Archives of Virology

Borealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported human infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of BRPV is unknown, although previous investigations have raised the possibility of wild small mammals transmitting the virus to humans, either through direct contact or via domestic cats and dogs. To understand which species may be involved in the maintenance and/or spillover of BRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and squirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai Peninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five species (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable virus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from historical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali National Park and Preserve in 1998 and 1999, 17 years before the first reported human case of BRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the hypothesis of local human infections through multiple spillover events. These findings suggest C. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in Alaskan wild small-mammal populations for at least 25 years. Our study highlights the potential of museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. Further museum- and field-based sampling will clarify the true geographic range of BRPV, which is closely related to Old World orthopoxviruses and may be circulating beyond North America.

Tomato brown rugose fruit virus (Tobamovirus fructirugosum) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus was reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of tomato fruits from local markets to investigate the variability of the virus in the country. We explored the different pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein, and the coat protein genes. Then, we used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with the ones from the world. We found that genetic diversity is low, which is consistent with the global situation. No signatures of diversifying selection were detected across the analyzed genes. However, the virus sequences from Morocco showed a clear geographic structure, suggesting that geographic factors probably combined with agricultural practices may contribute to shaping the population structure of ToBRFV in Morocco.

Recent advances in sequencing technology and transcriptome mining have revealed highly divergent hepaciviruses in birds. However, only a limited number of avian hepaciviruses have been identified to date, leaving their diversity and evolutionary history poorly understood. Moreover, deep phylogenetic gaps among known avian hepaciviruses suggest that additional lineages remain undiscovered. Here, we screened publicly available RNA-seq data and identified three previously undescribed hepaciviruses from rock pigeon (Columba livia), rusty-margined flycatcher (Myiozetetes cayanensis), and Hispaniolan amazon (Amazona ventralis), named rock pigeon hepacivirus (RpHV), rusty-margined flycatcher hepacivirus (RfHV), and Hispaniolan amazon hepacivirus (HaHV). Although these three viruses meet the ICTV species demarcation criteria relative to their closest known relatives, the NS5B-based criterion was not satisfied between RfHV and HaHV. Notably, however, their genome sequence identity is low at 43.2%, and their hosts differ at the order level, suggesting that their classification warrants further consideration. Our phylogenetic analysis showed that avian hepaciviruses, including those found in this study, are monophyletic, but phylogenetic incongruence was observed between avian hepaciviruses and their hosts, suggesting past cross-species transmission among avian hepaciviruses. Overall, this study provides novel insights into the diversity and evolution of hepaciviruses.

Avian influenza viruses (AIVs) are endemic in the Americas and responsible for outbreaks in both domestic and wild birds that occasionally spill over into humans. We report the first known outbreak of AIV H9N2 in lesser rhea (Rhea pennata), also known as Darwins rhea, in the region of Puno-Peru. The animals in this study lived in an isolated conservation center located in remote highlands above 4,000 m.a.s.l. Between June and July 2025, a total of 46/92 animals were recorded sick, with symptoms including greenish diarrhea (100%), hyporexia (24%), dyspnea (76%), nasal discharge (42%), drowsiness (18%) and isolation from the flock (73%), and 94% later died. Gross pathology exams revealed septicemia characterized by severe hepatitis, pneumonia, tracheitis, enteritis, and encephalitis. Swab and necropsy samples tested positive for Influenza A by PCR and were later identified as H9N2 through whole genome sequencing. We generated complete H9N2 genomes for two individuals. No additional pathogens were found. Phylogenetic analysis across all eight segments revealed that the viruses were low pathogenicity H9N2 AIV strains of North American origin, which indicated this outbreak was a new introduction of the virus into South America. We also performed a comparative mutational analysis and identified multiple mutations previously associated with mammalian host adaptation, increased virulence, increased pathogenicity, and increased virus binding to 2-6 receptors, which may explain the high mortality rates observed despite the supposedly low pathogenicity of the strain. We also identified novel mutations specific to rhea viruses that will need to be experimentally validated. This is the first report of a natural H9N2 systemic infection in an avian host, highlighting a need for increased surveillance efforts for zoonotic influenza viruses with pandemic potential. Author SummaryAvian influenza viruses (AIVs) are endemic in the Americas and cause more than 7,600 infections annually in domestic and wild birds worldwide each year. We report detection of AIV H9N2 in lesser rhea during an outbreak that occurred in June-July 2025 in the Andean highlands of Puno in Peru. Multiple sick animals were reported with symptoms of respiratory and gastrointestinal disease and 94% of them later died. Samples collected tested positive for Influenza A and they were subtyped as H9N2 of low pathogenic origin from North America. This is the third time H9N2 enters South America from North America, presumably through wild birds, some of which migrate along the Pacific Flyway. Comparison with other H9N2 sequences revealed a total of 44 mutations of interest that may explain the elevated death rates observed. Surveillance in wild birds remains patchy at best and needs to be strengthened in order to prevent spillover events into other animals, including humans.

