This review reports the latest evidence on human gut “virome” composition and its function, possible future therapeutic applications in human health in the context of the gut microbiota, and attempts to clarify the role of the gut “virome” in the larger microbial ecosystem.

According to previous evidence on pathogenic viruses, the human gut harbours plant-derived viruses, giant viruses and, only recently, abundant bacteriophages. New metagenomic methods have allowed to reconstitute entire viral genomes from the genetic material spread in the human gut, opening new perspectives on the understanding of the gut virome composition, the importance of gut microbiome, and potential clinical applications.

Human gut microbiota is a complex ecosystem with several functions integrated in the host organism (metabolic, immune, nutrients absorption, etc.). Human microbiota is composed by bacteria, yeasts, fungi and, last but not least, viruses, whose composition has not been completely described.

Based on the new findings obtained through metagenomic methods, this review will focus on the composition of the human gut virome, its role in the gut microbiota ecosystem, and possible future clinical applications.

However, because gut viruses not amenable to culture with common microbiological techniques, the development of non-culture based metagenomic methods have allowed to reconstitute viral particles from single genetic sequences from almost every environment. This has moved our idea of gut viruses from a mere source of pathogens to a physiological component of the healthy human microbiota [].

This last subset of findings has been mostly unexpected because of the common representation of the gut virome as a source of pathogens. Enteroviruses, Norwalk, Rotaviruses are well known in daily clinical practice and are known to be responsible for common infectious gastroenteritis [].

Thus, the microbiologic environment has attracted attention and resources from the clinical and economic sectors of our society to achieve a better understanding of the microbiota ecosystem. These efforts have led to the discovery of other components of gut microbiota such as yeasts, fungi, archaea, and last but not least, viruses [].

The Western lifestyle is associated with serious metabolic sequelae (diabetes, obesity, metabolic syndrome, increased cardiovascular risk, etc.) []; this has driven clinical and basic researchers’ attention to the possible modulation of gut microflora through diet, antibiotics, and pre-/probiotics with encouraging results, however awaiting wider population-based studies [].

Before the surge of interest into neglected components of the “gut microbiota” (including fungi and viruses), studies on the “gut bacterial microflora” and its widespread and well known bacterial species have collected evidence on the microbiota's role in metabolic, gastrointestinal, immune diseases and, lately, in cancer development [].

Knowledge on the composition of the gut virome has evolved from a niche of the gut microbiome populated by pathogens only (e.g. Norwalk, Rotavirus, Enterovirus, etc.), responsible for gastroenteritis by direct damage of enterocytes or through alteration of ion and water secretion in the colon, to an enlarged list of undetectable giant viruses (derived mainly from protozoa and parasites), and more recently to plant-derived viruses and bacteriophages, thanks to new metagenomic methods [].

However, some emerging microbiological data on yeast composition and functions have clarified their subsequent clinical use in the modulation of gut microflora. In fact, Saccharomyces boulardii is currently used with significant efficacy over placebo in the treatment of post-infectious and post-antibiotic diarrhoea [].

Little is still known about commensal fungi, archaea and protozoa [].

Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health.

Indeed, the human microbiota also contains other more neglected components such as archaea, viruses, fungi, yeasts and other Eukarya (such as Blastocystis and Amoebozoa) [].

Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health.

Bacteria reach more than 1 kg of weight and account for more than 1100 species; Bacteroidetes and Firmicutes are the predominant phyla in adults, followed by Actinobacteri and Proteobacteria [].

More recently intestinal bacteria have been implicated in the pathophysiology of psychiatric diseases such autism []. In fact, gut bacteria seem to interact with the central nervous system (CNS) via the enteric nervous system through the endocannabinoid system. Thus gut microbiota can affect the neuro-psychiatric state of the host and, conversely, the CNS is able to affect its composition through food intake regulation [].

Starting from these observations, the possible functions of gut microbiota were quickly related to other organs/apparata. The previous association between spontaneous bacterial peritonitis and small bowel bacterial overgrowth in liver cirrhosis [] has led to the understanding of the microbial molecular patterns triggering inflammation and fibrosis in liver diseases such as non-alcoholic liver disease (NAFLD) and its complications, i.e., non-alcoholic steatohepatitis (NASH), liver cirrhosis and hepatocellular carcinoma (HCC) []. Moreover, as in a vicious cycle, the role of microbial pathogen molecular patterns (PAMPs, e.g. gram-negative polysaccharide, LPS) in NAFLD pathophysiology has been linked to those of MetS, which is frequently concomitant [].

Specifically starting from the observations of an obese/lean gut microbiota associated with overweight or lean status it became clear how microbiota manipulation by diet was possible and how microbiota could be responsible not only for overweight but also for the chronic inflammatory state typical of the metabolic syndrome (MetS) []. However diet and the gut microbiota's role in obesity pathogenesis is not simply causative as was initially expected. In fact, a recent observation by Ridaura et al. has showed how co-housing mice with an obese twin's microbiota with mice containing the lean co-twin's microbiota prevented the development of increased body mass and obesity-associated metabolic phenotypes (greater polysaccharides metabolism and proteins degradation) in obese cage mates. More interestingly, an obesogenic diet (high in saturated fats and low in fruits and vegetables) counteracted the protective effect of the lean gut microbiota observed during lean and obese mice co-housing []. The role of diet in gut microbiota modulation is strengthened by the recent metagenome-wide association study by Qin et al. in type 2 diabetic patients, with a mainly diet-associated insulin resistance status; the Authors showed that these patients have a peculiar decrease in some butyrate-producing bacteria, an increase in various opportunistic pathogens and an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance [].

