Virus Evolution

Swine Influenza A virus (swIAV) poses constant health threat for both humans and animals. This study comprehensively analysed the genetic diversity and evolution of European swIAVs from 2013 to 2022. To address existing classification gaps, an easily accessible full-genome genotyping tool based on Nextclade was developed alongside a global nomenclature system. Application of this tool to European sequence data revealed unprecedented genetic diversity, successfully identifying 150 swIAV genotypes mixing segments of both swine and human origin. Bayesian phylogeographic models, combined with economic data, identified that the trade of live weaner pigs for fattening drove the transboundary spread of swIAVs across Europe. The study detected repeated human-to-swine reverse-zoonotic events across multiple European countries. These findings clearly highlight the role of commercial pig movements in viral dissemination and emphasize the critical importance of harmonized genomic surveillance to monitor swIAV viral evolution to inform global pre-pandemic preparedness strategies.

TThe discovery of virophages and polinton-like viruses (PLVs) sparked studies of their hyperparasitic relationships with their associated giant viruses and hosts. To date, every cultured virophage and PLV depends on an Imitervirales helper virus. The freshwater alga Chrysochromulina parva is infected by viruses in the Imitervirales and Algavirales, and recent work showed that the PLV, CpV-PLV Moe, replicates via co-infection with the Imitervirales host CpV-BQ3. The other known PLVs, CpV PLV Larry and CpV PLV Curly, were subsequently isolated alongside CpV-BQ1, a putative Algavirales virus, raising the possibility that they depend on an Algavirales helper. To determine the nature of Larry and Curlys hyperparasitic relationships, infection experiments were conducted with each PLV and either CpV-BQ3 or CpV-BQ1. These experiments demonstrated that that neither Larry nor Curly replicated when introduced to C. parva alone, nor during mixed infections that included the Imitervirales virus BQ3. In contrast, the Algavirales virus CpV-BQ1 supported replication of both PLVs, with each increasing roughly 1000-fold over the course of infection while CpV-BQ1 replication was simultaneously reduced up to 100-fold. Together, these findings provide the first culture based evidence that PLV replication can be supported by an Algavirales helper and demonstrate that C. parva supports at least two distinct hyperparasitic relationships spanning different viral orders within the class Megaviricetes.

Zika virus is a mosquito-borne flavivirus that caused a major epidemic in the Americas in 2015 and 2016 and is associated with congenital birth defects and neurological disease. Like other flaviviruses, it depends on conserved sequence and RNA structural elements that carry out essential steps in its life cycle and in evading host immune defenses. Public sequence databases and annotation tools give free access to Zika sequence data, but comparable structural information is sparse. We built a computational model of Zika sequence and secondary structure for use with the VADR software package, which validates and annotates Zika sequences for GenBank submission and assigns each sequence to a genetic lineage following a published classification scheme. On a test set of 1254 Zika sequences, 97.2% passed validation, and the model's annotations agreed with those of an existing annotation tool (VIGOR4) for 99.3% of the sequences compared. We also extended VADR to draw secondary structure diagrams of each sequence's structural elements in its linear and circular conformations using R2DT, and we submitted a newly characterized Zika pseudoknotted RNA element to the Rfam database. Together these freely available resources provide secondary structure information for every Zika sequence.

Broad-range RT-qPCR assays are essential for detecting highly diverse viruses within virus families, but designing suitable primers remains challenging because of extensive sequence diversity and biases in public sequence databases. Here, we present a semi-automated workflow that combines sequence clustering and varVAMP to streamline pan-RT-qPCR primer design. The workflow reduces sequence redundancy, improves the representation of viral diversity, and facilitates the identification of broadly reactive primer candidates. We demonstrate the workflow using the arboviral genera Orthoflavivirus and Alphavirus as proof-of-concept datasets, showing its applicability to large and genetically diverse viral sequence collections. The workflow provides a practical, scalable, and reproducible framework for developing broad-range primer candidates and may support surveillance, diagnostics, and preparedness for emerging or divergent viruses.

