Viruses

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus which is transmitted via cell-containing blood fluids or from mother to child via breastfeeding, leading to severe diseases such as adult T-cell leukemia/lymphoma (ATL) and neuroinflammation. Most studies focus on virus detection in peripheral blood due to limited access of tissue material, especially in infants. Thus, our understanding of viral distribution in organs, in particular along the oral route of transmission, is still a critical gap in HTLV-1 research. Methodology/Principal FindingsHere, we present an analysis of tissues from a non-human primate (NHP) colony (olive baboon: Papio anubis) naturally infected with the closely related counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1). Various organs and tissues of the oropharyngeal and gastrointestinal tract including tonsils, stomach, small intestine and colon were analyzed for the presence or absence of STLV-1. Beside TaqMan qPCR measuring relative copy numbers, we established a highly sensitive and precise droplet digital (dd) PCR protocol to measure absolute copy numbers of viral Tax DNA. Tax DNA was detectable in the tonsils in two NHPs, but to a greater extent in stomach in four NHPs. We also found Tax in parts of the small intestine, i.e. duodenum and Peyers patches, in a NHP with high blood proviral load. Conclusion/SignificanceThese data provide a quantitative analysis of STLV-1 Tax in the gastrointestinal tract and are, to our knowledge, the first indication of STLV-1 detection in stomach tissue of naturally STLV-1-infected asymptomatic NHPs. Although it is unclear how infection occurred - from mother-to-child, sexual or via animal bites - our study suggests that these parts of the gastrointestinal tract might either serve as site of virus transmission or as viral reservoir. Author summaryHuman T-lymphotropic virus type 1 (HTLV-1) is a human oncogenic retrovirus being transmitted via cell-containing body fluids such as breast milk, blood, or semen. The estimated number of infected people is around 10 to 20 million. To study the viral distribution and persistence of HTLV-1 in different organs in the oropharyngeal and gastrointestinal tract, a suitable in vivo model is essential. In this study, tissues of non-human primates (NHPs, baboon: Papio anubis) naturally infected with the closely related simian counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1), were analyzed for the presence or absence of the viral gene Tax. We identified the presence of STLV-1 Tax in the stomach, duodenum and Peyers patches by using two different detection methods: TaqMan-based qPCR and the more sensitive droplet digital PCR (ddPCR). Tax could be detected in tonsils in two NHPs only, but in stomach in four NHPs. Together, this is the first time that STLV-1 Tax was detected in stomach tissue of STLV-1-infected asymptomatic NHPs, highlighting the importance of investigating viral persistence and/or viral reservoirs of primate T-lymphotropic viruses independently of the entry route.

Background & Aims: The origin of hepatitis D virus (HDV) and related deltaviruses remains elusive. Contrarily, hepatitis B virus, HDV's helper virus, and related hepadnaviruses display long-term association with primates. Current data suggest cross-order host shifts as a common mechanism in deltavirus ecology, but corroborative evidence is lacking. Here, we aimed to elucidate the genealogy of primate deltaviruses. Methods: We screened 961 non-human primate (NHP) liver specimens obtained in Brazil between 2017-2023 for deltaviruses and hepadnaviruses. Complete deltaviral genomes were obtained via overlapping nested RT-PCR, cloned, expressed in vitro and analyzed via immunoblot and immunofluorescence analysis. Anti-deltaviral antibodies in NHP and vampire bats were detected via immunofluorescence analysis. Results: We detected deltaviruses in two NHP. Complete deltaviral genomes exhibited common features including high self-complementarity, genomic and antigenomic ribozymes, and a delta antigen open reading frame. NHP deltaviruses were phylogenetically related to viruses found in common vampire bats. The NHP deltaviruses replicated in vitro without the expression of a large delta antigen. We did not detect anti-deltaviral antibodies in NHP sera (0/249), in contrast to sera from common vampire bats (7/112; 6.2%, 95% CI: 1.8-10.7), indicating viral circulation in bats. Ancestral state reconstruction suggested a bat origin of NHP deltaviruses. Targeted screening excluded a coinfecting hepadnavirus in the deltavirus-positive animals but led to the discovery of a hepadnavirus, corroborating a non-recent introduction of hepadnaviruses into the primate stem-lineage. Conclusions: Our data are consistent with deltaviral cross-order host shifts and suggestive of the susceptibility of primates to reservoir-bound deltaviruses, lending credibility to a zoonotic origin of HDV.