The main components of gut microbiota are bacteria, fungi, yeasts, archaea and viruses []. While the Human Gut Microbiome project has shed new light on the entire human intestinal bacterial composition, the last three decades of microbiological/clinical research have helped to understand how food, pre-/probiotics, antibiotics can modulate the intestinal bacteria quali-/quantitative pattern resulting in different microbial-host functions [].

Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health.

Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health.

After birth the human intestine is progressively colonized by several microbial strains that fluctuate and change during our lifespan according to anatomical, dietary and nutritional status changes (e.g. obese, anorexic, lean nutritional status), environmental (e.g. climate, familial composition, life-style, working place, etc.), pathological (gastro-intestinal and systemic infections), and pharmacological factors (e.g. use of antibiotics, prokinetics, laxatives, probiotics) [].

Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health.

3. Human gut virome composition

3 Reyes A.

Semenkovich N.P.

Whiteson K.

et al. Going viral: next-generation sequencing applied to phage populations in the human gut. 13 Lozupone C.A.

Stombaugh J.I.

Gordon J.I.

et al. Diversity, stability and resilience of the human gut microbiota. As mentioned above the concept of the existence of a “gut virome” is, paradoxically, very recent [] although the presence of viruses as pathogenic organisms in human intestine has been known and documented for more than a century [].

13 Lozupone C.A.

Stombaugh J.I.

Gordon J.I.

et al. Diversity, stability and resilience of the human gut microbiota. 15 Lagier J.C.

Million M.

Hugon P.

et al. Human gut microbiota: repertoire and variations. 15 Lagier J.C.

Million M.

Hugon P.

et al. Human gut microbiota: repertoire and variations. 16 Beatty J.K.

Bhargava A.

Buret A.G. Post-infectious irritable bowel syndrome: mechanistic insights into chronic disturbances following enteric infection. Table 1 Known virotypes according to culturomics and metagenomics. Virus type Genome type Environment Associated disease Eukaryotic virus Rotavirus, Astrovirus, Calicivirus, Norovirus, Hepatitis E virus, Coronavirus and Torovirus, Adenovirus (serotypes 40 and 41) All RNA except Adenovirus (DNA) Human small bowel and colon Gastroenteritis (small bowel epithelium and the absorptive villi disruption, with consequent malabsorption of water and an electrolyte imbalance) (all the mentioned eukaryotic viruses) Adenoviridae, Picornaviridae and Reoviridae (genus enterovirus) RNA Human intestine Unknown (all the mentioned viruses) Plant derived virus Pepper mild mottle virus (PMMV), oat blue dwarf virus, Grapevine asteroid mosaic-associated virus, Maize chlorotic mottle virus, Oat chlorotic stunt virus, Panicum mosaic virus, Tobacco mosaic virus RNA Plants and human faeces Pathogenic for plants

Non pathogenic for humans (all the mentioned plant derived viruses) Giant virus (>300 kb) Mimiviridae, Mamaviridae, Marseilleviridae, Poxviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Asfaviridae DNA Human faecal protists, amoebae in lake, river and seawater Pneumonitis, Children diarrhoea (Mimiviridae only) Prophages Myoviridae, Siphoviridae, Podoviridae, Tectiviridae, Leviviridae, Inoviridae dsDNA Human faeces specimens Unknown (all the mentioned prophages) Virus (<145 kb) Microviridae family (Microvirus, Gokushovirinae, Alpavirinae, Pichovirinae) ssDNA Seawater, human gut bacteria Unknown (all the mentioned Microviridae viruses) dsDNA: double stranded DNA; ssDNA: single stranded DNA. Thus, the description of the gut virome composition can begin with these pathogenic viruses ( Table 1 ), whose viral particles were discovered by microbiologists mainly because they could be cultured []. Norwalk, Rotavirus and Enterovirus are the well-known agents of gastroenteritis in man []. The reason we consider linking these pathogens with the gut virome is that the infection of the gut is responsible for enterocyte and bacterial microflora changes. These can affect the host not only in the acute phase of the infection with gastrointestinal complaints such as nausea, vomiting, diarrhoea and weight loss, but also in the long-term with persistence of symptoms and the possible eliciting of functional gastrointestinal disorders such as functional dyspepsia and post-infectious irritable bowel syndrome [] ( Table 1 ).

17 Li J.

Jia H.

