Viral infection can initiate a multitude of physiological changes in host cells that can contribute to ME/CFS development (Fig. 1). Viral pathogens frequently associated with ME/CFS are also known to alter various molecular processes in host cells that define clinical conditions of ME/CFS (Table 3). Three of the most frequently discussed molecular processes, namely immune cell alterations, mitochondrial modulation and autoimmunity, are described in brief within this section.

Fig. 1 Schematic diagram showing various viral pathogens potentially associated with ME/CFS and possible molecular mechanisms altered by these pathogens that can contribute to ME/CFS development Full size image

Table 3 Possible viral contributions towards ME/CFS Full size table

Virus-induced alterations to immune cells

One commonality among the viruses linked to ME/CFS appears to be the ability to establish persistent infections. In order to do that, viruses must bypass and evade immune cells, and in doing so may alter immune cell functions. While several viruses may contribute to ME/CFS, the immunomodulatory capacity of the viral species, perhaps paired with its ability to establish persistent infection, may underlie its pathological potential in the setting of ME/CFS. Elevated activity of antiviral enzyme 2′-5′-oligoadenylate synthetase (OAS) in PBMC from patients with ME/CFS has been observed. This protein is induced by IFN-α and IFN-β, and degrades viral RNA and inhibits virus replication. Therefore, it plays an important role in the response against viral infections. The level of OAS correlates with severity of ME/CFS, suggesting that a chronic virus infection could be the cause of ME/CFS [151]. Moreover, proof of humoral immune response activation by herpesviruses-encoded dUTPases in patients with ME/CFS has been published [85].

The establishment of a persistent infection is influenced by immunosuppression and activated immune complexes, which may cause chronic inflammation [15, 152, 153]. Chronic immune system activation is accompanied by alterations in regulation of cytokine production [154], and stimulated lymphoid cells express or induce the expression of various cytokines in other cells that can set the stage for pathological manifestations [155]. Low-level inflammation and activation of cell-mediated immunity is observed in ME/CFS cases and the high level of TNF-α correlates with several clinical symptoms; therefore, an increase of inflammatory mediators might explain symptoms of the disease [156]. Likewise, it is possible that a viral infection causes dysfunction in cellular immunity, which consequently induces viral reactivation. Subsequently, viral proteins facilitate cytokine secretion, resulting in appearance of typical ME/CFS symptoms, such as fatigue, fever, sleep and cognitive disorders [7]. Chronic pain may be caused by inflammatory signals that are spread by glial cells activated by inflammatory cytokines and neuronal stimulation [153, 157].

In establishing persistence, viruses may induce immune disturbances directly and indirectly; for instance, they may infect cells involved in mediation of cellular and humoral immune response, and through indirect interactions, they might alter cell surface receptor expression as well as cytokine and chemokine expression levels, leading to local inflammation [158]. Infection of cells by enteroviruses affects cellular miRNA expression, which may result in dysregulation of immune pathways and cytokine production, and these viruses can reduce expression of type I and III interferon, which are primarily produced by natural killer (NK) and T cells [159,160,161]. Herpesviruses like HHV-6 are also able to alter expression of cellular miRNAs in various cell types including NK cells [162, 163], as well as cellular expression of NK cell receptors [164], and they may express their own miRNAs that aid in immune evasion [165]. Downregulated cytotoxic activity of NK cells in ME/CFS could be linked to a decreased expression level of NK cell activating receptor NKG2D [166], the ligand of which is down-regulated by HHV-6B [164]. In addition, the expression of viral homologs of cytokines/chemokines and cytokine/chemokine receptors by herpesviruses can impact immune pathways [167, 168]. Other herpesvirus-induced alterations to ligand/receptor signalling between NK and associated cells could also contribute to the pathogenic mechanism of the viruses in ME/CFS are described [169].

Enteroviruses, as well as most of the herpesviruses, use mechanisms that disturb first class major histocompatibility complex (MHC) molecules presenting virus antigen [167, 170, 171]. The HHV-7 U21 gene product interrupts viral antigen presentation to cytotoxic T cells that causes MHC class I molecules degradation in lysosomes. Besides interfering with the host cytotoxic T cell response, U21 lessens NK cell cytotoxicity [172]. Such changes of immunological parameters as a decreased count of CD3+ and CD4+ T cells, an increase of CD95+ and a decrease of CD4+/CD8+ ratio are observed in patients with a concurrent active HHV-6 and HHV-7 infection [64]. Other studies also show CD4+ T cell response to HHV-6 [173].