Hydrangea ringspot virus (HdRSV) is an emerging plant virus infecting ornamental hydrangea species worldwide, yet its genomic diversity and host associations remain poorly understood. To expand the available genomic resources and assess HdRSV variability, we screened 210 publicly available Hydrangea spp. transcriptomes from diverse tissues, complemented with four newly generated H. macrophylla transcriptomes from Colombia. Viral genomes were assembled from infected samples and analyzed to infer phylogenetic relationships, lineage distribution, and relative viral RNA abundance. Two well-supported phylogenetic lineages (HdRSV-L1 and HdRSV-L2) were recovered from both full-genome and replicase coding sequence (CDS) analyses. HdRSV was detected across all host tissues examined, with the highest median viral loads in roots, followed by stems and leaves. H. macrophylla harbored both viral lineages, while H. serrata was exclusively infected by HdRSV-L1. Cultivar-level analysis revealed marked differences in viral abundance, with lineages showing distinct tissue preferences but no co-infection patterns, except in the Bailer cultivar. Comparative analysis of the replicase CDS identified a single lineage-defining nonsynonymous mutation (C1578T; Thr[->]Ile), fixed in 90% of HdRSV-L2 genomes, corresponding to a polar-to-nonpolar amino acid change potentially associated with structural adaptation. Together, these findings provide the most comprehensive overview to date of HdRSV genomic diversity, host and tissue distribution, and molecular variation, offering new insights into the evolution and epidemiology of this understudied plant virus.

Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by the peste des petits ruminants virus (PPRV), which is classified into four distinct genetic lineages (I-IV). A critical concern in the recent epidemiological history of PPRV is the rapid and widespread expansion of lineage IV (LIV) across West Africa over the past decade. This dominance suggests a potential adaptive advantage of circulating LIV strains in the regions current epidemiological context. In this study, we obtain the genome sequence of 26 new PPRV samples, including historical (pre-2000) and many recent African LIV isolates, offering the first opportunity to investigate the evolutionary history of LIV in Africa and identify genetic events potentially associated with its recent spread. Phylogenomic analyses implemented on a dataset of 167 curated PPRV genome sequences reveal that the most ancestral LIV group comprises strains circulating in Sub-Saharan Africa (designated clade LIVssa), providing robust evidence for an African origin of lineage IV. Our results further indicate that PPRV strains linked to the recent West African expansion of LIV belong to a specific LIVssa subgroup, termed NigB. We identified multiple signatures of selection pressure within the LIVssa sublineage, particularly in the NigB cluster. Several amino acid substitutions unique to LIVssa or NigB were detected, some of which may impact protein function and warrant prioritised investigation. Additional genomic data are required to confirm the association between the NigB group and the ongoing spread of LIV in West Africa. The evolutionary adaptations observed in LIVssa - potentially enhancing transmission efficiency, host range or pathogenicity - could undermine current disease control strategies in regions where PPR poses significant threats to food security and local economies. Author SummaryPeste des petits ruminants virus (PPRV) infects sheep and goats across Africa, Middle East, Asia and Europe, causing disease with major impact on global economy and food security. One genetic lineage of PPRV, called lineage IV (LIV), is at the origin of most recent expansion of the distribution of the disease, including replacement of other lineages in areas of African where PPRV is historically present. Here, we generated genome sequences from PPRV LIV isolates from different dates and places to study the evolution of this genetic lineage and explore whether its recent spread can be associated with the appearance of new mutations in the virus genome. Our results provide evidence that the PPRV LIV originated in Sub-Saharan Africa and identify mutations present only virus isolates currently spready in new regions of Africa. Further research should investigate the impact of these mutations on protein functions and capacity of transmission of PPRV.

Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2) represents a major threat to the global swine industry. The epidemiological dynamics of PRRSV-2 are characterized by the recurrent annual emergence of dozens of variants. Long-distance spread of PRRSV-2 is largely driven by animal shipments. Spatiotemporal dynamics of PRRSV-2 in the USA have been explored; however, how fast variants spread to new regions after their emergence remains unclear, and this information could improve preparedness. To address this, we analyzed 14,835 sequences, retrieved from the Morrison Swine Health Monitoring Project (MSHMP), representing 156 variants sampled from 2015 to 2024, covering the five major swine-producing regions in the USA: the Upper Midwest (UM), Lower Midwest (LM), Atlantic Seaboard (AS), Northeast (NE), and Great Plains (GP). Time to spread was assessed using the time-to-dispersal event analysis and waiting time analyses. Genetic diversity was measured using Hill numbers. The UM had the highest variant richness (n=123), followed by the LM (n=47), AS (n=35), NE (n=45), and GP (n=38). Of the 62 variants that initially emerged in the UM, 17 later spread to other regions. The UM also received the highest number of variant introductions (n=24), followed by LM (n=14), NE (n=14), AS (n=4), and GP (n=7), highlighting regional differences in connectivity and risk. Our results suggest faster dispersal corridors among interior regions (e.g., GP to UM and LM to UM, [~]1.2-2.0 years) and slower for coast to interior pathways (AS to interior, [~]2-3 years). These findings may help anticipate the risk of PRRSV-2 variant introduction and provide more accurate dispersal time estimates, which are useful for improving epidemiological models and disease preparedness.

Avian influenza virus (AIV) epidemiology is well-documented in temperate regions but remains poorly understood in isolated ecosystems like tropical oceanic islands. On these islands, seabirds nest in dense interspecific colonies where the role of different species as reservoirs and dispersers of AIV may vary greatly. Here, we examine the role of noddies (Anous spp.) as potential reservoirs for low pathogenic AIV and evaluate their potential as sentinel species for highly pathogenic AIV introduction on tropical oceanic islands. We analyzed blood samples from 11 seabird species across eight islands in the southwestern Indian Ocean (2015-2020). Noddies exhibited high, stable seroprevalence (30-45%), comparable to reservoir host species in temperate regions. The detection of two N7-positive noddies, sampled the same year on two distinct islands, provided direct molecular evidence that AIV actively circulates on these island colonies. While most other species showed low exposure, Bridled Terns (Onychoprion anaethetus) had exceptionally high seroprevalence (80%), though their reservoir status requires further investigation due to limited sampling. Given noddies consistent exposure and regional distribution, we recommend prioritizing islands with large noddy populations for AIV surveillance. Continued investigation of viral dynamics within and among islands is now called for to elucidate the ecological drivers of AIV maintenance and transmission.

