Potential Molecular Mechanisms Of Chronic Fatigue in Long Haul COVID And Other Viral Diseases Part 1

Abstract

Historically, COVID-19 has emerged as one of the most devastating diseases of humankind, which creates an unmanageable health crisis worldwide. Until now, this disease has cost millions of lives and continues to paralyze human civilization’s economy and social growth, leaving enduring damage that will take an exceptionally long time to repair. While a majority of infected patients survive after mild to moderate reactions after two to six weeks, a growing population of patients suffers for months with severe and prolonged symptoms of fatigue, depression, and anxiety. These patients are no less than 10% of total COVID-19-infected individuals with distinctive chronic clinical symptomatology, collectively termed post-acute sequelae of COVID-19 (PASC) or more commonly long-haul COVID. Interestingly, Long-haul COVID and many debilitating viral diseases display a similar range of clinical symptoms of muscle fatigue, dizziness, depression, and chronic inflammation. In our current hypothesis-driven review article, we attempt to discuss the molecular mechanism of muscle fatigue in long-haul COVID, and other viral diseases as caused by HHV6, Powassan, Epstein–Barr virus (EBV), and HIV. We also discuss the pathological resemblance of virus-triggered muscle fatigue with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

Cistanche can act as an anti-fatigue and stamina enhancer, and experimental studies have shown that the decoction of Cistanche tubulosa could effectively protect the liver hepatocytes and endothelial cells damaged in weight-bearing swimming mice, upregulate the expression of NOS3, and promote hepatic glycogen synthesis, thus exerting anti-fatigue efficacy. Phenylethanoid glycoside-rich Cistanche tubulosa extract could significantly reduce the serum creatine kinase, lactate dehydrogenase, and lactate levels, and increase the hemoglobin (HB) and glucose levels in ICR mice, and this could play an anti-fatigue role by decreasing the muscle damage and delaying the lactic acid enrichment for energy storage in mice. Compound Cistanche Tubulosa Tablets significantly prolonged the weight-bearing swimming time, increased the hepatic glycogen reserve, and decreased the serum urea level after exercise in mice, showing its anti-fatigue effect. The decoction of Cistanchis can improve endurance and accelerate the elimination of fatigue in exercising mice, and can also reduce the elevation of serum creatine kinase after load exercise and keep the ultrastructure of skeletal muscle of mice normal after exercise, which indicates that it has the effects of enhancing physical strength and anti-fatigue. Cistanchis also significantly prolonged the survival time of nitrite-poisoned mice and enhanced the tolerance against hypoxia and fatigue.

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Keywords IFNγ, Microglia, CD4+ and CD8+ T cells, Mitochondria

Introduction

The role of viral infection in muscle fatigue has been debated for a long in the field of ME/CFS [1]. ME/CFS is a chronic inflammatory disease characterized by severe muscle weakness, fatigue, pain, lightheadedness, and brain fog [2]. One of the most debilitating symptoms of ME/CFS is post-exertional malaise (PEM), in which a patient suffers severe muscle fatigue and cognitive-, and orthostatic- exertions after mild exercise. This severe worsening of symptoms can cause a patient to be bedridden for a long time ranging from 24 hours to several months [3, 4]. Although the underlying molecular mechanism of severe muscle fatigue in ME/CFS is not known, a growing body of evidence suggests that intracellular inflammation and exaggerated productions of inflammatory mediators might contribute to the pathogenesis of muscle fatigue by promoting the degeneration of skeletal muscle cells and also inhibiting the differentiation of muscle progenitor cells [5, 6]. However, it is not known how the inflammation is initiated. In this context, a “ hit-and-run” mechanism of viral infection could be critical in which a transient viral infection is considered to potentiate a series of inflammatory events causing a sustained immunological disturbance [7]. A “virus reactivation theory” could be another mechanism [8], which suggests that the reactivation of viruses including EBV and HHV6 followed by a cascade of inflammatory events might contribute to the pathogenesis of ME/CFS [1]. Despite these competing hypotheses, the role of viral infection in the pathogenesis of muscle fatigue cannot be disregarded. Interestingly, a recent pandemic of COVID-19 also exhibits persistent symptoms of fatigue and weakness in approximately 10% of its survivors reiterating the potential role of virus infection in the pathogenesis of chronic fatigue syndrome [9]. Our current speculative review article discusses how HHV6, Powassan, EBV, HIV, and SARS-CoV2 viral infections adopt a common immunological mechanism that possibly leads to debilitating muscle fatigue.