Cai X.

et al. An integrated catalog of reference genes in the human gut microbiome. A recent paper by Li et al. offers a clear and advanced example of how metagenomics has changed the professional perspectives of microbiologists and translational researchers in the study of gut microbiome. Using first national, then intercontinental catalogues of reference genes of the human gut microbiome, in the last two decades, researchers used sequence reads and relative gene content to map the profile of the microbial species in biological samples. This is the fundamental principle of metagenomics which, instead of deep genome sequencing after sample culture, collects different sequences from various genetic materials (e.g. from human gut) and is able to use them to mark families, taxa and genera for both microbiome and virome. The knowledge of gene abundance levels can be also associated to different diseases in the attempt to setup a specific genetic marker. Another advantage of metagenomic analysis arises from the possibility to extract genetic material directly from faecal samples without any changes and/or contamination, which may arise during culture [].

18 Holtz L.R.

Cao S.

Zhao G.

et al. Geographic variation in the eukaryotic virome of human diarrhea. Very recently, thanks to these metagenomic methods, novel enteric eukaryotic viruses were found to be responsible for acute diarrhoea in children's small bowel enteropathy in developing areas of Australia. Interestingly these new data, confirmed by quantitative real time polymerase chain reaction, have shown that diarrhoea in children contains a higher abundance of viruses, many of them not previously known to be pathogenic, such as the Adenoviridae, Picornaviridae, Reoviridae families. Within the Picornaviridae family, Enterovirus were the most represented [].

19 Colsona P.

Fancelloa L.

Gimeneza G.

et al. Evidence of the megavirome in humans. In the past decades microbiological investigations have further discovered viruses infecting human intestinal parasites such as amebae (e.g. Mimiviridae, Mamaviridae, Marseilleviridae) from cooling towers, rivers, lakes, and seawater. These viruses are defined “giant” because of their dimensions. They are DNA viruses and their existence has been frequently doubted because they are undetectable by small-pore filtration ( Table 1 ). Some of the Mimiviruses have been associated with pneumonitis and diarrhoea in humans although evidence is controversial [].

20 Zhang T.

Breitbart M.

Lee W.H.

et al. RNA viral community in human feces: prevalence of plant pathogenic viruses. More recently plant viruses such as pepper mild mottle virus (PMMV), oat blue dwarf virus, grapevine asteroid mosaic-associated virus, maize chlorotic mottle virus, oat chlorotic stunt virus, panicum mosaic virus, and tobacco mosaic virus have been described [] ( Table 1 ).

20 Zhang T.

Breitbart M.

Lee W.H.

et al. RNA viral community in human feces: prevalence of plant pathogenic viruses. Their biological importance depends on their concomitant presence in plants as pathogens and as “commensals” in human faeces, explaining how food intake has conditioned and continues to condition human gut virome composition and enterocyte life-cycle. These data also suggest that these small plant-derived viruses can affect the intestinal bacterial quali-/quantitative composition and its functioning with consequences for the human host. There is no data on the presence of these viruses in patients with diarrhoea [].

3 Reyes A.

Semenkovich N.P.

Whiteson K.

et al. Going viral: next-generation sequencing applied to phage populations in the human gut. Finally yet importantly, intestinal bacteriophages have been discovered as the main component of the gut virome, accounting for about 90% of its composition [].

3 Reyes A.

Semenkovich N.P.

Whiteson K.

et al. Going viral: next-generation sequencing applied to phage populations in the human gut. 21 Mokili J.L.

Rohwer F.

Dutilh B.E. Metagenomics and future perspectives in virus discovery. Bacteriophages were one of the first microbiological entities characterized in the literature. Our knowledge about their life cycle is quite extensive; bacteriophages can be quite literally defined as “viruses of bacteria”. Bacteriophages are commonly known as bacterial “parasites”, who inject their genome in their host, integrating with its genetic material (prophage state) and inducing other phage particle synthesis with bacterial cell lysis (lytic state) [].

22 Roux S.

Krupovic M.

Poulet A.

et al. Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads. 23 Wommack K.E.

Colwell R.R. Virioplankton: viruses in aquatic ecosystems. 3 Reyes A.

Semenkovich N.P.

Whiteson K.

et al. Going viral: next-generation sequencing applied to phage populations in the human gut. 22 Roux S.

Krupovic M.

Poulet A.

et al. Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads. 24 Wegley L.

Edwards R.

Rodriguez-Brito B.

et al. Metagenomic analysis of the microbial community associated with the coral porites astreoides. Bacteriophages are viruses with double-stranded DNA (dsDNA) []. Single-stranded DNA (ssDNA) bacteriophages are mainly found among the Microviridae family and were initially considered secondary players in the environmental viral community because of their modest genome size [].

22 Roux S.

Krupovic M.

Poulet A.

et al. Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads. 24 Wegley L.

Edwards R.

Rodriguez-Brito B.

et al. Metagenomic analysis of the microbial community associated with the coral porites astreoides. 22 Roux S.

Krupovic M.

Poulet A.

et al. Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads. Microviridae are small icosahedral viruses with circular single-stranded DNA genomes [] and their members are divided into microviruses (genus Microvirus) and gokushoviruses (subfamily Gokushovirinae) []; more recently, a new sub-family, the Alpavirinae, was described ( Table 1 ). These viruses have been retrieved in bacteria belonging to two genera of the phylum Bacteroidetes: Prevotella and Bacteroides, with possible implications for human microbiota metabolism [].