Bats are major natural reservoirs for coronaviruses, yet complete viral genomes from South America remain scarce, limiting evolutionary and taxonomic understanding. Here, we conducted metatranscriptomic sequencing of coronavirus-positive bat samples collected across two ecologically distinct Brazilian biomes: the Atlantic Forest and the semi-arid Caatinga. We recovered seven complete or near-complete genomes belonging to Alphacoronavirus and Betacoronavirus. Phylogenetic and comparative similarity analyses of conserved replicase domains (3CLpro, NiRAN, RdRp, ZBD, HEL1), following International Committee on Taxonomy of Viruses (ICTV) demarcation criteria, revealed significant viral diversity. Within Alphacoronavirus, two genomes from Atlantic Forest phyllostomid bats (Artibeus lituratus and Carollia perspicillata) formed a deeply divergent sister lineage to Amalacovirus, exhibiting a mean amino acid similarity of 76.7% with the reference genome. Within Betacoronavirus, one genome from a Caatinga phyllostomid bat (Artibeus planirostris) clustered within the recently described Ambecovirus clade, displaying 75.9% mean amino acid similarity with mormoopid-associated reference sequences. Based on these divergence levels and non-recombinant genomic architectures, we propose two novel candidate subgenera, Phyllacovirus and Phyllobecovirus, alongside potential novel viral species. Furthermore, our findings demonstrate strong host-associated structuring and biogeographical partitioning of viral lineages across Neotropical biomes. Overall, this study expands the genomic landscape of South American bat coronaviruses and underscores the importance of continuous genomic surveillance at human-wildlife interfaces.

Porcine epidemic diarrhea virus (PEDV) was introduced into the United States (USA) in 2013 and has circulated ever since. PEDV causes significant losses in the swine industry, with the greatest impact observed during the first years following its introduction into the country. However, the evolutionary dynamics of the virus remain underexplored. Recently, analyses of the Spike protein subunit S1 suggested that the PEDV population currently circulating in the USA represents a fraction of its historical diversity and comprises only two clades restricted to specific geographic regions. These observations were based on a limited set of S1 sequences. Furthermore, our understanding of evolutionary dynamics at the genomic level is even more limited, as complete PEDV genomes have not been generated since 2017. Here, we characterized the evolutionary dynamics of PEDV populations in the USA using complete genomes and assessed the contribution of different evolutionary mechanisms to shaping population structure. We analyzed 338 complete genomes, of which 25 were obtained in this study, together with 623 Spike S1 sequences, of which 41 were obtained in this study. All sequences obtained in this study were assigned to the non-S-INDEL strain. No S-INDEL sequences were recovered in this study, although these viruses continue to circulate in the country. Our analyses revealed that most sequences sampled after 2023 formed a monophyletic clade divergent from sequences collected in previous years, suggesting an ongoing temporal population structuring. Population structure analyses further supported evidence of population differentiation over time. Recombination analyses indicated that sequences belonging to the contemporary clade shared a single recombination event, suggesting that this clade may have emerged through recombination. Time-scaled S1 phylogenies were consistent with previous findings, supporting the circulation of only two contemporary clades; however, one of these clades was detected for the first time outside its previously reported geographic range. Our findings document temporal genetic differentiation in US PEDV populations, consistent with continued viral evolution, and broaden the known geographic distribution of contemporary S1 clades. These results highlight the value of continued genomic surveillance to monitor changes in PEDV diversity and distribution and provide sequence data to inform vaccine research.