Hepatitis D virus (HDV) is a satellite virus that utilizes hepatitis B virus (HBV) as a helper for infectious particle formation. HDV was originally identified as a novel antigen in liver biopsies of HBV patients, and later studies showed the "delta" antigen (DAg) to be the sole protein encoded by HDV. Until the discovery of HDV-like agents in birds and snakes in 2018, HDV was a unique example of animal satellite viruses. We identified Swiss snake colony virus 1 (SwSCV-1) in the brain of a Boa constrictor, and through comparison we found the genome organization of SwSCV-1 to resemble that of HDV. However, in addition to the DAg open reading frame (ORF), the genome of SwSCV-1 includes another >500 nt ORF, "ORF2". To study whether the putative ORF2-encoded protein plays a role in the SwSCV-1 life cycle, we established an infectious clone of the virus with a point mutation in the methionine initiation codon of ORF2. The mutation did not significantly affect initiation of replication, establishment of persistent infection, or infectious particle formation upon superinfection with a helper virus. Using additional methods, we gathered further evidence confirming that ORF2 is not actively translated in boa constrictor cells. We further showed that unlike HDV, SwSCV-1 expresses a single form of the DAg. Although the proteins encoded by SwSCV-1 and HDV only include one and two forms of the DAg, respectively, whether other kolmioviruses express additional forms of DAg or related proteins in some cell types or host species merits further research. IMPORTANCEApproximately 40 years after the discovery of hepatitis D virus (HDV), satellite viruses with similar genome organization were found in various animals, thereby giving rise to family Kolmioviridae. HDV encodes a single protein, the delta antigen (DAg), which comes in small and approximately 20 amino acids longer large form. The genome of some HDV species and many of the newly found kolmioviruses contains additional open reading frames (ORFs), potentially enabling protein expression. Here, we studied the viral proteins expressed during Swiss snake colony virus 1 (SwSCV-1) infection of boa constrictor cells. Our findings show that unlike HDV, SwSCV-1 encodes only a single form of DAg. In addition, our study suggests that, like in HDV, the additional ORF in SwSCV-1 genome does not give rise to a protein. Although we could not demonstrate expression of additional viral proteins during SwSCV-1 infection, it is important to study the proteome of other kolmioviruses.

