Biology subjects

Greninger, A. L.

Publications and source records attributed to Greninger, A. L..

At least 19 recordsLinked to original sources

bioRxiv2026

H5N5 virus transmission is driven by repeated cross-Atlantic introductions and transmission among high latitude seabirds

In November 2025, the first global H5N5 infection was reported in a person in Washington State. Previously, these viruses had only been detected in seabirds and shorebirds near the Atlantic Ocean, suggesting potentially distinct ecology from dominant H5N1 strains. Using 354 global H5N5 genomes, we show that H5N5 viruses have been carried by high latitude seabirds and shorebirds across the Atlantic at least 9 times, with introductions more frequent in the fall and spring. We reconstruct a plausible path of cross-continental movement via Arctic or sub-Arctic regions, followed by a year of cryptic circulation prior to human spillover. These data show that H5N5 viruses have undergone far more intercontinental movements than previously recognized, and exhibit host and geographic patterns that are fundamentally distinct from dominant H5N1 lineages. The distinct host ecology, geography, and movement, paired with the capacity to infect a person, suggest that enhanced surveillance may be warranted.

evolutionary biology↗

bioRxiv2026

Single-Amplicon Sequencing of Coding-Complete Enteroviruses and Rhinoviruses Genomes from Clinical Specimens

Rhinoviruses and enteroviruses are among the most frequently detected respiratory viruses, yet routine diagnostics do not report species or genotype, and standard VP1 genotyping cannot resolve transmission chains or recombination. Recovering sequence beyond VP1 currently requires probe-based enrichment, deep metagenomic sequencing, or several overlapping amplicons. We developed a single-amplicon PCR and sequencing workflow that generates coding-complete genomes from both enteroviruses and rhinoviruses in a single reaction. We evaluated the protocol on 140 rhinovirus-positive respiratory specimens and a panel of 41 isolates spanning 26 enterovirus A-D types, comparing it with hybridization capture sequencing and relating its performance to viral load measured by RT-qPCR and droplet digital RT-PCR. Single-amplicon sequencing recovered 116 high-quality rhinovirus genomes, across 57 rhinovirus genotypes, compared with 113 from hybridization capture. Single-amplicon sequencing was associated with higher fidelity and more uniform sequencing depth compared to hybridization capture. The same primer configuration also recovered two unexpected coxsackieviruses (A6 and A16) from rhinovirus-positive clinical specimens, and the expected polyprotein-coding sequences from all 40 enterovirus isolates tested. Recovery increased with viral abundance, and a visible amplicon band predicted sequencing success. Single-amplicon sequencing reagents cost approximately three-fold less per sample than hybridization capture ($30 versus $85) and required one-third as much time. These findings support single-amplicon sequencing as a simple, cost-effective approach for genomic surveillance of enteroviruses and rhinoviruses from clinical specimens.

genomics↗

bioRxiv2026

EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase

Mosquito-transmitted alphaviruses, including chikungunya (CHIKV), Mayaro, Eastern equine encephalitis, and Venezuelan equine encephalitis viruses, cause severe arthritic or neurologic disease in humans. There are currently no approved antiviral treatments against alphavirus infection. Here, we report that the purine ribonucleoside EIDD-3525 is a potent inhibitor of alphavirus replication in vitro with broad spectrum antiviral activity against the Alphavirus genus. Using a trans-replicase assay, we find that EIDD-3525 blocks alphavirus RNA replication. Mutational profiling suggests that EIDD-3525 targets the alphavirus nsP4 RNA-dependent RNA polymerase that is conserved across medically important alphaviruses. In a pre-clinical mouse model of CHIKV infection and disease, oral treatment with EIDD-3525 reduced viral tissue burdens and the severity of virus-induced disease. Collectively, our findings support the continued development of the purine ribonucleoside EIDD-3525 as an orally available antiviral therapeutic for use against a broad group of alphaviruses.

microbiology↗

bioRxiv2026

Near real-time data on the human neutralizing antibody landscape to influenza virus in summer of 2026 shows antigenic advance of H3N2 subclade K region D mutants and H1N1 D.3.1.1 Sa mutants

