Oral-gut-brain axis virome / microbiome for periodontitis, irritable bowel disease and alzheimer's disease diagnostics

Abstract

The disclosure provides an evaluation of the virome in health and disease of the oral-gut-brain axis, in particular, periodontitis, irritable bowel disease (IBD) and Alzheimer's Disease (AD). For Alzheimer's disease, a striking feature was found via metagenomics of brain autopsy specimens - the presence of parvoviridae (comprised of erythoviruses and erythroparvoviruses) in the brains of individuals diagnosed with Alzheimer's Disease (AD) but not in brains from age-matched healthy individuals without a medical diagnosis of AD. For IBD, For Periodontitis and IBD, an analysis revealed the top 20 most abundant viral and bacterial species in saliva samples from individuals with periodontal disease or with a healthy periodontium and the top viral and bacterial species in stool samples from individuals with irritable bowel syndrome or with gastrointestinal health. Building upon these discoveries, a number of diagnostic methods and materials for AD and IBD are described herein.

Description

[0001] ORAL-GUT-BRAIN AXIS VIROME / MICROBIOME FOR PERIODONTITIS, IRRITABLE BOWEL DISEASE AND ALZHEIMER’S DISEASE DIAGNOSTICS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application No.63 / 663,849, filed June 25, 2024, entitled “ ORAL-GUT-BRAIN AXIS VIROME / MICROBIOME FOR PERIODONTITIS, IRRITABLE BOWEL DISEASE AND ALZHEIMER’S DISEASE DIAGNOSTICS”, which application is incorporated by reference herein. TECHNICAL FIELD The invention relates to methods and materials for observing pathologies such as Alzheimer’s and irritable bowel disease. BACKGROUND OF THE INVENTION The NIH Human Microbiome Project (HMP) has made groundbreaking contributions to further our understanding of the human bacteriome, opening new avenues of research in the biomedical and behavioral sciences and advancing healthcare delivery. In contrast, although viruses in our body outnumber bacterial cells, the human virome remains largely unexplored. The critical role of viruses in human health has long been recognized, underscored in recent years by the COVID- 19 pandemic and worldwide threats from emerging pathogenic viruses. However, our knowledge of zoonotic pathogens within the human body and, more broadly, a foundational understanding of what constitutes a healthy virome, is lacking. Addressing these knowledge gaps by performing a detailed mapping of the human virome in a diverse array of human cohorts across the lifespan has the potential to redefine our relationship with the viral world and improve our capacity to detect, diagnose, and discover viruses that may pose threats to human populations (1,2). Toward this goal, the Human Virome Program (HVP) is an ambitious undertaking that will reveal many critical aspects of viral communities in the human body across the life and health span. The HVP, together with groups focused on similar challenges, such as the Global Virome Project and other NIH-funded initiatives, will move the field forward for advancement of scientific knowledge and betterment of human health. In response to this initiative, we performed a pilot exploration to define the human virome of the oral–gut–brain axis in diverse healthy and diseased cohorts of different age groups. Viruses are the most abundant and diverse biological entities on our planet, with an estimated 1031particles, most of which are presumed to be bacteriophages (3). This conclusion is consistent with prior virome metagenomic sequencing studies that found most viral sequences do not align with information in databases and thus represent viral “dark matter” (4). Phages engage with their bacterial hosts via four main interactions: lytic growth (infecting and lysing host), lysogenic growth (stably persisting in host via either integration or episomal maintenance), pseudolysogeny (loose interaction between phage and host), and budding (infecting and preserving host cells while releasing new phage). Predominant taxa include tailed phages in the Caudoviricetes class (including phages previously in the order Caudovirales) (5,6) and Microviridae, icosahedral, non-tailed phages. However, within these groups, phages are highly diverse, and they play a myriad of crucial roles in shaping bacterial communities, including those in higher organisms, that we are only beginning to understand. Like phages, eukaryotic viruses can contain DNA or RNA genomes that are either double- or single-stranded. These genomes can range in size from a few kilobases (kb) to hundreds of kb or more and be encapsulated in either a protein capsid shell or one that is surrounded by lipid membranes. Morphologically, similar to phages, eukaryotic viruses adopt a number of different forms, including spherical (e.g., icosahedral), filamentous, bullet-shaped, pleomorphic, and tailed (3). Unlike phages, however, many of these viruses can infect human cells, causing acute infections and / or establishing long-term latency. Other viruses, such as Anelloviridae, can be considered commensal by engaging in benign colonization not associated with disease states. Prior studies of the human virome have revealed a complex and heterogeneous architecture, comprising approximately 1013particles per individual (3,7). Several key findings have emerged from initial attempts to define the virome in health and disease, including the existence of a spectrum of ecological niches that differ across the lifespan, complex interkingdom interactions between viruses and bacteria and with the host immune system, and the potential importance of commensal viruses (3,7–18). However, unlike the human microbiome, which has been well characterized through prior initiatives, such as the HMP, the human virome is largely unexplored, particularly within the oral cavity and brain. Available studies suggest that, in health, the virome is relatively stable over time. For example, 80–90% of the gut virome was found to persist over 1–2.5 years in small cohorts (n=1–10) (7,19). The oral virome showed similar stability in 5–9 individuals over 42–60 days (20,21). Even human blood from healthy individuals contains a stable community of commensal viruses (e.g., Anelloviruses) (22,23). However, higher levels of inter-individual and inter-group variability appear in the human virome across disease conditions. For example, breast milk from women infected with human immunodeficiency virus (HIV) contains a core bacterial microbiome / virome dominated by human cytomegalovirus species that is not present in milk from HIV-negative women or altered by immunosuppression (24). A unique bacterial profile associated with respiratory syncytial virus (RSV) infection was detected in pediatric cohorts, indicating the potential for interkingdom interactions (25). Similarly, data from humans with chronic oral diseases (e.g., periodontal disease) and animal models of the same condition point to a unique oral disease– associated virome (4,26). Beyond disease, other factors also impact the human virome, including type of birth delivery (i.e., vaginal vs. caesarian section), oral hygiene / health, diet (e.g., breastfeeding), genetics (as assessed by twin and mutation studies), geographic location (based on diverse cohort studies in China, Australia, Africa, and US), age and gender (3,4,27–29). In particular, several studies noted lifespan changes in the healthy human virome. It is generally thought that newborns are rapidly colonized at birth with pioneering bacteria carrying prophages, followed by successive waves of colonization by viruses, including lytic phages and eukaryotic viruses that are associated with diet and introduction to daycare (3,30). Studies of the hair virome in healthy humans also show differences across the lifespan highlighted by decreased diversity in early and later life (<18 and >65 years old) vs. adulthood (18–65 years old) (12). Phages and eukaryotic viruses are broadly and uniquely distributed in the body, with distinct viromes present at specific anatomical sites. The human oral cavity is a primary entry point for viruses and home to a diverse viral community. Most limited data on the human oral virome were derived from saliva samples (20,31,32) and, to a lesser extent, from dental plaque (33). Healthy humans contain approximately 108virus-like particles (VLP) per milliliter of saliva (32), with Caudoviricetes as the most abundant phage taxon in the oral saliva virome that also includes siphoviruses, myoviruses, and Streptococcus phages (20,21,34). Common eukaryotic viruses in the oral cavity of healthy adults include Papillomaviridae, Herpesviridae, Anelloviridae, and Redondoviridae (35–38). The gut virome is the best-studied and mostly highly colonized of all human viral communities, with approximately 109VLPs per gram of intestinal contents (Figure 1B). Phages are the most abundant members of the gut virome, in which Caudoviricetes and Microviridae are predominant (7,39–43), and the cross-assembly phage (crAss phage) lineage members podoviruses (now in the order Crassvirales) of the Caudoviracetes class being the most common (44–48). These phages infect bacterial species in the Bacteroidetes phylum that are abundant members of the gut microbiome. Eukaryotic viruses comprise a small proportion of the healthy gut virome and include the DNA lineages Anelloviridae, Geminiviridae, Herpesviridae, Nanoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae, Adenoviridae, and Circoviridae and RNA lineages Caliciviridae, Picornaviridae, Reoviridae, and Virgaviridae (49–53). Of note, with exception of coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most gut viruses lack lipid envelopes, a critical aspect of fecal–oral transmission (54,55). In contrast, the virome of the brain and central nervous system (CNS) in healthy humans is one of the least studied. The few prior investigations assessing viral communities within these regions have shown that, although they are commonly regarded as sterile, an estimated 104VLPs are present in each milliliter of cerebrospinal fluid (CSF) (56). Predominant members of the brain / CNS virome are phages, including myoviruses, siphoviruses, as well as podoviruses (56), whose natural hosts are unknown bacteria and archaea. Although Herpesviridae eukaryotic viruses were also detected in healthy CSF, these viruses — herpes simplex virus (HSV), human cytomegalovirus, varicella zoster — are best known for their roles in human disease and asymptomatic latency in the CNS (57,58). Collectively, these findings reveal distinct viral communities in various body sites, differing in composition and abundance. However, many critical knowledge gaps remain, most notably the full repertoire of viruses across the healthy lifespan. There is a need in the art for methods and materials useful for observing the human virome in healthy individuals, as well as individuals suffering from pathologies such as Alzheimer’s and irritable bowel disease. Towards this goal, we focused on mapping the human virome of the oral–gut–brain axis in several cohorts. These sites have been prioritized due to their central roles in human health and current lack of information about their viromes. The oropharyngeal site also provides easy access for sampling, is a critical entry point for infection, and is a key component of the oral– gut / oral–hepatic (59–64), oral–brain (65), gut–brain (66–72), and oral–gut‒brain signaling axes (4,27,28,73–75). SUMMARY OF THE INVENTION The virome of the brain and nervous system in healthy humans is one of the least studied. The few prior investigations assessing viral communities within these regions have shown that, although they are commonly regarded as sterile, an estimated 104VLP are present in each milliliter of CSF. Predominant members of the brain / central nervous system (CNS) virome are phages, including myoviruses, siphoviruses, and podoviruses, whose natural hosts are unknown bacteria and archaea. Although Herpesviridae eukaryotic viruses were also detected in healthy CSF, these viruses-herpes simplex virus (HSV), human cytomegalovirus, varicella zoster-are best known for their roles in human disease and asymptomatic latency in the CNS. As disclosed herein, we evaluated the virome in health and disease of the oral- gut-brain axis, in particular, periodontitis, irritable bowel disease (IBD) and Alzheimer's Disease (AD). For Alzheimer's disease, a striking feature was found via metagenomics of brain autopsy specimens - the presence of parvoviridae (comprised of erythoviruses and erythroparvoviruses) in the brains of individuals diagnosed with Alzheimer's Disease (AD) but not in brains from age-matched healthy individuals without a medical diagnosis of AD. This result was, then, validated by qPCR, showing significant higher levels of Parvovirus B19 in severe AD in both occipital and frontal lobes compared to healthy patients. Additionally, significant higher level of Parvovirus B19 in moderate AD compared