Mpox virus (MPXV) gained increased attention following the declaration of two Public Health Emergencies of International Concern (PHEICs) in 2022 and 2024. The rapid spread of MPXV and the increase in human-to-human transmission highlighted the need for improved diagnostic tools for characterizing infection patterns and transmission dynamics. While PCR is effective for detecting active infections, serological approaches can help identify previous or asymptomatic infections and support retrospective surveillance. However, many serological assays developed during recent outbreaks have not been evaluated in endemic settings such as the Democratic Republic of the Congo (DRC). This study aims to define antigen-specific serological cutoff values to differentiate MPXV-seroreactive individuals from those with other orthopoxvirus (OPXV) exposure or different vaccination histories, specifically for use in the DRC. Here, we analyzed 134 individuals, divided into six distinct cohorts with different exposures. Serum samples were tested using Mesoscale Discovery (MSD) to screen for five MPXV and vaccinia virus (VACV) orthologous antigens: A29L/A27L, A35R/A33R, B6R/B5R, E8L/D8L, and M1R/L1R. Receiver operating characteristic (ROC) analysis identified the best-performing antigens and established seroreactivity cutoff values. A binary composite rule was also evaluated to improve the classification of these results. We identified three MPXV antigens, E8L (cut-off=12.33 AU/mL), A35R (cut-off=5.22 AU/mL), and B6R (cut-off=9.77 AU/mL), that showed the strongest discriminatory performance in the dataset. Collectively, these three antigens form a significant panel that demonstrated clear separation between our mpox survivor cohort and other OPXV-exposed individuals. ImportanceEstablishing antigen-specific serological cutoff values for this assay using unique samples from endemic regions such as the DRC may improve future epidemiological and disease transmission surveillance efforts and contribute to broader efforts to ensure regionally appropriate cutoffs for serological assays.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes adverse pregnancy outcomes and high mortality in domestic and wild ruminants. The disease is caused by the RVF virus (RVFV), which is transmitted by mosquitoes from several genera, mainly Aedes and Culex. However, whether ruminants can become infected by horizontal virus transmission remains unclear. In addition, how the route of RVFV inoculation may influence RVF pathogenesis and the host immune response in animals is still largely unknown. With this aim, we conducted a comparative experimental study in which young sheep were either inoculated subcutaneously (SC) or intranasally (IN) with the virulent RVFV 56/74 strain. We then evaluated disease dynamics, viremia, virus excretion, tissue damage, and the humoral immune response. We also aimed to determine whether RVFV can be transmitted from infected to in-contact animals, and to assess whether the inoculation route may influence virus excretion and the likelihood of subsequent horizontal transmission. The results showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and an earlier seroconversion. In contrast, IN inoculated animals developed higher rectal temperatures, reached higher peak viremia, and developed a more robust neutralizing antibody response. They also exhibited increased concentrations of analytes indicative of moderate but more severe hepatic injury compared with the subcutaneous group, along with more pronounced histopathological damage in the central nervous system. These results demonstrate the influence of the route of inoculation on RVF pathogenesis and the host immune response. Our results also confirmed the horizontal transmission of RVFV between SC inoculated sheep and in-contact animals housed in the same room, a phenomenon not observed in the IN inoculated group. This finding underscores the influence of the inoculation route on virus transmission and the potentially significant role of horizontal transmission in RVF epidemiology and disease control. Author summaryAccording to the World Health Organization (WHO), RVFV is considered a priority pathogen due to its ability to strain animal and public health systems, especially in developing countries. RVF outbreaks have occurred across most of Africa and, since 2000, in the Arabian Peninsula. Evidence of RVFV circulation in North Africa further highlights the threat to Europe, where competent mosquito vectors are present. How the inoculation route shapes disease dynamics and hosts immunity is still largely unknown. Similarly, whether the virus can spread between infected and non-infected animals without competent vectors remains unclear. A comparative infection in which young sheep were inoculated SC or IN with the RVFV 56/74 strain showed that SC inoculated sheep had a shorter incubation period, an earlier onset of viremia, and earlier seroconversion. However, rectal temperature and peak viremia were higher in IN inoculated sheep, which also showed evidence of moderate but more severe hepatic damage, accompanied by greater central nervous system damage. Only the in-contact animals housed in the subcutaneous group became infected, demonstrating horizontal transmission. Our results show that the route of inoculation influences disease progression and that RVFV can be transmitted among sheep in the absence of mosquitoes.

The majority of emerging infectious diseases are zoonotic, having their origin in wildlife before spilling over into the human population. While small mammals are recognized as critical reservoirs for these viruses, their viral diversity remains largely uncharacterized across many African countries. We conducted molecular surveillance of synanthropic rodents and shrews in the Kibera informal settlement in Nairobi and the rural Taita Hills region of Kenya to detect and characterize potential zoonotic viruses. Tissue samples from 228 rodents and shrews were screened for six viral families using PCR assays. Rat hepatitis E virus (HEV) (Rocahepevirus ratti), a rodent-associated virus with potential for human spillover, was identified in Mus musculus and Rattus norvegicus from Kibera. NGS was conducted for the HEV positive samples, and we obtained two near-complete HEV genomes from Rattus norvegicus, which clustered within rodent-associated HEV genotypes in the phylogenetic analysis. The two sequences from the Rattus norvegicus cluster together, indicating a close genetic relationship. Paramyxoviruses belonging to the genera Jeilongvirus and Parahenipavirus were detected both from Taita and Kibera in nine different samples from Rattus norvegicus, Mus minutoides, Crocidura sp and Acomys ignitus. One paramyxovirus positive sample (Acomys ignitus) from Taita was selected for further sequencing with NGS, and a complete genome of a new jeilongvirus was assembled. Phylogenetic analysis of the detected viruses confirmed the close relation to previously known rodent-borne jeilongviruses but also revealed potentially novel jeilong- and parahenipavirus species. Our findings highlight the circulation of potentially zoonotic viruses in both urban and rural small mammals in Kenya. It emphasizes the necessity of continued genomic surveillance of zoonotic viruses to mitigate risks of their spillover into human populations. HighlightsO_LISurveillance reveals diverse rodent-borne viruses circulating in Kenya. C_LIO_LIRat-HEV was detected in Rattus norvegicus and Mus musculus from an urban low-income area. C_LIO_LIParamyxoviruses were detected across multiple rodent and shrew species, including novel Acomys ignitus jeilongvirus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/719784v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@86c94aorg.highwire.dtl.DTLVardef@10946adorg.highwire.dtl.DTLVardef@1ff45eforg.highwire.dtl.DTLVardef@4876f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

