Microbial Bioactives

1. Introduction

Viruses are now recognized as the most abundant biological entities on Earth, outnumbering cellular life across nearly all ecosystems—from the open oceans to extreme habitats such as permafrost and hot springs (Paez-Espino et al., 2016; Zhang et al., 2019). They exert profound influence on microbial communities, ecosystem functioning, and global biogeochemical cycles. Historically, viral research was constrained by culture-dependent techniques, which required co-cultivation of viruses with their cellular hosts. However, such methods provided only a limited view of viral diversity because the vast majority of viruses remain uncultivable. The advent of viral metagenomics has transformed this field by enabling direct sequencing of viral nucleic acids from environmental samples, thereby bypassing the cultivation bottleneck (Edwards & Rohwer, 2005; Breitbart et al., 2002).

Studies in extreme environments have revealed remarkable viral genetic diversity and complexity, offering insights into viral adaptation and evolution. Extremophiles—organisms capable of surviving in high salinity, acidity, temperature, or radiation—serve as hosts for diverse viruses that exhibit unique structural and genetic traits (Paez-Espino et al., 2016; Roux et al., 2019). Because viruses require living hosts for replication, their interactions shape host population dynamics, influence microbial diversity, and alter host genomes through horizontal gene transfer (Brüssow et al., 2004; Thingstad, 2000). These interactions are particularly consequential in extreme environments, where viruses regulate microbial development and contribute indirectly to global biogeochemical cycles, including carbon and nutrient turnover (Suttle, 2007; Weitz et al., 2018). Yet, despite their ecological importance, knowledge of viral diversity and prevalence in extreme habitats remains limited (Emerson et al., 2018; Roux et al., 2016).

The rise of metagenomics offers unprecedented opportunities to explore viromes—the total viral genetic content of a habitat. Unlike conventional virology, which focuses on isolated viruses, metagenomics provides a community-wide perspective, uncovering novel viral sequences and functions (Breitbart et al., 2002; Mokili et al., 2012). Many newly discovered viral sequences have no homologues in existing databases, underscoring the extent of “viral dark matter” (Roux et al., 2019; Pratama et al., 2021). This has major implications not only for understanding viral diversity but also for identifying novel proteins, enzymes, and bioactive molecules with potential biotechnological and medical applications (Guo et al., 2021).

Marine environments provide striking examples of viral abundance and influence. Estimates suggest that a milliliter of seawater can contain up to 15,000 viral genotypes (Suttle, 2007; Brum & Sullivan, 2015). These viruses regulate bacterial populations through infection and lysis, thereby controlling microbial community structure and driving nutrient recycling. Viral lysis releases dissolved organic matter, fueling microbial food webs and influencing global carbon cycling (Weinbauer, 2004; Roux et al., 2016). Similarly, soil viromes are now being revealed through metagenomics. Soil viruses, estimated at 107 to 108 particles per gram, modulate plant-microbe interactions and affect nutrient cycling processes, which has direct implications for agriculture and land management (Emerson et al., 2018; Starr et al., 2019).

Human-associated viromes also exemplify the ecological and biomedical significance of viral metagenomics. The human gut virome is dominated by bacteriophages, which influence microbiome composition and may modulate susceptibility to diseases, including Clostridium difficile infections and inflammatory disorders (Ott et al., 2017; Duerkop & Hooper, 2013). Discoveries such as crAssphage—one of the most abundant viruses in the human gut—highlight the power of metagenomics to uncover key viral players previously hidden from view (Díaz-Muñoz, 2019). These findings bridge ecology and medicine, demonstrating how viral ecology can inform human health and disease management (Monaco et al., 2016).

Despite rapid advances in sequencing technologies, challenges remain. Viral genomes are highly diverse, with small sizes, unusual structures, and rapid evolutionary rates, complicating assembly and classification (Pratama et al., 2021; Roux et al., 2019). Moreover, sampling bias persists, as much of Earth’s virome—especially from extreme environments—remains underexplored (Paez-Espino et al., 2016; Wommack & Colwell, 2000). Integrating multi-omics approaches—combining viral metagenomics with metatranscriptomics, metaproteomics, and single-virus genomics—holds promise for resolving functional roles of viruses across ecosystems (Brum & Sullivan, 2015; Gregory et al., 2016).

