM’s role in the thiolation of some transfer RNA (tRNA), and some viruses have developed diverse strategies to optimize tRNA utilization by using host tRNA for translation of viral proteins, packaging of virions, and priming reverse transcription reaction of their genome or some viruses encode their own tRNA (35). CHIKV infected Ae. aegypti showed enhanced levels of aae-miR-2b, leading to the downregulation of the URM, which controls the CHIKV replication in the vector (34) ( Figure 1 ).

In a study by Dubey et al., 2019, the authors studied the interactions of aae-miR-2944b-5p with CHIKV 3’ UTR. In addition, this miRNA targets cellular factor vacuolar protein sorting-13 (vps-13). ( Supplementary Table 1 ) Additionally, the cellular factor vps-13 contributes to balancing mitochondrial membrane potential in Ae. aegypti during CHIKV infection (36). The authors demonstrated that replication of CHIKV decreased in Aag2 cells (Ae. Aegypti cell line) and Ae. aegypti mosquitoes when vps-13 was silenced. Conversely, CHIKV replication increased in Aag2 cells when aae-miR-2944b-5b was silenced with anti-miR-2944–5p (36). In summary, research indicates that both aae-miR-2944b-5b and vsp-13 play crucial roles in the CHIKV lifecycle in insect hosts. Earlier studies have demonstrated that viruses like herpes simplex virus 1 (HSV-1) deplete the host’s mitochondrial DNA, while others, such as the human immunodeficiency virus (HIV), manipulate the host’s mitochondrial proteins to function entirely inside the host cell (37). The aae-miR-2944b-5p is restricted to insects and absent in mammals. The study shows that, by binding to the 3’UTR of CHIKV, aae-miR-2944b-5p downregulates CHIKV replication in mosquitoes. Also, it could maintain mitochondrial membrane potential during CHIKV infection by targeting cellular factor vps-13 (36). ( Figure 1 ) This study indicates that the mosquito cells may possess defense mechanisms that regulate viral proliferation, or the virus may exert control over the mosquito cells to ensure its survival within the cells. Thus, further studies are warranted to understand the detailed function and interactions of aae-miR-2b and aae-miR-2944b-5p. In summary, the findings suggest that manipulating the activity of specific miRNAs can be an effective method to restrict the transmission of arboviruses.

The saliva of mosquitoes consists of various proteins and enzymes that play roles in the modulation of blood coagulation, inflammation, platelet aggregation, and vascular constriction (38). It creates a favorable microenvironment for virus establishment and disease progression. Exosomes are the extracellular vesicles that are responsible for the transportation of miRNA in saliva and serum (39). A study by Fiorillo et al., 2022, demonstrated that certain miRNAs are enriched in the saliva and midgut of Ae. Aegypti (38). In another study by Maharaj et al., 2014, the authors identified 103 miRNAs in mosquito saliva using the next-generation sequencing platform. Several of these miRNAs were specifically expressed in the context of CHIKV infection. Authors used synthetic miRNA inhibitors to study the effect of inhibition of specific miRNA. They chose five distinct miRNAs (aae-miR-12, aae-miR-125, aae-miR-184, aae-miR-375, and aae-miR-2492) based on their relative abundance levels in CHIKV-infected saliva and prior studies. Aag-2 cells were treated with miRNA inhibitors, and the replication of CHIKV was assessed in these cells. Suppression of all five miRNAs exhibited declined CHIKV titer in Aag-2 cells. Notably, the suppression of aae-miR-184, aae-miR-375, and aae-miR-2492 resulted in a reduction in CHIKV titer in both Aag-2 (insect cells) and BHK-21 cells (Mammalian cells) (40). It is suggested that mosquito salivary miRNAs may potentially influence CHIKV pathogenesis across different species.

CHIKV mainly infects peripheral tissue but also affects the central nervous system in newborns and adults (41). The peripheral tissues are the first site of the virus encounter and the location of different immune cells (42). CHIKV initially replicates in the skin and fibroblast cells and then circulates via blood to the liver and joints (43). CHIKV infection leads to the activation of various signaling pathways in host cells (44–46). miRNAs are known to be involved in the regulation of viral infection, inflammation, and immune response (47). To date, the function of miRNA in CHIKV infection remains unclear.

Sharma et al., 2015, showed significant upregulation of hsa-miR-409–3p in CHIKV-infected fibroblast cells, and it was predicted to target topoisomerase IIβ (48) ( Supplementary Table 2 ). Topoisomerase IIβ is a DNA gyrase that has an essential role in the transcription and replication of DNA (49, 50).

Saxena et al., 2013, revealed that the cluster of hsa-miR-15b/16, hsa-miR-17–92, hsa-miR-23a/24, and hsa-miR-106b/miR25 were found to be upregulated in CHIKV-infected Human Embryonic Kidney 293 (HEK293T) cells (51). The miRNA clusters hsa-miR-17–92, hsa-miR-23a, and hsa-miR-106b regulate the TGF-b (JUN, SMAD6) signaling pathway (52, 53). TGF-b, endocytosis, and adherens junction pathways were upregulated during CHIKV infection, while the cell cycle, proteasome, and lysosome pathways were significantly downregulated (51). The hsa-miR-15/16 cluster targets BCL2 and triggers apoptosis (54). ( Supplementary Table 2 ) hsa-miR-15 and hsa-miR-16 act as regulators in Rheumatic arthritis (RA). The hsa-miR-15 was shown to be downregulated in arthritic synovial tissue, whereas hsa-miR-16 was upregulated in synovial fluid of RA patients (55, 56). Study showed that the apoptosis pathway and CHIKV-engulfed macrophages are the main contributors to prolonged arthritis (46). Studying the role of these miRNAs in CHIKV infection can aid in developing biomarkers to predict arthritis or treatments for CHIKV infection.