We recently reported the identification of endogenous viral elements (EVEs) originating from the Caulimoviridae family within the alfalfa (Medicago sativa L.) genome. Our subsequent identification of ubiquitous rhabdoviral elements in infected and healthy alfalfa tissues by high throughput sequencing prompted us to suggest that the alfalfa genome might be populated with integrated rhabdoviruses as well. Bioinformatics analysis using 26 publicly available alfalfa genomes proved the suggestion accurate. We found multiple non-retroviral segments of the Rhabdoviridae family belonging to the genera Betanucleorhabdovirus and Betacytorhabdovirus that appeared to be stable constituents of the host genome. In that capacity they could potentially acquire functional roles in alfalfas development and response to environmental stresses. We believe this study reveals the first documented case of rhabdoviruses integrated into the alfalfa genome.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

The yerba mate psyllid (Gyropsylla spegazziniana) poses a significant threat to yerba mate crops, causing extensive economic losses. While some ecological aspects as well as control strategies have been studied, its associated viral diversity remains unexplored. Here, by generating the first RNA high-throughput analysis (HTS) of this pest, we explored the G. spegazziniana virome, revealing novel and diverse RNA viruses. We characterized five new viral members belonging to distinct families, with evolutionary cues of beny-like viruses (Benyviridae), picorna-like viruses (Picornaviridae), and sobemo-like viruses (Solemoviridae); which were tentatively named Gyropsylla spegazziniana beny-like virus 1 (GSBlV1), Gyropsylla spegazziniana picorna-like virus 1 (GSPlV1), and Gyropsylla spegazziniana sobemo-like virus 1-3 (GSSlV1-3), respectively. Phylogenetic analysis of the bi-segmented and highly divergent sobemo-like viruses showed a distinctive evolutionary trajectory of its encoding proteins at the periphery of recently reported invertebrate Sobelivirales. The beny-like virus belonged to a cluster of insect-associated beny-like viruse; while the picorna-like virus clustered together with psyllid-associated picorna-like viruses. These results highlight the existence of a complex virome within G. spegazziniana and establish the basis for future studies investigating the ecological roles, evolutionary dynamics, and potential biocontrol applications of these viruses in the G. spegazziniana -yerba mate eco-systems.

Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like -2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

Hantavirids, specifically the members within the genus Orthohantavirus, represent a significant global public health threat, with bat-associated lineages challenging traditional reservoir paradigms. To investigate the genetic diversity of hantavirids in Southeast Asia, we conducted an expanded surveillance program in Lao PDR from May 2023 to October 2025 in bat populations and wild animals from local wet markets. Using molecular screening and deep sequencing to characterize hantavirids from bat populations and wild animals from local wet markets, we identified 20 positive samples across four bat species, recovering coding-complete genomes for multiple novel variants. Phylogenetic analysis confirmed that these viruses form a monophyletic group within Mobatvirus, resolving into two major subclades. The first subclade clustered with Quezon and Robina viruses found in fruit-eating bats. The second subclade further split into two lineages corresponding to Thakrong and Xuan Son viruses, which are associated with trident and leaf-nosed bats, respectively. Despite the strong host specificity observed, the detection of these viruses in a wet market, a critical interface for human-wildlife contact, indicates a potential zoonotic risk. These findings significantly expand the known diversity of mobatviruses in Laos and highlight the urgent need for serological surveillance in at-risk human populations to assess the potential for spillover.