HHV6 and chronic fatigue syndrome

The potential association between HHV6 and chronic inflammation was first introduced in 1992 by Buchwald et al. [10] when a cohort of 259 HHV6-infected patients was diagnosed with severe lymphocytic activation and cognitive impairment. Although, that study was controversial [11] to prove the link between chronic fatigue syndrome (CFS) and HHV6, in the same year, Kato et al. [12]

reported a case study with a 31-year-old woman who was initially admitted with CFS, was turned out to be positive with a high titer of anti-HHV6 antigen. Later on, a PCR-based study [13] identified strong upregulations of HHV6 A and B mRNAs in 7 of 13 CFS patients with high titer of HHV6 early antigen demonstrating a strong correlation between HHV6 infection and CFS. Furthermore, a strong upregulation of IgM antibody against HHV6 early antigen (EA) [14] in 93 of 154 CFS patients (60%) [15] established another possible link between HHV6 and CFS. Although the molecular mechanism of HHV6 infection and fatigue was still unclear, HHV6 was known to induce an acute immunosuppressive response. Although both HHV6-A and B strains infect CD4 + T helper and  CD8+ cytotoxic T cells [16], upon infection, HHV6 selectively suppresses the expression of IL12 and inhibits the T1 polarization of CD4 + T cells [16]. In infected CD4 + T cells, HHV6 also suppresses the proliferative response by downregulating the expression of IL2 [17] and augmenting cell cycle arrest [18]. All these events induce apoptotic signals to CD4+ T cells (Fig. 1). In response to these apoptotic T cells, macrophages perform phagocytosis and augment an anti-inflammatory “immunotolerant” microenvironment characterized by high levels of TGF-β and IL-10 [19]. In addition, a death response to CD4 + T cells causes acute suppression of anti-viral IFN-γ production [20, 21]. Interestingly, reduced IFN-γ and increased IL-10 are historically known to suppress inflammation [22, 23]. Therefore, the role of acute HHV6 infection in inducing inflammation seems elusive. One potential mechanism could be the escape of persistently infected CD4+ T cells from the above-mentioned acute apoptotic pathway that potentially stimulates an inflammatory response in macrophages and glial cells. A recent report also suggests that HHV6A directly stimulates the inflammation and migration of microglial cells via the activation of TREM2 and ApoE [24]. Therefore, active HHV6 infection, but not an acute immunosuppressive event, may be directly responsible for responsible for the microglial activation [25], and possible demyelination [26]. Furthermore, a recent study [27] identified that patients with demyelination in CNS displayed HHV6-immunoreactive oligoclonal bands in their cerebrospinal fluid indicating a potential link between multiple sclerosis (MS)-like encephalopathy and HHV6-infection.

Nevertheless, the upregulation of 8-hydroxy-2′- deoxyguanosine (8h2dg) [28], a DNA stress marker, in the  CSF of HHV6 encephalopathy patients and a significant recovery after FDA-approved ALS-drug Edarvone further confirmed the presence of encephalopathic response in HHV6 patients. That study demonstrated that 43.7% of HHV6 encephalopathy subjects had higher 8h2dg. Clinical symptoms such as muscle fatigue, sleep disturbance, problems with balance, impaired mobility, and seizures are pathological hallmarks of an encephalopathy [29, 30]. Therefore, combined with the mechanism of persistent immune activation, HHV6 infection could also trigger a CNS-specific stress response resulting in microglial inflammation [24], demyelination [31], oxidative stress [32], and neuronal damage [33], which might lead to the clinical manifestations of cognitive deficit, emotional disabilities, and muscle fatigue. HHV6 infection directly or indirectly triggers neurodegeneration. In an indirect mechanism, HHV6A promotes microglial expression of amyloid beta (Aβ) [24], the secretion of phospho-tau [24], and the induction of IL-1β. Moreover, HHV6 directly causes apoptosis of cerebellar Purkinje cells [34] suggesting its direct role in neurodegeneration. As a mechanism, the disruption of TLR4 signaling [35] and activation of TLR9 [36] followed by activation of nuclear factor κB (NF-κB) [37] might play key roles in inducing pro-inflammatory signaling events. HHV6 infection also profoundly contributes to central and peripheral demyelination. HHV6 virions directly infect oligodendroglial progenitor cells (OPCs) cause cell cycle arrest at the G1/S phase and inhibit its maturation to oligodendrocytes [38]. Other reports suggest that HHV-6A latency gene U94 directly inhibits migration and myelination of OPCs [39]. Similar to the situation in the CNS, HHV6 also induces peripheral demyelination by direct infection of the peripheral nervous system in the dorsal sensory ganglia [40, 41].