Flaviviruses contribute significantly to the global disease burden and are known to remodel host organelles extensively to their advantage. Centrosomal microtubule-organizing centres (MTOCs) having established role in cell division and signalling are also targeted by viruses. However, it remains uncertain whether they act as bystanders or actively engage in viral processes. Here, we elucidate the centrosome and cytoskeletal involvement during Japanese Encephalitis Virus (JEV) infection. Virus-free expression studies have identified a centrosome-targeting region within the C-terminal helicase domain that mediates the association of virus-derived structures with host MTOCs. When expressed exogenously, JEV-NS3 formed pericentriolar aggresomes resembling the distribution pattern of helicase-containing viroplasm in infected cells. Centriole depletion assays revealed the proviral role of centrosome facilitating JEV replication, where the viroplasm organization depends on centrosomal MTOCs, and vimentin cages. Additionally, microtubule disruption and dynarrestin blockade assays emphasized the roles of microtubules and dynein in concentrating NS3-containing vesicular packets towards centrosomes, highlighting the centrosome-cytoskeleton axis as a potential target for flaviviral intervention. SummaryThis study demonstrates that JEV helicase is directed to centrosomes via its CTHD domain, using them to initiate pericentriolar viroplasm formation. It also highlights the unappreciated function of centrosomes as a proviral hub, orchestrating cytoskeletal remodelling and viral factory organization. Graphical abstractJEV helicase is targeted to centrosomes to form viroplasm with the aid of microtubule and motor proteins. A) JEV lifecycle from receptor binding (Step 1) to endocytic internalization (Step 2) followed by uncoating and release of viral genome (Step 3) followed by replication within the ER derived Vesicular packets (Step 4). This vesicle packets harbors viral proteins that form replication complex, like helicase and replicase and the replicative intermediate, dsRNA. These vesicle packets with the aid of retrograde motor protein gets targeted towards the centrosome (Step 5). Ultimately multiple vesicle packets accumulate in the pericentrosomal region, where it forms a separate compartment enclosed by vimentin and tubulin cage (Step 6). Inset A, B and C depict the effect of cytoskeletal perturbations on Viroplasm formation. B) Disruption of microtubules using Nocodazole completely abolishes pericentriolar viroplasm organization. C) Centrinone mediated centrosome depletion markedly reduces both the number and size of pericentriolar viroplasm. D) Dynarrestin mediated retrograde microtubule transport significantly decreases the reorganization of vesicle packets to form the viroplasm in the pericentriolar region. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740276v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@17a6354org.highwire.dtl.DTLVardef@f208eorg.highwire.dtl.DTLVardef@1224fcaorg.highwire.dtl.DTLVardef@1a48bab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sicyoidochytrium minutum DNA virus (SmDNAV) was isolated several years ago from a protist host of family Thraustochytriaceae of the class Labyrinthulomycetes. This virus shared little similarity to other viruses in gene content and protein sequences, albeit seemingly belonging to the phylum Nucleocytoviricota. By extensive searches in genomic and metagenomic sequence databases, we identified numerous long contigs related to the SmDNAV genome and analyzed proteins shared by these putative viruses. Phylogenetic analyses place these viruses within the class Megaviricetes, outside of all established orders, and as a sister group to the clade combining families Mamonoviridae and Manesviridae. Homologs of SmDNAV proteins were found in association (either integrated or co-sequenced) with other Labyrinthulomycetes and Rhodophyta protists from diverse marine and freshwater environments. Consequently, we propose SmDNAV as the prototype member of a new order, provisionally named Ariadnavirales, within class Megaviricetes, phylum Nucleocytoviricota. Members of Ariadnavirales have lost most of the genes encoding components of the replication and transcription systems that are otherwise conserved in nucleocytoviricots, suggestive of transition to genome replication and expression dependent on the host nucleus.

