Review

An Update on RNA Virus Discovery: Current Challenges and

Future Perspectives
Humberto Debat * and Nicolas Bejerman *

Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias, Instituto Nacional de Tecnología

Agropecuaria (IPAVE-CIAP-INTA), Unidad de Fitopatología y Modelización Agrícola CONICET-INTA,
Córdoba 5020, Argentina
* Correspondence: [email protected] (H.D.); [email protected] (N.B.)

Abstract
The relentless emergence of RNA viruses poses a perpetual threat to global public health,
necessitating continuous efforts in surveillance, discovery, and understanding of these
pathogens. This review provides a comprehensive update on recent advancements in RNA
virus discovery, highlighting breakthroughs in technology and methodologies that have
significantly enhanced our ability to identify novel viruses across diverse host organisms.
We explore the expanding landscape of viral diversity, emphasizing the discovery of
previously unknown viral families and the role of zoonotic transmissions in shaping the
viral ecosystem. Additionally, we discuss the potential implications of RNA virus discovery
on disease emergence and pandemic preparedness. Despite remarkable progress, current
challenges in sample collection, data interpretation, and the characterization of newly
identified viruses persist. Our ability to anticipate and respond to emerging respiratory
threats relies on virus discovery as a cornerstone for understanding RNA virus evolution.
We address these challenges and propose future directions for research, emphasizing the
integration of multi-omic approaches, advanced computational tools, and international
collaboration to overcome barriers in the field. This comprehensive overview aims to
guide researchers, policymakers, and public health professionals in navigating the intricate
landscape of RNA virus discovery, fostering a proactive and collaborative approach to
anticipate and mitigate emerging viral threats.

Academic Editor: Ahmed El-Shamy Keywords: RNA virus discovery; viral evolution; metagenomics; zoonotic spillover; virus
Received: 11 June 2025 surveillance; emerging respiratory viruses; SARS-CoV-2 variants; pandemic preparedness
Revised: 9 July 2025
Accepted: 11 July 2025
Published: 15 July 2025

Citation: Debat, H.; Bejerman, N. An 1. Introduction

Update on RNA Virus Discovery:
The perpetual threat posed by RNA viruses to global public health demands contin-
Current Challenges and Future
Perspectives. Viruses 2025, 17, 983.
ual advancements in our ability to identify, characterize, and understand these dynamic
https://doi.org/10.3390/ pathogens. This review provides an updated perspective on the evolving landscape of
v17070983 RNA virus discovery, reflecting recent breakthroughs in technology and methodologies
Copyright: © 2025 by the authors.
that have expanded our capacity to uncover novel viruses across diverse host organisms.
Licensee MDPI, Basel, Switzerland. The acceleration of viral discovery is not merely a reflection of improved detection methods
This article is an open access article but also a testament to the inherent complexity and adaptability of RNA viruses [1].
distributed under the terms and Over the past decade, the acceleration of virus discovery has been driven by a synergy
conditions of the Creative Commons
between technological innovations in high-throughput sequencing, metagenomics, and
Attribution (CC BY) license
bioinformatics, and a growing understanding of biological factors such as host–pathogen
(https://creativecommons.org/
licenses/by/4.0/).
co-evolution and ecological dynamics, revolutionizing our approach to viral surveillance.

Viruses 2025, 17, 983 https://doi.org/10.3390/v17070983

Viruses 2025, 17, 983 2 of 16

These tools have enabled researchers to explore previously inaccessible niches, revealing an
unprecedented diversity of RNA viruses [2]. The discovery of novel viral families and the
identification of their reservoir hosts have illuminated the intricate relationships between
viruses and their environments, with zoonotic transmissions playing a pivotal role in the
dynamics of viral emergence [3].
This review probes into the expanding landscape of viral diversity, emphasizing the
implications of RNA virus discovery for our understanding of disease emergence and
the potential for pandemics. Despite the remarkable progress achieved, significant chal-
lenges persist in the field of viral discovery, ranging from limitations in sample collection
to the complexities of data interpretation and the characterization of newly identified
viruses. Addressing these challenges is crucial to bolstering our preparedness for emerging
viral threats [4].
We explore the current state of knowledge regarding the factors influencing the dy-
namics of RNA viruses, with a particular focus on the intersection of viral ecology, host
interactions, and the human–animal interface [5]. As we embark on an era of unprece-
dented viral discovery, it is imperative to critically assess the gaps in our understanding and
chart a course for future research endeavors [6]. In this context, we propose a roadmap for
advancing the field, advocating for the integration of multi-omic approaches, updated com-
putational tools, and collaborative international efforts to navigate the intricate landscape
of RNA virus discovery.
All in all, this review aims to provide a comprehensive introduction to the current
state of RNA virus discovery, offering insights into the breakthroughs, challenges, and
future perspectives that shape our ongoing efforts to unveil the viral dark matter landscape.
Through this exploration, we strive to facilitate a proactive and collaborative approach that
will enhance our ability to anticipate, understand, and ultimately mitigate the impact of
emerging viral threats on global health.

2. Results
2.1. Technological Advancements in RNA Virus Discovery
The field of RNA virus discovery has undergone a revolutionary transformation, pri-
marily driven by unparalleled technological advancements. High-throughput sequencing
and metagenomic techniques have transcended traditional barriers, enabling researchers to
venture into unexplored ecological landscapes with unprecedented precision [7]. These
technological innovations have not only expedited the identification of known viruses but
have also unearthed a plethora of previously undiscovered RNA viruses, expanding our
understanding of the viral microcosm (Table 1).
The advent of next-generation sequencing platforms has ushered in an era of genomic
exploration, facilitating the simultaneous analysis of vast viral populations within com-
plex biological samples (Figure 1). Metagenomic approaches, coupled with advancements
in sequencing technologies, empower researchers to conduct holistic surveys of diverse
environments, from remote ecosystems to human clinical samples (Table 2). This synergy
has resulted in the discovery of a myriad of RNA viruses, ranging from well-characterized
pathogens to entirely novel viral entities [8–11]. Furthermore, portable sequencing plat-
forms like Oxford Nanopore Technologies’ MinION have revolutionized field-based dis-
covery due to their affordability and real-time capabilities. Concrete examples of its impact
include its use in the rapid, culture-independent whole-genome sequencing of Nipah virus
during outbreaks [12], its application in identifying recombinant enteroviruses responsible
for outbreaks [13], and its utility in detecting novel plant viruses like macluraviruses and
potyviruses in yam plants [14]. In addition, the application of these techniques has led
to the discovery of novel RNA viruses in invertebrate hosts, such as ants, highlighting
Viruses 2025, 17, 983 3 of 16

the continuous expansion of the RNA virosphere in understudied taxonomic groups [15].
Similarly, recent advancements in unbiased metagenomics have enabled the identification
of entirely new lineages within the Orthornavirae kingdom, significantly broadening our
understanding of RNA virus evolution and diversity across a wide range of hosts [16].

Table 1. Timeline of key technological advancements in RNA virus discovery.