Taken together, both the central and peripheral mechanisms of HHV6-induced demyelination result in the progressive loss of nerve conduction to the synaptic terminal at the neuromuscular junction resulting in muscle weakness and fatigue (Fig. 1).

Powassan virus encephalitis and chronic fatigue

Powassan virus (POWV) encephalitis was first reported in 1958, when the titer of POWV, a neuroinvasive arbovirus, was detected from the brain autopsy of a young boy who died in Powassan, Ontario [42]. It is a tickborne flavivirus-induced [43, 44] disease that displays a wide spectrum of neuroinflammatory responses [45] in the brain and spinal cord including compromised blood–brain barrier integrity, enhanced infiltration of inflammatory T cells [46, 47], severe microglial activation [47], and demyelination [48] of oligodendrocytes resulting neuronal toxicity. While it is not known if POWV can induce a similar acute immunosuppressive mechanism as seen in HHV6, a recent study [49] demonstrated that there is a robust proliferation of reactive T1 cells in the spleens of the POWV-infected mice. This finding suggests that, in contrast to HHV6, POWV acutely induces the inflammatory response in the early phase of infection. During the acute phase of infection, there is an activation of innate immunity (Fig. 2) for the protection against POWV infection.  One such mechanism includes the activation of B cells and the subsequent expression of IgM antibodies. Indeed, elevated IgM antibodies have been identified in both CSF and sera of acute POWV-infected patients [44]. IgM antibody directly induces cytotoxicity of virus-infected cells. Another protective mechanism could include the acute activation of natural killer (NK) cells and natural killer T (NKT) cells [50] followed by the release of antiviral cytokine IFNγ (Fig. 2). Although this mechanism is yet to be established in POWV infection, another tickborne bacterial disease, namely Lyme disease [51] has been shown to directly activate NK cells in tick-borne encephalitis. Although a direct association of POWV with NK cells has yet to be established, infections of other flaviviruses such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV), have been shown to cause direct activation of NK cells [52]. However, POWV directly infects macrophages (Fig. 2) at an early stage in the tick-feeding site [53, 54], which potentially triggers the activation of NK and NKT cells to produce IFNγ causing a cytotoxic response in POWV virions (Fig. 2). The activation of cytotoxic T cells followed by the secretion of perforin and granzyme B could be another mechanism [55] for the cytotoxicity of virus-infected cells (Fig. 2). However, acute infection followed by sustained activation of innate immune response and IFNγ production could activate antigen-presenting cells as well. The activations of macrophages, dendritic cells, and microglia due to severe IFNγ production, could switch on downstream cell-based adaptive inflammatory response causing severe neuroinflammation (Fig. 2).

Similar to other tick-borne diseases such as Lyme disease, acute POWV illness presents with a diverse spectrum of clinical symptoms [56] including fever, pain, headache, and muscle weakness. Treatment paradigms are largely symptomatic and supportive thus contributing to the unpredictable course of illness over time. Interestingly, one of the most common clinical manifestations of POWV encephalitis is muscle fatigue. Encephalitis is often associated with increased demyelination [57] of peripheral nerves [58] which in turn causes impairment of ion conduction through sensory neurons [59] resulting in abnormalities in neuromuscular function [60].