Viral metagenomics has revolutionized virology by revealing the immense, previously hidden diversity of viruses and their roles in microbial ecology. By applying an ecological framework, researchers can better understand viral diversity, distribution, and interactions, as well as their consequences for ecosystem stability, global nutrient cycles, and human health (Roux et al., 2016; Hurwitz et al., 2013). This introduction sets the stage for examining how viral metagenomics contributes to our understanding of microbial community dynamics in both natural and host-associated ecosystems, with a special emphasis on extreme environments. An ecological framework illustrating how viral metagenomics integrates viral diversity, distribution, host interactions, and ecosystem-level processes across environmental and host-associated systems is presented in Figure 1.

Figure 1. Conceptual framework of virus ecology illustrating key components: interactions, diversity, dynamics, and distributions. The outer ring highlights major research domains such as evolution, host association, aquatic systems, soil ecology, and molecular mechanisms, while the inner ring emphasizes functional aspects including immune system, microbiome, nutrient cycling, photosynthesis, and virulence.

The primary objectives of this research are to investigate the diversity and composition of viral populations across marine, soil, and human-associated ecosystems using metagenomic approaches, providing a comprehensive understanding of viral community structures in distinct environments. It also aims to analyze how viruses influence microbial community dynamics and regulate key biogeochemical processes, thereby contributing to ecosystem functioning and nutrient cycling. Furthermore, the study seeks to evaluate the potential ecological and health implications of virome diversity, with particular attention to extreme environments and host-associated microbiomes where viral interactions may play crucial roles in maintaining ecological balance and impacting host health.

2. Materials and Methods

2.1 Study Design and Reporting Framework

This study was conducted as a systematic review to synthesize existing evidence on viral metagenomics and its ecological and human health implications across marine, soil, and human-associated ecosystems. The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines to ensure transparency, reproducibility, and methodological rigor throughout the study selection and synthesis process. The complete study selection process, including reasons for exclusion at each stage, is presented in the PRISMA flow diagram (Figure 2).

Figure 2. PRISMA 2020 flow diagram illustrating the identification, screening, eligibility assessment, and inclusion of studies in the systematic review of viral metagenomics across ecological and human-associated ecosystems.

The methodological framework was designed to identify, screen, and critically evaluate peer-reviewed studies that applied metagenomic approaches to investigate viral diversity, viral–host interactions, and ecological or health-related outcomes across diverse environments.

2.2 Literature Search Strategy

A comprehensive literature search was conducted across multiple electronic databases, including Web of Science, Scopus, PubMed, SpringerLink, and ScienceDirect, to capture a broad range of studies relevant to viral metagenomics. Additional records were identified through Google Scholar and manual screening of reference lists from key review articles and highly cited papers to ensure completeness (Figure 2).

Searches were performed using combinations of controlled vocabulary terms and free-text keywords related to viral metagenomics and ecological contexts. Core search terms included:

• viral metagenomics
• virome
• environmental virology
• marine virome
• soil virome
• human gut virome
• virus–microbe interactions
• ecosystem dynamics

Boolean operators (AND/OR) were applied to refine search results, and database-specific filters were used where appropriate. The search was limited to peer-reviewed articles published in English, with no strict temporal restriction in order to capture foundational and recent developments in the field.

2.3 Eligibility Criteria

Studies retrieved from the initial search were evaluated based on predefined inclusion and exclusion criteria.

Inclusion criteria:

• Original research articles or high-quality reviews applying metagenomic or viromic approaches
• Studies investigating viral diversity, composition, or function in marine, soil, or host-associated ecosystems
• Articles reporting ecological, evolutionary, or health-related implications of virome dynamics
• Peer-reviewed publications available in full text

Exclusion criteria:

• Studies relying solely on culture-based virology without metagenomic analysis
• Articles lacking sufficient methodological detail
• Conference abstracts, editorials, commentaries, or non-peer-reviewed literature
• Studies focused exclusively on single clinical case reports without broader ecological relevance

2.4 Study Selection Process

All records identified through database searching were imported into a reference management system, where duplicate entries were removed. The remaining records underwent a two-stage screening process.