Human seasonal influenza evolves rapidly, necessitating twice yearly decisions about whether to update the strains in the vaccine. To help inform this decision, we have been using high-throughput sequencing-based neutralization assays to make twice yearly measurements of how recent human sera neutralize current human H3N2 and H1N1 strains. Here we provide the third installment in this series of measurements by reporting 52,268 titers representing neutralization of 148 viral strains by 355 human sera collected between April and August of 2026. Our measurements show that new H3N2 subclade K strains with mutations in antigenic region D and new H1N1 subclade D.3.1.1 strains with mutations in antigenic region Sa (such as G155E) have reduced neutralization by human sera, with notable heterogeneity in the impact of some of these mutations across sera from different individuals. This paper is accompanied by an interactive summary (https://jbloomlab.github.io/flu-seqneut-2026/summary.html) that enables detailed exploration of the results, and all titer data are publicly available for further analysis to aid vaccine antigen selection and studies of viral evolution.

microbiology↗

bioRxiv2026

Six years of clinical herpes simplex virus genotypic acyclovir resistance testing confirms common resistance mechanisms and identifies novel mutations

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations. IMPORTANCEAcyclovir-resistant herpes simplex virus infections remain an important clinical concern, especially for immunocompromised patients, but testing for acyclovir resistance has traditionally been limited to lengthy and laborious phenotypic testing. Since 2020, the University of Washington Clinical Virology Laboratory has provided genotypic acyclovir resistance testing. This testing provides faster turnaround times but relies on prior mutation characterization to identify resistance making it important to continually evaluate and report identified mutations. The clinical experience with this testing provides valuable insight into resistance mechanisms, identifies common polymorphisms, and highlights novel mutations for further characterization.

microbiology↗

bioRxiv2026

Lineage-specific tprK diversification and Treponema pallidum transmission dynamics in Buenos Aires, Argentina

BackgroundSyphilis rates are rising globally, with increases in congenital syphilis in South America particularly concerning. The characterization of contemporary South American Treponema pallidum (Tp) strains is crucial to syphilis vaccine development, yet few genomic epidemiology studies have focused on this region. Here, we performed whole genome sequencing (WGS) of Tp from Buenos Aires, Argentina, as well as deep sequencing of the hypervariable tprK locus, which is critical to Tp immune evasion. MethodsPeople with primary, secondary, or congenital syphilis were enrolled at two clinics in Buenos Aires between October 2018 and January 2023, including individuals associated with intra-household transmission. Hybrid capture WGS was performed and a core genome phylogeny generated. K-mer-based methods using full-length tprK PacBio long reads were used to uncover differences in diversity and detect Tp transmission. FindingsTp genomes were recovered from 70 individuals in Buenos Aires and primarily belonged to globally dominant SS14 sublineage-1 and Nichols sublineage-8, as did Tp from Brazil (n=8). Peruvian samples (n=3) all belonged to sublineage-1. Two individuals from Argentina had co-infections with Nichols- and SS14-lineage strains. Macrolide resistance via A2058G occurred in 27/70 (38.6%) samples. Across 56 samples, tprK allelic diversity was significantly increased in secondary syphilis, oral lesions, and SS14-lineage strains compared to primary syphilis, anogenital lesions, and Nichols-lineage strains, respectively. Increased diversity in SS14-lineage strains is driven by an enhanced repertoire of V7-specific donor sequences. tprK sequences from intra-household transmissions were more similar than unrelated samples with identical core genomes. InterpretationTp circulating in South America is closely related to dominant global sublineages. Increased tprK diversity in the SS14 lineage may influence Tps ability to escape host immunity. tprK profiling is a promising tool to elucidate syphilis transmission networks. This study underscores the utility of genomics to yield insights into Tp pathogenesis. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAlthough whole genome sequencing (WGS) and genomic epidemiology have contributed to an understanding of the global diversity of Treponema pallidum (Tp), very few strains from South America have been sequenced to date. On January 6th, 2026, we performed a PubMed search including terms "syphilis genomic epidemiology", "South America", "Treponema pallidum", and "Argentina". We excluded studies of ancient Tp samples found in South American archeological sites. A single Tp sample from Argentina was originally sequenced in 2016 and included in subsequent analyses of global diversity. Nine Peruvian samples were previously sequenced in a study of global diversity on six continents. Thirty-three samples from Cali, Colombia were predominantly SS14-lineage and genotypic macrolide resistance was found in half of strains. Additionally, studies of Tp in Buenos Aires using a multi-locus sequence typing approach have shown circulation of strains belonging to both Nichols and SS14 lineages, with an increasing rate of macrolide resistance over time. Five previous studies have examined tprK in clinical specimens and consistently shown increased tprK diversity in specimens associated with secondary syphilis compared to primary syphilis lesions. One study has shown that loss of tprK donor cassettes is associated with reduced tprK diversity, consistent with results from in vitro experiments. A single study has shown that tprK sequence content is more similar in samples with a suspected epidemiologic link, though no prior studies have looked at tprK diversity in the context of known syphilis transmission events. Added value of this studyThis study is the first to use WGS to comprehensively examine Tp transmission in a large South American city, yielding 96 samples from 70 individuals. We also add the first contemporary Tp genomes from Brazil. In contrast to findings from England, Australia, and other high-income countries, no Tp sublineages are associated with demographic groups or sexual networks in Buenos Aires. Two of seventy patients were co-infected with both Nichols- and SS14-lineage strains, showing the previously unappreciated frequency of conditions that permit inter-strain recombination-driven diversification of Tp. This study also reveals novel aspects of Tp pathogenesis, including lineage-specific differences in tprK diversity. We also develop methods for the analysis of tprK relatedness between samples and demonstrate that tprK sequences are more similar in samples from individuals within intra-household syphilis transmission chains compared to those from epidemiologically unrelated individuals. Implications of all the available evidenceTp strains circulating in Buenos Aires are genetically similar to those circulating worldwide and in Brazil and Peru but are noteworthy for the low (but rising) rate of macrolide resistance. Lineage-specific patterns of tprK antigenic variation could result in differences between Nichols- and SS14-lineage strains interactions with the host immune system. Finally, we show that tprK profiling holds promise to identify samples from within a syphilis transmission chain and could play an important role in public health.