Based on a recent statistical report by the CDC [61], half of the people who survive severe POWV encephalitis continue to suffer from long-term muscle weakness and fatigue following their acute infection phase. Sometimes, a severe and chronic infection of POWV can cause complete paralysis in one side of the body, described clinically as hemiplegia [61]. Complete ophthalmoplegia [61] with loss of eye muscle function in both eyes is also common in POWV patients. A detailed electroencephalogram (EEG) study indicated that severe demyelination of white matter in the temporal lobe may contribute to the loss of downstream neuronal function controlling peripheral muscle movement. Another literature reports significant infiltration of POWV in the ventral horn of the spinal cord [62] that may also contribute to the demyelinating response in the peripheral nervous system and be a potential cause of the severe weakness of peripheral muscle tissue observed clinically. Taken together, these reports suggest that muscle fatigue in POWV-infected patients is possibly the result of a combination of factors including a severe demyelinating response in both the brain and spinal cord, increased expression of IFNγ, the infiltration of inflammatory T cells through the BBB, and microglial activation.

Epstein–Barr virus (EBV) infection and muscle weakness

EBV is a DNA herpes virus that primarily spreads through oral secretions and infects resident B lymphocytes (Fig. 3) in the oropharyngeal epithelium [63]. Upon infection, EBV transforms B cells into B cell lymphoblastoid cells that eventually enter into the follicle, and expand to form a germinal center (GC) [64]. The host’s protective response becomes very active at that stage, which elicits a cytotoxic response from NK cells, CD8+, and  CD4 + T cells (Fig. 3). Infected memory B cells remain latent during this stage. However, following a secondary infection, these memory B cells rapidly convert to plasma B cells. Although B cells are the primary target of EBV infection, T cells can also be infected by EBV [65]. These lymphocytes can penetrate BBB [66] and engage with microglia (Fig. 3). In some cases, EBV directly infects microglia [67]. Upon infection, extrachromosomal episomes of EBV [68], modulate the host immune response by triggering the expression of a wide range of inflammatory cytokines such as IFN-γ, TNF-α, and IL-2 [69], NF-κB [70], and proliferation of inflammatory T lymphocytes. Another possible mechanism of CNS inflammation is molecular mimicry, by which homology between EBV nuclear antigen-1 (EBNA-1) and the host’s myelin basic protein (MBP) elicits the activation of autoreactive T cells [71]. While most EBV infections are asymptomatic, infections during adolescence and adulthood frequently cause reactivation and mononucleosis [72]. Over 50% of patients with infectious mononucleosis manifest the triad of fever, lymphadenopathy, and pharyngitis [73]. Other symptoms include splenomegaly [74], and hepatomegaly [75]. Leucocytosis, atypical lymphocytosis, and elevated liver enzymes are also reported during EBV infection [76].

Recent studies demonstrate that muscle pain and fatigue can follow EBV infection and remain following the resolution of other acute symptoms. According to White et al. [77], in a cohort of 108 subjects, a subset of patients with EBV-induced glandular fever having throat and neck gland swelling was reported to display a distinct physical and mental fatigue, excessive sleep, psychomotor retardation, poor concentration, and anhedonia. The direct association of EBV infection and the pathogenesis of myalgic encephalomyelitis and chronic fatigue syndrome (ME/CFS) has been reported anecdotally for many years, and more clearly following the identification of increased EBV-induced gene 2 (EBI2) expression in PBMC samples from a subgroup of ME/CFS patients [78]. Moreover, upregulations of EBI2-associated early growth response genes known as EGR1, EGR2, and EGR3 in PBMCs of ME/CFS patients further reinforced the hypothesis that EBV infection could be directly linked to long-term muscle fatigue and pain experienced by the patient population [79]. In line with this idea, previous studies using animal models demonstrated that physical stress-induced immobility and restraint may cause the upregulation of EGR1 and other immediate early genes in the CNS [80, 81]. Chronic EBV infection is often reported in patients with polymyalgia rheumatica with periodically disabling fatigue [82], and patients with primary fibromyalgia with progressive symptoms of fatigue [83]. According to a recent case study [84], EBV-infected CD8+ cytotoxic T cells were found to have infiltrated the skeletal muscle tissue of 19 years old male suffering from chronic and active EBV infection suggesting a direct role of EBV infection in the cytotoxicity of skeletal muscle tissue. In some patients, acute EBV infection also caused severe myocardial necrosis with marked lymphocytic infiltration [85] suggesting a direct role of EBV-infected CD8+T cells in acute cytotoxicity [86] of cardiac tissue [87]. Although it is not yet completely understood how EBV infection may be responsible for the development of long-term muscle fatigue, there exists clinical evidence for the development of other chronic illnesses [88] following acute EBV infection including multiple sclerosis (MS) [89, 90] and, to some extent, systemic lupus erythematosus (SLE) [91, 92]. Taken together, it is now becoming evident that EBV infection and its subsequent reactivation in humans can result in the potentiation of a chronic inflammatory response in peripheral muscle tissue, and the infiltration of infected peripheral lymphocytes into the CNS.

These events eventually lead to the presentation of the cardinal clinical symptoms of ME/CFS which include fatigue, muscle weakness, dysautonomia, and neurocognitive impairment. The potential relationship between chronic EBV infection and MS-like encephalopathy was further corroborated with a study by Jilek et al. [93], in which a patient with acute EBV infection was reported to display a severe myelin oligodendrocyte glycoprotein (MOG)-specific immune response accompanied with clinical signs of encephalopathy.

Collectively, muscle fatigue is a common clinical manifestation of EBV infection and reactivation and there exist multiple potential molecular pathways that may underlie clinical symptoms including the infiltration of peripheral EBV-infected CD4+ T cells followed by reactive microgliosis, oligodendroglial demyelination, the direct infiltration of CD8+T cells and the subsequent cytotoxic response that might cause the weakness in skeletal muscle tissues (Fig. 3).

Human immunodeficiency virus (HIV) infection and muscle weakness

Chronic HIV infection is often associated with severe progressive neuromuscular weakness resulting in a steady decline of muscle strength [94] and muscle mass [95], which can lead to chronic movement impairment [96–98] and debilitating long-term disability. As a molecular mechanism, mitochondrial abnormality [99] has been often cited in the muscle tissue of HIV patients. Studies have identified HIV RNA in mitochondria of mitochondria of muscle tissue collected from acute HIV-infected patients [100]. The HIV tat protein has been shown to bind and alter mitochondrial membrane potential inducing mitochondrial death [101] and is a noteworthy molecular mechanism that may underlie the clinical features of severe fatigue and a loss of muscle tissue in these patients. A specific interaction between the HIV viral protein R and the mitochondrial permeability transition pore complex (PTPC) has recently been demonstrated by Jacotot and colleagues [102]. In their work, they found that PTPC-dependent permeabilization of mitochondrial membrane activates apoptosis and cytotoxicity [103] in muscle tissue. Apart from a mitochondrial impairment, a chronic inflammatory response such as activation of inflammatory T cells, gliosis, and demyelination are also critical factors [104–107] for the progression of neuromuscular weakness in HIV patients (Fig. 4). HIV virions directly infect macrophages [108] and microglia [105] and upregulate the expressions of inflammatory cytokines such as IL-1β, IL6, and TNF-α [109];   and chemokines such as CCL2, CCL5, and CXCL12 [110, 111]. Expressions of other neurotoxic factors such as NO [112, 113] and ROS [114] are also stimulated through this pathway. These factors contribute to the apoptosis of oligodendrocytes, the primary myelinating cells in CNS. The study suggests that the severity of myelin damage and white matter abnormality is often positively correlated with microglial activation [115]. Oligodendrocytes provide critical trophic support to the neuronal cells by covering axons with myelin membranes, which is crucially important for maintaining cellular functions and electrical conduction [116]. Therefore, microglial activation [117] followed by oligodendroglial injury [118] indirectly triggers neuronal damage in HIV patients [119, 120].

In a direct mechanism, HIV surface protein gp120 has been shown to interact with neurons [121] via the CXCR4 receptor. Upon interaction, gp120 stimulates the activation of NF-κB [122] in the neuron. GP120-mediated activation of NF-κB is reported to produce ROS [123] and stimulates the formation of rod-shaped actin-cofilin conjugated proteinopathy inclusions [121] causing neurodegeneration. In the peripheral nervous system, Schwan cells also undergo apoptosis via a similar mechanism. The interaction between CXCR4 of Schwan cells and gp120 of HIV causes exocytosis of lysosome and release of ATP [124]. Gp120 also triggers the release of TNFα upon binding to CXCR4 on Schwan cells [125]. TNFα potentially stimulates TNFR1-mediated apoptosis in Schwan cells and peripheral neurons causing neuropathy.

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