Diet can influence the virome indirectly, by modulating the bacteriome and thereby altering phage composition, and directly, by activating or inhibiting bacteriophage functions (Howard et al., 2024). In a mouse model of obesity, a high-fat/high-sucrose diet enriched temperate Caudoviricetes phages in the mucosa compared to the lumen and eliminated spatial distinctions between compartments (Kim and Jin, 2016). A high-fat diet alone reduced Siphovirus-like morphotype and increased Microviridae, with viral communities showing greater virulence toward bacterial hosts (Schulfer et al., 2020). Among direct dietary effects, prophage modulation is particularly relevant. Fructose metabolism by Lactobacilli produces acetic acid, which triggers prophage activation (Mahmoud et al., 2025). Boling and colleagues (Boling et al., 2020) screened common foods and identified several prophages inducers in Bacteroides thetaiotaomicron and Enterococcus faecalis, including aspartame, stevia, and bee propolis. Conversely, certain substances suppress prophage activation (e.g., cinnamon, oregano, and specific flavonoids) or reduce phage infectivity (e.g., tea extracts, cranberry juice, and ascorbic acid) (Marongiu et al., 2021). At the population level, virome variation reflects region-specific diets and other environmental conditions associated with geographical location, including urbanization-related lifestyle factors. In a large Chinese cohort, geography was the primary driver of gut DNA virome composition, exerting a stronger influence than the bacterial microbiome; urban residents showed reduced virome diversity, potentially increasing susceptibility to chronic inflammatory diseases (Zuo et al., 2020). Much of this effect is attributed to population-specific dietary patterns, which, together with host factors such as age, sex, and ethnicity, shape distinct viral signatures. Comparative studies confirm that viral species correlate with regionally defined diets, subsistence strategies, and lifestyles, with hunter-gatherer displaying higher virome diversity than urban populations (Rampelli et al., 2017). Population-specific viral markers have also been identified: Torque teno midi virus (TTMDV) 2 and HHV7 in the Hadza, Betapapillomavirus NC015692.1 and TTMDV2 in the Matses, Human papillomavirus type 178 in Italians, and HHV 8 in the US population. A dedicated mention is warranted for probiotics, which can be integrated into the daily diet. Lactobacillus and Bifidobacteria are among the most abundant and well-studied genera. They counteract pathobionts colonization through antibiotic-like compounds, ecological competition, immune stimulation, and reinforcement of intestinal mucosal integrity. By modulating bacterial communities, probiotic may also influence viral replication niches, phage–bacteria dynamics, and overall gut ecosystem homeostasis (see Vitetta et al., 2018 for review).

Host genetics influence gut virome composition primarily through modulation of immune responses and barrier integrity. Polymorphisms in immunity-related genes can alter viral recognition and susceptibility (Govender and Ghai, 2025), while physical and chemical barriers, such as the intestinal mucosa, regulate viral access and colonization (Buhner et al., 2006; Moehle et al., 2006; Muise et al., 2009). Host-encoded restriction factors, including APOBEC3 cytidine deaminases, shape viral evolution and persistence in a tissue-specific manner; for example, APOBEC3B expression in the colon is linked to mutational biases in parvovirus B19 (Pyöriä et al., 2024). Genetic background also affects genomic stability and chromatin architecture, influencing viral genome integration even in non-cancerous tissues for EBV, HHV-6B, and Merkel cell polyomavirus (Pyöriä et al., 2024). Beyond these mechanisms, ethnicity and heritable factors broadly shape virome diversity and functional potential, as shown in comparisons between Chinese and Pakistani populations, where virome profiles also co-varied with bacterial microbiota (Yan et al., 2021).

