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Review

Mucosal Viruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Missing Piece of the Puzzle?

by
Krishani Dinali Perera
1,
Paige Cameron
1,
Tayyibah Sarwar
1 and
Simon R. Carding
1,2,*
1
Food, Microbiome and Health Research Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, UK
2
Norwich Medical School, University East Anglia, Norwich NR4 7UQ, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(22), 11161; https://doi.org/10.3390/ijms262211161
Submission received: 19 October 2025 / Revised: 11 November 2025 / Accepted: 15 November 2025 / Published: 19 November 2025
(This article belongs to the Special Issue Advances in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), 2nd Edition)
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Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic condition without a definitive aetiology, no reliable diagnostic test, and no proven effective treatment. Despite most patients reporting a post-viral onset of illness, findings to date are conflicting on whether a single virus or multiple viral triggers are involved. Most studies to date have focused on detecting viruses in blood and circulating immune cells with relatively few investigating the presence of viruses in mucosal sites. In this review, we propose that this represents a critical gap in understanding the pathophysiology of ME/CFS knowledge, as mucosal tissues are primary entry points for most pathogens and often serve as reservoirs where viruses may persist. Consequently, they represent ideal niches for identifying persistent infections in ME/CFS. Emerging evidence from saliva and other mucosal samples in ME/CFS patients is consistent with this proposal and that latent viruses can persist and periodically reactivate in mucosal tissues from where they can potentially contribute to immune dysregulation, chronic inflammation, and increased symptom severity that defines ME/CFS.

1. Introduction

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition affecting an estimated 70 million individuals worldwide [1]. It is characterised by persistent extreme fatigue, impaired sleep, dysautonomia, post-exertional malaise (PEM), and immune dysfunction. Overall, individuals with ME/CFS are more functionally impaired than those with other chronic conditions including multiple sclerosis, cancer, and rheumatoid arthritis [2,3]. ME/CFS can severely impact a person’s ability to work, attend school, participate in social activities, and perform daily tasks, with 75% of patients unable to work and 25% of individuals so severely affected that they become confined to their house or bed [3,4,5,6]. The absence of definitive biomarkers, reliance on self-reported symptoms, and heterogeneous pathologies further hinder progress in developing effective diagnostics and treatments.
A majority of patients report a post-infectious onset, often following a viral illness [7], with research suggesting that ME/CFS is a post-viral fatigue syndrome (PVFS) attributable to virus-driven dysregulation in immune function, neuroendocrine abnormalities, and impaired cellular energy metabolism [8,9,10,11]. Consistent with this hypothesis, persistent viral infections, including Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6), influenza, and enteroviruses, have been described in numerous studies of ME/CFS, although findings remain inconsistent. Establishing a viral origin and identifying consistent disease mechanisms are particularly challenging due to several obstacles: difficulties in comparing viral persistence between ME/CFS patients and healthy controls, the presence of latent viruses in asymptomatic individuals, underpowered cohort sizes, dependence on single samples or single time points which fails to recapitulate the natural progression of a virus infection or fluctuations and peaks of viraemia, and variability in testing methods [12,13]. Moreover, most of these studies have relied on blood samples, yielding inconsistent results.
Many blood-based virome studies to date report no significant differences in viral prevalence, viral load, or seroprevalence of viruses such as HHV-6, human herpesvirus 7 (HHV-7) or EBV between ME/CFS patients and healthy controls (Table 1). Importantly, the majority of high-throughput sequencing and broad virome analyses (Table 1) also show no distinct virus signature or increased abundance in ME/CFS blood samples. Although some studies report associations with viral reactivation or elevated antibody responses for herpesviruses and other viruses occasionally, these findings are not consistently replicated across cohorts. These variabilities can be attributed to differences in patient cohorts and diagnostic criteria (case definitions used), the sensitivity and specificity of detection methods and sample types (plasma, serum, PBMC), and the complexity of viral latency/reactivation states. Also, these studies overlook compartmentalisation and persistent mucosal viruses, which may obscure their contribution to ME/CFS disease mechanisms. Overall, the current literature underscores the heterogeneity of ME/CFS populations and the challenges and constraints of definitively linking viruses to ME/CFS aetiology based on blood-based virological markers alone [12].
Studies that have focused on mucosal viral persistence as a potential contributor to ME/CFS pathology are discussed in detail in Section 3. Mucosal viruses can persist within tissues such as the gastrointestinal (GI) and respiratory tracts, where they can evade immune surveillance and be undetectable in blood-based analyses. This hypothesis is further supported by comparisons with the PVFS, post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID, where clinical symptoms substantially overlap with ME/CFS diagnostic criteria, in which mucosal viral reservoirs have been described [34,35,36]. The aim of this review is to synthesise current evidence of mucosal viral persistence in ME/CFS, while discussing methodological approaches to study this important yet underexplored viral niche. Investigating mucosal viral reservoirs offers promising opportunities to clarify disease aetiology, establish biomarkers, and inform the development of targeted therapeutics, including re-purposing of licenced anti-viral drugs for ME/CFS.

2. Mucosal Immune System

The mucosal immune system maintains a delicate balance between pathogen surveillance and responsivity and immune tolerance to the complex communities of microbes (microbiomes) that cohabit mucosal sites across the body. It comprises a network of tissues collectively known as the mucosa-associated lymphoid tissue (MALT), which includes nasopharyngeal-associated lymphoid tissue (NALT), bronchus-associated lymphoid tissue (BALT), gut-associated lymphoid tissue (GALT), and other areas such as the conjunctiva-associated lymphoid tissue (CALT). The reproductive tract and genital mucosae, although lacking organised lymphoid follicles as in Peyer’s patches [37,38] contains specialised immune cells which in the female reproductive tract are regulated by sex hormones [39].
Within MALT, commensal microbes (the microbiota) and IgA play crucial roles in mucosal defence by restricting pathogen colonisation and neutralising them in the lumen, preventing their adherence to boundary epithelial cells, while limiting inflammatory responses [40,41]. The NALT serves as the first line of defence against airborne pathogens, with the connected BALT of the lower respiratory tract and lungs becoming activated during respiratory inflammation and infection. GALT, within the GI-tract (GIT), contains organised lymphoid structures such as Peyer’s patches, mesenteric lymph nodes, and isolated lymphoid follicles, and is one of the most well-studied and documented MALTs. Peyer’s patches are the primary site of mucosal immune responses within GALT, where B cells undergo activation and class switching, leading to the maturation of IgA-secreting plasma cells [42]. The intestinal epithelial barrier (IEB) provides an additional layer of protection and is composed of tight junctions, a mucus layer, and the lamina propria, which harbours diverse immune cell populations. Specialised epithelial cells further strengthen this barrier, where goblet cells secrete mucins that form a sterile mucus layer coating all epithelial surfaces, while Paneth cells produce antimicrobial peptides (AMPs), lysozymes, and defensins [43,44]. Innate lymphoid cells (ILCs), particularly ILC3, are abundant in mucosal tissues and produce cytokines such as IL-22 and IL-17 [45]. These cytokines promote epithelial cell differentiation, stimulate AMP secretion, and recruit neutrophils, thereby supporting epithelial repair and antimicrobial defence [45]. The lamina propria also contains adaptive immune cells, including different subtypes of CD4+ T helper cells (e.g., Th1, Th2, and Th17 cells, which secrete IL-17) and FoxP3+ regulatory T cells (Tregs), which maintain immune tolerance [46].
Mucosal barrier integrity can be compromised, particularly in the GIT, because of microbial dysbiosis, exposure to toxins, drugs or food/water-borne pathogens resulting in increased IEB permeability (“leaky gut”) and mucus thinning, reducing barrier functionality of the protective mucosal layer. This enables viral particles and microbial products to translocate across the epithelium and gain access to the systemic circulation, triggering immune activation, which, if sustained, leads to chronic inflammation (Figure 1) [47]. This pathway is relevant to ME/CFS, where IEB dysfunction, immune activation, and persistent low-grade inflammation are present and can contribute to disease pathophysiology [48]. Some viruses, such as EBV and HHV-6 [49], exploit mucosal immune architecture to establish long-term persistence within the host. Viral reactivation can provoke immune responses without achieving clearance, potentially sustaining the chronic immune activation frequently observed in ME/CFS patients.

3. Common Mucosal Viruses Associated with ME/CFS

Most ME/CFS patients recall an acute viral infection before disease onset [7,28], consistent with a viral aetiology. Historical outbreaks of infectious diseases have been followed by increased reports of ME/CFS-like symptoms, including polio virus, EBV, enteroviruses and influenza [53,54,55,56]. While eukaryotic viruses have been associated with ME/CFS or PVFS, most of these studies have relied on blood and analysis of peripheral immune cells. Several epidemiological and clinical observations (Table 2) suggest that mucosal viruses may contribute to disease onset and persistence.

3.1. Herpesviruses

Herpesviruses, such as EBV, HHV-6, and HHV-7, are double-stranded DNA viruses that can establish long-term latency following an acute infection. EBV causes infectious mononucleosis by infecting oropharyngeal epithelial cells and B lymphocytes and later establishes lifelong latency in memory B cells [65]. During asymptomatic reactivation, often triggered by psychological stress, EBV sheds in the oropharynx [66]. EBV-infected individuals show vulnerability to PVFS with a proportion fulfilling ME/CFS diagnostic criteria [67]. Of note, the vulnerability to post-EBV fatigue is more strongly associated with pre-existing symptoms and functional limitations than with initial immune or infection indicators [67], explaining perhaps why a subset of EBV-infected individuals might develop ME/CFS. Notably, elevated EBV-specific antibodies have been detected in the saliva samples of ME/CFS patients compared to controls [34], establishing the virus’s potential link to ME/CFS. In addition, sputum analysis of ME/CFS patients found that 85% released high viral loads of EBV, compared to 50% of age-matched healthy controls [59].
Other herpesviruses can persist in salivary glands and mucosal tissues, with HHV-6 DNA in ME/CFS patients being linked to mitochondrial dysfunction and altered immune responses [68]. HHV-7 shares tropism with EBV and HHV-6, though its role in ME/CFS remains less well studied. Importantly, findings regarding the association of mucosal HHV-6 with ME/CFS remain inconsistent. Herpesviruses including HHV-6, HHV-7 and EBV, along with papillomaviruses were more frequently detected in saliva from both ME/CFS patients and healthy controls with no significant difference in prevalence between the groups [31]. Another study reported no significant difference in HHV-6 or human cytomegalovirus (HCMV) viral loads between ME/CFS patients and healthy controls in sputum samples [59]. Similarly, a study investigating HHV-6 and HHV-7 in saliva samples found no difference in viral load between chronic fatigue syndrome (CFS) patients and healthy controls [21]. In contrast, a six-month longitudinal study of ME/CFS, found that those with severe symptoms exhibited more frequent and higher levels of HHV-6B and HHV-7 DNA in saliva compared to healthy controls [58]. In a subset of these patients, peaks in viral load coincided with symptom flare-ups across neurological, immune, autonomic, and other systems, suggesting that herpesvirus reactivation may contribute to disease pathology [58]. This finding indicates the importance of longitudinal sampling to accurately detect and capture the dynamics of viraemia, and suggests that in some ME/CFS patients, HHV-6B and HHV-7 may reactivate in mucosal tissues, potentially exacerbating symptoms. These observations are consistent with findings obtained from blood and plasma samples [18,27,29,33].