to healthy patients. Further, we also found human endogenous retroviruses to be significantly higher in samples coming from those diagnosed with AD. Lastly, specific periodontal pathogenic bacteria, namely Treponema denticola was detected at different levels in all human brain regions (hippocampus, and frontal and occipital regions) examined for both AD and healthy specimens. For Periodontitis and IBD, an analysis revealed the top 20 most abundant viral and bacterial species in saliva samples from individuals with periodontal disease or with a healthy periodontium and the top viral and bacterial species in stool samples from individuals with irritable bowel syndrome or with gastrointestinal health. The most abundant viral species in saliva samples from healthy or diseased cohorts included Streptococcus phages and the most abundant bacterial species included Rothia, Schaalia, Streptococci, Veilonella, Prevotella, Phocaeicola, Parabacteroides, and Neisseria. Building upon the discoveries disclosed herein, we designed a number of methods and systems for diagnosing moderate to severe Alzheimer’s (AD) in a subject as well as methods and systems for diagnosing irritable bowel disease (IBD) in a subject. Embodiments of the invention include, for example, methods of diagnosing moderate to severe Alzheimer’s (AD) in a subject, the methods comprising: obtaining a brain tissue from the subject; and then observing the presence or absence of parvovirus B19 in the brain tissue, wherein the presence of parvovirus B19 in the brain tissue indicates a diagnosis of moderate or severe AD. In typical embodiment of the invention, the method comprises a qPCR method. Embodiments of the invention include further observing the presence or absence of adeno-associated dependoparvovirus and / or Torque Teno Virus. In illustrative embodiments of the invention, parvovirus B19 (and / or Torque Teno Virus 29) is present in the brain tissue in amounts that are at least 2, 5, 10, 25 or 50 fold higher than amounts of parvovirus B19 (and / or Torque Teno Virus 29) observed in brain tissues from an age-matched healthy control. Typically, the brain tissue is from an occipital or a frontal lobe. Embodiments of the invention include systems for diagnosing moderate to severe AD, the systems comprising brain tissue from a subject (e.g., brain tissue from an occipital or a frontal lobe) combined with a detection agent selected to bind a parvovirus B19 polypeptide or polynucleotide present in the sample. Typically in such systems, the detection agent comprises a polynucleotide probe that hybridizes with parvovirus B19 (and / or Torque Teno Virus) polynucleotides in the sample. Optionally such systems further include a detection agent selected to bind an adeno- associated dependoparvovirus and / or a Torque Teno Virus present in the sample. Embodiments the invention also include methods of diagnosing irritable bowel disease (IBD) in a subject, the methods comprising: obtaining a fecal sample from the subject; and then observing the presence or absence of tupanvirus in the fecal sample, wherein the presence of a virus identified herein in the brain tissue indicates a diagnosis of IBD. Certain embodiments of the invention further comprise observing the presence or absence of multiple types of viruses such as tupanvirus, moumouvirus and / or streptococcus phages. In typical embodiments of the invention, the method comprises a qPCR method. In some embodiments of the invention, the methods comprise the metagenomic sequencing using a Twist Comprehensive Viral Research Panel. In certain embodiments of the invention, the tupanvirus (and optionally moumouvirus and / or streptococcus phages) is present in the brain tissue in amounts that are at least 2 fold higher than amounts of tupanvirus and / or streptococcus phages observed in fecal samples from an age-matched healthy control. Embodiments of the invention further comprise systems for diagnosing irritable bowel disease (IBD), the system comprising a fecal sample from a subject combined with a detection agent selected to bind a moumouvirus and / or a tupanvirus and / or streptococcus phage polypeptide or polynucleotide present in the sample. Optionally such systems further include a detection agent selected to bind an moumouvirus and / or streptococcus phage present in the sample. Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Summary of the tissues samples and methods used in this study. Figure 2. Virome profiles for human oral, gut, brain specimens using the TWIST viral capture. Metagenomic libraries were subjected to virus hybridization capture with the TWIST Comprehensive Virus Research Panel kit before sequencing. A) Percentage of mapped reads aligned to the database of human and animal viral genomes in healthy human primary teeth (N=10 per group), stool samples (N=10 per group), and brain specimens (N=17 per group). Samples were sequenced after bulk genomic DNA isolation (left), DNA after host depletion (middle) or total RNA isolation (right). B) Taxonomy virome profile heatmaps for brain, stool and teeth samples listed in panel A stratified by nucleic acid isolation (bulk DNA, depleted DNA, RNA). C) Taxonomy heat map of the viruses found in the brain specimens of all cohorts (N=8 for Healthy; N=23 for Moderate AD; N=84 for Severe AD; N=16 unspecified AD). Figure 3 –Torque Teno Virus (TTV) 10, 18 and 24 are significantly increased in healthy brain tissues, while TTV 29 in significantly increased in severe AD patients’ brains. Data are represented as A) percentage of brain specimens positive for TTV10, 18, 24, 29 according to the sequencing data; B) total reads abundance (RPMKF); and C) qPCR validation for TTV10, 18, 24 and 29. * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001 and **** indicates p ≤ 0.0001 between marked samples. (N=8 for Healthy; N=23 for Moderate AD; N=84 for Severe AD; N=16 for Unspecified AD). Figure 4. Significant increased Parvovirus B19 levels in Occipital and Frontal lobe brain tissues correlate with Alzheimer’s Disease (AD) progression. A) Parvovirus B19 abundance in human brain specimens (healthy, moderate and severe AD specimens; all regions combined) using the TWIST viral capture assay. B) Parvovirus B19 abundance in Frontal, Hippocampus and Occipital regions of healthy, moderate and severe AD human brain specimens using the TWIST viral capture assay; C) Parvovirus B19 qPCR validation of the same human brain (Healthy, moderate and severe AD; all regions combined) specimens; D) Parvovirus B19 qPCR validation in Frontal, Hippocampus, and Occipital regions of healthy, moderate and severe AD human brain specimens. E) Parvovirus B19 qPCR detection in the NIH NeuroBioBank / University of Miami cohort of hippocampus samples against mock samples that underwent DNA isolation / purification and qPCR analysis similarly to the tested hippocampus tissues. F) Correlation between virome sequencing reads abundance (RPKMF) to qPCR abundance 40-Ct)2; Coverage of Parvovirus B19 genome highlighting different Single Nucleotide Polymorphism (SNPs) profiles together (G) and from individual brain specimens (H), highlighting the first batch of sequenced samples (Top grey samples) versus the second batch of sequenced samples (bottom black samples). * indicates p ≤ 0.05; ** indicates p ≤ 0.01, *** indicates p ≤ 0.001 and **** indicates p ≤ 0.0001 between marked samples. (N=8 for Healthy; N=23 for Moderate AD; N=84 for Severe AD; N=16 for Unspecified AD). Figure 5. Confocal microscopy using RNAscope in situ hybridization identifying Parvovirus B19 DNA in human brain specimens. Parvovirus B19 is indicated in red, while brain cells are in blue. White and Yellow boxes highlight cells positive for Parvovirus B19-specific RNAscope signal, while blue boxes highlight regions representing the absence of parvovirus B19-specific RNAscope signal in healthy samples. Yellow boxes indicate regions further magnified to determine the location of the viral signal (N=4; 4 separate unique patients). Figure 6. Distinct saliva virome in child after 4- and 9-month post-partum compared to mother’s saliva virome. A) Relative abundance of top 20 viral species in saliva samples from healthy mother-child dyads from the same household. Each stacked bar plot represents the proportional abundance of a single viral and bacterial species within the community. B) Euclidean clustering Z-scores of the viruses found in mother-child dyads. (N=5 per Dyad) Figure 7. Multiple Displacement Amplification (MDA) enhances the sequencing coverage of 10-15 years stored healthy saliva samples, allowing more species to be identified. Relative abundance of viral (Top) and bacterial (Bottom) communities in unamplified and MDA assayed saliva samples (Individuals 3, 4 and 5). Each of the stacked bar plots illustrates the relative abundance of the top 20 abundant viruses present in saliva samples. (N=3). Figure 8. Periodontal disease saliva samples contain reduced levels of streptococcal phages compared to healthy patients. A) Relative abundance of top 20 viral species in saliva samples from healthy and periodontal disease patients; B) Relative abundance of top 20 viral species in saliva samples from healthy and periodontal disease patients; C)NMDS analysis for viral species; D) NMDS analysis for bacterial species; E) Differential number of viral species between healthy and periodontal disease samples; F) Differential number of bacterial species between healthy and periodontal disease samples. (N=5 per group). Figure 9. IBS stool samples contain reduced levels of Carjuvirus communis compared to healthy patients. A) Relative abundance of top 20 viral species in stool samples from healthy and IBS patients; B) Relative abundance of top 20 viral species in stool samples from healthy and IBS patients; C) NMDS analysis for viral species; D) NMDS analysis for bacterial species; E) Differential number of viral species between healthy and IBS samples; F) Differential number of bacterial species between healthy and IBS samples. (N=5 per group). Figure 10. Significant higher abundance of Human Endogenous Retroviruses were found in Moderate and Severe AD brain samples compared to healthy patients. Relative abundance of microbiome species identified in healthy and AD brain tissues using non-enriched shotgun sequencing – top 20 most abundant virus (A), bacteria (B), fungi (C) and archaea (D) in brain tissues from healthy individuals as well as patients with moderate AD and Severe AD. TaqMan-based qPCR validation of HERV W and K (E) in all brain regions combined for healthy and AD brain tissues, total HHV6a / 6b (F) stratified by brain region (Hippocampus and Occipital), HHV4 / EBV (G) in occipital lobe, F. nucleatum (H) in frontal lobe, T. forsythia (I) in hippocampus, and E. coli (J) stratified by brain region (Hippocampus and occipital lobe). Simpson and Shannon diversity indexes (K) based on the species abundance, and Venn diagram (L) of viral and bacterial species. * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001 and **** indicates p ≤ 0.0001 between healthy and marked samples. (For Panels A-D, N=4 for Healthy; N=3 for Moderate AD; N=10 for Severe AD; For Panels E-H, N=8 for Healthy; N=23 for Moderate AD; N=84 for Severe AD). Figure 11. Primary teeth individual-levels indicate significant lower abundance of caudoviracetes sp. in their virome. A) viral, B) bacterial, C) fungi and D) archaea relative abundance content using non-enriched shotgun metagenomic sequencing. TaqMan-based qPCR validation E) E. coli, F) HHV 4 / EBV, G) HHV 6a / 6b and H) HHV 7. * indicates p ≤ 0.05; ** indicates p ≤ 0.01; and **** indicates p ≤ 0.0001 between Subject A vs Subjects B and C. (N=3 for Subject A and B; N=4 for Subject C). Figure 12. Relative abundance of microbiome species in human stool. A) virus, B) bacteria, C) fungi and D) archaea (D) in healthy and IBS stool based on non- enriched shotgun metagenomic sequencing. TaqMan-based qPCR validation of E) E. coli and F) F. nucleatum in healthy and IBS stool samples. * indicates p ≤ 0.05; ** indicates p ≤ 0.01; and **** indicates p ≤ 0.0001 between healthy and IBS samples. (N=5 per group). Figure 13. Main findings of the initial step of the Human Virome Project (HVP) across the lifespan / healthspan and in different body sites. DETAILED DESCRIPTION OF THE INVENTION Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. In the description of the preferred embodiment, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and reagent substitutions may be made without departing from the scope of the present invention. The human oral cavity is a primary entry point for viruses and home to a diverse viral community. Most limited data on the human oral virome were derived from saliva samples and, to a limited extent, from dental plaque. These studies have shown healthy humans contain approximately 108 virus-like particles (VLP) per milliliter of saliva. Caudoviricetes is the most abundant phage taxon in the oral saliva virome, including