Period/Year Milestone/Advancement
Foundational Sequencing and Bioinformatics
Completion of the Human Genome Project, establishing large-scale sequencing capabilities.
Early 2000s
Widespread adoption of Sanger sequencing and initial development of foundational
bioinformatics tools.
First-Generation Next-Generation Sequencing (NGS)
Introduction of high-throughput sequencing platforms (e.g., 454 Life Sciences, Illumina
Mid 2000s
Genome Analyzer), enabling parallel sequencing and significantly reducing costs and time
per base.
Metagenomics and Metatranscriptomics Emergence
Development and widespread application of unbiased metagenomic and
Late 2000s–Early 2010s
metatranscriptomic approaches, allowing for the discovery of novel viruses without prior
cultivation.
Third-Generation Sequencing and Portability
Commercialization of long-read sequencing technologies (e.g., Pacific Biosciences, Oxford
Mid 2010s
Nanopore Technologies’ MinION), offering real-time data, portability, and improved
resolution for complex genomes.
Advanced Computational and Single-Cell Approaches
Significant advancements in single-cell sequencing, enabling analysis of viral presence and
gene expression at individual host cell resolution. Increased integration of Artificial
Late 2010s–Early 2020s
Intelligence (AI) and Machine Learning (ML) for tasks like viral host prediction and
classification. Development of large-scale cloud-based bioinformatics infrastructures (e.g.,
Serratus) for petabase-scale data analysis.
Integrated Omics and Global Collaboration
Expansion of multi-omics integration (genomics, transcriptomics, proteomics) for a holistic
Recent/Ongoing
view of virus–host interactions. Growth of global data-sharing initiatives and collaborative
networks accelerating virus discovery and surveillance efforts.

Recent breakthroughs in single-cell sequencing technologies offer a nuanced perspec-

tive on RNA virus discovery. By dissecting individual host cells, researchers can discern
viral genomes with unprecedented resolution, unveiling the genetic diversity within in-
fected populations [17]. For instance, single-cell sequencing has demonstrated its ability to
detect viral transcripts and identify infected cell types in human skin biopsies for viruses
like Merkel cell polyomavirus and human papillomaviruses [18]. It has also been applied
to study the heterogeneity of influenza virus infections at the single-cell level, revealing
how genetic variations contribute to diverse immune responses [19], and to identify novel
host–virus interactions for giant viruses in marine environments [20]. This level of granu-
larity proves invaluable in understanding viral quasispecies dynamics, host interactions,
and the adaptive evolution of RNA viruses, providing a finer lens through which to explore
the intricacies of viral biology.
Viruses 2025, 17, 983 4 of 16

Figure 1. A schematic representation outlining the key stages in the contemporary workflow for
RNA virus discovery. The process begins with diverse sampling from clinical, environmental, vector,
or wildlife sources to capture a broad range of viral reservoirs, followed by RNA extraction and
library preparation, which includes RNA fragmentation, adapter ligation, and quality control to
ensure high-quality input for sequencing and post processing (e.g., FastQC, Trimmomatic). In the
sequencing and assembly phase, raw reads generated from high-throughput platforms are processed,
for instance, using k-mer–based approaches (e.g., SPAdes, MEGAHIT) to reconstruct viral genomes.
The resulting viral contigs then enter the bioinformatics analysis pipeline, where viral sequences
are annotated, quantified, and compared against reference databases using tools such as BLAST,
Diamond, Kraken, LucaProt or Serratus to identify known viral elements and potential novelties.
Phylogenetic placement (e.g., IQ-TREE, RAxML) enables preliminary classification into known,
unclassified, or novel viral groups. Virus characterization incorporates functional and structural
annotation through domain prediction (e.g., RdRp, helicase, capsid), protein structure modeling
(e.g., using AlphaFold or HHpred), and receptor-binding inference to understand viral biology
and potential pathogenicity. This multi-step workflow illustrates the integration of field sampling,
molecular biology, high-throughput sequencing, and computational tools required to advance RNA
virus discovery in the post-genomic era.

2.2. Bioinformatics and Computational Tools

The deluge of sequencing data demands robust bioinformatics and computational
tools for effective analysis. Advanced algorithms and machine learning models are in-
strumental in deciphering complex viral genomes, predicting potential host ranges, and
discerning patterns of viral evolution [9]. Specific machine learning models such as deep
learning models, random forests, and support vector machines (SVMs), alongside dedicated
bioinformatics tools like VIRify, VirHostNet, and DeepViral [21], are instrumental in deci-
phering complex viral genomes, predicting potential host ranges, and discerning patterns
of viral evolution. Beyond mere identification, AI/ML approaches are increasingly vital for
functions such as predicting host tropism, identifying potential zoonotic reservoirs, and
even inferring viral pathogenicity based on genomic features and protein structures (e.g.,
Viruses 2025, 17, 983 5 of 16

AlphaFold for protein modeling). The integration of these tools into RNA virus-discovery
pipelines not only accelerates the identification process but also enhances our ability to
extract meaningful insights from the vast genomic datasets generated. Notably, Serratus
re-analyzed petabase-scale sequence data from public databases to facilitate the discov-
ery of over 130,000 new RNA viruses, including several novel coronaviruses [22]. This
innovative system was crafted to streamline ultra-high-throughput sequence alignment
on a petabase scale. The researchers embarked on an exhaustive exploration, analyzing
a vast dataset comprising 5.7 million biologically diverse samples, collectively amounting
to 10.2 petabases. Focusing on the hallmark gene RNA-dependent RNA polymerase, this
methodological approach yielded remarkable results, uncovering more than 105 previously
unknown RNA viruses. This significant discovery exponentially expanded the breadth of
our understanding of viral species by roughly an order of magnitude, but more importantly,
it provided a user friendly platform to foster and democratize virus discovery beyond the
global north.

Table 2. Key challenges and future perspectives in RNA virus discovery.

Challenges Perspectives
Sample Collection
Multi-Omics Integration
Remote access, degradation, contamination, and lack of
Combining genomics, transcriptomics, and proteomics to
standardized methods limit the quality and scope of
illuminate virus–host dynamics. (See Section 2.4.)
samples. (See Section 2.5.1.)
Data Overload and Interpretation AI-Powered Discovery
Discriminating real viral sequences from noise in large Machine learning models to enhance virus classification,
metagenomic datasets remains difficult. (See host prediction, and outbreak risk assessment. (See
Section 2.5.2.) Section 3.4.)
Viral Characterization Portable Sequencing Platforms
Functional and biological validation lags behind genomic On-site and real-time virus detection through
identification due to lack of isolates and models. (See ultra-portable, affordable sequencing technologies. (See
Section 2.5.2.) Section 2.1.)
Taxonomic Uncertainty
One Health and Ecological Frameworks
Novel lineages challenge existing classification schemes,
Integrated views of human, animal, and environmental
demanding more flexible and dynamic frameworks. (See
health to contextualize virus emergence. (See Section 2.4.)
Section 2.5.2.)
Ethical and Legal Issues Global Equity and Capacity Building
Sample ownership, informed consent, and fair benefit International collaboration, open-access data, and
sharing are unresolved, especially in biodiverse regions. inclusive training to democratize discovery. (See
(See Section 3.3.) Section 3.3.)