Since emerging in 2020, clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has disseminated globally, causing substantial infection and mortality in wild birds and poultry. Additionally, numerous spill-over events into mammals have occurred, including mass mortalities and sporadic human infections. Such events can drive the acquisition of mammalian-adaptive mutations, which further increase the risk to humans; the most well characterised mutation is the glutamate to lysine change at amino acid position 627 in the polymerase basic 2 protein (PB2-627K). Across avian influenza viruses, PB2-627K enhances polymerase activity and replication in mammalian cells, increasing zoonotic and pandemic potential. However, bioinformatic analyses indicate that PB2-627K remains rare among clade 2.3.4.4b H5N1 viruses circulating in avian populations. Here, we investigated the impact of PB2-627K on viral fitness in avian hosts to assess the potential for mammalian-adapted viruses to re-establish in birds, mimicking infection of avian species following scavenging on mammalian species where PB2-627K has already emerged. Using a representative H5N1 virus (European genotype AB), PB2-627K increased polymerase activity and replication in human cells without compromising replication in avian cells. A mammalian-derived PB2-627K H5N1 isolate from a fox exhibited comparable replication kinetics and transmission efficiency in chickens and ducks to a closely related PB2-627E virus. Notably, PB2-627K remained genetically stable at consensus level following infection and transmission in both avian hosts. These findings demonstrate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian populations, potentially elevating zoonotic risk and underscoring the importance of surveillance for such mutations in birds.

Ancestral recombination graphs (ARGs) represent the coalescent history at every locus in the genome, subject to mutation and recombination. Modelling viruses using the coalescent with recombination remains less explored, due to the potential for complex, multi-scale dynamics. Using perturbative methods, we establish mathematical bounds that describe the balance between inter-host and within-host coalescence, and demonstrate that the highly-recombinant Epstein-Barr Virus (EBV) coalesces predominantly at the inter-host level. We then modify SINGER, a tool from diploid genetics, for haploid ARG inference with fine-grained variable recombination rates. We provide insight into the paradox of high recombination rates and slow linkage disequilibrium decay, as ARG-based methods potentially allow us to disentangle epistatic effects from shared ancestry, and illustrate this using the coalescent topologies of EBV's host cell-entry glycoproteins. Our findings demonstrate that ARG-based methods recover gene-level selective structure, and provide a general method for analysing virus phylodynamics in the limit of small quantities.

Oysters are vital to global aquaculture and marine ecosystems, yet, their associated RNA viruses remain largely unexplored. Here, we analysed ~1,000 publicly available metatranscriptomic datasets from 13 oyster species across 14 countries and 5 continents to characterize the oyster RNA virome (ORV). We identified 1,515 distinct RNA viruses putatively representing 17 viral orders and 766 species-level viral operational taxonomic units (vOTUs), with ~62% being previously undocumented putative species. These viruses not only populate extant branches of RNA virus evolution, but also represent previously unrecognized families and genera, and include viruses that putatively infect bacteria, fungi, protists, plants, invertebrates, and vertebrates. Based on sequence similarity, we found many calicivirus-like, orthoreovirus-like, astrovirus-like, and picornavirus-like viruses that do not infect invertebrates or microbes; thus, oysters accumulate diverse viruses from the surrounding water, and can potentially be interrogated as reporters of viruses circulating in the environment.

Urban waterfowl are important natural reservoirs of viruses that can affect wildlife, livestock and human health, yet the diversity and spatial distribution of these viral communities remain poorly understood. Here, we characterised the faecal viromes of urban waterfowl sampled from 13 locations across the length of Aotearoa (New Zealand). We identified 96 avian viruses (50 species) spanning nine viral families, including 33 previously undescribed viruses. Picornaviruses were the most widespread viral group, while several viruses were closely related to those previously identified in Australian waterfowl, indicating that some viral lineages are likely distributed across the broader Australasian region. Despite sampling locations spanning more than 1,500 km, virome composition exhibited little evidence of geographic structure, with little difference in viral diversity or community composition between sampling locations. Network analyses similarly demonstrated extensive sharing of virus species among sampling locations, indicating little geographic structuring of virome composition among urban waterfowl. In addition, we recovered the complete genome of a low-pathogenic A(H3N8) influenza A virus from mallards (Anas platyrhynchos) in the most southern region of Aotearoa. These findings reveal extensive viral diversity and widespread connectivity among urban waterfowl, providing new insights into viral ecology and the potential for widespread virus distribution across Aotearoa.