Bacteriophages modulate bacterial communities through transposition, induction, and horizontal gene transfer (Keen and Dantas, 2018). Specific Caudoviricetes show positive associations with lactic acid bacteria and negative correlations with Bacteroides (Mayneris-Perxachs et al., 2022), whereas Microviridae display the opposite pattern. Age- and species-specific co-occurrence patterns were demonstrated in Macaca fascicularis, linking classical phage families (Myovirus-like morphology, Siphovirus-like morphology, Podovirus-like morphology) to core bacterial phyla across life stages (Tan et al., 2021). In disease contexts, enrichment of these phage groups correlates with dysbiosis-associated taxa in chronic kidney disease, implicating viral contributions to disease progression (Zhang et al., 2025).

Broad-spectrum antibiotics disrupt bacterial hosts and can induce temperate phages (Ubeda et al., 2005; Misson et al., 2023), while phage-encoded antibiotic resistance genes facilitate the spread of resistance within the microbiome (Pfeifer et al., 2021), increasing virulence and resistance gene load (Chopyk et al., 2023). Antiviral treatments such as antiretrovirals reduce target virus loads and reshape gut viral diversity (Bhagchandani et al., 2024; Liang and Bushman, 2021). Immunosuppressants promote reactivation of CMV, BK virus, EBV, HHV-6A, and alter levels of immune-sensitive commensal viruses such as Anelloviridae (Fujimoto et al., 2020; Giacconi et al., 2020; Rosiewicz et al., 2024). Chemotherapy exerts cytotoxic and immunosuppressive effects promoting the expansion of latent or opportunistic viruses (Henze et al., 2022; Opzoomer et al., 2019; Yang et al., 2024).

The impact of smoking on the gut virome remains largely unexplored, but evidence from other mucosal sites underscores its relevance. Lung virome analyses show significant differences between smokers and nonsmokers: although 29% of viral populations were shared, several phage taxa varied markedly. Prevotella phages were more abundant in smokers, whereas Lactobacillus and Gardnerella phages were more prevalent in nonsmokers. Rare phage populations also showed group specificity (Gregory et al., 2018). Preliminary gut data indicate smoking-associated shifts in virome composition and functional potential, including altered abundance of vOTUs enriched in auxiliary metabolic genes (Istvan et al., 2024).

Persistent viral antigens-derived from chronic herpesvirus infections, reactivated latent viruses, and lifelong exposure to commensal viruses such as Anelloviridae, contribute to chronic stimulation of innate and adaptive immunity. This sustained antigenic load drives expansion of terminally differentiated CD8⁺ T cells, contraction of the naïve T cell pool, and impaired antiviral responses (Rodriguez and Parra-Lopez, 2024; Hu et al., 2025; Mangiaterra et al., 2024). The progressive rise of TTV viremia with age (Giacconi et al., 2020; Giacconi et al., 2018) exemplifies how reduced immune control enables viral persistence and may serve as a readout of immune exhaustion as observed in other chronic infections (Eslami et al., 2025; Hu et al., 2025). These processes converge on core Geroscience mechanisms including loss of proteostasis, impaired intercellular communication, and dysregulated immunity (Mittelbrunn and Kroemer, 2021; Fortunato et al., 2025).

Inflammaging creates a pro-oxidant, cytokine-rich environment that favors prophage induction in gut bacteria (Mahmoud et al., 2025). Prophages can naturally shape gut bacterial populations and, when activated, contribute to the loss of beneficial intestinal symbionts, thereby worsening dysbiosis (Nanda et al., 2015). Increased lytic activation releases bacterial cell-wall components, toxins, and immunostimulatory DNA, amplifying mucosal and systemic inflammation (Mahmoud et al., 2025). Prophage induction also results in the production of numerous phage particles capable of penetrating the intestinal epithelial layer (Nguyen et al., 2018). Once across the barrier, intestinal phages can directly interact with host immune cells, enhancing the production of chemokines and cytokines (Seo and Kweon, 2019; Federici et al., 2021). This self-reinforcing loop-chronic inflammation promoting prophage activity, which in turn fuels inflammation, links virome dynamics to age-associated barrier dysfunction, metabolic dysregulation, and immune remodeling.

