Archives of Virology

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.

Background: Klebsiella pneumoniae is a key causative agent of multidrug resistant nosocomial infections. Bacteriophages offer a promising alternative treatment, but the diversity of Klebsiella species requires the characterisation of a broad range of phages, particularly the poorly characterised jumbo phages. Materials and Methods: Phage BMCPR_005026 was isolated from sewage against Klebsiella pneumoniae. Whole genome sequencing, transmission electron microscopy and host range analysis against a diverse set of 40 clinically relevant Klebsiella strains were used to characterise the phage. Results: The virion had a myovirus morphology and genomic analysis placed it within an undefined family in Caudoviricetes where it forms a new species within the genus Alcyoneusvirus. BMCPR_005026 could form plaques on 3 of the 40 Klebsiella strains tested (7.5%) and formed halos. Conclusion: BMCPR_005026 represents a new species of phage within the genus Alcyoneusvirus with activity against 3 Klebsiella KL-types.

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.

Recent zoonotic coronavirus outbreaks have sparked an interest in understanding coronaviruses circulating in animal reservoirs. While bats are recognised as major reservoirs for these viruses, coronaviruses have also been detected in a wide range of terrestrial small mammals, particularly rodents. However, coronavirus diversity in these mammals, specifically in Europe, is still poorly characterised. In this study, we detected and characterised coronaviruses in terrestrial small mammals and bats from Belgium, France, Germany, and Ireland. We screened tissue samples from rodents and shrews and environmental bat guano using pan-coronavirus real-time and conventional PCRs, with positive results confirmed by sanger sequencing. Among 2667 bat and 1152 terrestrial mammal samples, we detected 59 positive samples, 36 from bats and 23 from rodents, from which we recovered 60 coronavirus sequences, spanning both Alphacoronavirus and Betacoronavirus lineages. Through next generation sequencing we identified one Pipistrellus pygmaeus bat guano sample carrying two distinct alphacoronaviruses, representing Nyctacovirus and Pedacovirus subgenera. Among rodents, most coronavirus-positive samples originated from a zoo in Belgium, where several CoV lineages co-circulated. The identified rodent coronaviruses clustered among previously described rodent-associated lineages, some of which also include human and other mammal-derived coronavirus sequences. Consistent with previous studies, we found sarbecoviruses in horseshoe bats (particularly Rhinolophus ferrumequinum and R. hipposideros) and merbecoviruses in Plecotus auritus bats, and these viruses are distantly related to the human coronaviruses of the respective families. Similar to bat coronaviruses, some rodent coronaviruses strains seemed to be association with specific rodent species. In conclusion, our study enhances the understanding of coronaviruses in rodents and bats, revealing potential hotspots for these viruses and their implication for human and animal health. These findings underscore the necessity for continued surveillance of coronaviruses in wildlife reservoirs to mitigate future zoonotic spillover risks.

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.

Low pathogenic avian influenza A(H7N7) virus detected in a poultry worker in Taiwan, 2026 was related to viruses circulating in Asian wild birds and possessed PB2 E627K, a mammalian adaptation marker. Enhanced surveillance for H7 viruses in poultry and rapid characterization of human cases are essential to assess pandemic potential.

dUTPases are essential enzymes for nucleotide metabolism as they catalyze the hydrolysis of dUTP to dUMP, thus preventing the toxic incorporation of uracil into DNA. dUTPases are found in all domains of life, including bacteriophages, where they serve multiple functions to support phage propagation. Phage-encoded dUTPases reduce cellular dUTP levels and provide precursors for de novo dTTP synthesis and the synthesis of modified nucleotides, which are important to avoid DNA-based phage defense systems. In this study, we focused on the dimeric dUTPase Gp135, encoded by the Mosigvirus phage AV110, and examined its effect on growth, cell morphology, DNA replication and cell division of AV110s host, extended-spectrum beta-lactamase-producing E. coli. We observed that Gp135 significantly inhibited growth by disrupting DNA replication and cell division. This resulted in cytoplasmic voids, cell elongation, and ultimately cell death. We further demonstrated the diversity of phage-encoded dUTP/dCTPases through phylogenetic analysis, identifying four distinct clusters: one comprising the most common dUTPases, which form trimers; two more divergent clusters comprising dimeric dUTP/dCTPases; and one new cluster of more diverse, uncharacterized dUTPases and dNTPases. Overall, our results highlight the evolutionary and functional diversity of phage-encoded dUTP/dCTPases, with the Mosigvirus AV110 dUTP/dCTPase illustrating their role in supporting phage propagation.