In the first stage, titles and abstracts were independently screened to exclude clearly irrelevant studies. In the second stage, full-text articles were assessed for eligibility based on the inclusion and exclusion criteria. Discrepancies during the screening process were resolved through discussion and consensus.

The study selection process is summarized using a PRISMA flow diagram, illustrating the number of records identified, screened, excluded, and ultimately included in the qualitative synthesis.

2.5 Data Extraction and Synthesis

Data were systematically extracted from eligible studies using a standardized extraction framework. Extracted information included:

• Study objectives and ecosystem type
• Sample source and environmental context
• Metagenomic sequencing platforms and analytical approaches
• Viral diversity metrics and dominant viral groups
• Reported virus–host interactions
• Ecological, biogeochemical, or health-related implications

Due to heterogeneity in study designs, sequencing technologies, and analytical pipelines, a qualitative narrative synthesis approach was employed rather than a quantitative meta-analysis. Findings were grouped thematically according to ecosystem type (marine, soil, human-associated) and ecological function, allowing for structured comparison across studies.

2.6 Assessment of Study Quality and Methodological Limitations

The methodological quality of included studies was assessed by examining clarity of sampling design, sequencing depth, bioinformatic rigor, and transparency of data reporting. Particular attention was given to potential sources of bias, including sampling limitations, incomplete viral genome recovery, and database dependency in viral annotation.

Limitations related to viral “dark matter,” host prediction uncertainty, and uneven ecosystem representation were critically considered during synthesis to ensure balanced interpretation of results.

2.7 Data Presentation and Visualization

Key findings from the reviewed studies were summarized using descriptive tables and conceptual figures to highlight patterns in viral abundance, diversity, and ecological roles across ecosystems. Tables were used to compare viral distributions and applications of viral metagenomics, while schematic figures illustrated ecological frameworks and virus–host interaction networks synthesized from the literature.

3. Understanding the Hidden Networks of Virus–Microbe Interactions

3.1 The Importance of an Ecological Framework for Viral Metagenomics

Ecology aims to explain the diversity, distribution, and dynamics of biological systems by examining how organisms interact with their biotic and abiotic environments (Paez-Espino et al., 2016). The enormous variety and impact of viruses in ecosystems, including soils, oceans, and even glaciers, are being revealed by viral metagenomics (Emerson et al., 2018). The relationships between viruses and their hosts, competitors, and the resulting effects on host metabolism can now be examined beyond the limited number of virus-host combinations traditionally studied (Suttle, 2007; Thingstad, 2000). Researchers are beginning to explore the indirect impacts of viral infections on biogeochemical cycles (Roux et al., 2016), the effects of viruses’ presence on hosts even without active infection (Secor et al., 2015), and the cascading consequences of viral infection on host-associated communities (Duerkop & Hooper, 2013).

Viral metagenomics holds the potential to advance virology in environmental and medical contexts. It enables the study of host range diversity and virus-virus interactions, including coinfections of the same host (Díaz-Muñoz, 2019). Monitoring viruses that could spill over to humans by expanding their host range has demonstrated connections between environmental and medical virology (Monaco et al., 2016). The identification of diverse viruses from host-associated microbial communities has been facilitated by advances in metagenomic techniques (Paez-Espino et al., 2016). A landmark discovery in this context is the cross-assembly phage (crAssphage) and its relatives, which co-occur with the Bacteroidetes phylum in the human gut, comprising up to 90% of gut virome reads (Pride et al., 2012).The capacity of opportunistic pathogens, such as Clostridium difficile and vancomycin-resistant Enterococcus, to establish in the gut has been linked to viral community composition and dynamics (Ott et al., 2017). Viral-mediated modulation of microbial communities also affects the manifestation and progression of diseases, such as AIDS (Monaco et al., 2016) and severe respiratory infections (Lysholm et al., 2012). Ecological interactions between bacteriophages (the primary constituents of the commensal microbiota) can drive these effects through direct parasitism, lysing hosts, and altering community structure, or via horizontal gene transfer, which affects genetic diversity and function within bacterial populations (Chatterjee et al., 2020; Chatterjee et al., 2021).

Viral metagenomics also advances our understanding of viral molecular mechanisms. Studies on phage-bacteria interactions and microbial communities reveal how phages influence microbial composition, dynamics, and evolution (Breitbart, 2012; Weinbauer, 2004). These insights facilitate lateral gene transfer (Brüssow et al., 2004) and have applications in medical, industrial, and environmental contexts (Soffer et al., 2017; Chang, 2020). Integration of viral metagenomics with high-throughput technologies, such as single-cell genomics or metaproteomics, enables comprehensive cataloging of virus-host interactions across diverse microbial communities (Paez-Espino et al., 2016; Brum & Sullivan, 2015). By filling gaps in public databases with sequences from diverse ecosystems, viral metagenomics improves viral diagnostics, pathogenesis understanding, and development of preventative or therapeutic measures. Guidelines such as MIUViG (Minimum Information about an Uncultivated Virus Genome) provide ecological context to genomic sequences, enhancing database utility (Roux et al., 2019).

3.2 Metagenomic Approaches to Viral Diversity and Their Distributions

Studying viral diversity and distribution requires viral genome assembly and detection. Metagenomic identification relies on genome assembly, whereas lineage-specific marker genes—such as RNA-dependent RNA polymerase or viral hallmark genes—can aid in detecting particular viral genomes (Zhang et al., 2019; Pratama et al., 2021). Genome assembly can be performed from double-stranded (ds) or single-stranded (ss) DNA, or cDNA derived from RNA. Nucleic acids can be obtained directly from samples or from virus-like particles (VLPs) separated via centrifugation or filtration. Each method has distinct advantages and limitations, depending on the goal of viral detection (Table 1).

Table 2. Applications and Implications of Viral Metagenomics

A major consideration is whether to focus on whole samples or only on VLPs. Whole samples contain abundant nucleic acids from bacterial, archaeal, and eukaryotic cells, complicating viral genome assembly (Trubl et al., 2020). Therefore, VLPs are often separated and concentrated prior to sequencing. Techniques for VLP separation vary depending on material type and have been successfully applied to seawater (Breitbart et al., 2002), soil (Emerson et al., 2018), fecal matter (Mokili et al., 2012), plant tissues (Hily et al., 2018), and animal tissues (Dunay et al., 2018). Common approaches include centrifugation and size-dependent filtration, followed by DNase/RNase treatment or density gradient centrifugation using CsCl or sucrose, and sometimes OptiPrep™ gradients for purifying VLPs (Roux et al., 2016; Guo et al., 2021).

Alternatively, single VLP isolation can be performed to conduct viral single amplified genome (vSAG) sequencing (Hurwitz et al., 2013; Pratama et al., 2020). VLPs are sorted via flow cytometry into droplets or beads and amplified using multiple displacement amplification before sequencing. This approach allows high-resolution assembly, especially for segmented, multicomponent, or satellite viruses, which are challenging to resolve in conventional metagenomic assemblies (Emerson et al., 2018). While VLP sequencing improves coverage of viral genomes and detection of rare viruses, it may miss nonencapsidated viruses, such as proviruses or retroviruses integrated into host genomes. Whole-sample metagenomics or metatranscriptomics can complement VLP sequencing, capturing viral genomes that might otherwise be missed. The choice of strategy depends on whether the study focuses on total viral diversity or specific viral populations or stages (Roux et al., 2016; Zhang et al., 2006).

4. Results and Synthesis of Evidence

4.1 Synthesizing Evidence on Virome Characterization

Across the reviewed literature, virome analysis consistently emerged as a foundational approach for understanding viral diversity and function within complex microbial ecosystems. Studies applying viral metagenomics collectively demonstrate that viromes represent highly diverse genetic reservoirs, often dominated by previously uncharacterized viral sequences (Breitbart et al., 2002; Paez-Espino et al., 2016). Rather than relying on culture-based detection, the reviewed studies used metagenomic sequencing to infer viral presence, diversity, and functional potential directly from environmental and host-associated samples.

Evidence synthesized from marine, soil, and human-associated systems shows that virome characterization typically involves enrichment or computational recovery of viral sequences from complex microbial datasets, followed by comparative bioinformatic analyses (Edwards & Rohwer, 2005; Zhang et al., 2019). Across studies, shotgun metagenomic sequencing enabled the detection of both abundant viral taxa and rare viral populations that would otherwise remain undetected. Importantly, many studies reported that a substantial proportion of recovered viral sequences lacked homologs in existing databases, reinforcing the concept of extensive “viral dark matter” across ecosystems.

Functional annotation of viral genomes, as reported in multiple studies, revealed genes associated with host metabolism, nutrient cycling, and ecological adaptation (Hurwitz et al., 2013). Comparative virome analyses across environments allowed investigators to infer virus–host associations, ecological drivers of viral distribution, and evolutionary relationships. Collectively, the reviewed evidence demonstrates that virome analysis provides an integrative lens through which viral diversity, ecological roles, and evolutionary dynamics can be systematically interpreted rather than experimentally generated.

4.2 Synthesized Impacts of Viruses on Microbial Communities

The reviewed studies consistently identify viruses as major regulators of microbial community structure and ecosystem stability across aquatic, terrestrial, and host-associated environments (Suttle, 2007; Thingstad, 2000). Viral-mediated control of microbial populations—often described as “top-down regulation”—was repeatedly reported as a key mechanism maintaining microbial diversity and preventing dominance by single taxa.

In marine ecosystems, multiple studies documented the role of bacteriophages in shaping bacterial community composition and driving nutrient recycling through host lysis (Weinbauer, 2004). Similarly, soil-focused investigations highlighted the influence of viruses on microbial turnover, nutrient availability, and plant–microbe interactions, emphasizing their indirect contribution to ecosystem productivity (Starr et al., 2019). These multi-scale virus–host and community interactions, ranging from individual viral entities to population- and ecosystem-level dynamics, are conceptually illustrated in Figure 3.

Figure 3: Metagenomic Approaches to Viral Diversity and Their Distributions. Viruses interact at various levels and thus can be studied from individual entities to larger-scale populations and communities through Viral metagenomics. Hence, viral metagenomics is facilitating the study of viruses within an ecological framework, including studying Viral diversity and distributions across ecosystems, and viral dynamics across variable ranges of potential host diversity and in a Multitude of environments where they can have positive or negative effects.

Beyond population control, horizontal gene transfer (HGT) emerged as a recurrent theme across the reviewed literature. Viral-mediated gene exchange was shown to accelerate microbial evolution by transferring genes associated with metabolic adaptation, virulence, and stress tolerance (Brüssow et al., 2004; Hambly & Suttle, 2005). Evidence from aquatic and eukaryotic microbial systems further demonstrated that large DNA viruses contribute to genetic innovation beyond prokaryotic hosts (Cottrell & Kirchman, 2016).

Together, these findings support a consensus view in the literature that viromes function as ecological regulators by integrating microbial mortality, genetic exchange, and nutrient cycling. This triad of interactions underpins ecosystem resilience and biogeochemical stability across diverse habitats (Breitbart, 2012).

4.3 Applications of Viral Metagenomics: Evidence Across Ecosystems

The systematic literature indicates expanding applications of viral metagenomics across medical, agricultural, and environmental domains. In clinical contexts, reviewed studies highlight how virome analyses have informed the development of phage-based therapies, particularly in response to rising antibiotic resistance (Berger et al., 2013). Metagenomic screening enables the identification of phages lacking undesirable virulence or resistance genes, improving therapeutic safety and efficacy. A synthesized overview of the major application domains, key insights, and broader implications of viral metagenomics across ecosystems is presented in Table 2.

Table 2. Applications and Implications of Viral Metagenomics

Human gut virome studies consistently demonstrated associations between phage community composition and microbial dysbiosis, with implications for inflammatory and infectious diseases (Sato et al., 2020). However, the literature also reflects variability in phage impacts, with some studies reporting significant microbiome restructuring and others observing limited phylogenetic disruption (Volant et al., 2016; Duerkop, 2021). These contrasting findings underscore the context-dependent nature of phage–microbiome interactions.

Environmental and agricultural applications were similarly prominent. Soil virome studies linked viral activity to carbon release, nutrient cycling, and plant-associated microbial regulation (Firestone & Pringle, 2019). Wildlife and environmental surveillance studies demonstrated the value of viral metagenomics in detecting spillover risks and monitoring ecosystem health under environmental change (Pulliam et al., 2019).

Overall, the reviewed evidence supports viral metagenomics as a versatile tool for ecosystem monitoring, therapeutic discovery, and ecological forecasting, particularly when integrated with functional and ecological modeling frameworks.

5. Integrated Discussion

Synthesized evidence across ecosystems confirms that viruses are not merely passive biological entities but active drivers of microbial ecology, evolution, and ecosystem function. Marine studies consistently reported extraordinarily high viral diversity, reinforcing the role of viruses in controlling microbial turnover and global carbon cycling (Suttle, 2017; Gregory et al., 2019). These findings align with broader ecological models describing viral regulation of oceanic food webs.

Soil-based studies further expanded this paradigm, demonstrating that terrestrial viromes influence plant health, nutrient availability, and ecosystem resilience. The consistency of these findings across geographically and environmentally distinct studies highlights the universality of viral ecological functions (Pratama & van Elsas, 2018).

Human-associated virome research revealed equally significant implications. The reviewed literature suggests that gut viruses modulate microbial balance and host immune responses, potentially contributing to disease susceptibility or protection (Shkoporov & Hill, 2019). The discovery of abundant yet previously unknown viral taxa in human samples emphasizes how much of the virome remains unexplored.

6. Future Directions and Methodological Challenges

The reviewed literature collectively emphasizes the need to expand viral metagenomic research across underrepresented ecosystems, particularly extreme and rapidly changing environments (Wommack et al., 2015). Longitudinal studies were repeatedly identified as essential for understanding temporal virome dynamics and ecosystem stability (Roux et al., 2016).

Integration of multi-omics datasets—including metatranscriptomics and metaproteomics—was highlighted as a priority for resolving functional virus–host interactions (Brum & Sullivan, 2015). In parallel, advances in machine learning and artificial intelligence were proposed as critical tools for managing large datasets and improving host prediction accuracy (Ren et al., 2020).

Despite these opportunities, significant challenges persist. Sampling bias, bioinformatic complexity, and the dominance of unclassified viral sequences remain major limitations (Paez-Espino et al., 2016; Roux et al., 2019). Additionally, the lack of cultivable viral hosts continues to hinder experimental validation of metagenomic discoveries.

7. Conclusion

This systematic review demonstrates that viral metagenomics has fundamentally reshaped our understanding of viral diversity and ecological function across marine, soil, and host-associated ecosystems. Synthesized evidence reveals viruses as central regulators of microbial communities, nutrient cycling, and ecosystem resilience, with direct relevance to environmental sustainability and human health. Although challenges remain in functional characterization and host assignment, continued methodological integration and analytical innovation promise to further illuminate the hidden virosphere. Ultimately, advancing our understanding of viromes offers critical insights into ecosystem stability, disease dynamics, and future biotechnological applications.

Author contributions

A.I.A.S.M. conceived and designed the study, developed the review framework, conducted the systematic literature search, performed data synthesis, and drafted the original manuscript. D.A.A.A. contributed to study methodology, critical evaluation of the literature, and substantial review and editing of the manuscript. M.M.A. provided supervision, validated the scientific content, and contributed to critical revision and final approval of the manuscript. All authors read and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Acknowledgment

The authors gratefully acknowledge the academic and research support provided by King Faisal University, Imam Abdulrahman Bin Faisal University, and King Abdulaziz University. The authors also thank colleagues and peers for their valuable discussions and constructive feedback during the preparation of this systematic review.

References