In vitro studies show that a possible mechanism of action for B19V is direct virus interaction with cells leading to more aggressive fibroblast functionality and degradation of cartilage matrix. Moreover, the activity of capsid protein VP1 affects arachidonic acid metabolism promoting inflammatory reactions, and the B19V non-structural NS1 protein also stimulates pro-inflammatory cytokine production, causing local inflammation [174] that might account for such ME/CFS clinical manifestations as fatigue, lymphadenopathy, joint pain, and muscle pain [68, 175, 176]. Enteroviruses [177, 178] and herpesviruses may also contribute to these signs and symptoms through up-regulation of pro-inflammatory cytokines [179].

Some viruses, like RRV, are able to infect macrophages using an antibody-dependent enhancement mechanism, suppressing antiviral genes and thereby resulting in replication [146]. The antiviral response to RRV is diminished by suppression of transcription factor protein complex activity [147]. In the case of acute RRV infection, functional polymorphisms in cytokine genes may affect the influence of inflammatory cytokines in the CNS and resulting neurocognitive manifestations [150].

Viruses and mitochondrial modulation

ME/CFS is considered to be a mitochondrial disease [180]. Some of the characteristic features of ME/CFS involve altered adenosine triphosphate (ATP) homeostasis [181, 182], increased reactive oxygen species (ROS) [183], changed mitochondrial metabolism [184, 185], and modulation of mitochondrial DNA content [182, 186, 187]. Many of the aforementioned viruses modulate host mitochondria in a variety of ways that can potentially present plausible explanations regarding the involvement of these viruses in ME/CFS. Viruses have evolved distinctive strategies to alter mitochondrial metabolism and bioenergetics, which may allow enhanced viral replication and provide anti-viral defence.

HSV-1 infection decreases cellular ATP levels and mitochondrial membrane potential [188], and HSV-1 anti-apoptotic protein gJ has been shown to induce ROS formation [189]. In vitro experiments in mammalian cells have shown rapid and complete degradation of host mitochondrial DNA by HSV-1 [190]. Evidence of mitochondrial dysfunction has been associated with post-infective fatigue after EBV infection [191], and EBV is known to alter mitochondrial dynamics through direct interaction [192]; the immediate early protein of EBV, Zta, interacts with mitochondrial single stranded DNA binding protein, leading to reduced mitochondrial DNA (mtDNA) replication and enhanced viral DNA replication [193]. HHV-6B improves viral infection through direct interaction of its U95 protein with human GRIM-19 protein [194], and the virus increases oxidative stress during persistent infection by reducing glutathione reductase activity [195]. During productive infection, HHV-6A can cause apoptosis through a caspase-dependent pathway accompanied by altered mitochondrial morphology and lower transmembrane potential [163, 196]. Similarly, latent CMV has been associated with greater oxidative damage [197]. HHV-8 modulates mitochondrial antiviral signalling via its interferon regulatory factor 1 [198]. In addition, porcine and canine parvovirus infection induces depolarization of the mitochondrial membrane, damage to the organelle’s structure [199], and ROS accumulation [200, 201]. Enterovirus 71 also induces ROS accumulation [202] for its successful replication.

Viruses and autoimmune signature

Autoimmune signature in ME/CFS has recently become a subject of intense research [29, 203]. Viral pathogens can contribute to autoimmune diseases in variety of ways. Because the viruses associated with ME/CFS are also commonly detected among healthy individuals, it may be the case that underlying immune dysfunction in the host acts as a predisposing factor in development of the disorder. Viruses may trigger immune dysregulation, but an individual may also be predisposed to either an exceptionally strong acute infection, an inability to completely clear the virus, or both. Subsequently, the abnormal immune profile post-acute infection may allow for continuous reactivation and incomplete clearance of pathogens, resulting in tissue damage and an overactive yet ineffective immune response leading to inflammation and autoimmune changes. The absence of strong viremia indicates that the viruses that are present are likely quite tissue-specific, and data suggests that the viral activity consists of greater “latency-associated replication”, as was noted for EBV [72]. The success of valganciclovir (active against CMV and HHV-6) and valacyclovir (active against HSV, VZV, and EBV) [86, 204] as well as rituximab and immunoadsorption of ß2 autoantibodies in subsets of ME/CFS patients suggests that both the viral activity itself and the immune response against/resulting from viral infection may contribute to the signs and symptoms of the illness [205, 206].

Enteroviruses, particularly B coxsackieviruses, are implicated in type 1 diabetes mellitus, which they may contribute to through dysregulation of host microRNAs [207] and induction of greater oxidative stress, inflammation, and proinflammatory M1 macrophage activity [208]. Chronic enterovirus-associated systemic or local inflammation, as a consequence of a weak innate immune response and virus-induced tissue damage, may result in bystander activation of auto-reactive cells. On the other end of the spectrum, an overactive innate immune response may also result in inflammation during the initial acute infection, leading to the same outcome [209]. This scenario has been proposed for enteroviruses in type 1 diabetes, but the theory may be more broadly applicable to other viruses and autoimmune conditions. Activation of auto-reactive bystander cells has also been proposed as a pathogenic factor in systemic lupus erythematosus [210], as has molecular mimicry.

The viruses discussed herein are capable of activating NK, B, and T cells, modifying expression of their cellular transcripts, altering cytokine production, and affecting signalling pathways integral to the proper functioning of the immune response, potentially producing a pro-inflammatory environment and autoimmune activity. For example, NK cells induce non-destructive inflammation in response to beta cell infection by coxsackie B4 enterovirus [211] and are activated by acute B19V infection [212], but NK and T cells are diminished in number during severe acute infection of enterovirus 71 [213]. HHV-6A-infected T cells express many miRNAs associated with inflammation and autoimmunity [214], and persistent HHV-6A infection is associated with altered NK cell profiles in cases of Hashimoto’s thyroiditis and acute necrotizing encephalopathy. These infections were characterized by incomplete clearance of the virus and a greater abundance of peripheral CD56bright NK cells [215, 216]. This environment of heightened cytokine secretion paired with the ineffectual clearance of infection may activate auto-reactive T and B cells, thereby contributing to autoimmunity. Indeed, the cellular activation of the CD56bright NK cells correlated with autoantibody levels in subjects with Hashimoto’s thyroiditis.

Interactions between the viruses and innate and adaptive immune cells are dynamic and vary temporally, across viral species, and between individual patients. Accordingly, activity of these cells has been found to vary across studies of ME/CFS and even within studies, according to the time-point [217]. Although cytokine expression and immune cell phenotypes have differed [62, 218], dysregulation of immune cell networks occurs in ME/CFS patients, and a pro-inflammatory milieu appears to predominate. Additionally, it is clear that patients’ immune responses against viruses differ from unaffected individuals. While healthy volunteers did not exhibit changes in cellular proliferation after vaccination with influenza strains, for example, ME/CFS patients displayed significantly higher cellular proliferation in response to stimulation compared to baseline [219]. Baseline T cell proliferation was lower than controls, and post-vaccination proliferation was higher in ME/CFS patients compared to controls, although these findings were not statistically significant. Lower proliferative responses were observed among other ME/CFS patients as well [220], and decreased cytotoxicity of NK and T cells [217], as well as increased levels of cytokine producing CD56bright NK cells [221], have been documented, although findings have not been consistently confirmed between studies.

Failure to produce IgG antibodies against EBV EBNA-1 has been noted, as has low levels of antibody secreting cells specific to EBNA-1 and VCA and an impaired B cell memory response to the virus [72]. Responses of EBV-specific B- and T-cells were suppressed, which has been suggested to result from frequently reactivated EBV and lymphocytic exhaustion. In contrast, reduced CMV and HSV specific B-cell responses were not identified. On the other hand, the presence of antibodies against EBV DNA polymerase and EBV-encoded dUTPase has been detected in the serum of a subset of ME/CFS patients but not in controls [84], and the IgG response to EBV EBNA-6 peptides has been heightened in ME/CFS [73]. Sequences of amino acids in the EBNA-6 repeat region were found to be homologous to the human lactoperoxidase precursor (LPO) and thyroid peroxidase precursor (TPO) proteins as well as the two enzymes ornithine transcarbamylase (OTC) and phosphofructokinase-1 (PFK-1), which have metabolic functions. Of these, only LPO peptide IgG levels correlated with EBNA-6 peptide and protein IgG. However, antigenic mimicry producing cross-reactivity among these proteins is considered as a possible factor behind ME/CFS pathogenesis. Analysis of genetic associations has revealed that the EBV protein EBNA-2 and its related human transcription factors are associated with many autoimmune risk loci, including those involved in MS, rheumatoid arthritis, type 1 diabetes, and systemic lupus erythematosus [222]. NF-kB was similarly associated.

Although systemic immune dysfunction has been observed, local virus-associated inflammation of nervous structures resulting in altered CNS and peripheral nervous system signalling has also been hypothesized as a mechanism behind ME/CFS [223, 224], and neuroinflammation has been observed in patients [225]. EBV [226], HSV [227], HHV-6 [228], CMV [229], VZV [230], enterovirus [231], and B19V [232] can all infect the brain. Notably, HHV-6A is able to induce neuroinflammation in the absence of active replication in a mouse model [233]. HHV-6A has also been found to impair myelin repair in vitro and in vivo using mouse with extensive loss of myelin [234].

HHV-6 has been strongly linked to autoimmune diseases like Hasimoto’s thyroiditis [235, 236], connective tissue disorders and MS [237]. Selective reactivation of HHV-6 has also been shown in patients with autoimmune connective tissue diseases [238]. MS patients are frequently detected with high levels of HHV-6A/B-specific IgG and IgM in the serum and CSF [239]. Myelin basic protein (MBP), one of the auto-antigens implicated in MS pathology shares amino acid sequence homology with the U24 protein from HHV-6 [240]. Hence molecular mimicry is considered as one of the potential mechanisms for HHV-6 mediated autoimmune diseases. Clinical cases showing increased glutamic acid decarboxylase (GAD) antibodies and HHV-6 infection has been reported where antiviral therapy improved patient’s clinical condition [241].

HHV-6B has weaker ties to autoimmunity, but it is thought to contribute to common symptoms of severe drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DIHS/DRESS) when it reactivates strongly during the course of the syndrome. The immune response to the viral activity is considered as potentially more destructive than the viral activity itself and is implicated in visceral organ damage [242]. Notably, initial regulatory T cell (Treg) expansion in this illness contrasts with subsequent exhaustion of Treg cells during remission, which appears to set the stage for the autoimmune sequelae that commonly occur afterward [243]. Autoimmune thyroiditis and diabetes mellitus frequently arise post-DIHS/DRESS, as does chronic fatigue. HHV-6 has been detected in Treg cells in a case of likely DIHS/DRESS [244], and HHV-6A [245] and HHV-6B [246] are also able to induce the development of Treg cells. Both upregulated and reduced levels of Tregs have been reported in ME/CFS [217, 221, 247].

The molecular link between B19V infection and autoimmune disorders is not very clear. However, B19V infection has been associated with development of autoimmune antibodies including rheumatoid factor [110, 248, 249], antinuclear antibody, anti-mitochondrial antibody, smooth muscle antibody, and gastric parietal cell antibody [250]. Recently, B19V infection has been shown to increase levels of cytokines like IL-4, IL-10, IL-12, IL-2 and TNFα in the plasma of rheumatoid arthritis patients [251]. B19V induced upregulation of IL-6 has also been noted for its potential role in autoimmunity [252]. Two of the B19V proteins, a proline-rich small protein [253] and the NS1 protein [254], which also function as a transcription regulator have been intensely studied because of their role in viral pathogenesis. These proteins also contribute to host cell immune modulation through their involvement in cell survival pathways [254, 255]. B19V has been implicated in clinical cases of systemic lupus erythematosus and rheumatoid arthritis where various auto-antibodies could be detected in patients. Intravenous immunoglobulin (IVIG) has been reported to reduce symptoms in some patients with persistent B19V infection, but IVIG administration has also resulted in unexpected worsening of symptoms in a ME/CFS patient [256]. This may indicate that, in some cases, the immune response against persistent infection contributes to the development of clinical symptoms more so than infection-induced tissue damage, and IVIG may contribute to increased inflammation. Heightened viral replication was also detected in this case, which was suggested to be a result of antibody-dependent enhancement.

At least in a subset of patients, the mitochondrial dysfunction and elements of autoimmunity that characterize ME/CFS may be linked to viral pathogenesis. Lack of extensive analysis of molecular mechanisms linking viral pathogens to ME/CFS restricts our understanding of this disease. Future studies need to focus on this aspect of ME/CFS research.