The bean thrips, Caliothrips phaseoli, is an emergent pest affecting peanut production. Thus, gaining a deeper understanding of the naturally occurring viral populations associated with this insect is of major importance, as such knowledge could support the design of novel pest management strategies that are both ecologically responsible and sustainable over the long term. In this study, we performed the first RNA high-throughput sequencing (HTS) analysis of C. phaseoli to unravel its virome, discovering a wide number of novel RNA viruses. Our HTS analysis revealed the presence of 31 viral segments, corresponding to 18 viruses, including double-stranded RNA viruses as well as positive- and negative-sense single-stranded RNA viruses. All these are novel viruses and cover multiple viral lineages belonging to diverse families. Overall, these results indicates that C. phaseoli hides a complex virome. The findings presented here provide an important framework for subsequent studies aimed at understanding the ecological significance, evolutionary patterns, and possible use of these viruses as components of biologically based management programs for this emerging peanut pest.

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.

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.

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

Infectious bronchitis virus (IBV) is an economically important avian gammacoronavirus. IBV uses sialic acids and heparan sulfate for cellular attachment, but the host proteins governing its infection remain poorly defined. Here, we investigated the roles of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) during IBV Beaudette (IBV BD) infection. ACE2 knockout impaired IBV replication in DF-1 cells, whereas ACE2 reconstitution restored infection. Furthermore, ACE2 overexpression enhanced infection of DF-1 and primary chicken embryo kidney cells, supporting the role of chicken ACE2 as a proviral host factor. In contrast, TMPRSS2 exerted a restrictive function on IBV BD infection as demonstrated in TMPRSS2 KO DF-1 cells and upon pharmacological inhibition, which significantly increased IBV BD infection in DF-1 and Vero cells. RNA sequencing revealed significant changes in lipid metabolism and membrane-related processes following TMPRSS2 knockout and pharmacological inhibition. These findings highlight the opposing roles of ACE2 and TMPRSS2 in IBV BD infection, emphasizing the combined impact of proviral host factors, protease activity, and the cellular membrane environment during gammacoronavirus infections.

Introduction: Acute gastroenteritis (AGE) is a cause of childhood morbidity and may be associated with human adenovirus (HAdV) infection. This study aimed to investigate the occurrence of HAdV in fecal samples from children with AGE in Salvador, Bahia, Brazil, as well as to molecularly characterize the identified strains and evaluate their cytopathic effects in cell culture. Methods: A total of 131 samples were analyzed using the nested polymerase chain reaction (Nested-PCR) technique. Molecular characterization was performed through Sanger sequencing and phylogenetic analysis, while Vero cell cultures were used to investigate the occurrence of cytopathic effects. Results: Of the samples analyzed, 13 (9.9%) tested positive for HAdV, while 118 (90.1%) were negative. The highest detection frequency was observed in children aged three years or younger, particularly those under one year of age. Phylogenetic analysis identified variants related to HAdV-F41 and HAdV-B3, revealing genetic differences and distinct clustering patterns compared with reference sequences. In cell culture, cytopathic effects were observed four days after inoculation, although further analyses are required to confirm productive viral replication. Conclusion: These findings demonstrate the presence of HAdV in the samples studied and contribute to understanding its genetic diversity and circulation in Salvador, highlighting the importance of epidemiological surveillance and further studies on its role in the etiology of childhood AGE.

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.

Urinary tract infections (UTIs) are frequently caused by Escherichia coli and are increasingly associated with antimicrobial resistance, highlighting the need for alternative therapies. Although phage therapy is a promising approach, the rational selection of phages and phage cocktails remains challenging, particularly when detailed genomic information on the infecting strain is unavailable. In this study, we isolated 18 phages from environmental samples collected in Sherbrooke, Quebec, Canada. Genome sequencing identified 15 pure lytic phages representing diverse taxonomic groups and revealed prophage-derived contaminants in three isolates, underscoring the importance of genomic validation during phage discovery. Host range was determined using efficiency-of-plating (EOP) assays against UTI-associated E. coli isolates. Based on host-range overlap and cumulative EOP values, we developed a phenotype-based framework for phage cocktail design and classified combinations as redundant or complementary. Complementary cocktails composed of phages with distinct host ranges and high infection effectiveness consistently outperformed redundant combinations in time-kill assays, demonstrating that host-range diversity and lytic effectiveness are independent properties that should be considered separately during cocktail design. To evaluate cocktail performance under physiologically relevant conditions, the most effective combinations were tested in artificial urine. Both cocktails exhibited reduced activity compared with standard laboratory media, indicating that environmental conditions strongly influence phage-host interactions. Together, our results demonstrate that host-range overlap and cumulative infection effectiveness provide a practical framework for identifying effective phage combinations in the absence of detailed bacterial genomic information and may facilitate the rational development of phage therapies for UTIs.