Eurasian avian like H1N1 (EAavH1N1) and influenza D viruses (IDV) with their ongoing evolution and zoonotic potential are a serious threat to animal and human health. Using experimental infection of pigs, we characterized and compared their pathogenesis, and immune responses. EAavH1N1 induced rapid viral clearance, early immune activation, including robust systemic and mucosal antibody responses and increased IFN{gamma} and TNF production. This heightened immune response was associated with more severe pathology of the upper and lower respiratory tract. In contrast, IDV infection resulted in prolonged viral shedding and higher viral titres, with delayed and attenuated cellular immune responses. Single cell transcriptomic analysis of lung further indicated early and persistent suppression of antiviral and innate immune pathways during IDV infection. These findings demonstrate that EAavH1N1 and IDV exhibit distinct viral kinetics, immune activation profiles, and lung responses, providing insight into differences in transmission dynamics, disease severity, and immune control among influenza virus types in swine.

Bovine coronavirus (BCoV) is an important contributor to the respiratory disease complex in cattle; however, integrated genomic and epidemiological data describing currently circulating respiratory BCoV strains in the United States remain limited. The objective of this study was to monitor respiratory BCoV at the genomic level and analyze its epidemiological patterns over a five-year period. A total of 4,505 respiratory samples submitted to a diagnostic laboratory between January 2020 and November 2025 were analyzed, of which 693 (15.38%) tested positive for BCoV. Positivity was highest in young calves (0-40 days; 20.0%) and declined significantly with increasing age based on logistic regression analysis. Temporal trend analysis using LOESS smoothing and the Mann-Kendall test showed no significant monotonic change in BCoV detection during the study period. Co-infection analysis indicated that BCoV was commonly detected with other viral respiratory pathogens, while bacterial pathogens predominated in many samples. Lung tissues from infected cattle were screened by RT-PCR, and selected samples with high viral loads were subjected to next-generation sequencing. Complete genome sequencing identified four respiratory BCoV isolates ([~]31 kb), all clustering within genotype GIIb with recent U.S. strains. Comparative genomic analysis revealed several amino acid substitutions in structural and non-structural proteins that may influence viral attachment, replication, and tissue tropism. These findings provide updated epidemiological and genomic insights into respiratory BCoV circulating in U.S. cattle.

Climate change and ecosystem collapse promote geographic expansion of vector-borne diseases, as witnessed by the recent incursions into Spain of the virus responsible for Crimean-Congo hemorrhagic fever (CCHFV). CCHFV is maintained in a tick-vertebrate cycle, principally involving ticks of the genus Hyalomma. Faced with the spread of Hyalomma ticks, and therefore the threat of a natural introduction of CCHFV into Western Europe, appropriate surveillance tools and control measures need to be implemented. It is both within and by the tick that CCHFV is maintained and spread in the environment. Despite prolonged portage of the virus, the tick is not overtly affected by CHFV infection. One of the prerequisites in conceiving control strategies is to understand the molecular mechanisms that intimately link the virus to its arthropod host. Despite the central role of the tick in the biology of CCHFV, these mechanisms are ill-defined, owing in part to the constraints associated with handling CCHFV-infected ticks in biosafety level 4 containment. In this study, we established the network of interactions between the S segment of the RNA genome Hazara virus (HAZV), a BSL-2 model of CCHFV, and Hyalomma proteins using ChIRP-MS technique. We identified 166 tick proteins, 21 of which have been described as RNA-binding proteins. Gene ontology and pathway enrichment analyses revealed that the S segment RNA interacts predominantly with mitochondrial proteins that belong to various mitochondrial metabolic pathways.