siphoviruses, myoviruses, and Streptococcus phages. Common eukaryotic viruses in the oral cavity of healthy adults include Papillomaviridae, Herpesviridae, Anelloviridae, and Redondoviridae. The gut virome is the best-studied and mostly highly colonized of all body viral communities, with approximately 109 VLPs per gram of intestinal contents. Phages are the most abundant members of the gut virome; Caudoviricetes and Microviridae are predominant, with the cross-assembly phage (crAssphage) lineage members (podoviruses now in the order Crassvirales) of the Caudoviracetes class being the most common.103-107 These phages infect bacterial species in the Bacteroidetes phylum, abundant members of the gut microbiome. Eukaryotic viruses comprise a small proportion of the healthy gut virome and include the DNA lineages Anelloviridae, Geminiviridae, Herpesviridae, Nanoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae, Adenoviridae, and Circoviridae and RNA lineages Caliciviridae, Picornaviridae, Reoviridae, and Virgaviridae. Of note, with exception of coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), most gut viruses lack lipid envelopes, which is thought to be important for fecal-oral transmission. In contrast, the virome of the brain and nervous system in healthy humans is one of the least studied. The few prior investigations assessing viral communities within these regions have shown that, although they are commonly regarded as sterile, an estimated 104 VLP are present in each milliliter of CSF. As disclosed herein, we evaluated the virome in health and disease of the oral- gut-brain axis, in particular, periodontitis, irritable bowel disease (IBD) and Alzheimer's Disease (AD). For Alzheimer's disease, a striking feature was found via metagenomics of brain autopsy specimens - the presence of parvoviridae (comprised of erythoviruses and erythroparvoviruses) in the brains of individuals diagnosed with Alzheimer's Disease (AD) but not in brains from age-matched healthy individuals without a medical diagnosis of AD. This result was, then, validated by qPCR, showing significant higher levels of Parvovirus B19 in severe AD in both occipital and frontal lobes compared to healthy patients. Additionally, significant higher level of Parvovirus B19 in moderate AD compared to healthy patients. Further, we also found human endogenous retroviruses to be significantly higher in samples coming from those diagnosed with AD. Lastly, specific periodontal pathogenic bacteria, namely Treponema denticola was detected at different levels in all human brain regions (hippocampus, and frontal and occipital regions) examined for both AD and healthy specimens. For Periodontitis and IBD, the analysis revealed the top 20 most abundant viral and bacterial species in saliva samples from individuals with periodontal disease or with a healthy periodontium and the top viral and bacterial species in stool samples from individuals with irritable bowel syndrome or with gastrointestinal health. The most abundant viral species in saliva samples from healthy or diseased cohorts included Streptococcus phages and the most abundant bacterial species included Rothia, Schaalia, Streptococci, Veilonella, Prevotella, Phocaeicola, Parabacteroides, and Neisseria. For saliva samples from healthy mother-child dyads the most abundant viral species included Streptococci phage and the bacterial species included Rothia and Streptococci species. Comparative analyses of these saliva data at 4 months post- delivery revealed a conserved structure of the viral community between mothers and their newborns. Moreover, as children aged, they established a more diverse viral community. The bacterial community revealed a similar trend. In stool samples, the most abundant viral species included Carjivirus communis and the most abundant bacterial species included Phocaeicola, Parabacteroides, Neisseria, Haemophisus, Faecalibacterium, and Bacteroides. Interestingly, comparative assessments among both sample types (saliva and stool) indicate that inflammation alters the evenness of viral communities, as indicated by BrayCurtis Dissimilarity analysis. When compared to healthy controls, this alteration may lead to a reduction in the diversity of the oral commensal virome. In contrast, an increase in diversity within the stool is observed. Notably, certain commensal viral species demonstrate a shared presence across both oral and gut inflammatory sites, including moumouvirus, streptococcus phages, and tupanvirus. In contrast, the diversity within bacterial communities appears to be only minimally affected by inflammation, evident in both oral and gut diseases. In particular for IBD, Bacteriophage species were also significantly depleted in the IBD patients, compared to healthy samples. Notably, no distinct bacterial clustering was observed between periodontal and irritable bowel diseased groups and their respective healthy counterparts. This finding was further confirmed by the Simpson indices for both saliva and stool samples, aligning with findings reported in other studies. These observations and our earlier studies suggest that inflammation may have a more pronounced impact on the composition of viral communities than on bacterial communities. The human virome is largely uncharacterized, including lifespan-associated virome changes. Therefore, we mapped the oral-gut-brain axis virome / microbiome in healthy versus disease cohorts stratified by age. Non-targeted shotgun sequencing, targeted viral capture, multiple displacement amplification (MDA), qPCR, and imaging were used to map the viromes and microbiomes in 1) healthy primary teeth, 2) saliva from healthy mother-child dyads, 3) saliva from patients with healthy or diseased periodontium, 4) stool from patients with irritable bowel syndrome (IBS) and healthy controls, and 5) brain autopsy specimens from aged healthy individuals and those with Alzheimer’s Disease (AD). In primary teeth, non-targeted sequencing identified bacteriophages, Caudoviricetes, crAss-like phages, and Siphoviridae ctK012 species. Targeted sequencing revealed human herpes, papilloma, and avian and porcine viruses. In saliva from mother-child dyads and adults with a healthy or diseased periodontium, the most common viruses were streptococcal phages, Rothia phages, and torque teno virus. The shared community between mothers and newborns changed as children later developed a more diverse microbiota. In stool specimens, non-targeted sequencing revealed more bacteriophages in healthy versus IBS specimens. Targeted sequencing revealed that stool specimens contained human herpes and gyro viruses as well as gamma retro, beta polyoma, lenti, and chaphamaparvo viruses, particularly in stool samples from healthy donors. In aged brains, non-targeted sequencing identified human endogenous retroviruses, which were higher in AD patients—who also had more fungi, archaea, and bacteria. Targeted sequencing showed that torque teno virus predominated in healthy brain specimens, but parvovirus B19 was found only in AD patients. We identified viruses and microbes common across the oral-gut-brain axis, including herpesviruses, as well as distinct viral communities in each site that were associated with health or disease. The disclosure presented herein allows artisans to determine what are significantly higher (or lower) populations of a virus in a patient sample as compared to populations of that virus in aged matched healthy controls. Illustrating this, as shown in Tables 2-6 below, artisans can focus on viruses such as the illustrative 5 viruses: Parvovirus B19 and four species of Torque Teno Viruses (TTV10, TTV18, TTV24, and TTV29). In Tables 2-6, for each virus, two evaluation metrics were used: (1) presence / absence in brain tissue (reported as “positivity” in the table), and (2) actual viral quantification. Both metrics include results from sequencing and qPCR validation. The data in Tables 2-6 shows the detection of significantly higher amounts of Parvovirus B19 and Torque Teno Virus 20 (TTV29) in AD brains as compared to aged-matched healthy controls. In studies of Parvovirus B19 Positivity: Both sequencing and qPCR data show that B19 is 3- to 4-fold more frequently detected in moderate and severe AD brains compared to healthy controls. Sequencing suggests B19 is ~40-fold more abundant in moderate / severe AD vs. healthy, while qPCR shows ~4.5-fold higher levels in moderate / severe AD compared to healthy. Similarly, in studies of TTV29 Positivity: Moderate and severe AD samples were 70- to 200-fold more frequently detected than controls, in TTV29 Quantification: 20- to 93-fold higher viral abundance in disease compared to healthy. In contrast, in studies of TTV10, TTV18, and TTV24 Positivity: their detection dropped from half to completely absent in moderate / severe AD compared to healthy controls (set as 100%). In TTV10, TTV18, and TTV24 Quantification: values fell from ~17% abundance to undetectable levels in disease groups, compared to healthy controls (set as 100%). Embodiments of the invention include, for example, methods of diagnosing moderate to severe Alzheimer’s (AD) in a subject, the methods comprising: obtaining a brain tissue from the subject; and then observing the presence or absence of parvovirus B19 in the brain tissue, wherein the presence of parvovirus B19 in the brain tissue indicates a diagnosis of moderate or severe AD. Typically, the brain tissue is from an occipital or a frontal lobe. Embodiments of the invention include methods of diagnosing moderate to severe Alzheimer’s (AD) in a subject, the methods comprising: obtaining cerebrospinal fluid (CSF) from the subject; and then observing the presence or absence of parvovirus B19 in the CSF, wherein the presence of parvovirus B19 in the CSF indicates a diagnosis of moderate or severe AD. Embodiments of the invention include methods of diagnosing moderate to severe Alzheimer’s (AD) in a subject, the methods comprising: obtaining saliva from the subject; and then observing the presence or absence of parvovirus B19 in the saliva, wherein the presence of parvovirus B19 in the saliva indicates a diagnosis of moderate or severe AD. In typical embodiment of the invention, the method comprises a qPCR method. Embodiments of the invention include further observing the presence or absence of adeno-associated dependoparvovirus and / or Torque Teno Virus. In illustrative embodiments of the invention, parvovirus B19 (and optionally adeno-associated dependoparvovirus and / or Torque Teno Virus 29) is present in the patient sample (e.g., brain tissue or CSF) in amounts that are at least 2, 5, 10, 25 or 50 fold higher than amounts of parvovirus B19 (and optionally adeno-associated dependoparvovirus and / or Torque Teno Virus 29) observed in a patient sample from an age-matched healthy control. Typically in these methods, the one or more adeno- associated dependoparvovirus and / or Torque Teno Virus and / or other virus disclosed herein is selected to be indicative of brain health (see, e.g., Tables 2-6 below). Embodiments of the invention include systems for diagnosing moderate to severe AD, the systems comprising brain tissue (or CSF) from a subject (e.g., brain tissue from an occipital or a frontal lobe) combined with a detection agent selected to bind a parvovirus B19 (and optionally adeno-associated dependoparvovirus and / or Torque Teno Virus) polypeptide or polynucleotide present in the sample. Typically in such systems, the detection agent comprises a polynucleotide probe that hybridizes with parvovirus B19 polynucleotides in the sample. Optionally such systems further include a detection agent selected to bind an adeno-associated dependoparvovirus and / or a Torque Teno Virus present in the sample. Embodiments the invention also include methods of diagnosing irritable bowel disease (IBD) in a subject, the methods comprising: obtaining a fecal sample from the subject; and then observing the presence or absence of tupanvirus in the fecal sample, wherein the presence of parvovirus tupanvirus and / or moumouvirus and / or streptococcus in the brain tissue indicates a diagnosis of IBD. Certain embodiments of the invention further comprise observing the presence or absence of multiple virus species. In typical embodiments of the invention, the method comprises a qPCR method. In some embodiments of the invention, the methods comprise the metagenomic sequencing using a Twist Comprehensive Viral Research Panel. In certain embodiments of the invention, the tupanvirus and / or optionally moumouvirus and / or streptococcus is present in the brain tissue in amounts that are at least 2 than amounts of tupanvirus and / or moumouvirus and / or streptococcus observed in fecal samples from an age-matched healthy control. Embodiments of the invention further comprise systems for diagnosing irritable bowel disease (IBD), the system comprising a fecal sample from a subject combined with a detection agent selected to bind a