Beyond their application in data analysis, the emerging potential of AI agents is

poised to transform the entire RNA virus discovery workflow through automation and
acceleration. These intelligent systems can revolutionize the field by automating tedious
data curation and preprocessing tasks, optimizing experimental design (e.g., through
active learning for targeted sampling), and even generating novel hypotheses for viral
origins or host interactions. For instance, AI-driven platforms can sift through vast public
genomic datasets, identify subtle patterns indicative of novel viral families, and prioritize
samples for further experimental validation, dramatically reducing the time from raw data
to actionable insights [23]. This shift towards AI-powered automation promises to enhance
efficiency, reduce human error, and enable researchers to explore previously intractable
scales of data, thereby significantly accelerating the pace of RNA virus discovery and
outbreak preparedness.
Viruses 2025, 17, 983 6 of 16

Technological progress in RNA virus discovery is further amplified by global collabo-

rations and open data-sharing initiatives. International networks of researchers, epidemiol-
ogists, and public health organizations collaborate to pool resources, share methodologies,
and collectively analyze data [24]. This collaborative approach accelerates the pace of
discovery and ensures a comprehensive understanding of the global viral landscape.

2.3. Unraveling Novel Viral Families

The exploration of RNA virus diversity has led to the revelation of novel viral families,
challenging existing paradigms and expanding the intricate tapestry of viral taxonomy [25].
Beyond the identification of individual viruses, the discovery of previously unknown
viral families has redefined our understanding of the fundamental building blocks of the
virosphere, emphasizing the dynamic nature of viral evolution and adaptation.
Recent breakthroughs in RNA virus discovery have necessitated a reevaluation of
taxonomic frameworks. The identification of novel viral families poses challenges to
traditional classification systems, prompting a paradigm shift in our conceptualization of
viral diversity [26,27]. The taxonomic implications of these discoveries highlight a need for
adaptive classification strategies that can accommodate the ever-expanding pool of newly
identified viruses. The discovery of novel viral families not only broadens the spectrum of
known viruses but also provides unique insights into the evolutionary dynamics of RNA
viruses. Comparative genomics and phylogenetic analyses of these newfound families
shed light on the origins, divergence, and adaptive strategies employed by diverse viral
lineages [26]. Understanding their evolutionary trajectories is crucial for deciphering the
mechanisms underpinning their emergence and persistence in various ecological niches.

2.4. Ecological and Zoonotic Dimensions of RNA Virus Discovery: Implications for One Health
and Public Health Preparedness
The discovery of novel RNA viral families is continually expanding our understanding
of the complex ecological relationships between viruses and their hosts [28]. These findings
reveal not only the hidden breadth of viral diversity but also the subtle and dynamic
interactions that viruses maintain within ecological networks. Each new viral lineage
provides a unique lens through which to examine the evolutionary strategies of replication,
transmission, and persistence, while also offering insights into the broader mechanisms
shaping host–pathogen co-evolution.
Beyond their genomic novelty, these newly identified viral families underscore the
functional diversity embedded within the virosphere [29,30]. Characterizing the replica-
tion strategies, structural features, and host interactions of these viruses is essential to
understanding their ecological roles. Importantly, determining host range and tropism
informs our ability to assess cross-species transmission potential and zoonotic risk—critical
parameters for anticipating disease emergence.
The ecological significance of these discoveries extends well beyond virology, intersect-
ing with key questions in evolutionary biology, biodiversity conservation, and ecosystem
health. Viruses, as integral components of ecosystems, can influence population dynamics,
species interactions, and even nutrient cycles. Zoonotic transmission is a central narrative
in the evolutionary trajectory of many RNA viruses, including several of public health
concern [31–33]. Zoonotic transmissions form a dynamic interface where the spheres of
animal and human health intersect. As viruses navigate this boundary, they encounter
diverse hosts, each presenting a unique set of challenges and opportunities for adapta-
tion [34]. Identification of reservoir hosts is pivotal in understanding the dynamics of
zoonotic transmissions [35]. Beyond the initial spillover event, there is a role of reservoir
hosts in the amplification and maintenance of viruses within natural ecosystems.
Viruses 2025, 17, 983 7 of 16

Zoonotic transmissions are influenced by a complex interplay of evolutionary forces

acting on both viruses and their hosts [36]. Evolutionary pressures, such as immune evasion
and adaptation to new environments, shape the genetic makeup of viruses and influence
their ability to cross species barriers [37].
Human activities and alterations in land use significantly influence the landscape
of zoonotic transmissions [38–40]. There is a substantial impact of anthropogenic factors,
including deforestation, urbanization, and agricultural practices, on the frequency and
intensity of zoonotic events. Understanding these influences is crucial for predicting and
mitigating the risk of viral spillover amidst environmental changes.
The evolving field of RNA virus discovery carries significant implications for our
understanding of disease emergence and requires a fundamental change in global pan-
demic preparedness [41]. There is an imperative to adapt public health strategies to the
evolving nature of RNA viruses and the associated challenges in mitigating the impact of
potential pandemics.
The rapid pace of RNA virus discovery and the expanding catalog of viral diver-
sity demand a fundamental shift in public health strategy, from reactive containment to
proactive, adaptive response frameworks [42]. As newly discovered RNA viruses require
adaptation in traditional approaches of surveillance, diagnostics, and intervention, public
health systems must evolve to incorporate flexibility, foresight, and effective utilization
of genomic data. Integrating viral discovery data into vaccine development pipelines,
therapeutic design, and diagnostic assay development is essential to address the evolving
nature of emerging and re-emerging pathogens.
Central to this adaptation is the adoption of data-driven decision-making processes.
Real-time integration of genomic surveillance, predictive modeling, and risk mapping
enables public health authorities to design more effective, context-specific interventions.
Rather than relying solely on established clinical case definitions or historical pathogen pro-
files, public health preparedness can now incorporate insights from novel virus detection,
viral evolution trends, and environmental triggers. The One Health approach, emphasizing
the interconnectedness of human, animal, and environmental health, offers an essential
framework for managing emerging infectious diseases [42]. Zoonotic spillovers rarely
arise from isolated events; they are ecological phenomena shaped by biodiversity loss,
land-use change, wildlife trade, and global mobility. As such, addressing them requires
interdisciplinary collaboration across virology, veterinary medicine, ecology, epidemiology,
and environmental science.
The global nature of emerging RNA virus threats also necessitates robust international
cooperation [24,43]. Effective pandemic preparedness depends on the establishment and
maintenance of cross-border networks for genomic surveillance, open-access data shar-
ing, resource allocation, and coordinated response strategies. Platforms that support the
timely sharing of viral sequences, epidemiological reports, and functional annotations
can significantly reduce the delay between discovery and intervention. In this context,
collaboration becomes both a scientific and geopolitical necessity. As our technical capacity
to detect and interpret viral diversity expands, so too must our ethical frameworks. Ethical
considerations surrounding RNA virus discovery include questions of informed consent,
benefit sharing, and the responsible use of genetic information, particularly when research
is conducted in biodiversity-rich but resource-limited regions [24]. Firstly, ethical data
sharing is paramount and encompasses adherence to international agreements such as the
Nagoya Protocol. This ensures that benefits arising from the utilization of genetic resources,
including viral sequences, are shared fairly and equitably with the countries providing
those resources. Secondly, the responsible use of viral genomic data originating from
low-income countries requires careful consideration. This involves preventing exploitation,
Viruses 2025, 17, 983 8 of 16

ensuring data security, and guaranteeing that research outcomes directly benefit the source
communities and nations. Thirdly, issues pertaining to informed consent and ownership
when working with indigenous communities are critical. Respect for tribal sovereignty,
cultural sensitivities, and the right to self-determination must guide all interactions, ensur-
ing free, prior, and informed consent for sample collection and data use, and recognizing
community ownership over their biological resources and associated traditional knowledge.
It is essential to ensure that the benefits of virus discovery are equitably distributed and that
the rights of individuals and communities are respected. Balancing global health protection
with justice, transparency, and respect for sovereignty is key to fostering trust and ensuring
the long-term sustainability of collaborative research.