Phages modulate bacterial networks involved in essential metabolic pathways such as short-chain fatty acid (SCFA) production, sulfur metabolism, and vitamin biosynthesis (Jessen and Bui, 2025). Auxiliary metabolic genes encoded by bacteriophages, particularly those enriched in centenarians, can enhance microbial metabolic flexibility and resilience (Johansen et al., 2023). Conversely, virome-driven shifts in Lactococcus or SCFA-producing taxa may impair epithelial integrity, mitochondrial metabolism, and neuroimmune communication, (Tetz et al., 2018; Yadav et al., 2022), contributing to metabolic reprogramming a central mechanism of aging.

Distinct viral communities, most notably the circulating virome, mirror the functional state of the immune system and correlate with biological age, frailty, and mortality risk (Kananen et al., 2015; Giacconi et al., 2023; Cianfruglia et al., 2025). Viral reactivation patterns (e.g., herpesvirus, human endogenous retroviruses (HERVs), torque teno virus, TTV) further track with epigenetic drift, cellular senescence, and loss of homeostatic regulation (Pantry and Medveczky, 2017; Noppert et al., 2020; Mao et al., 2024; Fortunato et al., 2025). These signatures support the concept that the virome is both a sensor and a potential driver of aging trajectories (Momkus et al., 2025; Fortunato et al., 2025; Johansen et al., 2023; Cianfruglia et al., 2025).

Together, these domains outline a mechanistic model wherein the virome interacts dynamically with the immune system, microbial metabolism, epithelial barriers, and systemic inflammation, contributing to the trajectory of aging and age-related disease. This conceptual synthesis integrates the gut and circulating virome into the Geroscience paradigm, highlighting both causative pathways and biomarker potential while underscoring the need for longitudinal mechanistic studies.

Although less studied than the bacteriome, the virome is increasingly implicated in Alzheimer’s disease (AD) and Parkinson’s disease (PD). In AD, metagenomics revealed reduced phage richness, with a 9% decrease in Uroviricota and Siphovirus-like morphology and an 11% increase in Poxviridae; several Lactococcus phages also declined, potentially affecting bacterial diversity and lactate metabolism (Ghorbani et al., 2023). In mild cognitive impairment (MCI), Lactobacillus phages Sha1 and LF1, Klebsiella virus KP36, and Lactobacillus phage blL309 were enriched compared to controls, while specific phages (e.g., Clostridium phage vBC peS CP51, Lactococcus phages jm3 and ul36) showed moderate diagnostic accuracy (54%–58%) and were more abundant in individuals with more severe cognitive deficits (Chaudhari et al., 2023). Kazen et al. (2025) identified 28 phages exclusive to amnestic MCI due to AD and 8 exclusives to healthy controls, with abundance shifts linked to disruptions in viral-encoded metabolic pathways (ATP/ADP synthesis from inosine monophosphate, propionyl-CoA metabolism, and methanogenesis) and increased lysogenic phages. In PD, Bedarf et al. (2017) observed reduced bacteriophages and archaeal phages versus controls, with no differences in prophage and plasmid content; this was confirmed in a spouse-controlled cohort to reduce lifestyle confounding (Mao et al., 2021). Conversely, Duru et al. (2025) found reduced plasmid alpha diversity, significant beta diversity differences, enrichment of Microviridae and Tectiviridae, and increases in phages infecting Bifidobacterium and Ruthenibacterium. Classification models moderately distinguished PD from controls using plasmid profiles (AUC = 0.661) and improved using phage profiles (AUC = 0.746). Overall, phages remain the most influential virome component in shaping microbiota due to diverse bacterial interactions (Figure 3) (Tisza and Buck, 2021).

Beyond correlative associations, mechanistic links have been proposed. Declines or lifestyle-driven shifts in Lactococcus phages may alter Lactococcus populations and disrupt the balance between L- and D-lactic acid. L-lactate supports neuronal metabolism, memory, protein synthesis, synaptic remodeling, and axonal excitability, whereas D-lactate can impair neuronal uptake of L-lactate and hinder function (Ghorbani et al., 2023). In PD, a predominance of strictly lytic Lactococcus phages, despite stable overall abundance, was linked to a >10-fold reduction in Lactococcus species, potentially lowering microbiota-derived dopamine and increasing gut permeability. These changes may contribute to gastrointestinal symptoms, systemic inflammation, and the initiation of neurodegenerative processes (Tetz et al., 2018). Aggregates of α-synuclein in the enteric nervous system and evidence of gut-to-brain propagation via the vagus nerve further support a mechanistic bridge between virome-driven dysbiosis and central pathology (Braak et al., 2006; Kim et al., 2019).