Agrawal et al., 2020 studied the differential regulation of miRNA in CHIKV-infected primary human synovial fibroblast cells and observed that upregulation of hsa‐miR‐1264, hsa‐miR‐4717‐3p, hsa‐miR‐ 4299, and hsa‐miR‐21. The miRNA target prediction analysis indicates that hsa‐miR‐4717‐3p targets the AKT3 (57). ( Supplementary Table 2 ) Many viruses exploit PI3K/Akt/mTOR for survival in host cells (58). Reports indicate that CHIKV infection relies on the PI3K/Akt/mTOR pathway for its replication and survival in the Vero cells. (Origin: African green monkey kidney epithelial cells) (44). The PI3K/Akt/mTOR pathway plays a vital role in protein translation, metabolism, survival, and cell growth. AKT is a crucial signaling molecule for these pathways (59). It has a role in developing T cells and regulates transcription factors NF-κB and FOXO3, which are responsible for T cell activation (60, 61). During CHIKV infection, AKT activation leads to the mTOR’s phosphorylation, which activates S6K and 4EBP1, which is responsible for the translation of the viral and host mRNAs (44). The hsa-miR-21 also targets the AKT, PTEN, and PELI1 (62). PELI is an E3 ubiquitin ligase that regulates innate immunity by ubiquitylation of IRAK1 (63). Also, the TRIM and SOCS 7 are potential targets of hsa-miR-1264 and hsa-miR-4299, respectively (57). ( Supplementary Table 2 ) TRIM is an E3 ubiquitin ligase that targets the transcription factor IRF3, which is mainly involved in IFN- β production (64). The SOCS family of proteins inhibits cytokine-induced signaling through the JAK/STAT pathway (65). SOCS 7 is a less-studied member of this family, and it inhibits the growth hormone and prolactin signaling pathway via STAT3 and STAT5 proteins (66). Hence, this miRNA may suppress the inflammatory response in CHIKV infection by targeting host signaling pathways.

Selvamani et al., 2014 demonstrated the role of hsa-miR-146a in the CHIKV life cycle. The levels of hsa-miR-146a were increased in CHIKV-infected primary human synovial fibroblasts. Upregulation of hsa-miR-146a led to decreased expression of IRAK1, TRAF6, and IRAK2, ultimately inhibiting the function of nuclear transcription factor kB (NF-kB). The inhibition of nuclear transcription factor kB (NF-kB) may reduce proinflammatory cytokine production. ( Figure 2 ) Hence, CHIKV may be using hsa-miR-146a to modulate the antiviral response (45). Similarly, hsa-miR-146a has been reported to disrupt the type I interferon and RIG signaling pathways in the case of vesicular stomatitis virus (VSV) (68). Primary hsa-miR-146a and mature hsa-miR-146a have been detected in RA synovial tissue (69). This study indicates that hsa-miR-146a may modulate the host antiviral response to establish CHIKV pathogenesis in primary human synovial fibroblasts.

Kansakar et al., 2022 have demonstrated that hsa-miR-142 targets TIM-1 in Human Endothelial cells (70). TIM-1 serves as the receptor for several viruses, including CHIKV (71). Authors suggested that hsa-miR-142 may play an important role in the life cycle of viruses such as CHIKV (70). Overall, it is suggested that hsa-miR-142 may be exploited further as a biomarker as well as a therapeutic candidate.

López et al., 2020, studied the role of hsa-miR-124–3p in CHIKV-infected Huh7.5.1 cell line. The hsa-miR-124–3p binding site was identified on both CHIKV and SINV genomes. When hsa-miR-124–3p was overexpressed in Huh7.5.1 cells, a significantly increased CHIKV replication was observed. While inhibition of hsa-miR-124–3p led to a drop in CHIKV titer (67). ( Figure 2 ) However, the hsa-miR-124–3p is a nervous system-specific miRNA (72). Thus, it would be interesting to examine whether hsa-miR-124–3p is associated with neuropathology during CHIKV infection.

Scheel et al. (2017) analyzed the interaction of miRNAs with fifteen infectious RNA viruses, including CHIKV, using the AGO-CLIP (Argonaute-crosslinking immunoprecipitation) method and miRNA target chimera analyses. Their study suggests many miRNA can potentially interact with CHIKV genome such as miR-21, miR-122, let -7 and miR-181 (73). Patil et al. (2020) further predicted the presence of a hsa-miR-214–3p binding site on the CHIKV genome (74). However, the role of these miRNAs in the CHIKV life cycle is still unknown. In summary, these miRNAs could be exploited for the development of broad-spectrum therapeutics. Therefore, it is essential to study the role of miRNAs in both host signaling pathways and virus infection.