microbiology↗

bioRxiv2026

Integration of an anellovirus genome in the SKNO-1 acute myeloid leukemia cell line

Anelloviruses are highly diverse, ubiquitous single-stranded DNA viruses whose replication, cellular reservoirs, and mechanisms of persistence remain poorly understood. Here, we identify and characterize a Alphatorquevirus homin 29 genome stably integrated into an rDNA locus of human chromosome 21 in the acute myeloid leukemia cell line SKNO-1. Large-scale mining of NCBI Sequence Read Archive datasets revealed unusually high anellovirus k-mer abundance specifically in sequencing runs of the SKNO-1 cell line from multiple laboratories. De novo assembly of RNA-seq data recovered a 3.25 kb viral genome, and long-read PacBio sequencing confirmed its integration within the RNA45SN2 gene on human chromosome 21. Digital droplet PCR (ddPCR) quantified [~]0.5 viral genomes per cell, and RT-ddPCR detected transcripts from ORF1 and viral terminal repeats, consistent with transcription of the integrant. The ORF1 coding sequence encoded for a truncated but structurally conserved capsid protein retaining jelly roll and P-domain features but lacking the C-terminal domain. Analysis of public ChIP-seq data from the SKNO-1 cell line demonstrated broad occupancy of the BRD4 transcriptional coactivator across the viral genome along with focal enrichment of hematopoietic ETS family transcription factors within the integrants [~]300 bp untranslated region upstream of viral open reading frames. Together, these findings demonstrate stable integration, chromatin accommodation, and transcriptional maintenance of an Alphatorquevirus in a human leukemia cell line, providing a unique model to study host tolerance and transcriptional regulation of anellovirus DNA and informing our understanding of anellovirus hematopoietic cell tropism. IMPORTANCEAnelloviruses are a curious group of highly genetically diverse single-stranded DNA viruses that have been found ubiquitously among humans and are hypothesized to be a potential commensal human virus. Their omnipresence has been matched only by the dearth of our understanding of their basic biological processes - how they persist, how they replicate, and how they spread. Here we identify a naturally integrated Alphatorquevirus genome in the widely used AML cell line SKNO-1 and show that it is stably maintained, transcribed, and embedded within a rDNA locus on human chromosome 21. This discovery highlights the ability to anelloviruses to integrate into human DNA, generates hypotheses around transcription factors used in anellovirus gene transcription, and suggests anelloviruses can persist in myeloblasts.