2.5. Persisting Challenges in RNA Virus Discovery

Despite remarkable strides in technology and methodology, the field of RNA virus
discovery grapples with persistent challenges that demand innovative solutions and
a concerted research focus [30]. This section delves into the enduring obstacles, rang-
ing from sample collection intricacies to the complexities of viral characterization, and
highlights the need for ongoing advancements to overcome these hurdles and propel the
field forward.

2.5.1. Sample Collection and Data Interpretation

The discovery of novel RNA viruses depends fundamentally on the quality and di-
versity of biological samples collected across ecological and clinical landscapes. However,
obtaining representative and high-quality samples remains one of the most persistent
challenges in the field [30]. Field sampling in remote or biodiverse habitats, collection from
wildlife or vector populations, and handling of clinical specimens all involve logistical,
technical, and biosafety constraints. Factors such as sample degradation, contamination,
and limited access to cold-chain infrastructure frequently compromise data quality and
hinder downstream molecular analyses. Inadequate sampling not only limits the resolu-
tion of viral diversity but also skews our understanding of host–virus associations and
transmission dynamics.
These challenges are compounded by the ethical dimensions of sample acquisition,
which are becoming increasingly salient as virus discovery expands into global and often
vulnerable contexts [24]. Navigating the balance between scientific necessity and ethical
responsibility requires meaningful engagement with local communities, respect for indige-
nous knowledge systems, and adherence to principles of benefit-sharing and informed
consent. Particularly in regions rich in biodiversity and cultural heritage, virus discovery
efforts must be guided by equitable frameworks that recognize the rights of stakeholders
and promote long-term collaboration rather than extractive research practices.
Once samples are collected and sequenced, the complexity of data interpretation
emerges as a second major bottleneck in the virus-discovery pipeline [7,24,30]. The high-
throughput sequencing platforms used in metagenomic studies generate vast datasets,
often containing millions of reads from mixed-origin nucleic acids. Distinguishing gen-
uine viral sequences from background noise, laboratory contaminants, or host-derived
elements demands rigorous quality control and the application of specialized bioinfor-
matics pipelines. This is especially critical in the detection of low-abundance or highly
divergent viral genomes, which may lack close homologs in existing reference databases.
Moreover, even when novel viral sequences are confidently identified, their biolog-
ical relevance is not always clear. The challenge of interpreting functional significance,
such as inferring host range, transmission potential, or pathogenicity, from genomic data
alone remains unresolved in many cases. Advances in machine learning, k-mer–based
Viruses 2025, 17, 983 9 of 16

classification, and domain-level annotation tools are beginning to bridge this gap, but
substantial innovation is still needed to develop analytical frameworks capable of handling
the scale and diversity of modern viromic datasets. Strengthening each of these pillars
is essential to ensure that virus discovery continues to produce biologically meaningful,
socially responsible, and actionable insights into the RNA virosphere.

2.5.2. Viral Characterization Complexity

Identifying a novel RNA virus is only the first step in a much more complex endeavor:
understanding its biology, host range, and potential impact on human and animal health.
Comprehensive characterization of newly discovered viruses remains a significant chal-
lenge in the post-discovery phase [7,24,30]. Many viruses, particularly those identified
through metagenomics, are known only from partial or complete genome sequences, with
no corresponding cultured isolate or infectious clone. This lack of biological material limits
experimental validation and hinders the study of viral replication dynamics, host cell
interactions, tissue tropism, and pathogenicity. Even when isolation is possible, elucidating
the full spectrum of host–virus interactions often requires the integration of diverse experi-
mental systems, including animal models, cell culture, and in vitro binding assays, each
with its own constraints and biases. Standardized protocols for viral characterization are
inconsistently applied across laboratories, and the interpretation of findings can vary de-
pending on the biological system and context. These discrepancies limit the comparability
of results and pose barriers to building a coherent understanding of the virosphere.
Addressing these limitations requires more than technological advancement; it de-
mands a truly interdisciplinary approach to viral discovery. RNA virus research sits at the
intersection of virology, ecology, evolutionary biology, bioinformatics, structural biology,
and public health [24]. However, fostering effective collaboration across these disciplines
remains a nontrivial task. Differences in technical language, methodological assumptions,
and research priorities can hinder communication and coordination between teams. More-
over, disparities in infrastructure and expertise, especially between institutions in high-
and low-resource settings, exacerbate the difficulty of implementing shared standards and
best practices.
One key area in need of improvement is data interoperability. As viral datasets grow
in size and complexity, the need for standardized metadata, annotation protocols, and
interoperable platforms becomes increasingly urgent. Harmonizing data formats and es-
tablishing community-driven guidelines for viral genome curation and characterization
would enhance reproducibility and facilitate more meaningful comparisons across studies.
Ultimately, the success of virus discovery efforts depends on our ability to bridge disci-
plinary divides and integrate knowledge at multiple levels, from molecular mechanisms
to ecological context to public health relevance. Building collaborative frameworks that
support equitable, interdisciplinary engagement will be essential to fully characterize the
expanding RNA virosphere and translate discovery into actionable insight.

2.6. Future Directions and Collaborative Approaches

Anticipating the changing field of RNA virus discovery requires a proactive approach
and collaborative frameworks across disciplinary and geographical boundaries [24]. Here
we envision future directions for research, emphasizing the integration of advanced tech-
nologies and collaborative approaches to enhance our collective capacity to understand
viral ecosystems and proactively address emerging threats (Table 3).
Viruses 2025, 17, 983 10 of 16

Table 3. Emerging tools and innovations shaping the future of RNA virus discovery.

Innovation Impact On Virus Discovery

Enables detection of highly divergent RNA viruses from massive
Petabase-Scale Alignment
sequencing repositories using signature viral markers like RdRp.
Provides resolution at the level of individual infected cells, uncovering
Single-Cell Sequencing
within-host diversity and viral replication dynamics.
Ultra-Portable Sequencers (E.G., Facilitates real-time, field-based virus detection for outbreak response and
Minion) surveillance in remote locations.
Integrates ecological, host, and evolutionary data to identify high-risk
Predictive Spillover Modeling
interfaces for zoonotic emergence.
Automates and accelerates the functional classification of viral genomes,
Ai-Based Genome Annotation
improving throughput and reliability.
Focuses on uncovering unclassified or unculturable viruses, expanding
Viral Dark Matter Exploration
the known virosphere and redefining taxonomy.
Democratizes computational power, enabling global researchers to
Cloud-Based Discovery Pipelines
analyze viral metagenomic data at scale.
Allows for the synthetic reconstruction and functional testing of candidate
Synthetic Viromics
viruses to evaluate host range and pathogenicity.

2.6.1. Multi-Omic Integration and Predictive Modeling for Surveillance

A key direction for RNA virus discovery involves the convergence of technological
innovation and systems-level biology. A significant area of focus is the integration of multi-
omic approaches, genomics, metagenomics, transcriptomics, proteomics, metabolomics,
and beyond, to achieve a comprehensive understanding of host–virus interactions and the
ecological factors that shape viral emergence [29,44]. This multi-layered strategy enables
the simultaneous analysis of viral genomic content and host cellular responses, offering
insights into the molecular mechanisms of infection, immune evasion, replication strategies,
and viral adaptation across diverse environments and species.
By combining these -omics layers, researchers can generate more robust hypothe-
ses about virus function, host specificity, and transmission potential. Such integrative
frameworks also facilitate the identification of conserved molecular signatures or host
biomarkers associated with pathogenicity or zoonotic risk, critical features for surveillance,
diagnostics, and therapeutic development. Complementing this multi-omic perspective is
the growing need for powerful computational tools capable of processing and interpreting
the increasing volume of data now produced by high-throughput platforms [9,22,30,45].
Future directions in bioinformatics emphasize the deployment of machine learning and
artificial intelligence to support automated genome annotation, functional prediction, and
taxonomic classification. Deep learning models trained on virome-scale datasets are in-
creasingly used to identify viral sequences lacking homology to known taxa, anticipate
mutational trajectories, and predict potential receptor usage, all without requiring prior
experimental characterization.
Of particular importance is the role of predictive modeling in redefining virus dis-
covery into a proactive tool for surveillance and pandemic prevention [9,31,46,47]. By
integrating ecological, environmental, and epidemiological data, such as host distribution,
land use, climate variability, and immunological pressures, computational models can
forecast geographic regions and ecological interfaces where viral spillovers are most likely
to occur. These predictive frameworks enable public health authorities to allocate resources
more strategically, prioritize high-risk zones for targeted sampling, and implement early
interventions before human outbreaks arise. As these tools mature, they hold the potential
Viruses 2025, 17, 983 11 of 16

to not only accelerate discovery but to embed it within real-time, globally coordinated
surveillance systems that can anticipate and mitigate the impact of emerging RNA viruses.

2.6.2. Global Collaborations, Capacity Building and Data Sharing

As the discovery and surveillance of RNA viruses increasingly become global en-
deavors, the role of international collaboration and open data sharing is increasingly
critical. The complexity and speed of viral emergence, particularly in the context of respira-
tory pathogens with pandemic potential, demand a coordinated, transnational response
grounded in transparency, mutual support, and scientific exchange [24]. The future of
RNA virus discovery envisions a interconnected research environment in which scientists,
public health authorities, and institutions across sectors and regions work collaboratively
to detect, understand, and contain viral threats.
Key to this approach is the development of standardized and interoperable data-
sharing platforms that allow for the timely and secure exchange of genomic sequences,
epidemiological metadata, functional annotations, and surveillance findings. Harmonized
protocols for data curation, open-access repositories, and equitable sharing of benefits
are essential not only for accelerating research but also for building trust among partners
and enabling rapid, evidence-based responses to outbreaks. Removing administrative
and political barriers to international data flow, while respecting privacy, sovereignty,
and bioethical norms, is a necessary step in establishing a more cohesive global virology
community. Equally vital to the future of RNA virus discovery is the deliberate and
sustained investment in capacity building [24]. The burden of viral emergence is often
highest in regions with limited surveillance infrastructure, fewer trained personnel, and
restricted access to sequencing or computational resources. To address this disparity, global
initiatives must prioritize training programs, fellowships, workshops, and regional hubs of
excellence that equip researchers with the tools and knowledge needed to participate fully
in discovery efforts. Capacity building is a central component of equitable and effective
global health preparedness.
Fostering a diverse and globally distributed scientific workforce enhances the resilience
and responsiveness of the global virology efforts. Local researchers are best positioned
to detect and contextualize unusual viral signals within their ecological, cultural, and
epidemiological settings. Supporting their inclusion not only enriches the quality of virus
discovery but also empowers communities to manage their local health challenges and
engage meaningfully with global science.

3. Discussion
3.1. The Importance of Virus Discovery in Understanding the Evolutionary Challenges of
RNA Viruses
The evolutionary plasticity of RNA viruses underlies their capacity to adapt rapidly,
evade host defenses, and generate novel variants with pandemic potential. Nowhere is
this adaptability more evident than in the emergence of SARS-CoV-2 variants of concern,
which have repeatedly reshaped the trajectory of the COVID-19 pandemic [48]. The
continued appearance of immune-evasive or highly transmissible lineages underscores the
fact that virus discovery is not merely a foundational exercise in cataloging biodiversity; it is
a critical element of understanding and responding to the evolutionary challenges posed
by RNA viruses [49].
Systematic virus discovery efforts provide the necessary genomic context to interpret
evolutionary trajectories. By expanding the known diversity of viral sequences, particularly
from under-sampled hosts and environments, we enhance the phylogenetic framework
required to detect unusual divergence, recombination, or lineage expansion. This is partic-
Viruses 2025, 17, 983 12 of 16

ularly important for respiratory viruses, whose cross-species transmission events, often
from birds, bats, or rodents, may occur long before human outbreaks are recognized.
Surveillance-based discovery in animal reservoirs, environmental sources, and immuno-
compromised human hosts helps identify the evolutionary intermediates and precursors of
emerging pathogens [50].
For example, the emergence of SARS-CoV-2 variants such as Alpha, Delta, and Omi-
cron has been linked to prolonged intra-host evolution in immunosuppressed individuals,
a process that likely accelerated the accumulation of mutations in key viral proteins like
spike. Discovery pipelines that incorporate longitudinal sampling and deep sequencing in
such patients offer rare windows into within-host evolutionary dynamics. Similarly, identi-
fying novel betacoronaviruses and other RNA respiratory viruses in animal populations
enables earlier risk assessment based on shared receptor usage, transmission potential, and
genomic plasticity [51]. Moreover, virus discovery contributes directly to the identification
of genomic features associated with adaptation, virulence, and immune escape. Novel
RNA viruses characterized through metagenomics and structural prediction platforms
can expose conserved or convergently evolving elements, such as RNA-dependent RNA
polymerases, proteolytic cleavage sites, or glycosylation patterns on viral envelopes, that
are central to viral fitness and host range shifts [52]. These insights not only inform the
molecular basis of evolution but also refine targets for broad-spectrum therapeutics and
vaccine design.
In the context of emerging respiratory diseases, the strategic integration of virus dis-
covery into public health surveillance can act as an evolutionary early warning system.
Discovering and monitoring RNA virus diversity across ecological interfaces allows for
the identification of high-risk lineages before they acquire enhanced human transmissibil-
ity [53]. This anticipatory approach, grounded in evolutionary virology and empowered by
discovery science, is essential for pandemic preparedness.

3.2. Zoonotic Interfaces and the Ecology of Viral Emergence

Zoonotic transmission remains one of the most critical and complex elements in the
RNA virus emergence continuum [31,32]. As novel viruses are increasingly detected at
the junction of wildlife, livestock, and human populations, it becomes clear that spillover
events are not isolated anomalies but rather emergent properties of disrupted ecosystems.
Land-use changes, such as deforestation and agricultural intensification, alongside global
wildlife trade and urban encroachment, have profoundly altered host–pathogen dynamics.
These disruptions facilitate the crossover of RNA viruses, many of which possess a high
mutation rate and plastic genomes, enabling rapid adaptation to new hosts.
Yet, predicting which viruses may emerge remains a formidable challenge [31,54].
Reservoir host identification and ecological context are often poorly understood, espe-
cially in biodiverse regions where surveillance infrastructure is limited. Moreover, many
spillovers are likely cryptic, involving asymptomatic or low-virulence infections that es-
cape detection. To address these gaps, discovery efforts must go beyond cataloging viral
genomes and incorporate ecological, behavioral, and evolutionary data to assess risk in
a biologically meaningful way. Integrative approaches rooted in field ecology, environmen-
tal virology, and evolutionary modeling will be essential to anticipate and mitigate future
zoonotic events.

3.3. Strategic Preparedness and the Ethics of Discovery

The accelerating pace of RNA virus discovery carries with it both promise and respon-
sibility. While technological advances have dramatically expanded our capacity to detect
novel viruses, their utility for global health depends on the translation of genomic data into
Viruses 2025, 17, 983 13 of 16

actionable public health strategies [55]. Preparedness now requires adaptive, data-driven
frameworks that can respond to the velocity and complexity of emerging threats. This in-
cludes the refinement of pathogen risk-ranking models, investment in pan-viral diagnostics
and vaccines, and real-time integration of genomic surveillance into health systems.
However, alongside these strategic imperatives, ethical considerations must be fore-
grounded. Sampling in low-resource or biodiverse settings, often where novel viruses are
most likely to be found, raises questions around informed consent, benefit-sharing, and the
use of indigenous knowledge. Equally important is the governance of open-access data:
while transparency is vital for rapid response, there must be safeguards to prevent misuse
and ensure equitable participation in downstream research and innovation [24]. As RNA
virus discovery becomes more globalized and technologically sophisticated, frameworks
for ethical collaboration, data stewardship, and equitable capacity building will be central
to ensuring that the benefits of discovery are shared and its risks responsibly managed.

3.4. The Next Ten Years of RNA Virus Discovery

The landscape of RNA virus discovery is expected to undergo significant evolution in
the coming decade, driven by ongoing advancements and shifting research paradigms [24].
We stand positioned to delve deeper into enigmatic viral reservoirs, from the guano-laden
roosts of bats to the hydrothermal vents of the deep sea, aiming to unveil ancestral strains
and zoonotic hidden threats. The development of next generation of ultra-fast, ultra-
affordable, and ultra-portable sequencers is anticipated to democratize virus detection,
enabling real-time surveillance and rapid outbreak response that could lead to more imme-
diate bedside diagnostics and on-the-spot environmental monitoring [56,57]. Sophisticated
algorithms, empowered by artificial intelligence, are expected to enhance the deciphering of
the avalanche of genetic data, unveiling the subtle markers of novel viruses and predicting
their potential for zoonotic spillover or pathogenicity [9,31]. This shift in focus extends
beyond readily classified viral families, venturing into the uncharted territory of “viral dark
matter,” encompassing unculturable viruses, uncovering hidden reservoirs, and illumi-
nating the intricate interface of viral interactions within their host organisms [58]. Finally,
future efforts may enable the promise of personalized virus tracking, where individual
genetic profiles inform tailored risk assessments and preventive measures, empowering
a proactive approach to the ever-evolving viral landscape. In the coming decade, RNA virus
discovery is projected to move beyond mere identification, evolving toward a comprehen-
sive understanding of viral origins, evolution, and potential impact. This knowledge will
forge the foundation for the development of intelligent tools for prevention, detection, and
treatment, contributing to greater resilience in the face of the ever-shifting viral panorama.

4. Conclusions
Shaping the Future of RNA Virus Discovery and Global Health Resilience
In conclusion, this review has examined the complex and changing field of RNA
virus discovery, highlighting the significant impact of technological advancements, the
increasing understanding of viral diversity, and the critical role of zoonotic transmission
in shaping global health threats. Together, these insights emphasize the strong need for
adaptive and forward-looking strategies in pandemic preparedness. As the field continues
to evolve, meaningful collaboration emerges as an essential foundation, one that must span
disciplinary, institutional, and geopolitical boundaries.
This review highlights a dynamic and interdependent relationship between viruses,
hosts, and ecosystems, demanding integrated approaches that blend molecular insight
with ecological and epidemiological context. The enduring challenges, ranging from
sampling and data interpretation to viral characterization and equitable access, call for
Viruses 2025, 17, 983 14 of 16

a sustained, coordinated effort across the scientific community. RNA virus discovery will
increasingly rely on the integration of advanced innovation, inclusive global collaboration,
and predictive capacity.
As we move deeper into less-explored viral ecosystems, our commitment to ethi-
cal practice, scientific integrity, and community engagement must remain unwavering.
Fostering a collaborative and equitable research culture is not only ethically important,
it is essential for building global resilience to future pandemics. The roadmap outlined
in this review serves to highlight the importance of action for researchers, public health
leaders, and policymakers to co-create a more prepared, responsive, and inclusive frame-
work for RNA virus discovery, one that meets the challenges of today and addresses those
of tomorrow.

Author Contributions: Conceptualization, H.D. and N.B.; data analysis, H.D. and N.B.; writing—original
draft preparation, H.D.; writing—review and editing, H.D. and N.B. All authors have read and agreed
to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable for studies not involving humans or animals.

Informed Consent Statement: Not applicable for studies not involving humans.

Data Availability Statement: Not applicable for this review.

Acknowledgments: The authors gratefully acknowledge the institutional support of the Instituto
Nacional de Tecnología Agropecuaria (INTA). We sincerely hope that the current and unprecedented
funding cuts to INTA and the broader Argentine Science and Technology system do not compromise
the continuity of research, the vitality of our institutions, or the future of scientific progress in
our country.

Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.

References
1. Alvarez-Munoz, S.; Upegui-Porras, N.; Gomez, A.P.; Ramirez-Nieto, G. Key factors that enable the pandemic potential of RNA
viruses and inter-species transmission: A systematic review. Viruses 2021, 13, 537. [CrossRef] [PubMed]
2. Ankolekar, A.; Eppings, L.; Bottari, F.; Pinho, I.F.; Howard, K.; Baker, R.; Nan, Y.; Xing, X.; Walsh, S.L.F.; Vos, W.; et al. Using
artificial intelligence and predictive modelling to enable learning healthcare systems (LHS) for pandemic preparedness. Comput.
Struct. Biotechnol. J. 2024, 24, 412–419. [CrossRef] [PubMed]
3. Bassi, C.; Guerriero, P.; Pierantoni, M.; Callegari, E.; Sabbioni, S. Novel Virus Identification through Metagenomics: A Systematic
Review. Life 2022, 12, 2048. [CrossRef] [PubMed]
4. Becker, D.; Albery, G.; Sjodin, A.; Poisot, T.; Bergner, L.; Chen, B.; Cohen, L.E.; Dallas, T.A.; Eskew, E.A.; Fagre, A.C.; et al.
Optimising predictive models to prioritise viral discovery in zoonotic reservoirs. Lancet Microbe 2022, 3, e625–e637. [CrossRef]
5. Bolles, M.; Donaldson, E.; Baric, R. SARS-CoV and emergent coronaviruses: Viral determinants of interspecies transmission. Curr.
Opin. Virol. 2011, 1, 624–634. [CrossRef]
6. Borham, A.; Motaal, K.; ElSersawy, N.; Ahmed, Y.; Mahmoud, S.; Musaibah, A.; Abdelnaser, A. Climate Change and Zoonotic
Disease Outbreaks: Emerging Evidence from Epidemiology and Toxicology. Int. J. Environ. Res. Public Health 2025, 22, 883.
[CrossRef]
7. Chang, W.; Harvey, E.; Mahar, J.; Firth, C.; Shi, M.; Simon-Loriere, E.; Geoghegan, J.L.; Wille, M. Improving the reporting of
metagenomic virome-scale data. Commun. Biol. 2024, 7, 1687. [CrossRef]
8. Charon, J.; Olendraite, I.; Forgia, M.; Chong, L.; Hillary, L.; Roux, S.; Kupczok, A.; Debat, H.; Sakaguchi, S.; Tahzima, R.; et al.
Consensus statement from the first RdRp Summit: Advancing RNA virus discovery at scale across communities. Front. Virol.
2024, 4, 1371958. [CrossRef]
9. Chen, G.; Jiang, J.; Sun, Y. RNAVirHost: A machine learning-based method for predicting hosts of RNA viruses through viral
genomes. GigaScience 2024, 13, giae059. [CrossRef]
10. Chen, Y.; Wang, P.; Zhang, F.; Dai, H.; Jiao, X.; Wang, X.-Y.; Yu, Q.-W.; Kang, M.; Su, S.; Wang, D. Sensors for surveillance of RNA
viruses: A One Health perspective. Lancet Microbe 2025, 6, 101029. [CrossRef]
Viruses 2025, 17, 983 15 of 16

11. Cobbin, J.C.; Charon, J.; Harvey, E.; Holmes, E.C.; Mahar, J.E. Current challenges to virus discovery by meta-transcriptomics.
Curr. Opin. Virol. 2021, 51, 48–55. [CrossRef] [PubMed]
12. Rahman, M.M.; Miah, M.; Hossain, M.E.; Rahim, S.; Sultana, S.; Satter, S.M.; Islam, A.; Whitmer, S.L.M.; Epstein, J.H.; Spiropoulou,
C.F.; et al. Development of a culture-independent whole-genome sequencing of Nipah virus using the MinION Oxford Nanopore
platform. Microbiol. Spectr. 2025, 13, e0249224. [CrossRef]
13. Chien, Y.; Chen, F.; Wu, H.; Lin, C.; Chang, W.; Perera, D.; Yang, J.; Lee, M.; Liao, Y. Cost-effective complete genome sequencing
using the MinION platform for identification of recombinant enteroviruses. Microbiol. Spectr. 2023, 11, e02507-23. [CrossRef]
[PubMed]
14. Filloux, D.; Fernandez, E.; Loire, E.; Claude, L.; Galzi, S.; Candresse, T.; Winter, S.; Jeeva, M.L.; Makeshkumar, T.; Martin, D.P.;
et al. Nanopore-based detection and characterization of yam viruses. Sci. Rep. 2018, 8, 17879. [CrossRef]
15. Kleanthous, E.; Olendraite, I.; Lukhovitskaya, N.I.; Firth, A.E. Discovery of three RNA viruses using ant transcriptomic datasets.
Arch. Virol. 2019, 164, 643–647. [CrossRef]
16. Neri, U.; Wolf, Y.I.; Roux, S.; Camargo, A.P.; Lee, B.; Kazlauskas, D.; Chen, I.M.; Ivanova, N.; Zeigler Allen, L.; Paez-Espino, D.;
et al. Expansion of the global RNA virome reveals diverse clades of bacteriophages. Cell 2022, 185, 4023–4037. [CrossRef]
17. Dolja, V.V.; Koonin, E.V. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus
transfer. Virus Res. 2018, 244, 36–52. [CrossRef]
18. Zhou, L.; Leung, T.H. Detection by Single-Cell RNA Sequencing of Virally Mediated Skin Diseases. JID Innov. Ski. Sci. Mol. Popul.
Health 2025, 5, 100348. [CrossRef]
19. Russell, A.B.; Elshina, E.; Kowalsky, J.R.; Te Velthuis AJ, W.; Bloom, J.D. Single-Cell Virus Sequencing of Influenza Infections That
Trigger Innate Immunity. J. Virol. 2019, 93, e00500-19. [CrossRef]
20. Fromm, A.; Hevroni, G.; Vincent, F.; Schatz, D.; Martinez-Gutierrez, C.A.; Aylward, F.O.; Vardi, A. Single-cell RNA-seq of the rare
virosphere reveals the native hosts of giant viruses in the marine environment. Nat. Microbiol. 2024, 9, 1619–1629. [CrossRef]
21. Dominguez-Huerta, G.; Wainaina, J.; Zayed, A.; Culley, A.; Kuhn, J.; Sullivan, M. The RNA virosphere: How big and diverse is it?
Environ. Microbiol. 2023, 25, 209–215. [CrossRef] [PubMed]
22. Edgar, R.C.; Taylor, B.; Lin, V.; Altman, T.; Barbera, P.; Meleshko, D.; Lohr, D.; Novakovsky, G.; Buchfink, B.; Al-Shayeb, B.; et al.
Petabase-scale sequence alignment catalyses viral discovery. Nature 2022, 602, 142–147. [CrossRef] [PubMed]
23. Elrashedy, A.; Mousa, W.; Nayel, M.; Salama, A.; Zaghawa, A.; Elsify, A.; Hasan, M. Advances in bioinformatics and multi-omics
integration: Transforming viral infectious disease research in veterinary medicine. Virol. J. 2025, 22, 22. [CrossRef]
24. Esposito, M.; Turku, S.; Lehrfield, L.; Shoman, A. The Impact of Human Activities on Zoonotic Infection Transmissions. Animals
2023, 13, 1646. [CrossRef]
25. Geoghegan, J.; Holmes, E. Predicting virus emergence amid evolutionary noise. Open Biol. 2017, 7, 170189. [CrossRef]
26. Ghai, R.R.; Carpenter, A.; Liew, A.Y.; Martin, K.B.; Herring, M.K.; Gerber, S.I.; Hall, A.J.; Sleeman, J.M.; VonDobschuetz, S.;
Behravesh, C.B. Animal reservoirs and hosts for emerging alphacoronaviruses and betacoronaviruses. Emerg. Infect. Dis. 2021,
27, 1015–1022. [CrossRef]
27. Gorbalenya, A.; Lauber, C. Bioinformatics of virus taxonomy: Foundations and tools for developing sequence-based hierarchical
classification. Curr. Opin. Virol. 2022, 52, 48–56. [CrossRef]
28. Grange, Z.L.; Goldstein, T.; Johnson, C.K.; Anthony, S.; Gilardi, K.; Daszak, P.; Olival, K.J.; O’Rourke, T.; Murray, S.; Olson,
S.H.; et al. Ranking the risk of animal-to-human spillover for newly discovered viruses. Proc. Natl. Acad. Sci. USA 2021,
118, e2002324118. [CrossRef]
29. Hill, V.; Githinji, G.; Vogels, C.; Bento, A.; Chaguza, C.; Carrington, C.; Grubaugh, N. Toward a global virus genomic surveillance
network. Cell Host Microbe 2023, 31, 861–873. [CrossRef]
30. Holmes, E.C. The ecology of viral emergence. Annu. Rev. Virol. 2022, 9, 173–192. [CrossRef]
31. Holmes, E.; Krammer, F. Goodrum F Virology—The next fifty years. Cell 2024, 187, 5128–5145. [CrossRef] [PubMed]
32. Hou, X.; He, Y.; Fang, P.; Mei, S.; Xu, Z.; Wu, W.-C.; Tian, J.-H.; Zhang, S.; Zeng, Z.-Y.; Gou, Q.-Y.; et al. Using artificial intelligence
to document the hidden RNA virosphere. Cell 2024, 187, 6929–6942.e16. [CrossRef] [PubMed]
33. Kawasaki, J.; Suzuki, T.; Hamada, M. Hidden challenges in evaluating spillover risk of zoonotic viruses using machine learning
models. Commun. Med. 2025, 5, 187. [CrossRef] [PubMed]
34. Kokoris, M.; McRuer, R.; Nabavi, M.; Jacobs, A.; Prindle, M.; Cech, C.; Berg, K.; Lehmann, T.; Machacek, C.; Tabone, J.; et al.
Sequencing by Expansion (SBX)—A novel, high-throughput single-molecule sequencing technology. bioRxiv 2025. [CrossRef]
35. Koonin, E.; Kuhn, J.; Dolja, V.; Krupovic, M. Megataxonomy and global ecology of the virosphere. ISME J. 2024, 18, wrad042.
[CrossRef]
36. Koren, O.; Chavez, L. The land-use land-cover change–emerging infectious disease nexus reconsidered. BioScience 2025, biaf045.
[CrossRef]
37. Lauber, C.; Seitz, S. Opportunities and challenges of data-driven virus discovery. Biomolecules 2022, 12, 1073. [CrossRef]
Viruses 2025, 17, 983 16 of 16

38. Liu-Wei, W.; Kafkas, Ş.; Chen, J.; Dimonaco, N.J.; Tegnér, J.; Hoehndorf, R. DeepViral: Prediction of novel virus–host interactions
from protein sequences and infectious disease phenotypes. Bioinformatics 2021, 37, 2722–2729. [CrossRef]
39. Mandl, J.N.; Ahmed, R.; Barreiro, L.B.; Daszak, P.; Epstein, J.H.; Virgin, H.W.; Feinberg, M.B. Reservoir host immune responses to
emerging zoonotic viruses. Cell 2015, 160, 20–35. [CrossRef]
40. Marie, V.; Gordon, M. The (Re-)Emergence and Spread of Viral Zoonotic Disease: A Perfect Storm of Human Ingenuity and
Stupidity. Viruses 2023, 15, 1638. [CrossRef]
41. Martinez-Hernandez, F.; Fornas, O.; Lluesma Gomez, M.; Bolduc, B.; de la Cruz Peña, M.J.; Martínez, J.M.; Anton, J.; Gasol, J.M.;
Rosselli, R.; Rodriguez-Valera, F.; et al. Single-virus genomics reveals hidden cosmopolitan and abundant viruses. Nat. Commun.
2017, 8, 15892. [CrossRef] [PubMed]
42. Padhi, A.; Agarwal, A.; Saxena, S.K.; Katoch, C.D.S. Transforming clinical virology with AI, machine learning and deep learning:
A comprehensive review and outlook. VirusDisease 2023, 34, 345–355. [CrossRef]
43. Pandit, P.; Anthony, S.; Goldstein, T.; Olival, K.; Doyle, M.; Gardner, N.R.; Bird, B.; Smith, W.; Wolking, D.; Gilardi, K.; et al.
Predicting the potential for zoonotic transmission and host associations for novel viruses. Commun. Biol. 2022, 5, 844. [CrossRef]
44. Pauciullo, S.; Zulian, V.; La Frazia, S.; Paci, P.; Garbuglia, A. Spillover: Mechanisms, Genetic Barriers, and the Role of Reservoirs
in Emerging Pathogens. Microorganisms 2024, 12, 2191. [CrossRef]
45. Plowright, R.; Parrish, C.; McCallum, H.; Hudson, P.; Ko, A.; Graham, A.; Lloyd-Smith, J. Pathways to zoonotic spillover. Nat.
Rev. Microbiol. 2017, 15, 502–510. [CrossRef]
46. Posada-Cespedes, S.; Seifert, D.; Beerenwinkel, N. Recent advances in inferring viral diversity from high-throughput sequencing
data. Virus Res. 2017, 239, 17–32. [CrossRef]
47. Roux, S.; Matthijnssens, J.; Dutilh, B. Metagenomics in Virology. Encycl. Virol. 2021, 1, 133–140. [CrossRef]
48. Rosenberg, R. Detecting the emergence of novel, zoonotic viruses pathogenic to humans. Cell. Mol. Life Sci. 2015, 72, 1115–1125.
[CrossRef]
49. Santiago-Rodriguez, T.; Hollister, E. Unraveling the viral dark matter through viral metagenomics. Front. Immunol. 2022,
13, 1005107. [CrossRef]
50. Sharan, M.; Vijay, D.; Yadav, J.; Bedi, J.; Dhaka, P. Surveillance and response strategies for zoonotic diseases: A comprehensive
review. Sci. One Health 2023, 2, 100050. [CrossRef]
51. Šimičić, P.; Židovec-Lepej, S. A glimpse on the evolution of RNA viruses: Implications and lessons from SARS-CoV-2. Viruses
2022, 15, 1. [CrossRef] [PubMed]
52. Simmonds, P.; Adriaenssens, E.; Zerbini, F.M.; Abrescia, N.; Aiewsakun, P.; Alfenas-Zerbini, P.; Bao, Y.; Barylski, J.; Drosten, C.;
Duffy, S.; et al. Four principles to establish a universal virus taxonomy. PLoS Biol. 2023, 21, e3001922. [CrossRef] [PubMed]
53. Struelens, M.J.; Ludden, C.; Werner, G.; Sintchenko, V.; Jokelainen, P.; Ip, M. Real-time genomic surveillance for enhanced control
of infectious diseases and antimicrobial resistance. Front. Sci. 2024, 2, 1298248. [CrossRef]
54. Walensky, R.P.; Walke, H.T.; Fauci, A.S. SARS-CoV-2 variants of concern in the United States—Challenges and opportunities.
JAMA 2021, 325, 1037–1038. [CrossRef]
55. Wang, S.; Li, W.; Wang, Z.; Yang, W.; Li, E.; Xia, X.; Yan, F.; Chiu, S. Emerging and reemerging infectious diseases: Global trends
and new strategies for their prevention and control. Signal Transduct. Target. Ther. 2024, 9, 223. [CrossRef]
56. Yang, Z.; Shan, Y.; Liu, X.; Chen, G.; Pan, Y.; Gou, Q.; Zou, J.; Chang, Z.; Zeng, Q.; Yang, C.; et al. VirID: Beyond Virus
Discovery—An Integrated Platform for Comprehensive RNA Virus Characterization. Mol. Biol. Evol. 2024, 41, msae202.
[CrossRef]
57. Zhang, Y.; Chen, Y.; Wang, W.; Qin, X.; Holmes, E. Expanding the RNA Virosphere by Unbiased Metagenomics. Annu. Rev. Virol.
2019, 6, 119–139. [CrossRef]
58. Zhang, T.; Li, H.; Jiang, M.; Hou, H.; Gao, Y.; Li, Y.; Wang, F.; Wang, J.; Peng, K.; Liu, Y.-X. Nanopore sequencing: Flourishing in its
teenage years. J. Genet. Genom. 2024, 51, 1361–1374. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.