Profound Implications of the Virome for Human Health and Autoimmunity
Revolutionary research illuminates that a new frontier of personalized medicine lies in the virome. Rather than harbingers of disease, viruses are intrinsic to immune modulation and to disease susceptibility.
The microbiota, composed of the thirty-eight trillion bacteria that inhabit the corporeal body, have garnered unprecedented publicity in recent years (1). However, unbeknownst to most of the public, mammals are also populated by a staggering host of chronic viruses called the virome, which elicit significant effects upon disease susceptibility and physiological homeostasis (2).
Ranging from innocuous to potentially lethal, an enormous array of endogenous viral elements, RNA and DNA viruses that infect host cells, and viruses that infect the microbiota are present in all adult humans (14). The quantity of viruses present in fecal matter, in fact, rivals that of bacteria, at upwards of one billion viral particles per gram (3). Many viruses elude annotation, representing novel viruses that are yet to be classified (4, 5).
Latent or Stealth Infections Enable Viruses to Evade Immune Detection
Infection with multiple herpes viruses, for instance, is an inextricable part of the human condition, to which more than 90% of humans are subject (6). Because the ancestral herpes viruses infect birds, reptiles, and mammals, pioneering researcher and world-renowned expert in immunology, virology, and infectious disease, Dr. Herbert W. “Skip” Virgin IV states, “The herpes viruses have been studying you far longer than you have been studying the herpes viruses” (7). In fact, herpes viruses co-evolved down species-specific lines with the speciation of mammals in evolutionary history (7).
Following immune clearance of the primary infection, the herpes virus adopts a dormant state called latency by expressing an alternative gene set which inhibits its central lytic functions, one of the two cycles of viral reproduction (8). Latency enables the virus to hide from the immune system and permanently persist within the host (8). For example, after acute infection, herpes simplex virus type 1 (HSV-1) replicates in epithelial cells and migrates to the sensory neurons via nerve termini where it enters a latent phase in its stronghold, the trigeminal ganglion in the dura mater (8, 9).
Latency, which was formerly considered to be a parasitic state, renders the host vulnerable to subsequent reactivation of the virus and secondary infections at peripheral sites (8). Recurrent episodes of infection, ranging from cold sores to eyesight-threatening ocular herpes and neurological herpes encephalitis can occur with successive viral re-activations (9). As articulated by Aranda and Epstein (2015), “Latency is an adaptive phenotype that allows the virus to escape immune host responses and to reactivate and disseminate to other hosts upon recognizing danger signals such as stress, neurologic trauma or growth factor deprivation” (8, p. 506).
The Virome Protects Against Bacterial Infection
In a paradigm-shifting revision, however, researchers discovered that latency may confer health benefits for the host. Barton and colleagues (2007) found that mice that harbored latent infections with murine gammaherpesvirus 68 or murine cytomegalovirus, genetic analogs to human pathogens Epstein-Barr virus (the causative agent behind mononucleosis, or the “kissing disease”) and human cytomegalovirus (CMV), respectively, were resistant to bacterial infection by Listeria monocytogenes and Yersinia pestis (10).
The mechanism whereby this occurred was by virally-stimulated up-regulation of the antiviral cytokine interferon-gamma (IFNγ) (10). In turn, IFNγ created systemic activation of macrophages, a cell subset which are vital to the non-specific, innate immune defenses which are first deployed on the scene of pathogen invasion and can curtail bacterial infectivity (10).
Infection with a chronic virus effectively “upregulates the basal activation state of innate immunity against subsequent infections” and “may also sculpt the immune response to self and environmental antigens through establishment of a polarized cytokine environment” (10, p. 326).
Therefore, rather than being completely pathogenic, “our data suggest that latency is a symbiotic relationship with immune benefits for the host” (10).
The Virome Alters Disease Susceptibility
In genetically susceptible individuals, viruses can modify risk for chronic disease. For instance, lymphocytic choriomeningitis virus (LCMV) can inhibit development of diabetes in rodent models, whereas it exacerbates glomerulonephritis, or acute inflammation of the kidney, in certain inbred populations (11). In those with abnormalities in genes related to viral recognition, including toll-like receptor 7 (TLR7) and TLR9, early life infection with severe rhinovirus (the common cold) is strongly implicated in development of asthma (12, 13).
Epstein-Barr virus (EBV) levels are enriched in autoimmune patients with rheumatoid arthritis (RA), Sjogren’s syndrome, systemic lupus erythematous (SLE), and multiple sclerosis (MS) (14). Researchers speculate that chronic EBV infection could incite autoimmune disorders through mechanisms including molecular mimicry (the immune response becomes misdirected against self) or the bystander effect (self-tissues become caught in the cross-fire) (15, 16).