Longitudinal monitoring in a murine AD model showed a simplified viral ecological network, stable alpha diversity, and increased beta diversity over time compared with wild-type controls. Virome composition shifted dynamically, with elevated Lahndsivirus rarus at 3 months and increased Lactobacillus prophages Lj771, KC5a, and phi Jlb1 at 6 months, paralleling disease progression (Zhang et al., 2024).

In older adults (60–69, 70–79, ≥80 years), progressive virome changes were linked to metabolic alterations, including increased activity in tricarboxylic acid cycle VI and NAD salvage pathway III, reduced pyruvate fermentation, and associations with cognitive status. Cognitively impaired individuals showed fewer viral species and distinct virome composition, with higher Myovirus-like morphology and Podovirus-like morphology, lower Siphovirus-like morphology and Inoviridae, and presence of Tectiviridae (absent in controls). They also exhibited altered phage–bacteria–metabolism interactions, notably reduced anaerobic sucrose degradation consistent with depletion of carbohydrate-metabolizing bacteria, potentially contributing to neuroinflammation (James et al., 2024).

CMV is an ubiquitous β-herpesvirus that elicits strong, lifelong T-cell responses characterized by age-dependent memory inflation and immune evasion (Jergovic et al., 2019).

CMV is considered a major driver of immunosenescence, inducing clonal expansion of highly differentiated, senescent CD8⁺ T cells at the expense of the naïve T-cell pool, thereby reducing immune repertoire diversity (Rodriguez and Parra-Lopez, 2024). These CD8⁺ subsets express senescence markers (CD57, KLRG1) and display diminished effector capacity (Pickering et al., 2025). Longitudinal studies show that CMV-seropositive older adults frequently exhibit the immune risk profile, marked by low CD4/CD8 ratio, expanded CD8⁺ CD28^- T cells, and chronic inflammation, correlating with frailty, poor vaccine responsiveness, and increased mortality (Rodriguez and Parra-Lopez, 2024). These immune alterations fuel inflammaging and are associated with cardiovascular, oncological, and neurodegenerative diseases, underscoring CMV as a central determinant of the biology of aging (Weltevrede et al., 2016).

HHV-6 comprises two strains: HHV-6A and HHV-6B. While HHV-6B causes roseola and is linked to severe neurological conditions and transplant failure (Jaworska et al., 2010), the role of HHV-6A is less understood. However, HHV-6A has been detected at higher levels during relapses in a subset of relapsing-remitting MS patients (Álvarez-Lafuente et al., 2006) and, along with HHV-7, shows increased levels in multiple brain regions of AD patients (Readhead et al., 2018).

At the mechanistic level, HHV-6 reactivation may be facilitated by age-related immune decline and epigenetic drift. Viral genome integration into host telomeres, a hallmark of HHV-6 biology, has been linked to genomic instability, telomere dysfunction, and premature cellular senescence (Arbuckle et al., 2010; Pantry and Medveczky, 2017), potentially contributing to accelerated aging phenotypes. Moreover, chronic low-level HHV-6/7 activity has been implicated in persistent activation of innate immune pathways, including type I interferon signaling, fueling inflammaging. In neurodegeneration, HHV-6A reactivation has been correlated with enhanced amyloid precursor protein processing and neuroinflammatory cascades in AD brain tissue (Readhead et al., 2018). Overall, HHV-6/7 may represent underrecognized viral pathobionts linking aging, immunity, and neurodegeneration, though further research is needed to determine whether they act as causal drivers or bystanders.