Migration can increase population connectivity and gene flow but may also facilitate pathogen transmission among hosts and habitats. The way life-history strategies shape viral diversity in closely related freshwater fishes remains poorly understood. Here, we used metatranscriptomics to compare the viromes of two fish in the family Galaxiidae with contrasting life-history strategies: amphidromous [i]nanga (Galaxias maculatus) and river-resident Gollum galaxias (Galaxias gollumoides). We analysed 54 pooled libraries derived from 416 individuals collected across 27 sites spanning multiple catchments and fish-passage barriers in southern New Zealand. A total of 13 novel fish-associated viruses were identified from five viral families (Astroviridae, Circoviridae, Coronaviridae, Hepeviridae, and Picornaviridae). Virome composition differed markedly between species, with only a single virus-positive pool detected among [i]nanga, whereas Gollum galaxias harboured 11 novel viruses, with circoviruses dominating the virome. No fish-associated viruses were detected in post-larval juveniles of either species, and alpha diversity did not differ significantly among catchments, sites or positions relative to fish-passage barriers. However, beta diversity analyses revealed significant spatial structuring of viral communities among sites and catchments, indicating localised variation in virome composition. Contrary to our predictions, the migratory species exhibited substantially lower viral diversity than the river-resident species. The absence of detectable viruses in juveniles, combined with the greater viral richness of the longer-lived Gollum galaxias, suggests that host longevity and cumulative environmental exposure may be important drivers of virome assembly. These findings demonstrate that host life-history strategy influences virome composition and reveal unexpectedly low viral diversity in a globally widespread migratory galaxiid, providing new insights into virus-host ecology and connectivity in freshwater fishes.

Dengue virus (DENV) is genetically diverse, consisting of four antigenically distinct serotypes and extensive sub-serotype variation that can influence epidemiology and control. As DENV continues to expand globally, there is an urgent need to characterize the dynamics of distinct DENV genetic lineages, including their persistence, establishment, and spread. Using a standardized DENV lineage nomenclature system and 37,828 publicly available DENV genomes sampled from 2000-2024, we classified sequences across nine global regions to construct a comprehensive resource of regional lineage distributions over time. We found marked regional differences in lineage diversity, with higher lineage richness in Southeast and East Asia than in the Americas. We further showed that COVID-19-related disruptions were associated with decreased regional lineage persistence and detection of new lineages during 2020-2021, followed by rebounds after 2022 and, in some regions, substantial lineage turnover. Finally, we identified a small set of globally expansive (cosmopolitan) lineages that are over-represented in available sequences, including five first-order minor lineages detected in at least seven of nine regions: 3III_B.3, 2II_F.1, 2II_A.1, 1III_A.3, and 1I_K.3. Together, these results demonstrate how standardized lineage classification enables global tracking of emerging DENV diversity and supports targeted genomic surveillance.

The Democratic Republic of the Congo is experiencing a major outbreak of Ebola disease caused by Orthoebolavirus bundibugyoense (Bundibugyo virus), which was declared on May 15, 2026. To investigate the timing of emergence and early transmission, we generated near full length Bundibugyo virus genomes from 686 PCR-confirmed individuals across 22 health zones. We estimated the time of emergence as late February 2026 (95% highest posterior density interval [HPDI]: January 21 and March 25, 2026) and an evolutionary rate of 8.7X10-4 substitutions per site per year (95% HPDI: 7.6 and 9.9X10-4), with no evidence of mutations associated with rapidly growing lineages.

Communities living in biodiversity hotspots are at increased risk of pathogen exposure and spread due to insufficient access to nutrition, clean water, and sanitation. Viruses, in particular, have high epidemic and spillover potential, and widespread health impacts, yet, viral diversity and dynamics remain understudied in many high-risk regions. In Madagascar, a hotspot with unparalleled biodiversity, socioeconomic constraints, and a heavy burden of infectious diseases, ongoing healthcare initiatives provide opportunities to survey circulating viral diversity. Here, we used a metagenomic workflow to characterize the viromes of oral/nasal swabs from febrile individuals visiting a mobile healthcare clinic in southeastern Madagascar, of which 49% were malaria-positive (Plasmodium falciparum). We characterized sequences of human-infecting viruses from twelve families (Adenoviridae, Anelloviridae, Orthoherpesviridae, Papillomaviridae, Polyomaviridae, Astroviridae, Coronaviridae, Matonaviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, and Sedoreoviridae). These viral sequences generally showed high similarity to known lineages circulating globally, but multiple viruses considered rare or emerging were identified (e.g. enterovirus C105, enterovirus C117). While we did not detect a significant association between malaria status and pathogenic virus presence, younger individuals had higher odds of having a pathogenic virus. Nonpathogenic viruses in Anelloviridae were prevalent and diverse with richness ranging from 0 to 70. Individuals with P. falciparum or a pathogenic virus tended to share more anelloviruses. We also detected viruses that are protist-associated, plant-infecting, or with unknown hosts. Overall, our work documents extensive viral diversity in Madagascar, highlighting how metagenomic workflows can be used to build viral genomic resources for pathogen surveillance in regions with high infectious disease burden.

A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well-characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo. Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPC{Delta}28-68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPC{Delta}28-68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo, suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.

SARS-CoV-2 continues to circulate globally at high levels and to evolve into new variants. Recently, a new saltation variant harbouring a highly mutated spike protein, PJ.2.1, emerged in North America. Here, we provide a brief epidemiological and virological characterization of PJ.2.1 and cocirculating variants. We report that PJ.2.1 is spreading in the USA, Canada, and Spain and mainly infects individuals aged 60 years and older, as do most other variants, with the exception of BA.3.2, which preferentially infects individuals aged 0-9 years. PJ.2.1 was less susceptible to inhibition by an antibody against ACE2 than were the other variants, indicating increased ACE2 binding. In contrast, PJ.2.1 did not show augmented evasion of antibodies from vaccinees who had received the LP.8.1-adapted vaccine compared with most other variants analysed. These results suggest that augmented ACE2 affinity rather than increased antibody evasion might drive the current, rapid spread of PJ.2.1.

Vibrio coralliilyticus (V. coralliilyticus) is an emerging coral pathogen associated with tissue-loss diseases in reef-building corals and diseases in other marine organisms, such as oyster larvae. Despite the great ecological and economic significance of this pathogenic bacterium, characterization of bacteriophages infecting V. coralliilyticus remains limited, particularly of temperate phages. In this study, we describe the isolation and characterization of four novel phages - Vibrio phages Satoshi, Kolob, Ushijima, and Kane - obtained from coastal waters near L[a]ie, Hawaii, using the indigenous Hawaiian V. coralliilyticus strain OCN008 as the host. All four phages produced small, turbid plaques and encoded integrases, consistent with temperate lifestyles. The genome organization and taxonomic relationships of the viruses were examined using whole-genome sequencing and comparative genomics, while representative virion morphologies were examined by transmission electron microscopy. To the best of the authors' knowledge, these are the first V. coralliilyticus phages reported from Oahu, Hawaii. Comparative genomic analyses support the placement of all four phages within the class Caudoviricetes, but outside of any previously described orders, families, or genera within the class. Satoshi, Kolob, and Ushijima, along with previously described V. coralliilyticus phage CKB-S2, cluster within one genus-level lineage, while Kane, which has no close relatives in available databases, represents a distinct, previously undescribed lineage. All four phages were also found to contain homologs to experimentally established virulence factors, which may contribute to the pathogenicity of the host bacterium during lysogeny. Together, these isolates expand the diversity of phages infecting V. coralliilyticus and support the proposal of two new genera of Caudoviricetes: the genus Mokushimavirus, containing Satoshi, Kolob, Ushijima, and CKB-S2, and the genus Laievirus, containing Kane.

A critical step in the emergence of a virus is the ability of the viral protein to bind a host receptor and mediate cell entry. For many coronaviruses, this interaction occurs between the Spike S1 subunit and the human ACE2 receptor. Whether this binding interface can be computationally distinguished across unstudied viruses without experimentally resolved protein structures remains an open question. We predicted how 28 emerging coronaviruses may bind to human ACE2 using structural predictions, static interaction prediction programs, and molecular dynamics simulations. To screen the emerging coronaviruses, we predicted a library of S1 structures using AlphaFold. These predicted structures were then used to model the S1-ACE2 interaction with AlphaFold, ClusPro, and HADDOCK. We used known ACE2-binding sarbecoviruses as positive controls and coronaviruses that bind other receptors as negative controls to threshold predicted binding. Contact analysis quantified the predicted binding and revealed that these static interaction prediction methods varied in discriminative power. Less restrained static predictions separated binders from non-binders, whereas heavily restrained docking did not, potentially forcing an interaction where none should exist. This analysis highlighted an emerging coronavirus, Zhejiang2013, as a potential ACE2 binder. We used molecular dynamics simulations to further assess the static predictions and model the interaction over time. Overall, our results indicate that Zhejiang2013 exhibits dynamic interaction patterns consistent with ACE2 binding. Given that two ACE2-binding coronaviruses have caused global pandemics within the past two decades, identifying potential ACE2 binders is critical for early warning and pandemic preparedness.

Since 2020, highly pathogenic avian influenza (HPAI) A(H5Nx) clade 2.3.4.4b viruses have spread globally, causing extensive outbreaks in domestic and wild birds. Increased circulation has resulted in frequent spillover to mammals, and occasional mammal-to-mammal transmission. Although human infections remain rare, the zoonotic potential of these viruses continues to be a public health concern. We investigated six cases of HPAI in red foxes (Vulpes vulpes) in Norway during 2022-2024 and identified infections with H5N1 and H5N5 viruses. Pathological and virological investigations demonstrated systemic infection, with prominent lesions in the brain and/or lungs. Phylogenetic analyses showed high similarity between viruses detected in foxes and those concurrently circulating in wild birds. The mammalian adaptation marker PB2:E627K was identified in one H5N1 virus and as a minority variant in a second H5N1 virus.

The 2022 global mpox outbreak was characterized by an unusual accumulation of more than fifty APOBEC-signature substitutions in MPXV genomes. A previous study suggested that these substitutions preferentially occur within and near hairpins or cruciforms - stem-loop structures formed by inverted repeats. Here, we show that this conclusion resulted from an inappropriate statistical analysis and, using two substantially larger datasets, demonstrate that APOBEC-signature mutations are specifically enriched at TC motifs located at the 3' ends of hairpin loops, but not in other regions of hairpins/cruciforms or in their vicinity. We further identified a preference for stem-loop structures with longer stems and shorter loops, consistent with patterns previously reported for the APOBEC mutagenesis in cancer genomes. Our analysis also revealed a positive association between APOBEC-signature mutation propensity and hairpin stability in single-stranded DNA. Nevertheless, stem-loop structures with long stems and short loops are relatively rare in the MPXV genome, limiting their contribution to the genome-wide distribution of APOBEC-signature mutations to a predominantly local effect. In contrast, extended nucleotide context downstream of the TC motif showed strong genome-wide predictive value, with a marked preference for guanine-rich trinucleotide immediately downstream. Analysis of the upstream nucleotide context was consistent with the mutational specificity of APOBEC3A. Together, these results identify distinct structural and sequence determinants of the APOBEC-associated mutagenesis in MPXV and show that local hairpin/cruciform architecture and extended nucleotide context contribute at different genomic scales.