Bacteriophages of the order Crassvirales are currently believed to be the most prevalent dsDNA phages in the human gut virome, yet their global biogeography and genomic diversity remain poorly characterized due to an overrepresentation of industrialized Western studies in public repositories. In this study, we integrated computational metagenomics and molecular approaches to identify and validate the first complete Crassvirales genome from an Egyptian population. De novo assembly and viral profiling yielded a 101,034 bp circular genome (contig k141_108779) predicted to infect the non-industrialized gut symbiont Segatella copri. The genome displays the notable feature of amber stop codon reassignments (NCBI Genetic Code 15), where canonical (TAG) stop codons encode glutamine (Q). This alternative code increases coding density to 91%. Population-level PCR surveillance and Sanger dideoxynucleotide sequencing across 252 individual Egyptian fecal samples, pooled in 10 composites, confirmed the active circulation and local sequence heterogeneity of this lineage within the community. Phylogenomic and intergenomic similarity analysis demonstrated that the isolate shares less than 50% total average nucleotide identity with all recognized type strains. These data establish that this phage constitutes a novel species within a newly proposed genus inside the family Darmviridae. Our findings expand the known geographic distribution of crAss-like phages, highlight translational versatility among Segatella-infecting viruses, and emphasize the importance of expanding virome cohorts to underrepresented regions.

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.

Despite the global burden of hepatitis E virus (HEV) infection, host-virus interactions underlying HEV pathogenesis and progression to chronicity remain incompletely understood. This study aimed to characterize the host transcriptomic response to infection with HEV variants harboring different host- and virus-derived insertions in the hypervariable region (HVR), with a focus on innate immune activation. We performed bulk RNA sequencing on PHHs from three donors infected with in vivo-identified HEV-3 variants harboring different HVR insertions (RPS17, HVR duplication, SERPINA1, TRIM22). To compare their replication fitness, we quantified progeny virus titers and viral RNA reads in the cellular transcriptome. Host transcriptomic responses were analyzed in detail at multiple time points to assess virus-induced differential gene expression. To further characterize the response to HEV infection with different HVR insertion variants, we performed gene ontology analysis and identified individual HEV-induced genes. Progeny virus titers at 72 hours post infection (h p.i.) were comparable across all PHH donors and HVR insertion variants. Two donors mounted robust host responses to HEV infection, characterized by widespread gene deregulation and interferon stimulated gene (ISG) upregulation at 72 h p.i. The HVR duplication insertion variant consistently triggered a weaker host response compared to RPS17, SERPINA1 and TRIM22 variants, with significantly fewer differentially expressed genes and reduced ISG induction. Gene ontology term analysis revealed enrichment of antiviral pathways upon infection with the different insertion variants, which was less pronounced for the HVR duplication variant. These findings reveal that HEV HVR rearrangement variants elicit distinct host response patterns. Importantly, our results suggest a positive correlation of viral replication and host response induction. This indicates that the HVR may play a crucial role in shaping host-virus interactions.

Bunyaviricetes is a class of segmented negative strand RNA viruses (sNSV) that includes causative agents of severe zoonotic diseases resulting in hemorrhagic fever in humans and livestock. Hantaan virus (HTNV), family Hantaviridae, is an example of life-threatening bunyavirus transmitted by rodents which leads to Hantavirus hemorrhagic fever with renal syndrome in humans. Infection relies on the replication and transcription of their tripartite genomes through multifunctional RNA-dependent RNA polymerases (RdRp) in the context of ribonucleoproteins (RNPs) made of one viral RNA fragment, one RdRp and multiple copies of viral nucleoproteins. The RdRp, also known as L protein, performs replication by de novo initiation through a prime-and-realign mechanism while transcription initiates through a cap snatching mechanism, that engages a cap-binding domain and an endonuclease (EN) domain present in the L protein C and N terminal regions, respectively. In this work we show that HTNV L protein has a terminal nucleotidyl transferase (TNTase) activity over double-stranded RNA substrates (dsRNA) in vitro. We show that the TNTase activity is also present in other bunyaviral L proteins by comparing HTNV L protein activity with La Crosse (LACV, family Orthobunyaviridae) and Crimean Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) L proteins. Our results show that TNTase activity appears to be a general feature of Bunyaviruses and report the differences on substrate and nucleotide specificity for each family of virus.

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.

