Epstein-Barr virus (EBV) belongs to the Herpesviridae family and infects more than 95% of adults worldwide [1]. EBV infection often has not any symptoms and the virus establishes a lifelong latent infection. EBV is associated with many diseases, including Burkitt's lymphoma [2], nasopharyngeal carcinoma [3], and autoimmune diseases [4] such as systemic lupus erythematosus [5], Sjogren's syndrome [6], rheumatoid arthritis [7], and multiple sclerosis (MS) [8]. The principal role of EBV as the main environmental causal agent of MS is well established [9]. Infection of B cells with EBV triggers some events, which drive the infection into an autoimmune response in MS disease [10].

miRNAs are small non-coding antisense RNAs that are approximately 18–25 nucleotides in length. miRNAs attach to the 3′ untranslated region (3′UTR) or open reading frame (ORF) of target mRNAs and perform their biological functions miRNAs can control various cellular activities ranging from normal development and homeostasis to disorders and regeneration. This can be mediated by mRNA suppression, degradation, or interfering with mRNA translation [11], [12], [13]. EBV expresses 44 mature microRNAs (miRNAs) derived from 25 miRNA precursors encoded by two primary transcripts (BamH I fragment H rightward open reading frame1 CSF [BHRF1] and BamH I fragment A rightward transcript [BART]) which are important for cell survival and proliferation in the pre-latent phase of B cell infection. EBV miRNAs can regulate genes involved in cell apoptosis, antigen presentation, and recognition, as well as B cell transformation [14], [15], [16], [17], [18]. Results showed EBV infection triggers deregulation of host miRNAs to a different extent. Chronic infection can alter the expression of host miRNAs, which in turn can modulate the host inflammatory response [19], [20]. Deregulated host miRNAs can affect B cell function. The miRNA expression profile of B cells before and after EBV infection found that EBV miRNAs and host B cell miRNAs were dysregulated after EBV infection and involved in MS pathogenesis through their interaction with MS risk loci [21]. Another study identified the expression of EBV miRNAs in MS and explored potential targets for EBV miRNAs [22]. EBV miRNAs, including BART miRNAs, have essential functions in cancer growth, tumor invasion, and host immune surveillance [23].

Extracellular vesicles (EVs), such as microvesicles and exosomes are derived from endosomes and contain a complex lipid-bilayer membrane with surface-encoded proteins. Exosomes are a subtype of extracellular vesicles with 40–100 nm size [24], [25]. Exosomes are small vesicles that are secreted by most cell types and interna;ized into the receipient cells. These vesicles contain various molecules derived from the original cell, including DNA, RNA (coding and non-coding), proteins, transposable elements, and lipids. Exosomes mediate cell-to-cell communication locally and systemically and are present in various biological fluids such as breast milk, blood, serum, urine, saliva, amniotic, and synovial fluids [26], [27], [28]. The delivery and transfer of exosomes to their recipient cells are regulated by “Endosomal Sorting Complexes Required for Transport” (ESCRTs), Ca+2 channels, and cellular pH levels. Internalization is a temperature-sensitive process where cells rapidly uptake exosomes via endocytic pathways that subject exosomes to various intracellular compartments [29], [30]. Proteins, metabolites, and nucleic acids delivered by exosomes into recipient cells effectively alter their biological response [31]. Exosomes represent a novel mode of intercellular communication, which may play a major role in many cellular processes, such as immune response, signal transduction, antigen presentation, and tumor progression. Exosomes are of significant importance in the immune cell migration across the blood-brain barrier (BBB) and the formation of MS [32], [33], [34], [35], [36], [37]. Several studies revealed that exosomes derived from virus-infected cells contain viral small RNAs and miRNAs including BART miRNAs involved in pathogenesis of the diseases [20], [38], [39], [40], [41]. These observations highlight the importance of exosome-mediated EBV miRNAs transport and its subsequent gene regulation effects. Accordingly, characterizing the exosomes of cerebrospinal fluid (CSF) circulating in CNS, a potential site of pathogenesis, could be very reasonable.

Considering that EBV miRNAs expression profile is specific to the disease status and the virus life cycle and changes in the level of microRNAs indicate a change in viral activity [16], [42], [43], [44], [45], in this study, for the first time, we measured the level of ebv-miR-BART9-3p and ebv-miR-BART15 in CSF exosomes from relapsing-remitting MS (RRMS) patients. It is noteworthy that there is no any published report about the expression level of ebv-miR-BARTs in CSF RRMS patients, and we hypothesized that quantification of EBV-encoded miRNAs could address the association between MS and EBV infection. In this study, we elucidated the presence of ebv-miR-BART 9-3p and ebv-miR-BART 15 in CSF exosomes from untreated RRMS patients, as compared to the control group. Forte et al. found that EBV infection induces the expression of some miRNAs, including hsa-miR-21-5p and hsa-miR-146a-5p in B cells, which are involved in oncogenesis [46]. So, we measured the levels of hsa-miR21-5p and hsa-miR146a-5p, which are dysregulated in EBV infection. Additionally, we elucidated the variation in the concentration of cytokines involved in MS including interferon-gamma (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-β) and tumor necrosis factor-alpha (TNF-α).