genomics↗

bioRxiv2025

Developmental candidate GHP-88310/EIDD-3608 with high tolerability and oral efficacy in measles and respiratory paramyxovirus models

Orthoparamyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV), are a major health threat. We discovered an orally efficacious broad-spectrum inhibitor of orthoparamyxovirus polymerases. However, here we found that tolerability in higher mammals was limited. We report development of clinical candidate analog GHP-88310 (EIDD-3608), which combines improved oral efficacy with favorable tolerability in non-rodents (ferrets and dogs). GHP-88310 was active against HPIV3, Sendai virus (SeV), MeV, and related canine distemper virus (CDV). In 7-day tolerability studies, daily doses of 2,000 mg/kg were well tolerated. Pharmacokinetic analysis revealed altered plasma exposure of GHP-88310 compared to the original hit. In HPIV3-infected cotton rats, GHP-88310 lowered respiratory tract viral load. Dosing of ferrets infected with CDV, causing lethal measles-like disease, resulted in complete survival, reduction of viremia and shed viral load, and alleviated lymphocytopenia. Once-daily GHP-88310 was efficacious in the CDV-ferret and HPIV3-cotton rat models. The compound was sterilizing against HPIV3 at physiological concentrations in human airway epithelium organoids.

microbiology↗

bioRxiv2025

Susceptibility to Penicillin G and Ceftriaxone in Three Clinical Treponema pallidum Isolates is not Altered by Amino Acid Polymorphisms in the Tp0705 Penicillin Binding Protein

We demonstrated no differences in susceptibility to penicillin G and ceftriaxone in three modern T. pallidum isolates (UW244B, UW249B, and UW330B), each carrying a variant of the penicillin-binding protein (PBP) Tp0705. This suggests that these polymorphisms should not be a reason for concern when {beta}-lactams are prescribed for syphilis treatment. SUMMARYWe report that three modern Treponema pallidum isolates carrying amino acid polymorphisms at positions 506, 625, and 708 of the penicillin-binding protein (PBP) Tp0705 are equally susceptible to penicillin G and ceftriaxone.

microbiology↗

bioRxiv2025

Stabilized Full-Length Measles Fusion Protein Elicits Potent Immunity and Protection In Vivo

Measles virus (MeV) is a highly contagious pathogen that causes significant morbidity and mortality in populations with low vaccination coverage. Infection typically leads to immune amnesia and, in rare cases, fatal neurological disease. While current live-attenuated vaccines are highly effective, they primarily elicit neutralizing antibodies against the hemagglutinin (H) glycoprotein, with a less robust response to the fusion (F) protein, a key protein for viral entry. To improve the immunogenicity of the F protein, we designed and characterized stabilized, prefusion MeV F protein antigens. We engineered both soluble ectodomains (FECTO) and full-length, membrane-embedded proteins (FFL) with mutations that confer thermal stability. Cryo-electron microscopy confirmed that these engineered antigens faithfully maintain the native prefusion conformation. When evaluated in a cotton rat model, immunization with either FECTO or FFL constructs induced neutralizing antibodies and elicited protection against viral challenge. The most stable full-length construct (FFL 3M) elicited a more potent neutralizing antibody response than its ectodomain counterpart. Importantly, no evidence of vaccine-enhanced respiratory disease was observed. These findings establish that a thermostable, full-length F protein is a superior immunogen to its soluble ectodomain. This work presents a promising candidate for next-generation, non-replicating measles vaccines intended to complement current vaccination strategies and provide a safe option for immunocompromised individuals and others who cannot receive live-virus vaccines. One-Sentence SummaryA prefusion-stabilized, full-length measles Fusion glycoprotein immunogen induces strong neutralizing responses and offers protection against challenge with wild-type virus.

immunology↗

bioRxiv2025

De novo recovery of Ghana virus, an African bat Henipavirus, reveals differential tropism and attenuated pathogenicity compared to Nipah virus

Henipaviruses (HNVs) like Nipah (NiV) and Hendra (HeV) viruses represent severe zoonotic threats. Ghana virus (GhV), identified in 2012, is the only African bat henipavirus with a near-complete genome assembly. However, without isolates in culture, GhV biology, pathogenicity, and zoonotic potential remain poorly understood. Using reverse genetics, we recovered a full-length infectious clone of GhV at BSL-4 following rational reconstruction of its incomplete 3' leader and modification of a non-canonical transcriptional initiation site. GhV demonstrated restricted receptor tropism (ephrin-B2 but not ephrin-B3) and distinct innate immune antagonism. Replication was attenuated in primary human cells, but was enhanced in bat cells. In Syrian golden hamsters, GhV infection caused no disease or mortality. Furthermore, a chimeric NiV encoding the GhV receptor-binding protein was completely attenuated in vivo, implicating ephrin-B3 receptor usage as a critical determinant of HNV pathogenesis. These findings elucidate GhV zoonotic potential and inform strategies for virus surveillance and control. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/679836v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1e5addcorg.highwire.dtl.DTLVardef@1f96850org.highwire.dtl.DTLVardef@12f842eorg.highwire.dtl.DTLVardef@8f013c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Structural and Mechanistic Basis for Antibody Neutralization of the Measles Fusion Protein

Measles virus (MeV) is a highly contagious viral pathogen and remains a major global health threat. Resurgent infections, driven by insufficient vaccine coverage, waning herd immunity, and the vulnerability of immunocompromised individuals, highlight the urgent need for effective countermeasures. Monoclonal antibodies (mAbs) represent a promising strategy, both as antiviral agents and as probes of viral entry mechanisms. While most vaccine-elicited neutralizing antibodies target the hemagglutinin (H) protein, emerging evidence suggests that antibodies against the fusion (F) protein are also potent inhibitors. Still, there is insufficient information on the target sites and activities of antibodies against the F protein. Like other class I fusion proteins, MeV F exists in a metastable prefusion state that undergoes dramatic conformational changes during viral entry. Here, we selected four mAbs that recognize conformational patterns of F-prefusion and/or postfusion, characterized their epitopes, specificities, and antiviral activities. Structural analyses mapped antibody interactions onto pre- and postfusion F conformations, revealing that all three neutralizing mAbs are specific for the prefusion form, while the non-neutralizing mAb recognizes only the postfusion F. Biophysical and functional assays defined distinct mechanisms: neutralization occurs either by stabilizing the prefusion protein or by preventing the extended intermediate from completing fusion. We also describe a novel mechanism of neutralization in which an antibody prematurely triggers F activation but blocks the subsequent refolding required for viral entry. Together, these findings provide the first detailed mapping of neutralizing epitopes on the MeV F protein and establish a framework for the rational design of F-targeted intervention.

immunology↗

bioRxiv2025

Identification and Characterization of Novel Chikungunya Virus Polymerase Inhibitors

Chikungunya virus (CHIKV) and other alphaviruses in the Togaviridae family are positive-sense RNA viruses and major human pathogens, causing millions of infections worldwide. In humans, alphaviruses such as CHIKV, Mayaro and Ross River viruses typically cause arthritogenic disease characterized by debilitating arthralgia, joint inflammation, fever, and rash. Although a vaccine was recently approved for use against CHIKV, no vaccines are licensed against other alphaviruses. No antiviral treatments are available to prevent or treat infections by any alphavirus. To address this unmet need, we used a CHIKV nanoluciferase reporter virus to develop a high-throughput screening assay for novel small-molecule inhibitors. From this campaign, we identified several unique inhibitors of CHIKV replication. Mechanistic characterization of two inhibitors revealed that both target the nsP4 RNA-dependent RNA polymerase, while susceptibility profiling pinpointed unique nsP4 mutations that specifically confer resistance. In silico docking analyses indicated potential binding poses of the inhibitors near the polymerase active site. Collectively, these results define multiple chemotypes for further development and highlight novel molecular targets within nsP4 for CHIKV inhibition. IMPORTANCEChikungunya virus is a mosquito-borne pathogen that has caused millions of human infections worldwide, producing severe fever, rash, and long-lasting joint pain that can persist for months. Related viruses such as Mayaro and Ross River viruses also cause debilitating disease, yet no antiviral drugs are available to treat any infection caused by this family of viruses. In this study, we developed a high-throughput assay that allowed us to rapidly identify compounds capable of blocking chikungunya virus replication. We discovered new hit compounds that inhibit virus growth. In addition, we determined that two of the most promising hit candidates target the viral nsP4 polymerase. By identifying these novel inhibitors and characterizing both their mechanisms of action and resistance profiles, we have established the groundwork for future efforts to develop much needed therapies against chikungunya virus and related pathogens.