viral polypeptide or polynucleotide present in the sample. In certain embodiments of the invention, in order to investigate the feasibility of sequencing viromes and microbiota from minimal sample volumes, we employed multiple displacement amplification (MDA) on 25 μL of saliva samples using a protocol established in our group and this data demonstrates that this MDA approach successfully enriched the samples for high-coverage sequencing for both virome and bacteriome expression in human saliva samples. Results We performed metagenomic sequencing and virome / bacteriome / microbiome analysis on biospecimens from various human cohorts of sample collections along the oral-gut-brain axis (healthy primary teeth, saliva from healthy and periodontal disease patients, saliva from healthy mother-child dyad, stool from healthy and IBS patients, and brain tissues from healthy and AD patients). We used samples that have been stored for several years, namely desiccated primary teeth stored for 15–20 years, saliva stored for 14-21 years, stool for >5 years and fresh-frozen brain specimens for 9–15 years. The samples produced moderate / good-quality DNA, with TapeStation DNA Integrity numbers ranging from 3 to 8. This shows that archived specimens can be used for nucleic acid extraction for virome studies. We then proceeded to analyze the samples with three different sequencing methods, namely the metagenomic sequencing using TWIST viral capture, non-enriched metagenomic sequencing (with and without MDA Enrichment), and non-enriched shotgun metagenomic sequencing Metagenomic Sequencing using Twist Comprehensive Viral Research Panel Virome characterization poses multiple technical challenges. First, viruses can be of DNA or RNA in genetic origin. Second, given that viruses infect their host, sequencing viral and host (human) nucleic acids requires extensive sequencing depth to fully characterize viral content if host content is included. In order to determine the best approach for virome characterization, we evaluated and compared data generated using three nucleic acid input types: 1) bulk DNA, 2) DNA after host depletion and 3) bulk RNA for brain, stool and teeth samples (Figure 2A). We also performed these comparisons stratified by disease state for brain and stool specimens such that the brain specimens analyzed were collected from healthy subjects as well as from moderate Alzheimer’s Disease (AD) and advanced AD patients, while stool samples were obtained from healthy individuals and those with IBS. Nucleic acids extracted from teeth, stool, and brain samples, including bulk or host depleted DNA or RNA, yielded reads mappable to virome databases based on the TWIST viral capture pipeline (Figure 2A). Furthermore, using these two different DNA isolation techniques (bulk or host depleted DNA) revealed that bulk DNA yielded more mappable reads compared to host depleted DNA (Figure 2A). Analysis of the isolated nucleic acids (bulk DNA and RNA) with the TWIST viral capture platform revealed an average of 20,319, 624 quality reads from RNA from brain tissue, 16,576, 842 quality reads from bulk DNA from brain tissue, 28,428,683 quality reads from RNA from stool, 7,729, 020 quality reads from bulk DNA from stool, 210,410 quality reads from RNA from teeth, and 24,069,875 quality reads from bulk DNA from teeth. Overall, archived human primary teeth exhibited 0.1 to 10% mappable reads, stool exhibited <0.001 to 10% mappable reads, and the brain exhibited 0.1 to 10% mappable reads to a virome database based on bulk DNA. RNA revealed lower mappable reads. Analysis with the TWIST viral capture platform revealed the virome taxonomy profile for each tissue (Table 1). Brain specimens showed enrichment in erythroparvoviruses, dependoparvovirus, roseoloviruses, simplexvirus, alphapolyomavirus, alphatorqueviruses (torque teno viruses), while stool specimens contained human herpesviruses (roseolovirus genus), gyroviruses, as well as a variety of gammaretroviruses, betapolyomaviruses, lentiviruses, and chaphamaparvoviruses; most of which were present in healthy individuals without IBS (Table 1). Finally, analysis of primary teeth revealed the presence of human herpesviruses (roseolovirus and lymphocryptovirus genus), papillomaviruses, and avian and porcine retroviruses (Table 1). All the primary teeth exhibited beta herpesvirus 7 in the bulk DNA-based analysis. Bulk DNA from some of the teeth in all individuals were positive for human gamma herpes virus 4, in addition to a variety of beta and gamma papillomaviruses, and avian viruses (Figure 2B). A striking feature from this viral capture data included the presence of several parvoviridae (comprised of both erythoviruses and erythroparvoviruses) in the brains of individuals diagnosed with Alzheimer’s Disease (AD) but not in brains from age- matched healthy individuals without a medical diagnosis of AD (Figure 2B). To investigate this further, we analyzed an additional set of 82 samples obtained from the frontal, hippocampal, and occipital brain regions of Alzheimer’s disease (AD) patients. These samples were sequenced using the TWIST viral capture and the sequencing results indicate that high read counts of Anelloviridae, especially Torque Tenovirus (TTV), are enriched in healthy patients, while higher read counts of Parvoviridae correlate with AD progression. Parvovirus B19 appears to be the most abundant species in those with AD diagnosis, followed by Adeno-associated Dependoparvovirus A (AAV) (Figure 2C). A deeper analysis of individual TTV species revealed a significant increase of TTV 10,13, 15,16, 18, 22 and 24 species presence (Figure 3A-H) and read levels of TTV 10, 18 and 24 (Figure 3I-P) in healthy samples compared to moderate and severe AD samples across all brain regions combined. On the other hand, Parvovirus B19 levels were significantly increased in patients with moderate and severe AD (across all brain regions combined) compared to age- matched healthy controls (Figure 4A). When stratified by brain region, we observed a significant elevation of Parvovirus B19 levels in the frontal lobe of moderate and severe AD patients and in the occipital lobe of severe AD patients compared to age- matched healthy controls (Figure 4B). Validation using qPCR with TaqMan primers and probe specifically designed for human Parvovirus B19 confirmed the findings of elevated levels only in severe AD across all brain regions (Figure 4C) and only in the frontal lobe, demonstrating a similar significant increase in viral levels in moderate and severe AD patients compared to age-matched healthy patients (Figure 4D). Due to the unavailability of age-matched healthy hippocampal specimens, comparisons between healthy and AD hippocampal samples were not feasible. Additionally, no significant differences were detected in human Parvovirus B19 levels between moderate and severe AD hippocampal samples using TaqMan primers and probe (Figure 4D). We also obtained hippocampal brain tissue specimens from AD patients from the NIH NeuroBioBank to further evaluate the presence of Parvovirus B19. Validation using mock and no template control samples (qPCR water) processed through the same DNA extraction and qPCR workflows confirmed that the detected viral levels originated from the samples themselves and not from procedural contamination (Figure 4E). Next, we correlated the sequencing and qPCR data for Parvovirus B19 and observed a significant positive correlation between the two techniques for frontal, hippocampus and occipital brain regions (Figure 4F), indicating that these results are biologically meaningful. We further evaluation of single nucleotide polymorphisms (SNPs) of the Parvovirus B19 genome to check whether these positive data are indeed from unique individuals of the virus, which revealed unique SNPs profiles prevenient from each individual specimens (Figure 4G and H), thus, reflecting Parvovirus B19 genetic diversity across the specimen collection. All in all, these distinct SNP profiles, as well as negative mock controls for Parvovirus B19 highlight a greater likelihood of viral evolution and diversity amongst patients rather than a potential laboratorial contamination. To further validate these results, we examined four specimens (one healthy control and three severe cases—each representing a distinct brain region (i.e., frontal, hippocampus, and occipital lobes)), using RNAscope (Figure 5). RNAscope is an advanced in situ hybridization (ISH) microscopy technique that enables the detection and visualization of DNA and RNA molecules directly within intact cells and tissues. The method employs a unique “double Z” probe design that hybridizes to target sequences with high specificity and sensitivity, allowing for precise spatial localization of nucleic acids. The results show distinct localization patterns of Parvovirus B19 DNA in the brain sections – either perinuclear (Figure 5 B1, B2, C1 and D2) or distal from the cell nuclei (Figure 5 C2 and D1), indicating possible differences in viral trafficking, replication stages or cell-type-specific interactions within the brain. Non-Enriched Metagenomic Sequencing and Multiple Displacement Amplification (MDA) Enrichment Next, we analyzed the human microbiome across multiple cohorts using non- enriched whole-genome shotgun metagenomic sequencing, conducted at two independent facilities: (1) UCLA TCGB, which employed a read-centric k-mer profiling approach, and (2) Novogene Corp., which used a gene-centric analysis pipeline. We began with the read-centric k-mer profiling approach at UCLA TCGB, analyzing saliva samples from two distinct cohorts—adult saliva (from both healthy individuals and those diagnosed with periodontal disease) and healthy mother–child dyads—as well as the same stool samples previously analyzed using the TWIST viral capture method (Figure 2). Comparative analyses of mother-child saliva dyads show a conserved viral community structure among the top 20 most abundant viral species in individual mothers during prenatal and postpartum periods (Figure 6A). In newborns, the viral community was established by species originating from their mothers by 4 months of age, when they were exclusively fed with milk. However, by 9 months of age, following the introduction of complementary food, their viral communities evolved, incorporating new species. We further compared the composition of saliva samples across different groups (Figure 6B). Distinct clusters emerged based on Euclidean clustering: cluster 1 (prenatal vs postpartum in mothers) was characterized by a significant higher relative abundance of species from the Skunavirus and Wroclawvirus genera; cluster 2 (mothers at 4 months postpartum vs children at 4 months postpartum) was defined by significant elevated abundances of Lymphocryptovirus and Buchavirus genera; and cluster 3 (children at 4 months postpartum vs children at 9 months postpartum) was marked by an significant increased presence of genera from Latrobevirus and Roseolovirus. Overall, the numbers of species in mothers remained stable after childbirth, with a slight increase by 9 months postpartum. In contrast, children developed their viral communities primarily from maternal sources and experienced a slight reduction in viral species diversity after the introduction of complementary foods. To investigate the feasibility of sequencing viromes and microbiota from minimal sample volumes, we employed multiple displacement amplification (MDA) on 25 ^L of saliva samples using a protocol established in our group (92). Figure 7 demonstrates that this MDA approach successfully enriched the samples for high- coverage sequencing for both virome and bacteriome expression in human saliva samples. Compared to unamplified saliva specimens from individual 3 , for example, MDA-based profiling does show comparative virome and bacteriome representation. For individuals 4 and 5 , the MDA-based approach results in more diversified virome and bacteriome coverage than with the unamplified method. This may point to an unevenness in unamplified library assembly and potential bias in the resulting sequencing content. Next, we analyzed the virome and bacteriome of additional samples in human saliva samples from individuals with or without periodontal disease (Figure 8) and the same stool samples of patients with and without IBS previously examined using TWIST viral capture (Figure 9) using non-enriched whole-genome metagenomic sequencing. The top viral (Figure 9A) and bacterial species (Figure 9B) in stool samples from individuals with irritable bowel syndrome (IBS) or gastrointestinal health. Interestingly, comparative assessments among both sample types (saliva and stool) indicate that inflammation alters the evenness of viral communities, as indicated by Bray-Curtis Dissimilarity analysis (Figure 8C and 8C). When compared to healthy controls, this alteration may lead to a reduction in the diversity of the oral commensal virome (Figure 8C ). In contrast, an increase in