Another virus which alters disease risk is norovirus, a virus which is culpable for the vast majority of epidemic non-bacterial episodes of gastroenteritis (stomach flu) in humans (17). For instance, in mice harboring a mutation in the autophagy gene Atg16L1, which enhances predisposition to Crohn’s disease, intestinal pathology was induced when murine norovirus infection was present (11). When mice with the Atg16L1 mutation and murine norovirus were administered the toxic substance dextran sodium sulfate (DSS), which induces inflammatory bowel disease, there was an increased amount of DSS-induced colitis as well as the presence of DSS-induced villus atrophy signifying enhanced intestinal damage in a manner resembling Crohn’s disease (11).
In concert with the susceptibility gene, the virus induced aberrations in granule packaging in ileal Paneth cells, a specialized intestinal epithelial cell that secretes granules containing antimicrobial peptides and lysozyme, contents which change the intestinal environment (11, 18). These same Paneth cell abnormalities were observed in humans with the Atg16L1 mutation, which means that presumably norovirus could trigger Crohn’s disease expression in humans with this genetic propensity as well.
In addition, the combination of the virus plus the gene mutation led to a distinct profile of gene transcription. The authors conclude that the “virus-plus- susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease” (11, p. 1135). Stated differently, viruses can trigger disease onset in genetically vulnerable hosts.
The Virome Changes Genetic Expression and Autoimmune Risk
In the aforementioned study, the presence of the murine norovirus led to substantial changes in gene expression in the Atg16L1-mutant animals compared to the wild-type (normal) animals (11). For instance, there were complete inversions in the levels of expression for genes regulating carbohydrate and amino acid metabolism, intracellular protein traffic, and protein targeting and localization, indicating that genetic vulnerabilities may determine the way that viral infections influence our transcriptional identity (11).
These alterations in gene expression may elicit significant effects on the immunophenotype of the host. The immunophenotype is the basal level of activation of the immune system upon challenge with antigens, or immunogenic material against which an immune response is directed (19). Thus, changes in gene expression due to chronic viral infection may influence the way the immune system responds to future pathogenic invaders.
Differential expression of genes in response to viral infection may also influence susceptibility to and progression of chronic disease pathogenesis (19). Latent infection with gammaherpesvirus 68 in mice has been shown to produce differential expression of genes in the spleen, brain, and liver, leading to marked changes in the transcriptional status of organs of the host (19). Most modifications in gene expression occurred to immune-related genes, and in particular, it was demonstrated that latent viruses regulated expression of genes that conferred risk for autoimmune disorders including celiac disease, Crohn’s disease, and multiple sclerosis (7, 19).
Viral Infection Complements Immunodeficiency
Mutations in the Hoil-1 gene produce a disorder of both immunodeficiency and chronic inflammation, which renders people with risk alleles extremely susceptible to bacterial infections (7). In order to examine the implications of this mutation, MacDuff et al. (2015) studied mice with equivalent mutations, which died when infected by certain bacteria and parasites including Listeria monocytogenes, Toxoplasma gondii, and Citrobacter rodentium due to impaired production of pro-inflammatory cytokines that are required for resistance to these pathogens (20).
However, researchers state that latent murine herpesvirus 68 infection “rescued HOIL-1 deficient mice from lethality during Listeria infection and induced high levels of the protective cytokine, interferon-gamma (IFNγ)” (20, p. 3). IFNγ is a cytokine which the body produces upon viral exposure, which promotes neutralization of viruses with antibodies and killing of virally-infected cells by immune cells called cytotoxic T lymphocytes and natural killer (NK) cells (21).
Therefore, this virally-induced IFNγ production leads to a form of immunomodulation which can protect the host from bacterial infection.
Likewise, in mice with genetic mutations in immune-related genes encoding proteins for interleukin-6, an inflammatory intercellular signaling molecule, and caspases-1 and caspase-11, enzymes which function in programmed cell death, chronic herpesvirus infection dramatically protected these immunodeficient mice from Listeria monocytogenes infection (20). In other words, “chronic herpesvirus infection stimulates the immune system, and so allows it to compensate for the lack of cytokine production associated with various immunodeficiencies” (20, p. 2).
Differences in viral elements may account for why people with the same genetic predilection have vastly different clinical presentations. This is another example of how genes should not be equated with destiny, as expression of genetic mutations is influenced by environmental triggers, including viral elements. Thus, it is possible that infection with latent viruses, which develop a symbiotic relationship with the host, may be a future therapeutic strategy for favorably changing the clinical presentations of particular immunodeficiency-related genetic disorders.
Other Commensal Microbes Influence Viral Pathology
Researcher Herbert W. “Skip” Virgin IV and his colleagues developed the hypothesis that viral immunity and viral pathogenesis would be governed by ‘transkingdom metagenomic interactions’ (7). In other words, the interplay between all genetic sequences in or on the host, from either human genetic material or commensal microorganisms residing within the human body, would dictate the course of a viral infection.
Helminths, for example, which are parasitic worms that infect mammals, can promote viral replication by both inhibiting the antiviral effects of the cytokine interferon-γ (IFNγ) and by inducing production of the cytokine interleukin-4 (IL-4), both of which culminate in reactivation of the murine γ-herpesvirus infection (6). The helminth likewise activates the transcription factor Stat6, which elicits downstream changes that induce viruses to move from a latency phase to active infection (6). In this instance, the virus senses and responds to the immunological milieu of the host, which is influenced by the helminth.
Norovirus, the most prevalent cause of acute infectious gastroenteritis, is another example of a virus that can latently infect the human intestine (22). In fact, norovirus is present in 21% of people with immune deficiencies and is asymptomatically shed in the feces of 3-17% of humans, which can lead to the chronic norovirus epidemics (23).
Norovirus represents another example of a transkingdom interaction, as the bacterial microbiota in the gut can foster viral persistence of this viral subtype. This phenomenon was demonstrated by an experiment where antibiotic administration, which presumably decimated the microbiota, prevented persistent murine norovirus (MNoV) infection (24). However, restoration of the microbiota with a fecal transplant reversed the inhibition of persistent intestinal norovirus infection and led to viral reactivation in the lymph nodes, ileum, and colon as well as viral shedding in the stool (24).
The enteric microbiota, at a mechanistic level, can perpetuate the infectivity of viruses by “the direct facilitation of viral infection, including bacterial stabilization of viral particles and the facilitation of viral attachment to host target cells; and the indirect skewing of the antiviral immune response in a manner that promotes viral infection” (25, p. 197).
The effect of the microbiota on the viral infection, however, is mediated by the host immune system, and certain immune-related genes are required for the antibiotic-mediated suppression of the viral response. This is illustrated by data showing that with mice who were genetically manipulated to be deficient in certain genes, such as interferon-gamma, the antibiotics had no effect on decreasing viral persistence (24). Interferon-lambda, or type III interferon, a cytokine which is used to treat hepatitis C in humans, can both prevent establishment of persistent infection with intestinal norovirus and can cure persistent viral infection (26).
These examples represent evolutionarily conserved interactions between organisms of divergent kingdoms, such as bacteria and parasites, along with host molecules such as interferon, which influence the infectivity of chronic viruses.
Virome Alterations are Related to Autoimmune and Inflammatory Diseases
In a multi-center clinical study, researchers analyzed the viromes of cohorts with inflammatory bowel disease (IBD) compared to household controls (27). It is well-established that patients with Crohn’s disease and ulcerative colitis have diminished species richness and phylogenetic diversity in their gut microflora compared to healthy cohorts (27). However, when their viromes were sequenced, increased numbers of bacteriophages, or viruses that infect and multiply within bacteria, were found in the IBD populations (27).
In particular, signature bacteriophages were found to be IBD-subtype specific, with different viruses appearing in ulcerative colitis versus Crohn’s disease (27). In addition, a significant expansion of Caudovirales bacteriophages was observed in both ulcerative colitis and Crohn’s disease (27). Rather than virome changes occurring secondarily to microbiome changes, researchers speculate that a predator-prey relationship exists between the virome and microbiome (7). Within this paradigm, bacteriophage introduction changes the microbiome, shifting to a new equilibrium state of enhanced disease vulnerability (7). The researchers conclude that, “These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis…the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and [we] speculate that the enteric virome may play a role in other diseases” (27, p. 447).
Not only will this body of literature have implications for other disorders in which microbial dysbiosis, or bacterial imbalance, plays a role, but it also paves the way for the development of condition-specific probiotics and even provirotics, or viruses that elicit beneficial effects on the host. It also raises questions about the utility of probiotics already on the market, which may fall victim to infection with bacteriophages when ingested by the host, which could theoretically exacerbate some conditions.
Future Implications of the Virome
In summary, when examining the relationship between the genotype, or the genetic constitution of an organism, and the phenotype, or the observable characteristics resulting from the interaction between genes and environment, the virome must be taken into account (7). In the meta-genome, there are layers of interactions between bacteria, parasites, viruses, and host physiology, which can influence disease risk (7).
Viruses are essential to the convoluted and dynamic network of microorganisms that reside within the body (14). Early life infection with certain viruses has even been demonstrated to change the expression of genes related to vaccine responses in both mice and humans (7), which may account for why some individuals are more susceptible to vaccine injury than others.
Further, vaccinations may deprive the body of favorable immune-modulating effects of some viral infections. Contrary to the dualistic view of Western medicine, most viruses are neither innately good nor bad, as “one virus could have multiple adverse and beneficial immunomodulatory effects on the host that are dependent on the anatomical location, host genotype, and the presence of other infectious agents and commensal microbes” (14). This confirms what Louis Pasteur, the father of immunization and pasteurization himself, admitted on his death bed: that it is the biochemical context and physiological milieu that matters, rather than the infecting pathogen (Tracey, 2017).
This research represents a fundamental revisioning of what it means to be human, and an expansion upon Stanford’s Dr. Justin Sonnenberg’s hypothesis that humans may merely be elaborate vessels designed for the propagation of bacterial colonies. Human physiology and genetic expression is influenced by an amalgamation of organisms transcending phylogenetic designations. Because this field is still in its infancy, the virome represents uncharted terrain and an unexplored opportunity to delineate how viruses favorably and unfavorably modulate human biology.
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