Tick-borne encephalitis virus (TBEV) is a zoonotic flavivirus maintained in complex enzootic cycles involving ticks and vertebrate hosts. While vector-mediated transmission has been extensively studied, host-associated mechanisms, including vertical transmission and prolonged viral carriage in natural reservoir populations, remain poorly understood. Here, we investigated TBEV circulation, genetic diversity, and evidence for vertical transmission in bank voles (Clethrionomys glareolus) from a highly endemic region of North-eastern Poland. A total of 258 wild rodents were screened using molecular and serological approaches. TBEV RNA was detected in 14.3% of individuals, whereas seroprevalence was substantially lower (6.6%), revealing limited concordance between viral presence and humoral response under natural conditions. Near-complete genome sequences were obtained from 10 TBEV-positive individuals, representing the first near-complete TBEV genomes generated from wild rodents in Poland. All isolates belonged to the European subtype (TBEV-Eu), and phylogenetic analysis revealed two genetically distinct viral clades co-circulating within a narrow spatiotemporal window. Importantly, TBEV RNA and/or envelope protein were detected in embryos from naturally infected females, providing evidence consistent with vertical transmission in a wild reservoir host. Together, these findings suggest that vertical transmission and complex host infection dynamics may be underappreciated components of TBEV maintenance in natural reservoir populations. Our results highlight the need to integrate vertebrate host dynamics into models of TBEV ecology and support expanded wildlife-based surveillance to better understand and predict zoonotic risk.

Canine distemper virus (CDV; Morbillivirus canis) is a multi-host paramyxovirus primarily maintained in the order Carnivora. However, recent outbreaks in Neotropical primates suggest recurrent cross-species spillover into non-human primates. Between May and July 2022, seven marmosets (Callithrix penicillata) were found dead in urban and peri-urban areas of Montes Claros, Minas Gerais, Brazil. Viral metagenomic sequencing recovered two near complete CDV genomes directly from liver samples. Maximum likelihood and Bayesian phylogenetic analyses placed these genomes within the South American CDV lineage and supported at least three independent zoonotic introductions into Callithrix populations. Domestic dogs (Canis lupus familiaris) were inferred as the most likely source and the crab-eating fox (Cerdocyon thous) as a probable bridge host. Ancestral-state reconstruction identified a substitution at position 519 of the hemagglutinin (H) protein (R519I in the two primate clades and R519G in a singleton primate sequence). In silico modelling of the marmoset signaling lymphocytic activation molecule (SLAM) complex was consistent with a modest reorganisation of the receptor-binding head in the 519-substituted variants (ipTM = 0.82), generating a testable hypothesis that this residue may contribute to adaptation at the primate SLAM receptor interface and is associated with altered predicted docking orientation for the receptor. Together, these findings suggest that residue 519 is a candidate marker of CDV adaptation to Neotropical primates and emphasise the importance of integrating genomic surveillance with domestic dog vaccination efforts under a One Health framework to reduce the threat of CDV spillover to vulnerable wildlife populations.