Abstract Bacteriophage receptor-binding proteins (RBPs) determine host recognition and host range but are frequently missed by conventional annotation because they evolve rapidly and often lack recognizable sequence domains. Pseudomonas phage UF_RH1 is a lytic Septimatrevirus with a 42,567 bp double-stranded DNA genome encoding 57 predicted open reading frames, several of which remain annotated as hypothetical proteins. Here, we reanalyzed the UF_RH1 distal-tail region using comparative synteny, phage RBP specific prediction tools, structure-informed domain analysis, oligomeric-symmetry prediction, and independent physicochemical calculations. Two adjacent proteins, WCF59042.1 (317 aa) and WCF59043.1 (320 aa), emerged as the strongest candidates for functional reannotation. WCF59042.1 was classified by DepoScope as a tail-spike protein with probability 1.00 and contains a high confidence C terminal tail-spike-associated segment spanning residues 210 to 317. WCF59043.1 was independently classified as a tail-spike candidate by RBPdetect2 (probability 0.51) and showed a strong Seq2Symm prediction for C3 homo-trimeric symmetry (0.990). Comparative genomic analysis places these proteins at positions corresponding to DLP2 gp22 and gp23, two similarly sized hypothetical virion proteins immediately downstream of the tape measure gene in a conserved distal-tail morphogenesis cassette. Because the closely related phages DLP1 and DLP2 use type IV pili for adsorption, we propose a testable model in which WCF59042.1 functions as a proximal connector/adaptor and WCF59043.1 contributes a distal trimeric receptor binding element. The model is computational and does not establish direct binding to PilA or assembled pili. Together, the convergent sequence, structural, oligomeric, and syntenic evidence supports a previously unrecognized tandem distal tail receptor-binding module in UF_RH1 and provides experimentally tractable targets for defining Septimatrevirus adsorption and host range.

Introduction: Acute gastroenteritis (AGE) is a global public health problem and a major cause of mortality in low- and lower-middle-income countries. Human bocavirus (HBoV) is among the viral agents associated with this condition. Therefore, investigating the presence of HBoV in cases of AGE in Salvador, Bahia, as well as its prevalence and circulating strains, is important. Objective: To investigate the occurrence of HBoV infection during an AGE outbreak in Salvador. Methods: A total of 178 samples were analyzed for HBoV using nested polymerase chain reaction (nested PCR), and the amplification products were visualized by agarose gel electrophoresis. HBoV was detected in 5.7% of the samples analyzed. Conclusion: This study contributes to the understanding of HBoV epidemiology as a secondary agent associated with acute gastroenteritis during a norovirus outbreak in Salvador. Continued investigation and identification of the etiological agents involved in outbreaks are essential for evaluating the causes of AGE. These findings also highlight the need for broader diagnostic approaches and the importance of phylogenetic analysis for monitoring and controlling viral infections.

Thrips are known for being pests of many agricultural crops, as well as vectors of plant viruses. Yet despite their economic importance, genomic resources for this group remain limited, and no chromosome-level genome has been available for the genus Scirtothrips. One such species is the chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae). This invasive and highly polyphagous pest is established across much of the world and transmits several tospoviruses. Genomic resources would provide a new means to understand the traits that make thrips difficult to manage. Here, we present a chromosome-level genome assembly for S. dorsalis. The assembly is 266.5 Mb, with 96.9% of the sequence anchored in 16 chromosome-scale scaffolds. It contains 20,557 genes, and repetitive elements account for 28.8% of the sequence, which is comparable to those of other sequenced Thripidae. A species tree based on 3,019 single copy orthologs placed S. dorsalis as sister to Scirtothrips hansoni, with Scirtothrips most closely related to Frankliniella, Megalurothrips, and Odontothrips. The mitochondrial genome is a single circular molecule of 15,207 bp in which the sequence of the published mini circle is integrated, in contrast to the bipartite mitogenome previously reported for the same cryptic species.

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.

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.