EBV infects nearly the entire global population and establishes latency primarily in B lymphocytes, though it can also infect epithelial cells (Smatti et al., 2018). Most infections remain clinically silent due to effective immune control, although some individuals develop infectious mononucleosis or EBV-driven malignancies. Lifelong latency in memory B cells allows viral persistence, with subclinical reactivations fueling chronic inflammation and overt reactivation triggering lymphoproliferative disorders, autoimmune flares, or frailty in older adults (Hatton et al., 2014; Suzich and Cliffe, 2018; Noronha et al., 2021; Gadoth et al., 2024).

In older adults, EBV-driven antigenic pressure contributes to immune exhaustion, with expansion of dysfunctional T and NK cells expressing inhibitory checkpoints (Mangiaterra et al., 2024; Hu et al., 2025). This impairs both antiviral and tumor immune surveillance, consistent with the higher incidence of EBV-related malignancies in aging. Furthermore, EBV has been linked to late-onset autoimmunity, with evidence associating it with rheumatoid arthritis (Miljanovic et al., 2023) and MS in elderly cohorts (Bjornevik et al., 2023), highlighting EBV as a contributor to immune dysregulation in aging, extending its relevance beyond oncogenesis.

HSV-1 and HSV-2 are widespread, infecting approximately 64% and 13% of the global population, respectively (Birkmann and Saunders, 2025). These neurotropic viruses establish lifelong latency in sensory ganglia (Bello-Morales et al., 2018; Quadiri et al., 2024). HSV-1 is mainly linked to orolabial lesions and encephalitis, while HSV-2 is associated with genital infections (Hou et al., 2017). In older adults, latency becomes increasingly destabilized: impaired type I interferon responses, suboptimal dendritic cell function, and reduced virus-specific CD8⁺ T cell activity contribute to more frequent and severe reactivations (Srivastava et al., 2025).

Epidemiological and mechanistic studies further link HSV-1 reactivation to neurodegeneration, with recurrent viral episodes driving β-amyloid deposition, tau hyperphosphorylation, and neuroinflammation, thereby implicating HSV-1 as a potential pathobiont in AD within the broader framework of the aging virome (Protto et al., 2024; Cairns et al., 2025).

VZV, the etiological agent of herpes zoster (HZ), establishes lifelong latency in sensory ganglia after primary infection in childhood (Zerboni et al., 2014). Age-related immune decline can trigger reactivation, leading to HZ, which is associated with prolonged morbidity, including post-herpetic neuralgia, sensory deficits, and increased risk of stroke and myocardial infarction (Zhang et al., 2017; Curran et al., 2023; Bakradze et al., 2019). In older adults, T cell–mediated immunity against VZV declines, with expansion of senescent and exhausted VZV-specific CD4⁺ and CD8⁺ memory T cells despite preserved interferon-γ responses (Mangmee et al., 2025). HZ incidence rises with age, reflecting progressive loss of immune surveillance (Muñoz-Quiles et al., 2020). Vaccination with the recombinant zoster vaccine (RZV, Shingrix) provides durable protection, approximately 80% up to 10 years and ∼73% in adults ≥70 years, by sustaining VZV-specific T cell immunity and reducing subclinical viral reactivation (Strezova et al., 2022, 2025; Williams et al., 2025). Beyond preventing HZ, RZV exemplifies a Geroscience-based approach, mitigating chronic antigenic stimulation and inflammaging while enhancing immune resilience in aging populations.

Herpesvirus latency is governed by epigenetic mechanisms that progressively weaken with age. In HSV-1, the viral genome is maintained in heterochromatin (H3K9me3, H3K27me3); however, age-related oxidative stress and the decline of interferon responses compromise this silencing, while neuronal miRNAs regulating latency (e.g., miR-9) also become dysregulated (Messer et al., 2015; Tsai et al., 2022; Deng et al., 2024). In CMV, repression of the major immediate-early promoter by CCCTC-binding factor and bromodomain and extra-terminal domain proteins is relaxed during myeloid differentiation or under inflammatory conditions, facilitating viral reactivation (Groves et al., 2021; Groves et al., 2024). EBV latency programs rely on 3D chromatin topology and viral cofactors such as EBNA-LP, which are perturbed by age-related epigenetic drift, promoting oncogenic gene expression (Caruso et al., 2023; Lieberman and Tempera, 2025). In VZV, latency is maintained by minimal transcription of the VZV latency-associated transcript (VLT)/VLT-ORF63 under tight epigenetic control, which becomes less stable with age, increasing the likelihood of reactivation (Ouwendijk et al., 2020).

Herpesviruses are emerging as key targets of Geroscience-oriented interventions. The RZV remains the most successful example, providing durable protection against HZ and postherpetic neuralgia, while reducing inflammation from VZV reactivation (Strezova et al., 2022; Williams et al., 2025). For CMV, antivirals such as Letermovir and Maribavir have demonstrated reduced toxicity in frail populations (Shigle et al., 2020; Walti et al., 2023). EBV vaccine candidates (e.g., mRNA-1189) aim to prevent infectious mononucleosis and long-term immune complications, while HSV-1 latency-targeted approaches, including CRISPR-based genome editing and epigenetic inhibitors, are under investigation to reduce latent genomes or prevent viral reactivation (Amrani et al., 2024; Aubert et al., 2024).

Taken together, these findings demonstrate that, in older adults, viral pathobionts, including endogenous retroelements and herpesviruses, can profoundly influence aging, by promoting persistent immune activation, inflammaging, and immunosenescence. Understanding the molecular and epigenetic mechanisms that govern their latency and developing strategies to prevent reactivation in the setting of age-related immune decline, represent critical opportunities to foster healthier and more resilient aging trajectories.

The NGS technologies have prompted metagenomic studies over the last two decades, greatly increasing our understanding of how microorganisms influence human physiology and disease. Although most of the studies to date focused their attention on the bacterial counterpart, accumulating evidence indicates that the virome also contributes to human health and disease. However, it should be underlined that virome research poses distinct analytical challenges that must be considered during both study design and data analysis. Bacterial communities can be profiled through 16S rRNA amplicon sequencing, a simple and cost-effective approach that has generated large datasets and facilitated extensive characterization of the bacterial microbiome. In contrast, viruses lack a universal phylogenetic marker and require large-scale metagenomic approaches, which increase sequencing time and costs (Garmaeva et al., 2019; Sandybayev et al., 2022). Pre-analytical steps (collection, transport and storage) are critical to avoid contamination, an issue that is especially acute for low-biomass samples such as those used to profile viral communities. Contaminant DNA and cross-contamination can profoundly bias results; best practices and checklists have been proposed to mitigate these risks (Eisenhofer et al., 2019). In addition, DNase treatment, commonly used to remove non-encapsidated DNA, can inadvertently degrade viral genomes that are not fully protected within stable capsids, thereby introducing further bias in virome preparation. Once carefully collected, samples should be processed immediately for DNA extraction or, alternatively, frozen on dry ice and stored at −80 °C. This procedure helps preserve, and effectively “lock”, the microbial communities and their relative abundances at the time of collection; otherwise, ongoing microbial interactions may alter the sample composition, making it no longer representative of the in vivo state (Shkoporov et al., 2018).

Nucleic acid extraction represents another critical step. Viral communities can be characterized either through shotgun metagenomic approaches, which rely on the extraction of total DNA from the sample, or by focusing on isolated viral particles. Whole metagenomes are dominated by bacterial sequences, with viral reads often representing only a minor fraction, which explains why enrichment strategies are frequently required to obtain sufficient viral signal for virome sequencing. Approaches targeting isolated viral particles rely on enrichment procedures, such as ultracentrifugation or chemical precipitation, each of which introduces specific methodological biases (Manrique et al., 2016; Ramirez-Martinez et al., 2018; Mirzaei et al., 2021).

Another class of viral contaminants from laboratories environment are known as “kitome” and derive from spin-columns and other extraction kit reagents (Sabatier et al., 2020). These reagents-derived reads can significantly alter taxonomic profiling results. Regardless of these methodological differences, enrichment of viral particles offers the advantage of specifically targeting the viral fraction, thereby reducing the need for deep sequencing (with a positive impact on costs) and simplifying downstream bioinformatic analyses. However, this approach often involves multiple time-consuming steps, and the final yield may be insufficient for subsequent library preparation (Garmaeva et al., 2019). Phages typically represent the main component of virome studies. When total metagenomic DNA is extracted, however, the resulting dataset is overwhelmingly dominated by bacterial sequences, and viral reads often represent only a very small fraction. For this reason, most virome studies rely on enrichment approaches targeting viral particles, rather than on whole-metagenome sequencing, to obtain a sufficient viral signal for meaningful characterization. Nevertheless, most virome studies to date have relied on DNA-based approaches, potentially underestimating the role of RNA viruses. Similarly, single-stranded DNA viruses are often underrepresented due to challenges in their isolation and amplification biases (Roux et al., 2016). Genomic library preparation can be particularly challenging for low-biomass samples, such as those obtained from viral particle enrichments. In these cases, whole-genome amplification may be employed to increase yield, although this approach can introduce methodological biases (Garmaeva et al., 2019; Wang et al., 2023). For RNA and ssDNA viruses, additional steps are often required and should be optimized according to the sample type and the specific objectives of the study. Finally, sequencing depth must be carefully evaluated to ensure sufficient coverage for a comprehensive characterization of the metagenome and for the assembly of the highly diverse and often low-abundance viral fractions. Adequate depth is essential to recover complete or near-complete viral genomes, including its less abundant viral fractions. Emerging long-read sequencing technologies may help overcome current limitations by reducing assembly biases, while direct RNA sequencing approaches—by bypassing cDNA synthesis and its associated biases—hold promise for more accurate investigation of RNA viruses (Smith et al., 2022). The gut virome is extremely diverse, encompassing a wide range of viral morphologies, genome types, and sequence content. This diversity is a major driver of methodological biases, and no single approach can fully capture the virome, highlighting the need for complementary strategies to achieve a more complete characterization. In addition, it is important to acknowledge that enrichment-based approaches themselves introduce biases. Methods that isolate viral particles often select viruses based on physical characteristics such as size, capsid stability, or density, potentially underrepresenting or excluding certain viral taxa (Parras-Molto and Lopez-Bueno, 2018). Moreover, virome sequencing typically captures extracellular viral particles and thus does not account for integrated prophages, which are also poorly characterized in metagenomes and represent a particularly challenging component of the virome to study.

Taken together, these pitfalls highlight why, despite remarkable advances in sequencing technologies, our understanding of the human virome still lags far behind that of the bacterial microbiome. Overcoming these challenges will require not only technical improvements but also careful consideration of how methodological choices at every stage can profoundly influence biological interpretations.

While many of the findings discussed in previous sections are derived from metagenomic analyses, current virome databases remain incomplete, biased, and lack standardized protocols for data processing and interpretation. This limits the resolution with which we can explore the diversity, function, and host interactions of viral communities, particularly in aging populations where subtle shifts in virome composition may have significant biological consequences. These issues are exacerbated by the extreme sequence diversity of viruses and by the limited sampling available across ecosystems, which makes it difficult to compare viral sequences across studies and highlights the lack of a centralized, comprehensive viral database.

Recent studies have emphasized the substantial difficulties involved in detecting viral sequences within complex, mixed-community datasets, a task that remains notoriously problematic (Gregory et al., 2020). Major obstacles include the lack of standardized analytical pipelines, inconsistencies in sample processing, the vast underrepresentation of viral genomes in reference databases, the limited availability of culturable host microbes, and pronounced inter-individual variability. These challenges are even more pronounced for phage-specific databases, which remain particularly sparse, poorly curated, and less frequently updated. Together, these factors contribute to a fragmented and often incomplete picture of the human virome and its contribution to health and disease during aging. In addition, many predicted viral ORFs encode hypothetical proteins, making functional inference particularly challenging due to the scarcity of homologs in current reference databases.