3.2. Parvoviruses

Parvovirus B19 may play a role in ME/CFS pathogenesis. A study of GI biopsies found that Parvovirus B19 DNA was markedly higher in CFS patients across all biopsy sites [61]. The same study also showed that HHV-7 was abundant, while EBV and HHV-6 levels were lower in these duodenal biopsies compared to the stomach biopsies in both CFS patients and controls [61]. Co-infections were similarly prevalent in both groups [61]. Interestingly, HHV-6 and Parvovirus B19 were not detected in PBMC samples from these parvovirus-positive patients, although EBV and HHV-7 were occasionally detectable in PBMCs of biopsy-positive individuals [61]. The inability to detect Parvovirus B19 in PBMCs indicates that the virus may persist primarily in mucosal tissues rather than circulating immune cells. This emphasises the importance of examining tissue-specific viral reservoirs in understanding ME/CFS viral pathogenesis.

3.3. Coronaviruses

The overlap between long COVID and ME/CFS highlights the importance of identifying mucosal viruses and their potential roles in ME/CFS disease mechanisms. SARS-CoV-2 infection can lead to long COVID in some patients which is estimated to affect at least 65 million individuals worldwide [69]; with the majority meeting the diagnostic criteria for ME/CFS and being more functionally impaired than long COVID patients without ME/CFS [70,71,72,73,74,75,76,77,78]. This suggests that PVFS develops in a subset of patients following SARS-CoV-2 infection, potentially linked to the reactivation of persistent viruses. Saliva antibody profiling revealed elevated responses to latent viruses, including EBV, HHV-6, and human endogenous retrovirus-K (HERV-K), in both non-vaccinated ME/CFS patients and healthy controls 3–6 months after mild or asymptomatic SARS-CoV-2 infection [34]. IgG levels against EBV antigens (EBNA1 and VCA) were significantly higher in ME/CFS patients, suggesting enhanced viral reactivation [34]. These findings indicate that even mild infections may trigger latent virus reactivation, contributing to immune dysregulation in ME/CFS. Additionally, evidence of persistent or reactivated mucosal viruses months after SARS-CoV-2 infection supports the hypothesis that latent virus reactivation at mucosal sites may contribute to ME/CFS onset. Importantly, these elevated antibody levels were more prominent in saliva and not detectable systemically in plasma, highlighting the importance of assessing local mucosal immunity [34]. Similarly, most COVID patients with persistent long COVID symptoms, including fatigue, cognitive impairment, headaches, and sleep disturbances, showed evidence of EBV or HHV-6 reactivation, as indicated by the detection of viral DNA in saliva and the oropharynx [36]. Among this study cohort, patients with herpesvirus reactivation (EBV, HHV-6 or coinfection of EBV and HHV-6) experienced a more severe form of COVID-19, including higher rates of pneumonia and longer hospital stays, compared to patients without viral reactivation [36]. These findings emphasise that reactivation of herpesviruses may exacerbate the severity of long COVID symptoms in patients. Also of note, a Norwegian study found that individuals infected during the 2009 H1N1 influenza pandemic had more than a two-fold increased risk of developing ME/CFS compared to non-infected individuals, although laboratory confirmation of active influenza infection was limited [54]. Thus, respiratory viruses of epidemic potential could drive disease pathologies like those observed in ME/CFS. Further longitudinal studies are essential to track the disease course and determine if affected patients will eventually develop ME/CFS.

3.4. Picornaviruses

Enteroviruses are members of the Picornaviridae family, which are single-stranded RNA viruses with GI and respiratory tropisms. These viruses can persist in deep tissues, such as skeletal muscle and the brain, potentially provoking chronic low-grade inflammation and immune dysregulation [79]. Historical epidemics of ME/CFS have shown acute symptoms coinciding with enterovirus outbreaks [79,80,81,82]. Enterovirus RNA has been detected in stomach biopsies from CFS patients [60], as well as in throat swabs [57]. In one report, three patients with documented acute enterovirus infection subsequently developed ME/CFS where stomach biopsies taken years later revealed persistent, low-abundance enteroviral RNA and capsid protein [62]. This indicates the potential for enteroviruses to persist long-term in mucosal epithelial cells following acute infection and provides evidence that it may contribute to the pathophysiology of ME/CFS.

3.5. Adenoviruses

Adenoviruses are double-stranded DNA viruses that infect respiratory and GI epithelia. While acute adenovirus infections are often self-limiting, evidence suggests that adenoviruses can persist in the NALT, remaining latent for months to years [83]. Adenoviruses in the context of ME/CFS, however, remain understudied. Notably, COVID-19 infection may trigger adenovirus reactivation in the oral mucosa of ME/CFS patients, as indicated by elevated IgG levels against Human adenovirus (HAdV) detected in saliva following SARS-CoV-2 infection [35]. This IgG elevation was not detected in plasma [35]. Interestingly, in another study by the same group, HAdV was detected in sputum from a patient with severe ME/CFS and in a non-ME/CFS immunosuppressed individual, but not in other ME/CFS patients or healthy controls [59]. These results, while intriguing, are insufficient in determining whether HAdV contributes to the pathophysiology of ME/CFS, particularly in severe cases.

3.6. Retroviruses

Another important family of viruses that may contribute to immune dysfunction in ME/CFS are HERVs, such as HERV-K, which can be detected in a variety of tissues, including mucosal tissues [84,85]. HERVs are retroelements derived from ancient infections of germ cells by exogenous retroviruses during evolution and now comprise nearly 8% of the human genome (“fossil viruses”). They can contribute to the pathophysiology of human disease, with their expression linked to a wide range of conditions, including autoimmune diseases such as multiple sclerosis [86,87,88,89,90], cancers such as melanoma and prostate cancer [91,92,93,94], and inflammatory bowel disease [95]. HERV expression is tightly regulated but influenced by host antiviral immunity and exogenous viral infections, which can trigger (re)activation [96,97]. Relevant viruses that trigger (re)activation of HERV expression include SARS-CoV-2 [98,99,100,101,102], HHV-6 [88], EBV [89,103], and HIV [104], the consequences of which include impaired physical function, inflammation, disease severity, and subsequently immune senescence [105,106]. To date, findings regarding HERVs in ME/CFS are inconsistent. In one study, HERV-K transcript levels in PBMCs did not differ between healthy controls and ME/CFS patients, and no correlation was observed between HERV-K18 transcript levels and HHV-6 or HHV-7 viral copy number [21]. By contrast, differential expression of HERV-K, but not HERV-W, has been reported in the blood of patients with moderate ME/CFS [107]. Likewise, a genome-wide HERV expression profiling of PBMCs revealed distinct, disease-specific HERV signatures that distinguished ME/CFS patients from healthy controls and fibromyalgia patients with overlapping symptomology and chronic fatigue with ME/CFS, with the most pronounced HERV dysregulation correlating with symptom severity [108]. Overexpression of HERVs of the H, K, and W types has also been detected in a small cohort of fibromyalgia patients, with or without comorbid ME/CFS, with increased HERV expression levels correlating with interferon-β and interferon-γ expression levels [109]. These findings indicate a potentially disease-relevant dysregulation of HERVs in ME/CFS. Yet, evidence from mucosal samples remains sparse. One study revealed the presence of different HERVs (HERV-K, HERV-FRD and HERV-R) in duodenal biopsies from 8 out of 12 ME/CFS patients [63]. Supporting this evidence, saliva antibody profiling revealed elevated responses to HERV-K, along with other latent viruses including EBV and HHV-6, in both non-SARS-CoV-2 vaccinated ME/CFS patients and healthy controls months after mild or asymptomatic SARS-CoV-2 infection [34]. Given that mucosal areas are primary sites of viral persistence and immune activation, investigating HERV expression and antibody responses to HERVs in these compartments could provide new insight into the role of HERVs in ME/CFS.

3.7. Gastrointestinal Tract Viruses

Our own study of the GI (faecal) virome in ME/CFS and healthy individuals found that while the virome and bacterial diversity were similar between ME/CFS patients and controls, certain human viruses, including HHV-6A, papillomaviruses, coronavirus NL63, and adenovirus 54, were detected exclusively in ME/CFS samples [64]. This suggests subtle but distinct viral–bacterial host associations specific to ME/CFS that needs to be confirmed in larger studies.

3.8. Reproductive Tract Viruses

It is regrettable that the reproductive tract has received limited attention in ME/CFS research. Persistent viral infections in reproductive tissues have the potential to affect fertility, alter hormone production, increase the risk of viral transmission both vertically and sexually, and provoke local inflammation and immune activation. Numerous viruses are known to persistently infect and establish reservoirs in male and female reproductive tissues, including CMV, HHV-6, mumps virus, herpes simplex viruses (HSV-1 and HSV-2), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), hepatitis B and C viruses, as well Zika virus and Ebola virus [110,111,112]. The effects of SARS-CoV-2 on reproduction are still unclear. Notably, the immune-privileged nature of the male reproductive tract could facilitate viral persistence. Thus, it is possible that viruses capable of persisting in the reproductive tract are not only sexually transmissible but may also play a role in the development of ME/CFS. However, there is currently no evidence linking viral reservoirs in the reproductive tract to ME/CFS, highlighting the need for further investigation.
Given that ME/CFS is more prevalent in females [7,113,114], it is important to investigate the sex-specific differences in the reproductive tract that may contribute to disease susceptibility or progression. The female reproductive tract (FRT) is a microbially diverse environment, and the mucosal surfaces contain a spectrum of antimicrobial factors that serve as the first line of defence against invading pathogenic bacteria, viruses, and fungi [115]. The antimicrobial defences throughout the FRT are regulated in part by sex hormones, which influence their production by innate immune cells [115]. Most research on the FRT has focused on the bacteriome, while relatively few studies have investigated the viral components within this ecosystem. The FRT harbours abundant bacteriophages which constitute the majority of the prokaryotic virome, and this reflects the structure of the bacterial community which may influence disease-associated shifts in the microbiome [116,117]. A diverse range of eukaryotic viruses are also present in the FRT with Papillomaviridae, Anelloviridae, and Orthoherpesviridae viruses being the most prevalent [116]. DNA eukaryotic viruses in the FRT are linked to adverse outcomes such as preterm birth, inflammation, and cervical cancer [116] while little is known about RNA viruses. This again highlights the need for further research into the role of the FRT virome in health and disease, and particularly its possible contribution to the higher prevalence of ME/CFS in females.
Collectively, the findings from virus studies in ME/CFS underscores that virus persistence in mucosal sites is often ignored or understudied. Post-viral triggers such as SARS-CoV-2, and evidence of their persistence and reactivation support the assumption that mucosal viral reservoirs contribute to ME/CFS pathophysiology. Subtle differences in viral composition, as observed in the GI virome, and detection of specific DNA viruses exclusively in ME/CFS patients further suggest that viral–host interactions at mucosal sites could influence and be a driver of immune dysregulation, chronic inflammation, and symptom severity. The systematic investigation of mucosal viruses as potential drivers or modulators of ME/CFS is warranted for further study.

4. Mucosal Viral Persistence in ME/CFS: A Hypothesis

The evidence from ME/CFS, long COVID and other PVFS points towards a shared aetiology: that certain viruses remain active or reactivate in mucosal tissues, influencing both local and systemic immune responses. In ME/CFS sites such as the oral cavity, nasopharynx, gut, or reproductive tract can serve as long-term viral reservoirs. Similarly, persistence of replicating virus, or their nucleic acid/antigens are linked to long COVID [118]. As highlighted in Section 3, multiple viruses have been identified to persist in mucosal sites, with increases in viral load correlating with symptom flare-ups [58]. Notably, increased replication of persistent viruses following SARS-CoV-2 infection [34,35,36] was detected exclusively in saliva but not plasma samples [34], and patients with persistent viral reactivation exhibited more severe COVID [36]. These findings suggest that mucosal compartments may harbour clinically relevant viral activity often missed by blood-based studies, underscoring the need for more focused research on these mucosal reservoirs.
Viruses such as EBV and HHV-6 can remain active in mucosal or tissue reservoirs without being detectable in the circulation at the time of sampling. Since many of these virus suspects are common in healthy, asymptomatic individuals, detecting their presence in blood at a single time point does not accurately reflect their pathogenic potential. Overall, persistent viral activity in these sites can maintain the local immune system in a state of low-level activation, eventually disrupting normal regulation and contributing to immune exhaustion, fatigue, cognitive deficits, and post-exertional malaise. We have termed this the “mucosal viral persistence in ME/CFS” hypothesis (Figure 2). This hypothesis can explain why blood-based studies often yield inconsistent results.
Persistent virus infection in mucosal tissue drives chronic immune activation, producing pro-inflammatory cytokines and immune mediators. Chronic inflammation could disrupt tight junction proteins, and increased epithelial barrier permeability (“leaky barriers”) would allow viral particles, microbe-derived microvesicles, particulate antigens, and inflammatory mediators to access the systemic circulation, thereby exacerbating the immune response. Impaired mucosal immune response, microbiome dysbiosis, and persistent virus triggered inflammation can impact the autonomic nervous system through neuroendocrine pathways. Herpesviruses such as EBV and HHV-6 can also infect endothelial cells leading to endothelial dysfunction characterised by microvascular abnormalities and microclot formation, which may underlie cognitive impairments and neurological disturbances observed in ME/CFS [119]. Moreover, latent herpesvirus antigens could inhibit apoptosis of these infected endothelial cells, prolonging dysfunction and contributing to hypercoagulability, vascular pathology, and compromised vascular integrity [119]. Although mucosal virus reactivation might be sufficient to induce neuroinflammation, some neurotropic herpesviruses may directly reactivate within the central nervous system, further driving neuroinflammation and contributing to chronic relapse-recovery cycles by facilitating infection across a dysfunctional blood–brain barrier [118,120]. Additionally, persistent virus infections could disrupt the hypothalamic–pituitary–adrenal (HPA) axis, leading to abnormal cortisol production and stress response [121,122] where the severity of the disruption could be influenced by pre-existing HPA abnormalities or the magnitude of the response to the virus infection [122]. Oxidative stress induced by chronic inflammation may further impair mitochondrial function, contributing to overall fatigue and other hallmark symptoms of ME/CFS such as cognitive dysfunction, and post-exertional malaise.
It is important to acknowledge the absence of direct experimental or clinical evidence that demonstrates the causal mechanisms of mucosal virus persistence in ME/CFS. This significant knowledge gap underscores the need for future investigations involving mucosal sampling and multi-omics approaches to understand virus persistence, immune interactions and ME/CFS pathogenesis.

5. Methodological Directions

A multi-disciplinary approach is essential to investigate viruses within mucosal sites in well-defined clinically diagnosed cohorts of patients, and appropriately matched healthy and disease control cohorts and to validate the theory of mucosal virus persistence in ME/CFS. Importantly, non-invasive sample collection at mucosal sites offers the practical advantage of remote, home-based, sampling that enables severely affected ME/CFS patients to contribute to research studies. Furthermore, swab samples may accurately reflect viral communities in relevant tissues affected by ME/CFS pathophysiology, replicating viral presence and potential reactivation in the mucosal reservoirs that blood-based studies miss. In addition, repeated sampling to capture virus activity over time is feasible with such non-invasive, home-based sampling protocols, acknowledging that it can increase cost and participant burden. It is worth noting the limitations of mucosal swabs and, in particular, their typically low viral particle/nucleic acid yield, which can increase the risk of false negatives and require highly sensitive, carefully optimised, uniform and controlled methods for extraction and detection in addition to inclusion of appropriate control samples to distinguish genuine signals [123].
Quantitative PCR approaches are well-established methods for quantifying viral loads with high sensitivity for known viral sequences. Additionally, non-biassed metagenomic-based next-generation sequencing enables a comprehensive study of viral communities within samples, including de novo detection of viruses. Although comparatively and subjectively less sensitive than qPCR, metagenomic next-generation sequencing is valuable in characterising a multitude of viruses in complex biological samples, including the broader virome and uncovering co-infections or rare viral species. However, sample contamination is a particular concern for low biomass samples and highly sensitive sequencing methods require strict laboratory controls to exclude, for example, “kitomes” from downstream analyses. Importantly, detecting the presence of RNA viruses in virome analysis is challenging owing to RNA instability and limitations in sequencing approaches and data interpretation [124]; thus, their role is often underestimated.
By combining targeted detection methods with broader sequencing approaches and aligning sampling with disease onset and symptoms, a more comprehensive picture of how mucosal viral activity relates to ME/CFS can be established. This integrated approach would also help distinguish between transient exposure, latent infection, and active replication of mucosal viruses which may be associated with the pathogenesis of ME/CFS.

6. Conclusions

While recent advances have highlighted the role of viral infections, particularly EBV, HHV-6/7, influenza, and SARS-CoV-2, in the onset and chronicity of ME/CFS, much of the research has to date focused on blood-based investigations and single-time point analyses, leaving the role of the mucosal immune system largely unexplored. Emerging evidence from saliva, mucosal tissues, and longitudinal studies suggests that latent viruses can persist and periodically reactivate at mucosal sites, potentially driving immune dysregulation, chronic inflammation, and symptom severity via mechanisms involving gut–brain axis interactions and local immune responses. These findings, along with observations in long COVID and other PVFS, emphasises that viral “reservoirs” at mucosal surfaces may have lasting impacts beyond initial infection.
To address current knowledge gaps, future research should consider repeated mucosal sampling, combined virus detection methods including metagenomic sequencing, and comprehensive immune and symptom tracking to clarify whether mucosal viral activity is a causative agent, a consequence, or a bystander in ME/CFS. Confirming the clinical relevance of mucosal viral persistence could enable the use of non-invasive sampling methods, the discovery of robust biomarkers, and the development of targeted antiviral and immunomodulatory therapies, as well as drug repurposing (e.g., anti-retroviral therapy; ART) clinical trials. This could ultimately improve diagnosis and symptom management for individuals suffering from this complex and debilitating disease.

Author Contributions

Conceptualisation, K.D.P. and S.R.C.; investigation and writing, K.D.P., P.C., T.S. and S.R.C.; review and editing, K.D.P. and S.R.C. All authors have read and agreed to the published version of the manuscript.

Funding

K.D.P. is a Luna Nova Fellow, P.C. and T.S. were funded by Invest in ME Research (UK Charity number 1153730) undergraduate student bursaries. S.R.C. is supported by the BBSRC Institute Strategic Programme Grant BB/X011054/1.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Vardaman, M.; Gilmour, S. Letter: Time to correct the record on the global burden of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). J. Transl. Med. 2025, 23, 331. [Google Scholar] [CrossRef]
  2. Jason, L.A.; Ohanian, D.; Brown, A.; Sunnquist, M.; McManimen, S.; Klebek, L.; Fox, P.; Sorenson, M. Differentiating Multiple Sclerosis from Myalgic Encephalomyelitis and Chronic Fatigue Syndrome. Insights Biomed. 2017, 2, 11. [Google Scholar] [CrossRef]
  3. Nacul, L.C.; Lacerda, E.M.; Campion, P.; Pheby, D.; Drachler, M.d.L.; Leite, J.C.; Poland, F.; Howe, A.; Fayyaz, S.; Molokhia, M. The functional status and well being of people with myalgic encephalomyelitis/chronic fatigue syndrome and their carers. BMC Public Health 2011, 11, 402. [Google Scholar] [CrossRef] [PubMed]
  4. Pendergrast, T.; Brown, A.; Sunnquist, M.; Jantke, R.; Newton, J.L.; Strand, E.B.; Jason, L.A. Housebound versus nonhousebound patients with myalgic encephalomyelitis and chronic fatigue syndrome. Chronic Illn. 2016, 12, 292–307. [Google Scholar] [CrossRef] [PubMed]
  5. Unger, E.R.; Lin, J.-M.S.; Tian, H.; Natelson, B.H.; Lange, G.; Vu, D.; Blate, M.; Klimas, N.G.; Balbin, E.G.; Bateman, L.; et al. Multi-Site Clinical Assessment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (MCAM): Design and Implementation of a Prospective/Retrospective Rolling Cohort Study. Am. J. Epidemiol. 2017, 185, 617–626. [Google Scholar] [CrossRef] [PubMed]
  6. Vink, M.; Vink-Niese, F. Work Rehabilitation and Medical Retirement for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. A Review and Appraisal of Diagnostic Strategies. Diagnostics 2019, 9, 124. [Google Scholar] [CrossRef]
  7. Angelsen, A.; Schei, T. EMEA Survey of ME/CFS Patients in Europe: Same Disease, Different Approaches and Experiences. European ME Alliance (EMEA), 2024. Available online: https://www.europeanmealliance.org/documents/emeaeusurvey/EMEAMEsurveyreport2024.pdf (accessed on 28 August 2025).
  8. Arron, H.E.; Marsh, B.D.; Kell, D.B.; Khan, M.A.; Jaeger, B.R.; Pretorius, E. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: The biology of a neglected disease. Front. Immunol. 2024, 15, 1386607. [Google Scholar] [CrossRef]
  9. NIH Study Offers New Clues into the Causes of Post-Infectious ME/CFS|National Institutes of Health (NIH). Available online: https://www.nih.gov/news-events/news-releases/depth-study-finds-brain-immune-metabolic-abnormalities-linked-debilitating-chronic-disease (accessed on 28 August 2025).
  10. Sotzny, F.; Blanco, J.; Capelli, E.; Castro-Marrero, J.; Steiner, S.; Murovska, M.; Scheibenbogen, C. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome—Evidence for an autoimmune disease. Autoimmun. Rev. 2018, 17, 601–609. [Google Scholar] [CrossRef] [PubMed]
  11. Van Campenhout, J.; Buntinx, Y.; Xiong, H.-Y.; Wyns, A.; Polli, A.; Nijs, J.; Aerts, J.L.; Laeremans, T.; Hendrix, J. Unravelling the Connection Between Energy Metabolism and Immune Senescence/Exhaustion in Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Biomolecules 2025, 15, 357. [Google Scholar] [CrossRef]
  12. Obraitis, D.; Li, D. Blood virome research in myalgic encephalomyelitis/chronic fatigue syndrome: Challenges and opportunities. Curr. Opin. Virol. 2024, 68–69, 101437. [Google Scholar] [CrossRef]
  13. Rasa, S.; Nora-Krukle, Z.; Henning, N.; Eliassen, E.; Shikova, E.; Harrer, T.; Scheibenbogen, C.; Murovska, M.; Prusty, B.K.; European Network on ME/CFS (EUROMENE). Chronic viral infections in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). J. Transl. Med. 2018, 16, 268. [Google Scholar] [CrossRef]
  14. Buchwald, D.; Ashley, R.L.; Pearlman, T.; Kith, P.; Komaroff, A.L. Viral serologies in patients with chronic fatigue and chronic fatigue syndrome. J. Med. Virol. 1996, 50, 25–30. [Google Scholar] [CrossRef]
  15. Galbraith, D.N.; Nairn, C.; Clements, G.B. Evidence for enteroviral persistence in humans. J. Gen. Virol. 1997, 78, 307–312. [Google Scholar] [CrossRef]
  16. Reeves, W.C.; Stamey, F.R.; Black, J.B.; Mawle, A.C.; Stewart, J.A.; Pellett, P.E. Human herpesviruses 6 and 7 in chronic fatigue syndrome: A case-control study. Clin. Infect. Dis. 2000, 31, 48–52. [Google Scholar] [CrossRef]
  17. Ablashi, D.V.; Eastman, H.B.; Owen, C.B.; Roman, M.M.; Friedman, J.; Zabriskie, J.B.; Peterson, D.L.; Pearson, G.R.; Whitman, J.E. Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients. J. Clin. Virol. 2000, 16, 179–191. [Google Scholar] [CrossRef]
  18. Chapenko, S.; Krumina, A.; Kozireva, S.; Nora, Z.; Sultanova, A.; Viksna, L.; Murovska, M. Activation of human herpesviruses 6 and 7 in patients with chronic fatigue syndrome. J. Clin. Virol. 2006, 37, S47–S51. [Google Scholar] [CrossRef]
  19. Chapenko, S.; Krumina, A.; Logina, I.; Rasa, S.; Chistjakovs, M.; Sultanova, A.; Viksna, L.; Murovska, M. Association of Active Human Herpesvirus-6, -7 and Parvovirus B19 Infection with Clinical Outcomes in Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Adv. Virol. 2012, 2012, 205085. [Google Scholar] [CrossRef] [PubMed]
  20. Burbelo, P.D.; Bayat, A.; Wagner, J.; Nutman, T.B.; Baraniuk, J.N.; Iadarola, M.J. No serological evidence for a role of HHV-6 infection in chronic fatigue syndrome. Am. J. Transl. Res. 2012, 4, 443–451. [Google Scholar] [PubMed]
  21. Oakes, B.; Hoagland-Henefield, M.; Komaroff, A.L.; Erickson, J.L.; Huber, B.T. Human Endogenous Retrovirus-K18 Superantigen Expression and Human Herpesvirus-6 and Human Herpesvirus-7 Viral Loads in Chronic Fatigue Patients. Clin. Infect. Dis. 2013, 56, 1394–1400. [Google Scholar] [CrossRef]
  22. Miller, R.R.; Uyaguari-Diaz, M.; McCabe, M.N.; Montoya, V.; Gardy, J.L.; Parker, S.; Steiner, T.; Hsiao, W.; Nesbitt, M.J.; Tang, P.; et al. Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects. PLoS ONE 2016, 11, e0165691. [Google Scholar] [CrossRef] [PubMed]
  23. Bouquet, J.; Gardy, J.L.; Brown, S.; Pfeil, J.; Miller, R.R.; Morshed, M.; Avina-Zubieta, A.; Shojania, K.; McCabe, M.; Parker, S.; et al. RNA-Seq Analysis of Gene Expression, Viral Pathogen, and B-Cell/T-Cell Receptor Signatures in Complex Chronic Disease. Clin. Infect. Dis. 2017, 64, 476–481. [Google Scholar] [CrossRef]
  24. Blomberg, J.; Rizwan, M.; Böhlin-Wiener, A.; Elfaitouri, A.; Julin, P.; Zachrisson, O.; Rosén, A.; Gottfries, C.-G. Antibodies to Human Herpesviruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. Front. Immunol. 2019, 10, 1946. [Google Scholar] [CrossRef] [PubMed]
  25. Bouquet, J.; Li, T.; Gardy, J.L.; Kang, X.; Stevens, S.; Stevens, J.; VanNess, M.; Snell, C.; Potts, J.; Miller, R.R.; et al. Whole blood human transcriptome and virome analysis of ME/CFS patients experiencing post-exertional malaise following cardiopulmonary exercise testing. PLoS ONE 2019, 14, e0212193. [Google Scholar] [CrossRef]
  26. Cliff, J.M.; King, E.C.; Lee, J.-S.; Sepúlveda, N.; Wolf, A.-S.; Kingdon, C.; Bowman, E.; Dockrell, H.M.; Nacul, L.; Lacerda, E.; et al. Cellular Immune Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Front. Immunol. 2019, 10, 796. [Google Scholar] [CrossRef] [PubMed]
  27. Shikova, E.; Reshkova, V.; Kumanova, A.; Raleva, S.; Alexandrova, D.; Capo, N.; Murovska, M. Cytomegalovirus, Epstein-Barr virus, and human herpesvirus-6 infections in patients with myalgic encephalomyelitis/chronic fatigue syndrome. J. Med. Virol. 2020, 92, 3682–3688. [Google Scholar] [CrossRef]
  28. Domingues, T.D.; Grabowska, A.D.; Lee, J.-S.; Ameijeiras-Alonso, J.; Westermeier, F.; Scheibenbogen, C.; Cliff, J.M.; Nacul, L.; Lacerda, E.M.; Mouriño, H.; et al. Herpesviruses Serology Distinguishes Different Subgroups of Patients From the United Kingdom Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Biobank. Front. Med. 2021, 8, 686736. [Google Scholar] [CrossRef]
  29. Orlova, S.; Rudjko, G.; Orlova, O.; Dakukina, T. Detection of herpes viruses in patients with myalgic encephalomyelitis /chronic fatigue syndrome in Belarus. Pol. J. Appl. Sci. 2021, 6, 50–53. [Google Scholar] [CrossRef]
  30. Gravelsina, S.; Vilmane, A.; Svirskis, S.; Rasa-Dzelzkaleja, S.; Nora-Krukle, Z.; Vecvagare, K.; Krumina, A.; Leineman, I.; Shoenfeld, Y.; Murovska, M. Biomarkers in the diagnostic algorithm of myalgic encephalomyelitis/chronic fatigue syndrome. Front. Immunol. 2022, 13, 928945. [Google Scholar] [CrossRef]
  31. Briese, T.; Tokarz, R.; Bateman, L.; Che, X.; Guo, C.; Jain, K.; Kapoor, V.; Levine, S.; Hornig, M.; Oleynik, A.; et al. A multicenter virome analysis of blood, feces, and saliva in myalgic encephalomyelitis/chronic fatigue syndrome. J. Med. Virol. 2023, 95, e28993. [Google Scholar] [CrossRef]
  32. Domingues, T.D.; Malato, J.; Grabowska, A.D.; Lee, J.-S.; Ameijeiras-Alonso, J.; Biecek, P.; Graça, L.; Mouriño, H.; Scheibenbogen, C.; Westermeier, F.; et al. Association analysis between symptomology and herpesvirus IgG antibody concentrations in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis. Heliyon 2023, 9, e18250. [Google Scholar] [CrossRef] [PubMed]
  33. Rasa-Dzelzkaleja, S.; Krumina, A.; Capenko, S.; Nora-Krukle, Z.; Gravelsina, S.; Vilmane, A.; Ievina, L.; Shoenfeld, Y.; Murovska, M.; The VirA Project. The persistent viral infections in the development and severity of myalgic encephalomyelitis/chronic fatigue syndrome. J. Transl. Med. 2023, 21, 33. [Google Scholar] [CrossRef] [PubMed]
  34. Apostolou, E.; Rizwan, M.; Moustardas, P.; Sjögren, P.; Bertilson, B.C.; Bragée, B.; Polo, O.; Rosén, A. Saliva antibody-fingerprint of reactivated latent viruses after mild/asymptomatic COVID-19 is unique in patients with myalgic-encephalomyelitis/chronic fatigue syndrome. Front. Immunol. 2022, 13, 949787. [Google Scholar] [CrossRef]
  35. Hannestad, U.; Apostolou, E.; Sjögren, P.; Bragée, B.; Polo, O.; Bertilson, B.C.; Rosén, A. Post-COVID sequelae effect in chronic fatigue syndrome: SARS-CoV-2 triggers latent adenovirus in the oral mucosa. Front. Med. 2023, 10, 1208181. [Google Scholar] [CrossRef] [PubMed]
  36. Zubchenko, S.; Kril, I.; Nadizhko, O.; Matsyura, O.; Chopyak, V. Herpesvirus infections and post-COVID-19 manifestations: A pilot observational study. Rheumatol. Int. 2022, 42, 1523–1530. [Google Scholar] [CrossRef]
  37. Mestecky, J.; Moldoveanu, Z.; Russell, M.W. Immunologic Uniqueness of the Genital Tract: Challenge for Vaccine Development. Am. J. Reprod. Immunol. 2005, 53, 208–214. [Google Scholar] [CrossRef]
  38. Mestecky, J.; Fultz, P.N. Mucosal Immune System of the Human Genital Tract. J. Infect. Dis. 1999, 179, S470–S474. [Google Scholar] [CrossRef]
  39. Monin, L.; Whettlock, E.M.; Male, V. Immune responses in the human female reproductive tract. Immunology 2020, 160, 106–115. [Google Scholar] [CrossRef]
  40. Chapter 2—Components of the Immune System. In Primer to the Immune Response, 2nd ed.; Mak, T.W., Saunders, M.E., Jett, B.D., Eds.; Academic Cell: Boston, MA, USA, 2014; pp. 21–54. [Google Scholar] [CrossRef]
  41. Reinholdt, J.; Husby, S. IgA and Mucosal Homeostasis. In Madame Curie Bioscience Database; Landes Bioscience: Austin, TX, USA, 2013. [Google Scholar]
  42. Cerutti, A.; Cols, M.; Gentile, M.; Cassis, L.; Barra, C.M.; He, B.; Puga, I.; Chen, K. Regulation of mucosal IgA responses: Lessons from primary immunodeficiencies. Ann. N. Y. Acad. Sci. 2011, 1238, 132–144. [Google Scholar] [CrossRef]
  43. Barbara, G.; Barbaro, M.R.; Fuschi, D.; Palombo, M.; Falangone, F.; Cremon, C.; Marasco, G.; Stanghellini, V. Inflammatory and Microbiota-Related Regulation of the Intestinal Epithelial Barrier. Front. Nutr. 2021, 8, 718356. [Google Scholar] [CrossRef] [PubMed]
  44. Ouellette, A.J. Paneth cells and innate mucosal immunity. Curr. Opin. Gastroenterol. 2010, 26, 547. [Google Scholar] [CrossRef]
  45. Panda, S.K.; Colonna, M. Innate Lymphoid Cells in Mucosal Immunity. Front. Immunol. 2019, 10, 861. [Google Scholar] [CrossRef]
  46. Harrison, O.J.; Srinivasan, N.; Pott, J.; Schiering, C.; Krausgruber, T.; Ilott, N.E.; Maloy, K.J. Epithelial-derived IL-18 regulates Th17 cell differentiation and Foxp3+ Treg cell function in the intestine. Mucosal Immunol. 2015, 8, 1226–1236. [Google Scholar] [CrossRef] [PubMed]
  47. Di Vincenzo, F.; Del Gaudio, A.; Petito, V.; Lopetuso, L.R.; Scaldaferri, F. Gut microbiota, intestinal permeability, and systemic inflammation: A narrative review. Intern. Emerg. Med. 2024, 19, 275–293. [Google Scholar] [CrossRef]
  48. König, R.S.; Albrich, W.C.; Kahlert, C.R.; Bahr, L.S.; Löber, U.; Vernazza, P.; Scheibenbogen, C.; Forslund, S.K. The Gut Microbiome in Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS). Front. Immunol. 2022, 12, 628741. [Google Scholar] [CrossRef]
  49. Cohen, J.I. Herpesvirus latency. J. Clin. Investig. 2020, 130, 3361–3369. [Google Scholar] [CrossRef] [PubMed]
  50. Maidji, E.; Somsouk, M.; Rivera, J.M.; Hunt, P.W.; Stoddart, C.A. Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction. PLoS Pathog. 2017, 13, e1006202. [Google Scholar] [CrossRef]
  51. Garrec, C.; Arrindell, J.; Andrieu, J.; Desnues, B.; Mege, J.-L.; Omar Osman, I.; Devaux, C. Preferential apical infection of Caco-2 intestinal cell monolayers by SARS-CoV-2 is associated with damage to cellular barrier integrity: Implications for the pathophysiology of COVID-19. PLoS ONE 2025, 20, e0313068. [Google Scholar] [CrossRef]
  52. Moretti, S.; Schietroma, I.; Sberna, G.; Maggiorella, M.T.; Sernicola, L.; Farcomeni, S.; Giovanetti, M.; Ciccozzi, M.; Borsetti, A. HIV-1–Host Interaction in Gut-Associated Lymphoid Tissue (GALT): Effects on Local Environment and Comorbidities. Int. J. Mol. Sci. 2023, 24, 12193. [Google Scholar] [CrossRef] [PubMed]
  53. Hanson, M.R. The viral origin of myalgic encephalomyelitis/chronic fatigue syndrome. PLOS Pathog. 2023, 19, e1011523. [Google Scholar] [CrossRef]
  54. Magnus, P.; Gunnes, N.; Tveito, K.; Bakken, I.J.; Ghaderi, S.; Stoltenberg, C.; Hornig, M.; Lipkin, W.I.; Trogstad, L.; Håberg, S.E. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is associated with pandemic influenza infection, but not with an adjuvanted pandemic influenza vaccine. Vaccine 2015, 33, 6173–6177. [Google Scholar] [CrossRef]
  55. Underhill, R.; Baillod, R. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Organic Disease or Psychosomatic Illness? A Re-Examination of the Royal Free Epidemic of 1955. Medicina 2020, 57, 12. [Google Scholar] [CrossRef]
  56. Underhill, R.A. Myalgic encephalomyelitis, chronic fatigue syndrome: An infectious disease. Med. Hypotheses 2015, 85, 765–773. [Google Scholar] [CrossRef]
  57. Galbraith, D.N.; Nairn, C.; Clements, G.B. Phylogenetic analysis of short enteroviral sequences from patients with chronic fatigue syndrome. J. Gen. Virol. 1995, 76, 1701–1707. [Google Scholar] [CrossRef]
  58. Lee, J.-S.; Lacerda, E.M.; Nacul, L.; Kingdon, C.C.; Norris, J.; O’Boyle, S.; Roberts, C.H.; Palla, L.; Riley, E.M.; Cliff, J.M. Salivary DNA Loads for Human Herpesviruses 6 and 7 Are Correlated With Disease Phenotype in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Front. Med. 2021, 8, 656692. [Google Scholar] [CrossRef]
  59. Hannestad, U.; Allard, A.; Nilsson, K.; Rosén, A. Prevalence of EBV, HHV6, HCMV, HAdV, SARS-CoV-2, and Autoantibodies to Type I Interferon in Sputum from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. Viruses 2025, 17, 422. [Google Scholar] [CrossRef]
  60. Chia, J.K.S.; Chia, A.Y. Chronic fatigue syndrome is associated with chronic enterovirus infection of the stomach. J. Clin. Pathol. 2008, 61, 43–48. [Google Scholar] [CrossRef]
  61. Frémont, M.; Metzger, K.; Rady, H.; Hulstaert, J.; Meirleir, K.D. Detection of Herpesviruses and Parvovirus B19 in Gastric and Intestinal Mucosa of Chronic Fatigue Syndrome Patients. In Vivo 2009, 23, 209–213. [Google Scholar]
  62. Chia, J.; Chia, A.; Voeller, M.; Lee, T.; Chang, R. Acute enterovirus infection followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral persistence. J. Clin. Pathol. 2010, 63, 165–168. [Google Scholar] [CrossRef]
  63. De Meirleir, K.L.; Khaiboullina, S.F.; Frémont, M.; Hulstaert, J.; Rizvanov, A.A.; Palotás, A.; Lombardi, V.C. Plasmacytoid Dendritic Cells in the Duodenum of Individuals Diagnosed with Myalgic Encephalomyelitis Are Uniquely Immunoreactive to Antibodies to Human Endogenous Retroviral Proteins. In Vivo 2013, 27, 177–187. [Google Scholar]
  64. Hsieh, S.-Y.; Savva, G.M.; Telatin, A.; Tiwari, S.K.; Tariq, M.A.; Newberry, F.; Seton, K.A.; Booth, C.; Bansal, A.S.; Wileman, T.; et al. Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus–Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Int. J. Mol. Sci. 2023, 24, 17267. [Google Scholar] [CrossRef]
  65. Damania, B.; Kenney, S.C.; Raab-Traub, N. Epstein-Barr virus: Biology and clinical disease. Cell 2022, 185, 3652–3670. [Google Scholar] [CrossRef]
  66. Skuhala, T.; Židovec-Lepej, S.; Trkulja, V.; Radmanić, L.; Đaković Rode, O.; Špiljak, B.; Šimunović, L.; Rimac, M.; Dragobratović, A. Epstein—Barr Virus Salivary Shedding in Patients with Acute Infectious Diseases: A Pilot Study. Acta Stomatol. Croat. 2024, 58, 76–84. [Google Scholar] [CrossRef]
  67. Pedersen, M.; Asprusten, T.T.; Godang, K.; Leegaard, T.M.; Osnes, L.T.; Skovlund, E.; Tjade, T.; Øie, M.G.; Wyller, V.B.B. Predictors of chronic fatigue in adolescents six months after acute Epstein-Barr virus infection: A prospective cohort study. Brain. Behav. Immun. 2019, 75, 94–100. [Google Scholar] [CrossRef]
  68. Schreiner, P.; Harrer, T.; Scheibenbogen, C.; Lamer, S.; Schlosser, A.; Naviaux, R.K.; Prusty, B.K. Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. ImmunoHorizons 2020, 4, 201–215. [Google Scholar] [CrossRef]
  69. Davis, H.E.; McCorkell, L.; Vogel, J.M.; Topol, E.J. Long COVID: Major findings, mechanisms and recommendations. Nat. Rev. Microbiol. 2023, 21, 133–146, Correction in Nat. Rev. Microbiol. 2023, 21, 408. https://doi.org/10.1038/s41579-023-00896-0. [Google Scholar] [CrossRef]
  70. AlMuhaissen, S.; Abu Libdeh, A.; ElKhatib, Y.; Alshayeb, R.; Jaara, A.; Bardaweel, S.K. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and COVID-19: Is there a connection? Curr. Med. Res. Opin. 2023, 39, 1119–1126. [Google Scholar] [CrossRef]
  71. Annesley, S.J.; Missailidis, D.; Heng, B.; Josev, E.K.; Armstrong, C.W. Unravelling shared mechanisms: Insights from recent ME/CFS research to illuminate long COVID pathologies. Trends Mol. Med. 2024, 30, 443–458. [Google Scholar] [CrossRef]
  72. Ballering, A.V.; van Zon, S.K.R.; Olde Hartman, T.C.; Rosmalen, J.G.M.; Lifelines Corona Research Initiative. Persistence of somatic symptoms after COVID-19 in the Netherlands: An observational cohort study. Lancet 2022, 400, 452–461. [Google Scholar] [CrossRef]
  73. Bonilla, H.; Quach, T.C.; Tiwari, A.; Bonilla, A.E.; Miglis, M.; Yang, P.C.; Eggert, L.E.; Sharifi, H.; Horomanski, A.; Subramanian, A.; et al. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome is common in post-acute sequelae of SARS-CoV-2 infection (PASC): Results from a post-COVID-19 multidisciplinary clinic. Front. Neurol. 2023, 14, 1090747. [Google Scholar] [CrossRef]
  74. Dehlia, A.; Guthridge, M.A. The persistence of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) after SARS-CoV-2 infection: A systematic review and meta-analysis. J. Infect. 2024, 89, 106297. [Google Scholar] [CrossRef]
  75. Jason, L.A.; Dorri, J.A. ME/CFS and Post-Exertional Malaise among Patients with Long COVID. Neurol. Int. 2023, 15, 1–11. [Google Scholar] [CrossRef]
  76. Morita, S.; Tokumasu, K.; Otsuka, Y.; Honda, H.; Nakano, Y.; Sunada, N.; Sakurada, Y.; Matsuda, Y.; Soejima, Y.; Ueda, K.; et al. Phase-dependent trends in the prevalence of myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) related to long COVID: A criteria-based retrospective study in Japan. PLoS ONE 2024, 19, e0315385. [Google Scholar] [CrossRef]
  77. Davis, H.E.; Assaf, G.S.; McCorkell, L.; Wei, H.; Low, R.J.; Re’em, Y.; Redfield, S.; Austin, J.P.; Akrami, A. Characterizing long COVID in an international cohort: 7 months of symptoms and their impact. eClinicalMedicine 2021, 38, 101019. [Google Scholar] [CrossRef]
  78. Kedor, C.; Freitag, H.; Meyer-Arndt, L.; Wittke, K.; Hanitsch, L.G.; Zoller, T.; Steinbeis, F.; Haffke, M.; Rudolf, G.; Heidecker, B.; et al. A prospective observational study of post-COVID-19 chronic fatigue syndrome following the first pandemic wave in Germany and biomarkers associated with symptom severity. Nat. Commun. 2022, 13, 5104, Correction in Nat. Commun. 2022, 13, 6009. https://doi.org/10.1038/s41467-022-33784-x. [Google Scholar] [CrossRef]
  79. O’Neal, A.J.; Hanson, M.R. The Enterovirus Theory of Disease Etiology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Critical Review. Front. Med. 2021, 8, 688486. [Google Scholar] [CrossRef]
  80. Blattner, R.J. Benign myalgic encephalomyelitis (Akureyri disease, Iceland disease). J. Pediatr. 1956, 49, 504–506. [Google Scholar] [CrossRef]
  81. Chapman, N.M. Prior immune exposure can protect or can enhance pathology in the enteroviruses: What predicts the outcome? Virulence 2016, 8, 643–645. [Google Scholar] [CrossRef]
  82. Parish, J.G. Early outbreaks of “epidemic neuromyasthenia”. Postgrad. Med. J. 1978, 54, 711–717. [Google Scholar] [CrossRef]
  83. Boldogh, I.; Albrecht, T.; Porter, D.D. Persistent Viral Infections. In Medical Microbiology, 4th ed.; Baron, S., Ed.; University of Texas Medical Branch at Galveston: Galveston, TX, USA, 1996. [Google Scholar]
  84. Ahn, K.; Kim, H.-S. Structural and Quantitative Expression Analyses of HERV Gene Family in Human Tissues. Mol. Cells 2009, 28, 99–104. [Google Scholar] [CrossRef]
  85. Kim, T.-H.; Jeon, Y.-J.; Yi, J.-M.; Kim, D.-S.; Huh, J.-W.; Hur, C.-G.; Kim, H.-S. The Distribution and Expression of HERV Families in the Human Genome. Mol. Cells 2004, 18, 87–93. [Google Scholar] [CrossRef]
  86. Bhetariya, P.; Kriesel, J.; Fischer, K. Analysis of Human Endogenous Retrovirus Expression in Multiple Sclerosis Plaques. J. Emerg. Dis. Virol. 2017, 3. [Google Scholar] [CrossRef]
  87. Garcia-Montojo, M.; Dominguez-Mozo, M.; Arias-Leal, A.; Garcia-Martinez, Á.; Heras, V.D.L.; Casanova, I.; Faucard, R.; Gehin, N.; Madeira, A.; Arroyo, R.; et al. The DNA Copy Number of Human Endogenous Retrovirus-W (MSRV-Type) Is Increased in Multiple Sclerosis Patients and Is Influenced by Gender and Disease Severity. PLoS ONE 2013, 8, e53623. [Google Scholar] [CrossRef]
  88. Pérez-Pérez, S.; Domínguez-Mozo, M.I.; García-Martínez, M.Á.; García-Frontini, M.C.; Villarrubia, N.; Costa-Frossard, L.; Villar, L.M.; Arroyo, R.; Álvarez-Lafuente, R. Anti-Human Herpesvirus 6 A/B Antibodies Titers Correlate With Multiple Sclerosis-Associated Retrovirus Envelope Expression. Front. Immunol. 2021, 12, 798003. [Google Scholar] [CrossRef]
  89. Pérez-Pérez, S.; Domínguez-Mozo, M.I.; García-Martínez, M.Á.; Ballester-González, R.; Nieto-Gañán, I.; Arroyo, R.; Alvarez-Lafuente, R. Epstein-Barr Virus Load Correlates with Multiple Sclerosis-Associated Retrovirus Envelope Expression. Biomedicines 2022, 10, 387. [Google Scholar] [CrossRef]
  90. Schmitt, K.; Richter, C.; Backes, C.; Meese, E.; Ruprecht, K.; Mayer, J. Comprehensive analysis of human endogenous retrovirus group HERV-W locus transcription in multiple sclerosis brain lesions by high-throughput amplicon sequencing. J. Virol. 2013, 87, 13837–13852. [Google Scholar] [CrossRef]
  91. Agoni, L.; Guha, C.; Lenz, J. Detection of Human Endogenous Retrovirus K (HERV-K) Transcripts in Human Prostate Cancer Cell Lines. Front. Oncol. 2013, 3, 54271. [Google Scholar] [CrossRef]
  92. Agoni, L. Alternative and aberrant splicing of human endogenous retroviruses in cancer. What about head and neck? —A mini review. Front. Oncol. 2022, 12, 1019085. [Google Scholar] [CrossRef]
  93. Hahn, S.; Ugurel, S.; Hanschmann, K.-M.; Strobel, H.; Tondera, C.; Schadendorf, D.; Löwer, J.; Löwer, R. Serological response to human endogenous retrovirus K in melanoma patients correlates with survival probability. AIDS Res. Hum. Retroviruses 2008, 24, 717–723. [Google Scholar] [CrossRef]
  94. Schmitt, K.; Reichrath, J.; Roesch, A.; Meese, E.; Mayer, J. Transcriptional Profiling of Human Endogenous Retrovirus Group HERV-K(HML-2) Loci in Melanoma. Genome Biol. Evol. 2013, 5, 307–328. [Google Scholar] [CrossRef]
  95. Tovo, P.-A.; Ribaldone, D.G.; Galliano, I.; Caviglia, G.P.; Dini, M.; Veglio, V.; Calvi, C.; Montanari, P.; Pitoni, D.; Frara, S.; et al. Enhanced Transcription of Human Endogenous Retroviruses and TRIM28 Downregulation in Patients with Inflammatory Bowel Disease. Viruses 2024, 16, 1570. [Google Scholar] [CrossRef]
  96. Bao, C.; Gao, Q.; Xiang, H.; Shen, Y.; Chen, Q.; Gao, Q.; Cao, Y.; Zhang, M.; He, W.; Mao, L. Human endogenous retroviruses and exogenous viral infections. Front. Cell. Infect. Microbiol. 2024, 14, 1439292. [Google Scholar] [CrossRef]
  97. Chen, J.; Foroozesh, M.; Qin, Z. Transactivation of human endogenous retroviruses by tumor viruses and their functions in virus-associated malignancies. Oncogenesis 2019, 8, 6. [Google Scholar] [CrossRef]
  98. Charvet, B.; Brunel, J.; Pierquin, J.; Iampietro, M.; Decimo, D.; Queruel, N.; Lucas, A.; Encabo-Berzosa, M.d.M.; Arenaz, I.; Marmolejo, T.P.; et al. SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients. iScience 2023, 26, 106604. [Google Scholar] [CrossRef]
  99. Giménez-Orenga, K.; Pierquin, J.; Brunel, J.; Charvet, B.; Martín-Martínez, E.; Perron, H.; Oltra, E. HERV-W ENV antigenemia and correlation of increased anti-SARS-CoV-2 immunoglobulin levels with post-COVID-19 symptoms. Front. Immunol. 2022, 13, 1020064. [Google Scholar] [CrossRef]
  100. Kitsou, K.; Kotanidou, A.; Paraskevis, D.; Karamitros, T.; Katzourakis, A.; Tedder, R.; Hurst, T.; Sapounas, S.; Kotsinas, A.; Gorgoulis, V.; et al. Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients. Microbiol. Spectr. 2021, 9, e0126021. [Google Scholar] [CrossRef] [PubMed]
  101. Marston, J.L.; Greenig, M.; Singh, M.; Bendall, M.L.; Duarte, R.R.R.; Feschotte, C.; Iñiguez, L.P.; Nixon, D.F. SARS-CoV-2 infection mediates differential expression of human endogenous retroviruses and long interspersed nuclear elements. JCI Insight 2021, 6, e147170. [Google Scholar] [CrossRef] [PubMed]
  102. Petrone, V.; Fanelli, M.; Giudice, M.; Toschi, N.; Conti, A.; Maracchioni, C.; Iannetta, M.; Resta, C.; Cipriani, C.; Miele, M.T.; et al. Expression profile of HERVs and inflammatory mediators detected in nasal mucosa as a predictive biomarker of COVID-19 severity. Front. Microbiol. 2023, 14, 1155624. [Google Scholar] [CrossRef] [PubMed]
  103. Sutkowski, N.; Conrad, B.; Thorley-Lawson, D.A.; Huber, B.T. Epstein-Barr Virus Transactivates the Human Endogenous Retrovirus HERV-K18 that Encodes a Superantigen. Immunity 2001, 15, 579–589. [Google Scholar] [CrossRef]
  104. van der Kuyl, A.C. HIV infection and HERV expression: A review. Retrovirology 2012, 9, 6. [Google Scholar] [CrossRef]
  105. Liu, X.; Liu, Z.; Wu, Z.; Ren, J.; Fan, Y.; Sun, L.; Cao, G.; Niu, Y.; Zhang, B.; Ji, Q.; et al. Resurrection of endogenous retroviruses during aging reinforces senescence. Cell 2023, 186, 287–304.e26. [Google Scholar] [CrossRef]
  106. Nevalainen, T.; Autio, A.; Mishra, B.H.; Marttila, S.; Jylhä, M.; Hurme, M. Aging-associated patterns in the expression of human endogenous retroviruses. PLoS ONE 2018, 13, e0207407. [Google Scholar] [CrossRef]
  107. Rodrigues, L.S.; da Silva Nali, L.H.; Leal, C.O.D.; Sabino, E.C.; Lacerda, E.M.; Kingdon, C.C.; Nacul, L.; Romano, C.M. HERV-K and HERV-W transcriptional activity in myalgic encephalomyelitis/chronic fatigue syndrome. Autoimmun. Highlights 2019, 10, 12. [Google Scholar] [CrossRef]
  108. Giménez-Orenga, K.; Martín-Martínez, E.; Nathanson, L.; Oltra, E. HERV activation segregates ME/CFS from fibromyalgia while defining a novel nosologic entity. eLife 2025, 14, RP104441. [Google Scholar] [CrossRef]
  109. Ovejero, T.; Sadones, O.; Sánchez-Fito, T.; Almenar-Pérez, E.; Espejo, J.A.; Martín-Martínez, E.; Nathanson, L.; Oltra, E. Activation of Transposable Elements in Immune Cells of Fibromyalgia Patients. Int. J. Mol. Sci. 2020, 21, 1366. [Google Scholar] [CrossRef]
  110. Laudański, P.; Zyguła, A.; Czyżyk, A.; Olszak-Wasik, K.; Warzecha, D.; Wojtyła, C.; Kurzawa, R. Viral infection and its impact on fertility, medically assisted reproduction and early pregnancy—A narrative review. Reprod. Biol. Endocrinol. 2025, 23, 68. [Google Scholar] [CrossRef]
  111. Marci, R.; Gentili, V.; Bortolotti, D.; Lo Monte, G.; Caselli, E.; Bolzani, S.; Rotola, A.; Di Luca, D.; Rizzo, R. Presence of HHV-6A in Endometrial Epithelial Cells from Women with Primary Unexplained Infertility. PLoS ONE 2016, 11, e0158304. [Google Scholar] [CrossRef]
  112. van der Kuyl, A.C.; Berkhout, B. Viruses in the reproductive tract: On their way to the germ line? Virus Res. 2020, 286, 198101. [Google Scholar] [CrossRef] [PubMed]
  113. Samms, G.L.; Ponting, C.P. Unequal access to diagnosis of myalgic encephalomyelitis in England. BMC Public Health 2025, 25, 1417. [Google Scholar] [CrossRef] [PubMed]
  114. Vahratian, A.; Lin, J.-M.S.; Bertolli, J.; Unger, E.R. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in Adults: United States, 2021–2022; NCHS Data Brief: Hyattsville, MD, USA, 2023. [Google Scholar]
  115. Wira, C.R.; Rodriguez-Garcia, M.; Patel, M.V. The role of sex hormones in immune protection of the female reproductive tract. Nat. Rev. Immunol. 2015, 15, 217–230. [Google Scholar] [CrossRef]
  116. Honorato, L.; Paião, H.G.O.; da Costa, A.C.; Tozetto-Mendoza, T.R.; Mendes-Correa, M.C.; Witkin, S.S. Viruses in the female lower reproductive tract: A systematic descriptive review of metagenomic investigations. Npj Biofilms Microbiomes 2024, 10, 137. [Google Scholar] [CrossRef] [PubMed]
  117. Madere, F.S.; Monaco, C.L. The female reproductive tract virome: Understanding the dynamic role of viruses in gynecological health and disease. Curr. Opin. Virol. 2022, 52, 15–23. [Google Scholar] [CrossRef] [PubMed]
  118. Komaroff, A.L.; Dantzer, R. Causes of symptoms and symptom persistence in long COVID and myalgic encephalomyelitis/chronic fatigue syndrome. Cell Rep. Med. 2025, 6, 102259. [Google Scholar] [CrossRef] [PubMed]
  119. Nunes, J.M.; Kell, D.B.; Pretorius, E. Herpesvirus Infection of Endothelial Cells as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Viruses 2024, 16, 572. [Google Scholar] [CrossRef] [PubMed]
  120. Tate, W.; Walker, M.; Sweetman, E.; Helliwell, A.; Peppercorn, K.; Edgar, C.; Blair, A.; Chatterjee, A. Molecular Mechanisms of Neuroinflammation in ME/CFS and Long COVID to Sustain Disease and Promote Relapses. Front. Neurol. 2022, 13, 877772. [Google Scholar] [CrossRef]
  121. Papadopoulos, A.S.; Cleare, A.J. Hypothalamic-pituitary-adrenal axis dysfunction in chronic fatigue syndrome. Nat. Rev. Endocrinol. 2011, 8, 22–32. [Google Scholar] [CrossRef]
  122. Tomas, C.; Newton, J.; Watson, S. A Review of Hypothalamic-Pituitary-Adrenal Axis Function in Chronic Fatigue Syndrome. ISRN Neurosci. 2013, 2013, 784520. [Google Scholar] [CrossRef]
  123. Savela, E.S.; Viloria Winnett, A.; Romano, A.E.; Porter, M.K.; Shelby, N.; Akana, R.; Ji, J.; Cooper, M.M.; Schlenker, N.W.; Reyes, J.A.; et al. Quantitative SARS-CoV-2 Viral-Load Curves in Paired Saliva Samples and Nasal Swabs Inform Appropriate Respiratory Sampling Site and Analytical Test Sensitivity Required for Earliest Viral Detection. J. Clin. Microbiol. 2022, 60, e01785-21. [Google Scholar] [CrossRef]
  124. Debat, H.; Bejerman, N. An Update on RNA Virus Discovery: Current Challenges and Future Perspectives. Viruses 2025, 17, 983. [Google Scholar] [CrossRef]
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Figure 1. Conceptual schematic of the proposed role of persistent mucosal viruses in the aetiology of ME/CFS. Mucosal cells, such as epithelial cells, can serve as reservoirs for persistent viruses, potentially supporting viral replication under pathological conditions including stress, inflammation, microbiota dysbiosis, epithelial injury, or impaired immune surveillance [50]. These infected mucosal cells can release viral particles both apically (into the lumen) and basolaterally, promoting further infection and systemic spread [50,51,52]. Persistent mucosal viruses may also cross the disrupted gut intestinal epithelial barrier (leaky gut), activating dendritic cells and macrophages within the lamina propria, promoting CD4 Th1 and Th17 responses. Th1 cytokines, IFN-γ and TNF-α, activate M1 macrophages, driving the release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) into systemic circulation. These cytokines inhibit Th2 differentiation or the production of immune tolerance promoting cytokines resulting in a more pro-inflammatory state dominated by Th1/Th17 responses, which further exacerbates inflammation. Th17-derived IL-17 and IL-22 promote neutrophil recruitment and the release of antimicrobial peptides (AMPs) such as lysozymes and defensins with chronic neutrophil activation leading to epithelial damage and degradation and thinning of the mucus layer. Disruption of the epithelial barrier facilitates viral translocation which may promote a feed-forward inflammatory loop perpetuating local and systemic immune dysregulation observed in ME/CFS. (Created in BioRender. Perera, K. (2025) https://BioRender.com/rxje1b4, accessed on 10 November 2025).
Figure 1. Conceptual schematic of the proposed role of persistent mucosal viruses in the aetiology of ME/CFS. Mucosal cells, such as epithelial cells, can serve as reservoirs for persistent viruses, potentially supporting viral replication under pathological conditions including stress, inflammation, microbiota dysbiosis, epithelial injury, or impaired immune surveillance [50]. These infected mucosal cells can release viral particles both apically (into the lumen) and basolaterally, promoting further infection and systemic spread [50,51,52]. Persistent mucosal viruses may also cross the disrupted gut intestinal epithelial barrier (leaky gut), activating dendritic cells and macrophages within the lamina propria, promoting CD4 Th1 and Th17 responses. Th1 cytokines, IFN-γ and TNF-α, activate M1 macrophages, driving the release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) into systemic circulation. These cytokines inhibit Th2 differentiation or the production of immune tolerance promoting cytokines resulting in a more pro-inflammatory state dominated by Th1/Th17 responses, which further exacerbates inflammation. Th17-derived IL-17 and IL-22 promote neutrophil recruitment and the release of antimicrobial peptides (AMPs) such as lysozymes and defensins with chronic neutrophil activation leading to epithelial damage and degradation and thinning of the mucus layer. Disruption of the epithelial barrier facilitates viral translocation which may promote a feed-forward inflammatory loop perpetuating local and systemic immune dysregulation observed in ME/CFS. (Created in BioRender. Perera, K. (2025) https://BioRender.com/rxje1b4, accessed on 10 November 2025).
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Figure 2. A proposed mechanism linking latent mucosal viral reservoirs to systemic inflammation and neuroimmune symptoms observed in ME/CFS. Latent viruses such as EBV, HHV6/7, and enteroviruses persist in mucosal tissues. Reactivation triggered by internal or environmental stressors, combined with immune-mediated damage to the epithelial barrier, disrupts its integrity. Immune spillover occurs as local pro-inflammatory mediators (e.g., cytokines) and immune responses escape the mucosal compartment, leading to the release of cytokines and viral leakage into deeper tissues and bloodstream causing viraemia. The resulting systemic inflammation promotes chronic low-grade immune activation. Concurrent viral infection in endothelial cells (e.g., herpesviruses) may induce endothelial dysfunction and cause vascular abnormalities, further exacerbating systemic inflammation. Circulating neuroimmune factors could reach the central nervous system, causing neuroinflammation. Disruption of the hypothalamic–pituitary–adrenal (HPA) axis impairs cortisol production and stress regulation, often because of ongoing inflammation. Persistent viral activity and inflammation can also activate HERVs further amplifying immune dysregulation and contributing to chronic infection. All these factors drive key ME/CFS symptoms, including fatigue, cognitive dysfunction (brain fog), post-exertional malaise, and sleep disturbances. This model highlights how mucosal reservoirs may contribute to systemic immune dysregulation in ME/CFS. (Created in BioRender. Carding, S. (2025) https://BioRender.com/5bmmgg5, accessed on 10 November 2025).
Figure 2. A proposed mechanism linking latent mucosal viral reservoirs to systemic inflammation and neuroimmune symptoms observed in ME/CFS. Latent viruses such as EBV, HHV6/7, and enteroviruses persist in mucosal tissues. Reactivation triggered by internal or environmental stressors, combined with immune-mediated damage to the epithelial barrier, disrupts its integrity. Immune spillover occurs as local pro-inflammatory mediators (e.g., cytokines) and immune responses escape the mucosal compartment, leading to the release of cytokines and viral leakage into deeper tissues and bloodstream causing viraemia. The resulting systemic inflammation promotes chronic low-grade immune activation. Concurrent viral infection in endothelial cells (e.g., herpesviruses) may induce endothelial dysfunction and cause vascular abnormalities, further exacerbating systemic inflammation. Circulating neuroimmune factors could reach the central nervous system, causing neuroinflammation. Disruption of the hypothalamic–pituitary–adrenal (HPA) axis impairs cortisol production and stress regulation, often because of ongoing inflammation. Persistent viral activity and inflammation can also activate HERVs further amplifying immune dysregulation and contributing to chronic infection. All these factors drive key ME/CFS symptoms, including fatigue, cognitive dysfunction (brain fog), post-exertional malaise, and sleep disturbances. This model highlights how mucosal reservoirs may contribute to systemic immune dysregulation in ME/CFS. (Created in BioRender. Carding, S. (2025) https://BioRender.com/5bmmgg5, accessed on 10 November 2025).
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Table 1. Summary of blood-based virus studies in ME/CFS.
Table 1. Summary of blood-based virus studies in ME/CFS.
SampleDiagnostic
Criteria		CohortsDetection MethodViruses Studied *Association with ME/CFSRef.
SerumCenters for Disease Control and Prevention (CDC)548 chronic fatigue syndrome (CFS) patients and 30 healthy controlsImmunoassayHerpes simplex virus (HSV) 1/2
Rubella
Adenovirus
HHV-6
EBV
Cytomegalovirus (CMV)
Coxsackie B virus types 1–6		No difference in the seroprevalence of the viruses between CFS and healthy controls.[14]
SerumOxford8 CFS patientsPCR and sequencingEnterovirusEnteroviruses can persist in the serum of CFS patients.[15]
Serum and peripheral blood leukocytes (PBLs)CDC26 CFS patients and 50 healthy controlsPCR and antibody profilingHHV-6A/B
HHV-7		No difference in active or latent HHV-6A/B or HHV-7 infections between CFS patients and controls.[16]
Serum, plasma and PBMCCDC-1994 (“Fukuda”)35 CFS patients (27 severe CFS) and 25 healthy controlsPCR and immunoassayHHV-6
HHV-7
HHV-8		Frequent reactivation of HHV-6 detected in CFS patients.[17]
PBLs and plasmaFukuda17 diagnosed CFS patients, 12 unexplained CFS patients, and 20 healthy controlsPCR and immunoassayHHV-6
HHV-7		No difference in prevalence of HHV-6 or HHV-7 between the groups. CFS patients showed a significantly higher rate of dual HHV-6 and HHV-7 infection, increased plasma viraemia for HHV-7 and simultaneous reactivation of both viruses.[18]
PBLs and plasmaFukuda108 ME/CFS patients and 90 healthy controlsPCR and immunoassayHHV-6
HHV-7
Parvovirus B19		Active HHV-6, HHV-7 and parvovirus B19 infections and co-infection of these viruses show a high prevalence in ME/CFS patients compared to healthy controls.[19]
SerumFukuda72 CFS patients and 59 healthy controlsImmunoassayHHV-6A/BNo difference in antibody levels to HHV-6A/B between CFS patients and healthy controls.[20]
PBMCFukuda39 CFS patients and 9 healthy controlsRT-PCRHHV-6
HHV-7
Human endogenous retrovirus (HERV)-K18		No difference in HHV-6 or HHV-7 viral copy numbers or HERV-K18 transcripts between CFS patients and healthy controls.[21]
PlasmaCanadian Consensus Criteria (CCC)25 ME/CFS patients, and 25 healthy controlsMetagenomicsBroad virus diversityNo consistent viral associations detected in ME/CFS and contamination and batch effects confound results if controls are inadequate.[22]
Whole bloodCCC25 ME/CFS patients, and 25 healthy controlsMetagenomicsAnelloviruses
Pegivirus 1
Herpesviruses
Papillomaviruses		No difference in the viral composition between ME/CFS cases and controls.[23]
SerumCCC163 ME/CFS patients with/without comorbidities and 103 healthy controlsImmunoassayHHV-1 to HHV-7No significant difference in IgG reactivity to HHV-1 to HHV-7 between ME/CFS and controls, but there are subtle variations in antibody responses to HHV-1 and EBV antigens in ME/CFS.[24]
Whole blood CCC14 female ME/CFS patients and 11 matched sedentary controlsMetagenomicsEnterovirus A
Influenza A
Anelloviruses
Human herpesviruses		No differences in viral abundance or virome composition between ME/CFS patients with post-exertional malaise and controls before or after exercise.[25]
PlasmaCCC and Fukuda251 ME/CFS patients and 107 healthy controlsImmunoassayHSV-1/2
VZV
CMV
EBV
HHV-6		No difference in the seroprevalence of these viruses between ME/CFS patients and healthy controls.[26]
PBMC and plasmaFukuda58 ME/CFS patients and 50 healthy controlsPCR and immunoassayHHV-6
CMV
EBV		Active EBV infection is more common in ME/CFS patients, with no significant differences in active CMV or HHV-6 infections.[27]
PlasmaFukuda or CCC226 ME/CFS patients and 99 healthy controlsImmunoassayHSV-1/2
VZV
CMV
EBV
HHV-6		Herpesvirus serology could potentially distinguish different subgroups of ME/CFS patients based on their reported disease trigger (infection versus non-infection)[28]
PBMC and serumFukuda30 ME/CFS patients and 20 healthy controlsPCR and immunoassaysHHV-6
HHV-7
EBV		Higher frequencies of both latent and active HHV-6, HHV-7, EBV, and co-infections of these viruses detected in ME/CFS patients compared to healthy controls.[29]
Whole bloodFukuda134 ME/CFS patients and 33 healthy controlsPCRHHV-6A/BDisease severity in ME/CFS relates to persistent HHV-6 viral load.[30]
Plasma and PBMCFukuda and CCC391 ME/CFS patients and 292 healthy controlsPCR, and VirCapSeqHerpesviruses
Pegiviruses
Anelloviruses
HERVs		No difference in the prevalence or diversity of viruses between ME/CFS patients and healthy controls.[31]
PlasmaCDC or CCC222 ME/CFS patients and 46 multiple sclerosis (MS) patientsImmunoassayHSV-1/2
VZV
CMV
EBV
HHV-6		IgG antibody concentrations explained symptom associations in multiple sclerosis more robustly than in ME/CFS.[32]
Whole blood and plasmaFukuda200 ME/CFS patients and 150 healthy controlsPCRHHV-6A/B
HHV-7
Parvovirus B19		Persistent active infections or co-infections with HHV-6A/B, HHV-7, and parvovirus B19 are more frequent in ME/CFS patients compared to healthy individuals.[33]
* Refers to all viruses investigated in each study, including detection of viral antigens, antibodies, nucleic acids, or other viral markers as applicable. Specific viral components assessed (e.g., antigen vs. antibody) may vary by study and are detailed in the cited publications.
Table 2. Publications on persistent viral analysis in mucosal regions of ME/CFS patients.
Table 2. Publications on persistent viral analysis in mucosal regions of ME/CFS patients.
Mucosal
Sampling Site		Diagnosis
Criteria		CohortsDetection MethodViruses Studied *Association with ME/CFSRef.
Throat swabOxford175 throat swabs from CFS patientsPCR and sequencingEnterovirusesEnterovirus sequences found in some CFS patients.[57]
SalivaFukuda39 CFS patients and 9 healthy controlsRT-PCRHHV-6
HHV-7		No difference in HHV6 or HHV7 viral loads. No difference detected in human endogenous retrovirus-K18 (HERV-K18) levels in PBMCs.[21]
SalivaCCC and/or Fukuda14 ME/CFS-Mild/ Moderate, 16 ME/CFS-Severe Patients, and 16 healthy controlsddPCRHHV-6B
HHV-7
HSV-1
EBV		High viral loads of HHV-6B and HHV-7 observed in ME/CFS patients, and fluctuations in viral load correlated with specific ME/CFS disease phenotypes. No difference in EBV or HSV-1 viral loads.[58]
SalivaCCC95 ME/CFS patients (78 female) and 110 healthy controls (71 female)Antibody profilingEBV
HHV-6
HERV-K		COVID-19 triggers reactivation of latent herpesviruses (EBV, HHV-6) and HERV-K, with stronger reactivation and uniquely elevated EBV nuclear antigen antibodies in ME/CFS patients, who also show higher baseline EBV antibodies before SARS-CoV-2 infection.[34]
Saliva and oropharynx 88 post-COVID patients (68 with herpesvirus reactivation and 20 non-detectable herpesvirus DNA controls). 46/88 female and 42/88 male.RT-PCREBV
HHV-6		Herpesvirus reactivation is associated with post-COVID syndrome and chronic fatigue.[36]
SalivaCCC84 ME/CFS patients and 94 healthy controlsAntibody profilingAdenovirus
SARS-CoV-2		SARS-CoV-2 triggers Adenovirus reactivation in ME/CFS.[35]
Saliva and faecesFukuda and CCC106 ME/CFS patients and 91 healthy controlsVirCapSeqHerpesviruses and papillomavirusesNo difference in the prevalence of viral sequences between ME/CFS patients and healthy controls.[31]
SputumCCC 13 ME/CFS patients, 10 healthy, 4 elderly, and 2 immunosuppressed controlsRT-PCREBV
HHV-6
Adenovirus		High EBV viral load in ME/CFS patients. No difference in HHV-6 viral loads.[59]
Stomach biopsiesCDC165 patients, 22 healthy controls, and 12 patients with other gastric disordersImmuno-stainingEnterovirusesEnterovirus detected in stomach biopsies from 82% of CFS patients compared to 20% of controls.[60]
Gastric antrum and duodenum biopsiesFukuda48 CFS patients and 35 healthy controls. 78% patients and 66% of the controls were females.RT-PCRHHV-7
HHV-6
EBV
Parvovirus B19		High viral loads of Parvovirus B19 detected in ME/CFS patients.[61]
Stomach biopsies Follow-up of three patients with acute enterovirus infection, later developed ME/CFSRT-PCR Immuno-stainingEnterovirusesAcute enterovirus infection could lead to ME/CFS, and enteroviruses were detected in stomach biopsies years after initial infection.[62]
Duodenal and stomach biopsiesCCC and Fukuda12 ME patients and 8 healthy controlsImmuno-stainingHERVsImmunoreactivity to HERV proteins was observed in some ME/CFS patients.[63]
FaecesCCC and National Institute for Health and Care Excellence (NICE) 2007/CG53 guideline9 severely affected female ME/CFS patients and 8 healthy controls (3 males and 5 females)Virome profiling, metagenomicsMultiple
bacteriophages
HHV-6A
Papillomaviruses
Coronavirus NL63
Adenovirus 54		ME/CFS-specific viral–bacterial associations.[64]
* Refers to all viruses investigated in each study, including detection of viral antigens, antibodies, nucleic acids, or other viral markers as applicable. Specific viral components assessed (e.g., antigen vs. antibody) may vary by study and are detailed in the cited publications.
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Perera, K.D.; Cameron, P.; Sarwar, T.; Carding, S.R. Mucosal Viruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Missing Piece of the Puzzle? Int. J. Mol. Sci. 2025, 26, 11161. https://doi.org/10.3390/ijms262211161

AMA Style

Perera KD, Cameron P, Sarwar T, Carding SR. Mucosal Viruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Missing Piece of the Puzzle? International Journal of Molecular Sciences. 2025; 26(22):11161. https://doi.org/10.3390/ijms262211161

Chicago/Turabian Style

Perera, Krishani Dinali, Paige Cameron, Tayyibah Sarwar, and Simon R. Carding. 2025. "Mucosal Viruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Missing Piece of the Puzzle?" International Journal of Molecular Sciences 26, no. 22: 11161. https://doi.org/10.3390/ijms262211161

APA Style

Perera, K. D., Cameron, P., Sarwar, T., & Carding, S. R. (2025). Mucosal Viruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Missing Piece of the Puzzle? International Journal of Molecular Sciences, 26(22), 11161. https://doi.org/10.3390/ijms262211161

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