Mammarenaviruses (MaAv) cause persistent infections in diverse rodent reservoirs worldwide and several are zoonotic pathogens with an important public-health burden in their endemic regions. Moreover, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized pathogen of clinical significance in congenital infections and immunocompromised individuals. The lack of FDA-approved vaccines or antivirals for MaAv infections underscores the urgent need for novel anti-MaAv therapeutic strategies. Neutral sphingomyelinase 2 (nSMase2) was recently identified as a host factor contributing to LCMV multiplication, and its inhibitor cambinol exhibits dose-dependent antiviral activity against LCMV but the underlying mechanisms remain undefined. Here, we show that cambinol disrupts multiple stages of the LCMV life cycle. Cambinol inhibits the pH-dependent fusion event mediated by MaAv glycoprotein, a step required for completion of virus cell entry. It also reduces viral ribonucleoprotein (vRNP)-directed genome replication and transcription and impairs the budding activity of the virus matrix Z protein. Cambinol also inhibits sirtuins 1 and 2 (Sirt-1 and Sirt-2), two NAD+-dependent protein deacetylases with pleiotropic roles in cellular metabolism and stress responses, raising the question of whether cambinol anti-LCMV activity reflects nSMase2 inhibition alone or also involves sirtuin-dependent pathways. LCMV multiplication was significantly reduced in SIRT1, but not SIRT2, knockout (KO) cells, uncovering a pro-viral role for Sirt-1 in the LCMV life cycle. Consistent with this finding, LCMV vRNP activity and production of infectious progeny were reduced in SIRT1 KO cells. These findings identify Sirt-1 as a host factor required for optimal LCMV multiplication. Sirt-1 inhibitors are in clinical development for oncological and neurological indications, raising the possibility of repurposing Sirt-1 inhibitors as host-directed antivirals (HDAs) against human pathogenic MaAv. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/740332v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@d53c5forg.highwire.dtl.DTLVardef@16ea861org.highwire.dtl.DTLVardef@1f09addorg.highwire.dtl.DTLVardef@1472e6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Kolmioviruses (KoVs; family Kolmioviridae) are circular, single-stranded RNA viruses that have recently been discovered in a wide range of animal species. However, invertebrate-associated KoVs have so far been identified only in termites and their diversity and evolution remain largely unexplored. Here, we comprehensively analyzed publicly available animal RNA-seq datasets and identified six novel KoVs associated with one fish and five invertebrate species: coho salmon (Oncorhynchus kisutch [Walbaum, 1792]), spotted babylon (Babylonia areolata [Link, 1807]), Atlantic pearl oyster (Pinctada imbricata [Roding, 1798]), small emerald moth (Hemistola chrysoprasaria [Esper, 1795]), codling moth (Cydia pomonella [Linnaeus, 1758]), and cocoa pod borer (Conopomorpha cramerella [Snellen, 1904]).

Hypervirulent Klebsiella pneumoniae represents a growing clinical threat, particularly where multidrug resistance limits treatment options. Here, we report the isolation and characterization of three novel lytic phages from Benin (Kp1Bj_HH11_M23, Kp2Bj_LN294_M23, and Kp10Bj_LN54_14). These Myoviruses, belonging to the genera Marfavirus and Jiaodavirus, display broad host range activity against multidrug-resistant and hypervirulent K. pneumoniae strains. Genomic analysis confirmed the absence of virulence and antimicrobial resistance genes. The phages exhibit rapid adsorption, short latency periods, and high burst sizes (119-2208 PFU/cell). All three phages significantly inhibited biofilm formation and reduced established biofilms in vitro. Their stability across a wide range of temperatures and pH further supports their potential for therapeutic development. Together, these data highlight the value of locally sourced phages as candidates for tackling region-specific antimicrobial resistance challenges.

The current outbreak of Bundibugyo virus (BDBV) in Africa is a global public health concern particularly as there are no licensed medical countermeasures (MCM). Well characterized animal models that accurately replicate human BDBV infection are needed to develop effective MCM. We exposed 21 cynomolgus monkeys (CM) to BDBV to examine the progression and natural history of BDBV disease (BVD). BVD was more protracted than reported for Ebola and Sudan infection in CM with a lower lethality rate of 67% consistent with lower human BVD mortality rates. IHC and spatial proteomics identified CD209+, CD68+, and/or HLA-DR+ macrophages and dendritic cells as early targets of BDBV. These infected cells frequently colocalized with fibrin and infiltrating MPO+ neutrophils and S100A9+ myeloid-derived suppressor cells, consistent with the development of an active inflammatory response and early coagulopathy. Transcriptomic and proteomic analyses of the circulating immune response correspondingly reflected a cytokine-driven hyperinflammatory state in CM that succumbed to disease. Surviving animals resolved systemic inflammation by the study endpoint; however, BDBV antigen was identified in immune privileged tissues with lesion-associated inflammation aligning with known post-Ebola sequela in humans. This data should assist in identifying weaknesses in the disease course that can be exploited to develop new MCM.

Background: Madariaga virus (MADV), has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien province. Methods: We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. Additionally, by applying a catalytic, force-of-infection statistical model to two serosurveys from Darien province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. Findings: In 2017, MADV and VEEV IgM seroprevalence was 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%; VEEV: 16.8%; Una virus (UNAV): 16.0%; and Mayaro virus (MAYV): 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households raising questions about its vectors and enzootic transmission cycles. Insomnia was associated with MADV and VEEV infection, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in some regions. Interpretation: The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that, over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially.

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.

In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates.