Microbiome studies have to date almost exclusively focused on defining changes in intestinal bacterial population; recently however, the remit has broadened to assess the non-bacterial microorganisms of the intestine. Of particular interest are the viral components of the microbiota. Components of the virome, specifically bacteriophages, which make up 90% of the gut virome composition [
125] are primary drivers of bacterial diversity and influence community structure by both eliminating and introducing traits to their host species via the horizontal transfer of genes [
126,
127,
128]. So far, the impact of the intestinal virome in healthy and disease states has received very little attention, but in its unexplored functional role it is possible that the phages in the virome will have as yet unexplored, explored effects. These can be indirect effects resulting from changes to bacterial populations, or direct effects following stimulation of the immune system if they cross gut epithelial barriers and enter the host.
5.1. The Intestinal Virome
The virome has been shown to be more personalized and stable than bacterial intestinal communities [
129], with the profiling of viral communities in female monozygotic twins and their mothers finding limited intra-personal variation compared to high inter-personal viral variation [
130]. This variability has been attributed to both the presence of individual intestinal bacteria and the rapidly evolving nature of viral populations. The longitudinal sampling of a healthy male over a period of 2.5 years revealed that 80% of viral contigs, which are contiguous reads of sequenced DNA, persisted over that period. Variations over time were attributed to diversity-generating retroelements, a family of genetic elements that serve to diversify DNA sequences and CRISPR arrays, short segments of prokaryotic DNA within bacterial and archaeal genomes that can establish if a microbial population has been previously infected by phage [
130].
Virome sequencing has lagged behind bacterial microbiome studies; principally as a result of technical limitations [
131]. Recent viral metagenomic studies have displayed limited capacities to assign viral sequences to taxa, with current databases lacking appropriate depth to identify viruses, let alone link specific bacteriophages to individual bacteria or disease states. Shotgun metagenomics is a high throughput technique that allows direct analysis of random pieces of DNA sequences from the genomes of whole samples and initial reads have been used to assemble complete genomes of phages and viruses that infect humans [
132,
133]. However, metagenomic sequencing analyses has often ignored RNA viruses, whose role in the intestinal microbiome remain unclear and current isolation procedures which are primarily designed to isolate DNA viruses, may have overlooked the presence of RNA viruses in the intestinal virome [
134]. Prior attempts at characterizing the virome have only been able to assign 15% to 87% of virus-like particle sequences or contigs within sequence databases [
129,
130,
135]. This variability is a limitation of current taxonomic assignment criteria and we are only able to report on bacteriophages that are most closely related to taxa in current databases. This is thought to be due to rapid evolution of phages and their sequences, although it can also be due to the initial quality of sequence reads and the proportion that were trimmed prior to analysis. It is clear that each individual displays different viral sequence diversity, of which the full representation of the resident viruses can only be obtained with deeper sequencing approaches. Such techniques are required to enable the identification of regions of viral diversity that associate with the virulence of specific viruses or disease. This is a process that requires the compilation of complete reference databases for gene families in addition to reads that are long enough to be identifiable.
With increasing interest and expanding evidence of the role of phages in governing disease states and the potential use of phages in combatting disease [
136], interest has incorporated the role of viruses in normal human physiology, a prime area of research of which is the contribution of the intestinal microbiota in influencing the brain and behaviour as part of the gut-microbiota-brain axis.
5.2. The Gut-Microbiota-Brain Axis
A potential pathophysiological mechanism for ME/CFS development can be explained by the integrated microbiota-gut-brain-axis [
137], which describes the physiological links between the microbiota and the central nervous system (CNS), the autonomic and enteric nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. Increasing evidence indicates that the microbiota can, by as yet incompletely defined mechanisms and molecular mediators, communicate with the CNS via immune, neural and endocrine pathways, which are likely to have effects on cognitive function and behaviour [
138].
Microbial interactions with the enteric nervous system have been demonstrated extensively using germ-free mice, which allows the impact of colonising bacteria (coventionalisation) on mood and cognitive function to be tested [
139,
140]. The physiological mechanisms by which stress can alter the microbiota are thought to be due to its effects on intestinal physiology, altering gastrointestinal motility and secretions via reductions in the waves of electrical activity (migrating motor complexes) that sweep through the intestine in a regular cycle and trigger peristalsis, increasing intestinal permeability and reducing the regenerative capacity of the intestinal mucosa, all of which serve to alter the natural environment of intestinal microbiota. Different microbial niches within the intestinal environment initiate the release of stress hormones such as norepinephrine, which can influence inter-bacterial signalling, the growth of specific pathogens in addition to their ability to adhere to the intestinal mucosa [
141]. Sudo
et al. were the first to demonstrate differences in brain function between germ-free and specific pathogen free (SPF) mice, with germ-free mice displaying exaggerated hypothalamic-pituitary responses to mild restraint stress, through increased levels of adrenocorticotrophic hormone (ACTH) and corticosterone release compared to SPF mice [
142]. This was subsequently reversed upon conventionalisation in germ-free mice using faecal samples from the SPF mice. Sudo
et al. also demonstrated increased stress responses in germ-free mice colonised with enteropathogenic
E. coli, with subsequent reversal of stress-induced ACTH and corticosterone levels when germ-free mice were colonised with the human commensal
B. infantis; the effect of which was partially reversed when germ-free mice were colonized with intestinal microbes from SPF mice. Interestingly, Sudo
et al. displayed this reversibility in very young mice, indicating a potential stage in which neurones that regulate stress responses are sensitive to stimulation from the microbiota. Stress induced in early life has been demonstrated to lead to dysbiosis in germ-free mice and has been deemed to be a critical determinant of abnormal behaviours [
143]. Furthermore, Heijtz
et al. demonstrated the influence of the intestinal microbiota in influencing synaptic brain activity, with germ-free mice displaying increased motor activity and reduced anxiety in comparison with SPF mice with normal microbiota, indicating the influence of intestinal microbes in brain activity and psychiatric comorbidity [
140].
Further studies have demonstrated the impact of host microbiota in controlling the maturation and function of microglia in the CNS [
144]. Most significantly, Bercik
et al. conducted the first study to demonstrate the ability for behavioral traits to be transferred between mouse strains that displayed anxious and aggressive behaviors strains using fecal microbiota transplantation (FMT) [
145].
The study of associations between autistic spectrum disorder (ASD) and the intestinal microbiota has provided avenues for exploration in ME/CFS cohorts. Patients with ASD, like ME/CFS, have a high prevalence of gastrointestinal dysfunction [
146], which has also been linked to alterations in microbiota composition [
147,
148]. A study exploring the connection between the microbiota and brain function in ASD showed patients to have marked alterations in the microbiota, with specific alterations in various
Clostridium species [
149]. Hsiao and colleagues showed a gut-microbiota-brain connection in an adolescent mouse model of ASD (maternal immune activation, MIA), which exhibits intestinal barrier defects and microbiota alteration [
150]. Upon treatment of MIA offspring with
B. fragillis, intestinal permeability was corrected, microbial composition was altered and ASD-related deficits in communication and behaviour were reduced. MIA offspring also displayed an altered serum metabolomics profile, specifically key molecules in a tryptophan metabolism pathway, including increased levels of serotonin that were restored to normal upon treatment with
B. fragillis [
151]. This work suggests the role of probiotic bacteria and bacterial metabolites in preventing or causing neurodevelopment disorders respectively, suggesting an important role for the microbiota in the development of social behaviour in ASD. Of possible interest in ME/CFS research is the sleep-inducing substance derived from bacterial cell walls, Factor-S. Studies have suggested that intestinal bacteria are an important source of Factor-S, especially after Brown demonstrated normal sleep patterns to be disrupted after perturbation of the microbiota with oral antibiotics [
152].
Stress, or an organism’s ability to cope with environmental demands is well known to increase susceptibility to diseases that include gastrointestinal disorders [
153]. It is also well established that dietary and environmental stresses in turn, create different microbial niches affecting the localisation of the different microbiota populations which can particularly favour pathogenic bacterial species [
154]. This can lead to the release of stress hormones and cytokines such as IL-6 [
142,
155]. Bacteria too, can respond to and synthesize hormones and neurotransmitters with for example,
Lactobacillus and
Bifidobacteria species producing acetylcholine and/or gamma-amino butyrate (GABA);
Escherichia species producing norepinephrine, serotonin and dopamine; and serotonin produced by
Streptococcus and
Enterococcus species [
156]; all of which can contribute to the host’s regulation of mood, cognition, pain and anxiety.
There is clearly still much to be learned about the molecular connections and pathways involved in gut-microbiota-brain signalling and how perturbations of this communication pathway might give rise to disease states. The effect of the intestinal microbiota on the CNS and cognitive function represents exciting and potentially fruitful opportunities for future investigation and explaining the manifestation of the core symptoms of ME/CFS. Also, the advent of ‘biotics’ (probiotics and/or prebiotics) and FMT provides opportunities to manipulate the microbiota to improve or restore gut health and impact on how it influences the function of other organ systems, including the immune system and the CNS.