A further complication arises from database misclassification. In 2022, Chen et al. (2022) identified about 2 million exogenous sequences annotated as viral sequences in the Reference Sequence database, GenBank, and non-redundant databases. Authors found misclassified entries deriving from host genomic fragments, bacterial DNA and artificially designed vectors. Among laboratory-derived contaminants, they detected sequences containing functional cassettes originating from Simian Virus 40, CMV promoters and retroviral gag (group-specific antigen) and polymerase elements.

To prevent false positives in viromic profiles curated reference resources such as the Eukaryotic Viral Reference Database (EVRD) should be preferred. Alternatively, potential contaminants can be removed using additional controls, such as reference vector databases or tools like UniVec (The UniVec Database) and VecScreen (Schäffer et al., 2018).

Finally, even high-quality reference databases must contend with the lack of sequences from understudied species and with ongoing revisions in viral taxonomy by the International Committee on Taxonomy of Viruses. For example, the ICTV Master Species List release of 2021 updated the Orthonairoviridae family with 26 new species and renamed numerous species in pre-existing vOTUs (Relich and Loeffelholz, 2023). Moreover, the 2025 ICTV update introduced major revisions to phage taxonomy, further emphasizing the instability of current viral classification frameworks. In addition, phage–host linkage cannot be directly inferred and requires additional analyses (e.g., CRISPR spacers, co-abundance signals), which may provide limited or no results. To address both limitations, modern bioinformatics pipelines usually consider both reference-based classifiers and reference-free machine-learning approaches. Kraken, a taxonomic classifier for genomic and metagenomic sequences, relies on exact matches of k-mers (subsequences of length k from DNA or RNA sequences) against large nucleotide repositories to rapidly annotate known viruses. However, it cannot detect novel clades absent from its database (Wood and Salzberg, 2014). In contrast, reference-free tools such as VirFinder identify distinguishing k-mer signatures using logistic regression (Ren et al., 2017), while DeepVirFinder leverages convolutional neural networks trained on k-mer encodings to improve accuracy on ∼300-bp fragments (Ren et al., 2020).

Despite increasing efforts to curate and clean up viral reference sets, a large portion of reads in virome studies, ranging from 60% to 90%, remains taxonomically unassigned and it is commonly termed “viral dark matter” (Roux et al., 2015; Santiago-Rodriguez and Hollister, 2022). This gap reflects not only the lack of viral genomes in public databases but also the rapid evolution and ecological heterogeneity of viruses in nature. Together with the poor sampling of viromes across hosts and environments, these factors continue to limit our ability to compare viral communities across studies and age groups.

A further conceptual limitation in virome research is the difficulty in determining whether virome alterations observed in aging act as causal drivers of biological decline, reflect underlying immune dysfunction, or represent epiphenomena of bacterial dysbiosis. Ageing is accompanied by immunosenescence and chronic inflammation (Gadoth et al., 2024; Noronha et al., 2021; Cianfruglia et al., 2025; Giacconi et al., 2023), both of which may influence viral reactivation patterns and favor shifts in gut and circulating viromes (Jergovic et al., 2019; Mayneris-Perxachs et al., 2022; Yang et al., 2021; Lai et al., 2023). At the same time, age-related changes in the bacteriome, including reductions in Firmicutes and increases in Proteobacteria, strongly shape phage community structure (Francini et al., 2025), complicating the interpretation of phage expansions, which may either contribute to dysbiosis or simply mirror bacterial fluctuations (Fujimoto and Daichi, 2022; Yang et al., 2021). Because these processes occur simultaneously, cross-sectional virome studies cannot resolve temporal or causal relationships. Disentangling these competing hypotheses will require longitudinal sampling, multi-omic integration, and models capable of investigating mechanistic phage-host interactions (Shkoporov, 2019). Until such evidence is available, virome changes in ageing should be interpreted with caution, acknowledging both causal and reactive scenarios.