diversity within the stool is observed (Figure 9C). Notably, certain commensal viral species demonstrate a shared presence across both oral and gut inflammatory sites, including moumouvirus, streptococcus phages, and tupanvirus. In contrast, the diversity within bacterial communities appears to be only minimally affected by inflammation, evident in both oral and gut diseases. Notably, no distinct clustering was observed between periodontal (Figure 8C) and IBS groups (Figure 9C) and their respective healthy counterparts. This finding was further confirmed by the Simpson indices for both saliva (Figure 8C) and stool samples (Figure 9C), aligning with the findings previously reported in a separate study (93). These observations suggest that inflammation may have a more pronounced impact on the composition of viral communities than on bacterial communities (Figures 8D and 9D). Interestingly, we identified 201 viral species strictly correlated to oral health and 189 viral species strictly correlated to periodontal disease (Figure 8E) in the saliva samples. Similarly, 429 viral species were strictly correlated to GI health, while 274 were strictly correlated to IBS (Figure E) in stool samples. Afterward, a gene-centric analysis approach was used to analyze and further validate the microbiome of the same human brain, gut, and teeth samples assayed in Figure 2. In the brain tissues, the most prevalent brain viruses included human endogenous retroviruses (HERVs) that were significantly higher in samples coming from individuals with AD (Figure 10A). qPCR validation indicated that HERV W and K to be significantly higher in severe AD, compared to moderate AD in all brain regions combined (Figure 10E). The most abundant bacteria included Escherichia coli (E. coli), which was significantly higher in the healthy brain specimens versus those from individuals diagnosed with AD (Figure 10B); qPCR validation indicated the presence of E. coli only in the occipital and hippocampus of severe AD patients (Figure 10J). The most abundant brain fungi included Rhizopus and Hanseniaspora fungi that were again more abundant in samples coming from individuals diagnosed with AD (Figure 10C). The most abundant archaea specimens included uncultured Marine thaumarchaeote that were also more significantly abundant in individuals diagnosed with AD (Figure 10D). Thus, these data corroborate the viral capture data indicating significant alterations in metagenomic content in brain samples between healthy individuals and those with AD. No significant differences in Simpson or Shannon diversity indexes were observed among the groups (Figure 10K), still a trend indicating lower diversity and evenness in healthy brain samples, compared to moderate and severe AD can be seen. Using these data, we then evaluated for unique viral and bacterial species in each condition (i.e., healthy, moderate and severe AD) – Figure 10L). We found Siphoviridae sp. ctPAi1 to be only found in healthy brain samples, while 4 species (Myoviridae sp. ctiv53, Siphovidiae sp. ct6662, ctK0l2 and ctvGX2) to be unique in severe AD. No particular species were found to be uniquely associated in moderate AD. One unclassified bacteriophage species was found to be in common between healthy and severe AD groups, an unclassified Inoviridae species was in common between moderate and severe AD groups and 152 species in common among all the groups. For bacterial species, we found 1030 species to be in common among all the groups, 120 bacterial species to be unique to healthy brains, 242 species were found to be unique to severe AD, while no species found to be uniquely found in moderate AD. In addition, qPCR validation also revealed the presence in Human herpesvirus (HHV) 6a and 6b in the hippocampus of severe AD patient (Figure 10F); HHV4 (also known as Epstein-Barr virus (EBV)) in the occipital and frontal lobes of severe AD patients (Figure 10G), and Fusobacterium nucleatum (F. nucelatum) and Tannarella forsythia (T. forsythia), known periodontal pathogens, in the frontal lobe and hippocampus of severe AD patients). Next, we analyzed primary teeth samples from three individuals (Subjects A, B and C) for virus, bacteria, fungi and archaea content (Figure 11A-11D). The most abundant viruses included Bacteriophage species, Caudoviricetes species, Cr-Ass like species, and Siphoviridae species ctK012. We noted a significant increase in the Caudoviricetes species Subject A. The most prevalent bacterial species included E. coli and Lautropia mirabilis. Aggregatibacter aphrophilus were significantly more abundant in tooth samples from Subject A. The most ubiquitous fungi included Rhizopus delemar, Rhizopus arrhizus, and Hanseniaspora uvarum, all of which were all significantly less abundant in Subject A. Lastly, the most common archaea identified were uncultured marine thaumarchaeote KM3_53_F08 and Sulfolobus species A20-N-F8, both of which were less abundant in Subject A. No significant differences in Simpson or Shannon diversity indexes were observed among the subjects (Figure 11I). Evaluating for unique viral and bacterial species (Figure 11J) revealed 29 unique viruses and 560 bacterial species to be unique to Subject A; 8 viral species and 167 bacterial species to be unique to subject B and 1 viral species (Siphoviridae sp. ct9GL2) and 67 bacterial species to be unique to subject C.166 viral species and 1785 bacterial species shared among all 3 subjects. Similar to teeth samples, stool samples contained Bacteriophage and Caudoviricetes species (Figure 12A), while Bacteriophage species were significantly depleted in the IBS stool samples. The most abundant bacterial species in stool samples included Ruminococcus species CAG: 117 that was significantly higher in healthy stool that IBS samples (Figure 12B). The most common stool fungi included Rhizopus arrhizus, Hansenispora uvarum, Thecaphora frezi, and Batrachochytrium salamandrivorans; the latter of which displayed significantly higher levels in the IBS samples (Figure 12C). The most abundant archaea in stool samples were Methanobrevibacter smithi, uncultured marine thaumarchaeote KM3_53_F08, Candidatus Methanomassiliicoccus intestinalis and Unclassified archaeon were the most prevalent archaea in stool, with Candidatus Methanomassiliicoccus intestinalis was significantly more abundant in the IBS samples than in healthy stool (Figure 12D). No significant differences in Simpson or Shannon diversity indexes were observed among the subjects (Figure 12G), although a small trend towards higher diversity and evenness in IBS compared to healthy can be seen. Evaluating for unique viral and bacterial species (Figure 12H) revealed 115 unique viruses and 671 bacterial species to be unique to healthy stools, while 149 viral species and 1006 bacterial species to be unique to IBS. 174 viral species and 4204 bacterial species shared among between healthy and IBS stools. qPCR validation also revealed the presence of E. coli (Figure 12E) only in IBS samples and F. nucleatum (Figure 12F) in both healthy and IBS samples. Even though no significant differences were found between healthy and IBS for these species, a trend indicating higher levels of E. coli and lower levels F. nucleatum levels can be seen in IBS samples. Finally, the main viral findings obtained in this study—representing an initial step of the Human Virome Project (HVP)—are summarized in Figure 13. These outcomes encompass the virome profiles across the human lifespan (from newborn to elderly individuals) and healthspan, as well as across multiple anatomical sites, including brain, oral cavity and gut. Together, these data provide a foundational framework for understanding the spatial and temporal dynamics of the human virome Discussion As a result of the human microbiome project, it is now well known that there is a rich repertoire of bacterial microbes that colonize the humans body (3,27). However, less well known is that healthy humans are also colonized by a remarkable diversity of viruses that define the human virome (3). The human virome comprises bacteriophages (phages) that infect bacteria, viruses that infect other cellular microorganisms, such as archaea and fungi, viruses that infect human cells and transients viruses present in food (3). In response to the NIH Human Virome Project, we performed a pilot study to define the human virome of the oral–gut–brain axis in diverse healthy and diseased cohorts of different age groups, specifically primary human teeth, saliva from healthy mother-child dyads, saliva from patients with periodontal disease and healthy controls, stool from patients with irritable bowel syndrome and healthy controls, and brain autopsy specimens from individuals with Alzheimer’s Disease and age-matched controls. This study utilized three different sequencing facilities in which whole-genome shotgun metagenomic sequencing data from a wide variety of human tissues were compared to sequencing data after two specialized capture / enrichment techniques: 1) TWIST Viral Capture, to enrich in vertebrate viral species content; and 2) Multiple Displacement Amplification (MDA) Enrichment for working with of small amounts of input DNA. These capture and enrichment tools improve our ability to detect members of the microbiome community beyond that of traditional shotgun metagenomic sequencing protocols. The data derived from these capture, enrichment and amplification protocols dovetail well with whole-genome shotgun metagenomic sequencing, and the information derived from these approaches highlight their utility in expanding the data success in generating high quality data from precious archival tissues / samples maintained in long-term storage. In addition, we employed two complementary bioinformatic strategies, leveraging their distinct strengths. 1) A gene-centric approach, involving de novo assembly followed by gene prediction and homology searching (e.g., via DIAMOND against NCBI-NR), was utilized to link genetic sequences to putative functions and broad taxonomic origins, thereby illuminating the community's functional potential. Complementing this, 2) a read-centric k-mer profiling method (Kraken 2 / Bracken) was applied for rapid and sensitive taxonomic classification of individual sequencing reads. This second approach provided high-resolution taxonomic assignments, excelling at species-level differentiation and capturing overall community structure efficiently, including organisms potentially missed by assembly. While the gene- centric method offers functional context, it can be computationally demanding and less comprehensive taxonomically; conversely, k-mer profiling delivers taxonomic depth rapidly but lacks inherent functional data. By integrating these approaches, we harnessed the functional insights of assembly-based analysis with the taxonomic resolution and coverage of read-based classification, achieving a more robust and holistic understanding of the gut microbial and viral landscape than either method could yield in isolation. Given the high number of species from several different biospecimens / tissues, we were only able to analyze a limited number of taxa (i.e., the top 20 viruses, bacteria, fungi and archaea) across the human specimen collection, although an exhaustive and holistic analysis of the microbiome presented here is beyond the scope of this pilot study. The precise moment at which the microbiome colonizes the oral cavity after birth is still under debate (94). However, it is well established that the child’s early oral microbiome profile is dependent on the mode of delivery (i.e. vaginal vs. C-section) (94). A few days after delivery, these differences seem to shift and resemble more of the mother’s oral microbiome. In agreement with these findings, our analysis of mother-child dyad saliva samples showed that the child’s oral bacteriome transitions away from that of the mothers at 4-months post-delivery, while the virome shifted away at 9-months post-delivery compared to the mothers; likely as a result of exposure to different diets and introduction to daycare (3,89). In terms of the primary dentition, the literature has primarily shown that the salivary microbiome has significant higher alpha diversity compared to predentate infants, and to mixed and permanent dentition, with higher dominance by streptococci species (95). In contrast, our study here is the first report profiling of the microbiome / virome found within pulpal and dentinal tissues retrieved from the inner pulp chamber surfaces of exfoliated primary teeth. We found that the microbiome was dominated by E. coli, Lautropia mirabilis and Aggregetibacter aphrophilus, while the virome was dominated by bacteriophages and caudoviricetes species. Additional studies with larger cohorts on the microbiome of the primary dentition and pulpal and dentinal tissues are needed to further validate these findings. The salivary microbiome reflects the composition of bacteria shed from oral sites and circadian oscillations have been previously documented (96). The salivary microbiome / bacteriome of individuals with periodontal disease has been extensively studied and compared to healthy controls using PCR-based methods and 16S rRNA sequencing. However, only a few studies have used Next Generation Sequencing (NGS) to characterize the salivary microbiota in periodontal disease patients (96) and limited studies have examined the virome of periodontal disease. Lundmark et al. (97) found increased levels of T. forsythia, Filifactor alocis and Parvimonas micra in saliva from patients with periodontal disease compared to healthy controls, while Ly et al. (29) found significant differences in Firmicutes, Actinobacteria, and Bacteroidetes phages in saliva from patients with periodontal disease compared to healthy controls. Similarly, our study found higher counts of streptococci phages in saliva derived from patients with periodontal disease compared to healthy controls. However, we also found higher counts of Neisseria subflava and Hemophilus parainfluenza bacterial species in saliva samples from patients with periodontal disease compared to healthy patients. Further studies are needed to validate how these salivary virome signatures impact and influence periodontal disease. The gut microbiome is comprised of a myriad of microbial species and gut microbiome dysbiosis has been correlated to IBS. Although some differences have been reported in the gut microbiome / bacteriome of IBS patients compared to healthy ones, a distinct signature has yet to be defined (98). A meta-analysis of 23 IBS studies involving more than 1,300 subjects found lower levels of Lactobacillus and Bifidobacterium, and higher levels of E. coli and Enterobacter in subjects diagnosed with IBS compared to healthy ones (99). In terms of the gut virome, we found lower counts of Canjivirus communis and higher levels of tupanviruses in IBS patients compared to healthy subjects. Further studies are needed validate this gut virome signature of IBS and its influence on patient care and disease outcomes. Turning to brain microbiome, we found a unique and significant positive correlation between the presence of parvoviridae and AD severity in the occipital lobe of human brain autopsy specimens. These results were corroborated by using commercially available TaqMan-based primers and hydrolysis probes specific for Parvovirus B19, for not only confirming the results for the occipital lobe, but also in the frontal lobe of AD patients compared to healthy brains. TaqMan assays provide higher DNA amplification specificity than DNA binding dyes, such as SYBR Green, as only sequence-specific amplification is measured during PCR (100,101). However, additional samples from different sources / institutes are still necessary to validate these results. Sampling is a challenge, most notably with respect to brain tissues. We were unable to obtain age-matched hippocampus tissues from healthy individuals without a medical diagnosis of AD from both cohorts and further details from the NIH NeuroBioBank / University of Miami cohort, such as AD stage classification. Future work will require accessing healthy hippocampus tissues for further analyses. Parvovirus B19 has been previously associated with disease manifestations in the CNS, such as myalgic encephalomyelitis, progressive multifocal leukoencephalopathy, encephalitis, encephalopathy, or meningoencephalitis (102). To the best of our knowledge, this is the first report of a positive correlation between Parvovirus B19 presence in the brain tissues of AD patients, thus opening new opportunities for further research in the field, as well as potential implications and mechanisms of this virus with AD. progression At this time, there is no information regarding where the virus resides in the brain and its possible cellular targets and cell tropism effects in the brain (102). Epidemiological data on this topic continues to be heterogeneous and sparse (103). In this context, the role of Parvovirus B19 in neurological diseases, remains incompletely described and poorly understood (102). We hypothesize that Parvovirus B19 may target oligodendrocytes, causing demyelinization and neuroinflammation of the brain, followed by Tau phosphorylation entanglements and amyloid beta plaques formation (102,104), but this remains to be confirmed. Noteworthy, our study also corroborates the presence of E. coli in both healthy and AD brain samples. Previous studies reported the presence of E. coli’s DNA, LPS and pili proteins in the hippocampus and temporal lobes of AD patients (105,106). In particular, Zhan et al.(106) found E. coli’s DNA in 9 / 10 healthy control brain samples and in 9 / 13 of AD brain samples In our study, we found a significant decrease of E. coli levels in the occipital lobe from healthy individuals to severe AD patients. However, additional studies are still necessary to confirm the presence of this bacterium in AD patients. Our data also reveal a notable increase in the relative abundance of human endogenous retroviruses (HERVs), especially HERV-K and HERV-W, in AD patients compared to healthy controls. This finding is consistent with existing literature indicating that HERV reactivation may contribute to AD pathology through mechanisms such as neuroinflammation and tau hyperphosphorylation (107–109). Specifically, HERV-K expression has been shown to correlate with Toll-like receptor (TLR) expression in AD patients, suggesting a pathological link to neuroinflammation (110). In addition, the HERV-W envelope (Env) protein can activate TLR2 and TLR4, leading to NF-κB activation and proinflammatory cytokine secretion (111). It is worth noting that mouse models have demonstrated that antiretroviral therapy and the inhibition of reverse transcriptase significantly reverse microglial morphological activation and biological inflammation(112). Clinical studies have also indicated that antiretroviral therapies may reduce markers of neuroinflammation in mild cognitive impairment (113), underscoring the potential therapeutic value of targeting HERVs in AD. Additionally, aging (114) and certain microbial infections from herpesviruses (115), Herpesvirus 4 / EBV (116,117), SARS-CoV-2 (118), HIV (119), adenovirus (120) or Mycobacterium avium subspecies paratuberculosis (121), may further stimulate HERV activation, exacerbating disease progression. One of the biggest strengths of this study, namely the use of three different sequencing facilities and three different capture / enrichment techniques, may also be one of its limitations. Since each individual sequencing facility used unique techniques to capture the microbiome, our ability to perform reproducibility and cross-check analyses is limited. Checking for inter-laboratories reproducibility of next generation sequencing data, Pallarz et al. (122) concluded that reproducibility among independent external laboratories are high, given sufficient amount of reads are provided. For studies focusing on understanding and identifying human microbiome, the minimum requires number of reads is still debatable. For instance, Hillman et al. (123) recommends a minimum of 0.5 million reads per samples, but encourage readers to use 2 million reads per sample if budget allows, while Liu et al. (124) recommends a minimum of 15 million reads per sample for more stable species composition. In our report here, we obtained least 26 million raw reads per sample at each sequencing center. Another limitation in this study is the potential contamination of sequencing data with host DNA. To mitigate this, we evaluated potential impacts of an enzymatic host DNA depletion on the virome and compared the results of host-depleted against bulk DNA samples extracted from brain, stool and primary teeth. Overall, we found that sequencing using host-depleted DNA yielded a significant lower percentage of mappable reads for stool and teeth reads compared to bulk DNA and non-significant lower trends for brain samples, indicating a possible loss of overall virome read depth after host depletion, thus impacting further analysis. Notably, even after host- depletion (during extraction) and removing host reads (post-sequencing), we found significant higher HERV levels in AD patient samples compared to healthy ones. Indeed, this result may indicate higher HERV activation / expression in AD patients, given that: 1) sample genomic contamination is substantially minimized by the techniques employed in this study; and 2) previous studies have recently implicated chronic HERV activation to the progression of senescence, neurodegeneration and AD (110,114,125). One additional potential confounder in our study involves long-term sample storage (5 to 21 years), which could lead to degradation of the genetic material in the samples and overall loss of microbial diversity (126). To mitigate this issue, we employed MDA, a widely-used technology for amplifying limited input DNA amounts before sequencing(127–129). The advantages of MDA are higher fidelity and DNA yields compared to other thermal cycling techniques, such as PCR (127). Using MDA, we achieved enrichment of up to 173.5% and 13.7% in the number of species identified within the virome and bacteriome, respectively. However, MDA does show amplification biases and non-specific amplification when amplifying sub- nanogram amounts of template DNA (127,128). As a result, we observed average losses of 40.9% and 19.9% in species identification for the virome and bacteriome, respectively. Nonetheless, the MDA protocol can be further optimized to better accommodate such low-input samples and improve enrichment outcomes. In light of these challenges, methodological approaches utilizing MDA have expanded virome sequencing data from precious samples that have been stored for long periods of time. Conclusions These findings reveal distinct viral communities found in various body sites, including the brain, gut, and the oral cavity both in health and disease. Further, we found that Parvovirus B19 may be a potential new biomarker for AD. These data help validate existing and establish novel virome signatures for health from birth to adulthood and disease, especially periodontal disease, irritable bowel disease and AD. This study has also informed the field about helpful methodological approaches to expand virome sequencing data from precious samples that have been stored for long periods of time. Materials and Methods A summary of the tissues and methods described in this section can be found in Figure 1. Ethics Statement This study has been certified as exempt per 45 CFR 46.104 category 4. (University of California, Los Angeles IRB #24-000223 certified on March 5th, 2024). Sample Cohorts Unique Human Biospecimens from Healthy or Diseased Conditions We collected and analyzed biospecimens, as well as accompanying demographic and clinical data, from several unique human healthy and diseased cohorts to characterize the virome across the oral–gut–brain axis. As controversy exists over what constitutes health in humans (76–80), our operational definition of health was the absence of overt disease based on clinical / medical diagnosis. However, recognizing there are important social / ethical values that underlie the definition and conceptualization of “health,” as well as the use of the term “healthy human virome” in the singular rather than an appreciation of the wide heterogeneity that could exist within and among human individuals and groups (i.e., “healthspan”), we note the need for further consideration of this terminology. Primary Exfoliated Molar Teeth Biospecimens – Private Collection Primary exfoliated molar teeth were obtained from one of the authors who collected the specimens from family members that gave verbal consent for their use were gathered for the study. The desiccated teeth had been stored in separate containers for approximately 15-20 years and were garnered from three individuals (Individual A – N=3; Individual B – N=3; Individual C – N=4).From these specimens, pulpal and dentinal tissues were retrieved from the inner pulp chamber surface of the teeth using a dental drill on a slow speed setting and with a continuous circular motion. These tissues were then incubated in a 0.5M EDTA solution for decalcification overnight and subsequently processed by centrifugation for 5 minutes followed by extraction of total DNA, RNA, and depleted DNA with the QIAamp® DNA Mini kit (Qiagen, Netherlands), RNAeasy® Mini Kit (Qiagen, Netherlands) and HostZero Microbial DNA kit (Zymo Research, USA) respectively, and according to manufacturer’s instructions. Saliva Biospecimens – Longitudinal Biospecimens from Periodontal Disease Individuals Saliva samples collected from 2006 to 2009 as part of a single-center longitudinal study of patients with and without periodontal disease as previously described (81,82) were gathered for the study. Whole saliva was obtained from 47 adult patients with chronic periodontitis and 10 healthy controls. Clinical measurements, including probing depth, clinical attachment level, and radiographs were used to classify patients into healthy, mild and moderate chronic periodontitis. From this cohort, 5 healthy controls and 5 chronic periodontitis samples were selected for DNA and RNA extraction using QIAamp® DNA Mini kit (Qiagen, Netherlands) as specified by the manufacturer. Saliva Biospecimens – Longitudinal Biospecimens from Mother–Child Dyads Saliva samples collected as part of a longitudinal study Mother and Youth Access (MAYA) healthy cohort of mother–child dyads were gathered for the study. MAYA was a randomized clinical trial (U54 DE014251) conducted from 2002–2008 and aimed at reducing early childhood caries (ECC) among infants and toddlers in communities with high ECC rates, low socioeconomic status (SES), limited dental care access, and no optimally fluoridated municipal water (83,84). Data were collected at the San Ysidro Health Center, where 95% of patients are Latinx, 69% live below the federal poverty level, and 68% have caries, representing more than twice the national average (83,84). In total, 361 pregnant individuals in their second trimester with no pregnancy complications or antibiotic use were recruited; baseline dental exams, oral health counseling, and oral health questionnaires were administered. Saliva was sampled from all subjects at the prenatal visit. At 4 months postpartum, subjects returned with their babies for randomization; saliva was sampled from mother–child dyads at all after-birth visits (4-, 9-, 12-, 18-, 24-, 30-, 36-months). In total, 2,192 mother–child dyad saliva samples were collected. Five of these mother- child dyads were selected for the study, comprising pre-natal, 4- and 9-month post- delivery samples. Bulk DNA extraction was performed with the QIAamp® DNA Mini kit (Qiagen, Netherlands) according to manufacturer instructions. Stool Biospecimens - UCLA Goodman-Luskin Microbiome Center (GLMC) Biospecimen Bank Stool samples for collected at UCLA Health and stored at the UCLA Goodman- Luskin Microbiome Center (GLMC) Biospecimen Bank were gathered for the study. The GLMC Biospecimen Bank contains a total of 412 stools specimens from male and female patients of diverse backgrounds with a diagnosis of gastrointestinal (GI) health or GI diseases (irritable bowel syndrome (IBS), Irritable bowel disease (IBD) / ulcerative colitis / Crohn’s disease, celiac disease, chronic constipation, functional constipation, functional diarrhea, vulvodynia, colon cancer). Out of these, 5 stool samples from individuals with IBS and 5 stool samples from healthy controls without GI conditions were selected for the present analyses. Eligibility criteria for patients with IBS required them to be 18 years of age or older and to have a diagnosis according to the Rome III criteria (85). Patients were excluded if they had other GI diseases that could affect IBS symptoms, a history of cancer in the last five years, a GI infection reported within two weeks before evaluation, or if they had used antibiotics or probiotics that affect the gut in the previous 12 weeks. Healthy controls were individuals without any clinical diagnosis of major medical problems including but not limited to cardiovascular, pulmonary, GI, autoimmune, and psychiatric diseases. All healthy controls did not use prescription medications and did not use any probiotics or antibiotics within 3 months of their sample collection. DNA, RNA, and host-depleted DNA extraction for these specimens was performed with the QIAamp® PowerFecal® DNA Kit (Qiagen, Netherlands) and RNAeasy® Mini Kit (Qiagen, Netherlands), and HostZero Microbial DNA kit (Zymo Research, USA), respectively according to manufacturer’s instructions. Brain Biospecimens - UCLA Research Center Brain Bank Fresh frozen brain autopsy tissue specimens from individuals diagnosed with brain health or Alzheimer’s Disease (AD) collected and banked at the UCLA Research Center Brain Bank were gathered for the study. Brain tissues were obtained from subjects who underwent autopsy at UCLA between 1999-2019. Fresh sections were sampled from the frontal and occipital lobes and hippocampus and immediately frozen at -80°C. The remainder of the brain was fixed in 10% formalin and sampled according to the UCLA dementia autopsy protocol including specimen sections from the frontal, temporal, parietal, and occipital cortices, hippocampus, entorhinal cortex and amygdala, basal ganglia, brainstem, and cerebellum. Sections were assessed for neuropathologic changes of AD, amyloid plaques and neurofibrillary tangles, using standard diagnostic criteria (86–90). For this study, an initial cohort comprising N=4 healthy, N=3 moderate AD, and N=10 severe AD brain specimens from the occipital area of the brain were selected for both Twist Comprehensive Viral Research Panel and non-enriched whole-genome shotgun metagenomic sequencing. An amount of 150mg of each tissue was used for extraction of bulk DNA, RNA, and host-depleted DNA with the QIAamp® DNA Mini kit (Qiagen, Netherlands), RNAeasy® Mini Kit (Qiagen, Netherlands) and HostZero Microbial DNA kit (Zymo Research, USA) respectively, as recommended by the manufacturer. Bulk DNA, RNA, and host- depleted DNA were submitted for Metagenomic Sequencing using Twist Comprehensive Viral Research Panel (Baylor College of Medicine Alkek Center for Metagenomics and Microbiome Research), while only bulk DNA was submitted for Non-Enriched Whole-Genome Shotgun Metagenomic Sequencing (Novogene Corporation, US) – more details in the Sequencing Methods section. Next, a second cohort of brain samples comprised of frontal and hippocampus counterparts of the initial cohort (N=4 healthy, N=6 moderate AD, and N=20 severe AD) in addition to N=14 moderate and N=54 severe AD patients comprising the occipital, frontal and hippocampus regions. We did not have available tissue from the hippocampus area for healthy brain specimens. For these samples, an amount of 50mg of each tissue was used for extraction of bulk DNA using QIAamp® DNA Mini kit (Qiagen, Netherlands) as recommended by the manufacturer and submitted to Baylor College of Medicine Alkek Center for Metagenomics and Microbiome Research for Metagenomic Sequencing using Twist Comprehensive Viral Research Panel. A qPCR validation was also done in all of the first and second cohorts. In addition, 16 brain samples with unspecified severity of AD (labeled as AD Brains) were obtained from the NIH Neuro Biobank and used for qPCR validation of Parvovirus B19 levels. Sequencing Methods Metagenomic Sequencing using Twist Comprehensive Viral Research Panel Twist Comprehensive Viral Research Panel (Twist Biosciences, US) was used for unbiased enrichment of viral nucleic acids for DNA (both bulk and host-depleted) and RNA samples for Brain, Primary teeth and Stool samples. Viral capture, library preparation, quality control (QC) testing and next-generation sequencing were performed at the Baylor College of Medicine (BCM) Alkek Center for Metagenomics and Microbiome Research. Non-Enriched Whole-Genome Shotgun Metagenomic Sequencing and Multiple Displacement Amplification (MDA) Enrichment Samples were also sent to two different facilities for non-enriched whole-genome shotgun metagenomic sequencing. 1) Bulk DNA isolated from saliva (both periodontal disease and mother–child dyads cohorts) and stool samples were quantified and normalized for the library construction at the UCLA Technology Center for Genomics and Bioinformatics (TCGB). In addition to those samples, an aliquot of 25uL of N=3 mother-child dyads saliva samples with lower quality control (QC) levels were used for multiple displacement amplification (MDA) enrichement with the REPLI-g Single Cell Kit (Qiagen, US) following manufacturer’s guidelines. Metagenomic library preparation was conducted with KAPA HyperPrep Kits (Roche, US) according to the manufacturer’s protocol. Libraries were sequenced as a paired-end 300-cycle run on the Illumina NovaSeq X platform at expected sequencing reads at a depth of 100 million per sample. For each sample, the two FASTQ files specific for the forward and reverse paired-end reads were concatenated into one single FASTQ file that was subsequently used as input for our in-house whole genome sequencing and data analysis pipeline. Our in-house analysis pipeline utilized KneadData (version 0.6.1), a computational tool designed to perform quality control on metagenomic sequencing data. KneadData integrates the tools FastQC (version 0.11.9), Trimgalore (version 0.6.7), and Bowtie2 (version 2.4.2), to perform QC checks, quality filtering, and decontamination of host sequences, respectively. Raw reads were mapped against a set of reference genomes to quantify reads per organism. Kraken2 database (ccb.jhu.edu / software / kraken2 / ) was used for taxonomic classification of metagenomic sequencing data, and human reference genome (hg38) was utilized via the UCSC Genome Browser (genome.ucsc.edu) for alignment to the human genome. 2) Bulk DNA isolated from primary teeth and stool, and host-depleted DNA extracted from brain samples were quantified and sent to Novogene Corporation (USA) for library preparation and non-enriched whole-genome shotgun metagenomic sequencing. Upon arrival, samples were randomly sheared into short fragments and the obtained fragments were end-repaired, A-tailed and further ligated with Illumina (US) adapters. The fragments with adapters were ligated, PCR amplified, size selected, and purified. The resulting libraries were pooled and sequenced as paired-end, 300 cycle runs on the Illumina NovaSeq X Plus platform at depth of 100 million reads per sample. Next, Readfq was used for preprocessing raw data from the Illumina sequencing platform to obtain the clean data for subsequent analyses. Considering the possibility of host contamination in samples, the sequencing data were BLASTed using the host database via Bowtie2 (Version: 2.4.2) to filter out reads that may come from host origin before proceeding. Next, Megahit (v1.0.4) was used for metagenome assembly and Diamond (version 2.1.9) for alignment of Unigenes sequences with those of bacteria, fungi, archaea, and viruses extracted from NCBI's NR database taxonomy annotation. Finally, species were categorized into bacteria, fungi, archaea, and virus taxa using the Taxize package (v0.9.99) in R programming language (91). Real-Time Polymerase Chain Reaction (qPCR) Validation TaqMan-based real-time PCR (qPCR) was used for validating the presence of several viruses initially identified in the sequencing data: 1) the oral pathogens Treponema denticola (T.denticola), Fusobacterium nucleatum (F. nucleatum), Porphyromonas gingivalis (P. gingivalis) and Tannarella forsythia (T. forsythia); 2) Escherichia coli (E.coli), 3) human herpesvirus 4 (HHV4; also known as Epstein-Barr virus), 4) human herpesvirus 6a / 6b (HHV6a / 6b), and 5) human herpesvirus 7 (HHV7). Parvovirus B19, Torque Teno Virus (TTV) 10, TTV18, TTV24 and TTV29 was validated only in AD human brain specimens given the sequencing results. DNA was extracted from the specimens using the QIAamp® DNA Mini kit (Qiagen, Netherlands) according to the manufacturer’s instructions. qPCRs were performed using Taqman®primers and probes targeting the previously mentioned species in the QuantStudio 3 Real-time qPCR system (Applied Biosystems, USA). Results for all species, except the oral pathogens, were shown as reciprocal values of the obtained Ct values so that results are directly proportional to the viral and bacterial loads in the samples, given that no commercial DNA standard could be found for these species. For the oral pathogens, DNA standard curves were used to establish the copy numbers of each species, which are represented accordingly. Power Analysis Statistical Analysis Twist viral capture… Non-enriched shotgun whole-genome sequencing sequenced at UCLA TCGB… Non-enriched shotgun whole-genome sequencing sequenced at Novogene Corp. was evaluated via One-Way ANOVA, followed by post-hoc Dunnett’s multiple comparison test. Validation tests via qPCR were statistically analyzed via non- parametric Kruskal-Wallis test followed by the post-hoc Dunn’s multiple comparison test. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. CONCLUSION This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. References 1. Carroll D, Daszak P, Wolfe ND, Gao GF, Morel CM, Morzaria S, et al. The Global Virome Project. Science.2018 Feb 23;359(6378):872–4. 2. Jonas O, Seifman R. Do we need a Global Virome Project? Lancet Glob Health.2019 Oct;7(10):e1314–6. 3. Liang G, Bushman FD. The human virome: assembly, composition and host interactions. Nat Rev Microbiol.2021 Aug;19(8):514–27. 4. Martinez A, Kuraji R, Kapila YL. The human oral virome: Shedding light on the dark matter. 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[0002] Table 1. Top viral species of all the samples shown in Figure 2B. Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF

[0003] Sequence Species Genus Tissue Number of Average name detections RPKMF

[0004] Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF

[0005] Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF Sequence Species Genus Tissue Number of Average name detections RPKMF

[0006] Table 2: Parvovirus B19 Positivity Table 3: TTV10 Positivity

[0007] Table 4: TTV18 Positivity

[0008] Table 5: TTV24 Positivity

[0009] Table 6: TTV29 Positivity

Claims

CLAIMS:

1. A method of diagnosing moderate to severe Alzheimer’s (AD) in a subject, the method comprising: obtaining a brain tissue from the subject; observing the presence or absence of parvovirus B19 in the brain tissue, wherein the presence of parvovirus B19 in the brain tissue indicates a diagnosis of moderate or severe AD.

2. The method of claim 1, where the method is a qPCR method.

3. The method of claim 2, wherein parvovirus B19 is present in the brain tissue in amounts that are at least 2 fold higher than amounts of parvovirus B19 observed in brain tissues from an age-matched healthy control.

4. The method of claim 1, further comprising observing the presence or absence of one or more adeno-associated dependoparvovirus and / or Torque Teno Virus, wherein the one or more adeno-associated dependoparvovirus and / or Torque Teno Virus is selected to be indicative of brain health.

5. The method of claim 1, wherein the brain tissues is from an occipital or a frontal lobe.

6. A system for diagnosing moderate to severe AD, the system comprising brain tissue from a subject combined with a detection agent selected to bind a parvovirus B19 polypeptide or polynucleotide present in the sample.

7. The system of claim 6, wherein the detection agent comprises a polynucleotide probe that hybridizes with parvovirus B19 polynucleotides in the sample.

8. The system of claim 6, further comprising a detection agent selected to bind an adeno-associated dependoparvovirus and / or a Torque Teno Virus present in the sample.

9. The system of claim 6, wherein the brain tissues is from an occipital or a frontal lobe.

10. A method of diagnosing irritable bowel disease (IBD) in a subject, the method comprising: obtaining a fecal sample from the subject; observing the presence or absence of tupanvirus and / or moumouvirus and / or streptococcus in the fecal sample, wherein the presence of tupanvirus in the brain tissue indicates a diagnosis of IBD.

11. The method of claim 10, where the method is a qPCR method.

12. The method of claim 11, wherein virus is present in the brain tissue in amounts that are at least two fold higher than amounts of virus observed in fecal samples from an age-matched healthy control.

13. The method of claim 11, further comprising observing the presence or absence of multiple virus species.

14. The method of claim 11, wherein the method comprises metagenomic Sequencing using a Twist Comprehensive Viral Research Panel.

15. A system for diagnosing irritable bowel disease (IBD), the system comprising a fecal sample from a subject combined with a detection agent selected to bind a tupanvirus polypeptide or polynucleotide present in the sample.

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