molecular biology↗

bioRxiv2025

Impact of Mutations Affecting 4'-Fluorouridine Susceptibility on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthitogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 4-Fluorouridine (4-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4-FlU in a mouse model. Although mutations against 4-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4-FlU for the treatment of alphavirus encephalitis. ImportanceVEEV is one of several mosquito-spread viruses that can cause serious brain infections in people and animals. Unlike CHIKV, against which an approved vaccine exists, there are no countermeasures to prevent or treat VEEV infections or block its close relatives, EEEV and WEEV. 4-FlU inhibits many different RNA viruses, including VEEV, and is currently being developed to treat multiple viral infections. A major problem with small-molecule antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we identified mutations in VEEVs RNA-dependent RNA polymerase (nsP4) that confer reduced susceptibility to 4-FlU. We then engineered these mutations into full-length infectious clones and assessed if viruses encoding for these mutations were still pathogenic. In addition, we treated infected mice with 4-FlU and measured how well the compound inhibited virus replication and prevented severe disease, even when mice were infected with viruses harboring mutations that reduced susceptibility to 4-FlU. Although VEEV can develop moderate resistance to 4-FlU in vitro, administration of 4-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4-FlU. These results suggest that 4-FlU has strong potential as a future treatment for alphavirus infections like VEEV encephalitis.

microbiology↗

bioRxiv2025

Near real-time data on the human neutralizing antibody landscape to influenza virus to inform vaccine-strain selection in September 2025

The hemagglutinin of human influenza virus evolves rapidly to erode neutralizing antibody immunity. Twice per year, new vaccine strains are selected with the goal of providing maximum protection against the viruses that will be circulating when the vaccine is administered [~]8-12 months in the future. To help inform this selection, here we quantify how the antibodies in recently collected human sera neutralize viruses with hemagglutinins from contemporary influenza strains. Specifically, we use a high-throughput sequencing-based neutralization assay to measure how 188 human sera collected from Oct 2024 to April 2025 neutralize 140 viruses representative of the H3N2 and H1N1 strains circulating in humans as of the summer of 2025. This data set, which encompasses 26,148 neutralization titer measurements, provides a detailed portrait of the current human neutralizing antibody landscape to influenza A virus. The full data set and accompanying visualizations are available for use in vaccine development and viral forecasting.

microbiology↗

bioRxiv2025

Automated Annotation and Validation of Human Respiratory Virus Sequences using VADR

Accurate annotation of viral genomes is essential for reliable downstream analysis and public data sharing. While NCBIs Viral Annotation DefineR (VADR) pipeline provides standardized annotation and quality control, it only supports six viral groups to date. Here, we developed and validated 12 new reference sequence-based VADR models targeting key human respiratory viruses: measles virus, mumps virus, rubella virus, human metapneumovirus, human parainfluenza virus types 1-4, and seasonal coronaviruses (229E, NL63, OC43, HKU1). Model construction was guided by a comprehensive analysis of intra-species genomic and phylogenetic diversity, enabling the development of genotype-specific models associated with reference genomes that defined expected genome structure and annotation. Models were trained on 5,327 publicly available complete viral genomes and tested on 372 viral genomes not yet submitted to GenBank. VADR passed 96.3% of publicly available viral genomes and 98.1% of viral genomes not in the training set, correctly identifying overlapping ORFs, mature peptides, and transcriptional slippage as well as genome misassemblies. VADR detected novel viral biology including the first reported HCoV-OC43 NS2 knockout in a human infection and novel G and SH coding sequence lengths in human metapneumovirus. These VADR models are publicly available and are used by NCBI curators as part of the GenBank submission pipeline, supporting high-quality, scalable viral genome annotation for research and public health.

bioinformatics↗

bioRxiv2025

DEVELOPMENT OF A POTENT MONOCLONAL ANTIBODY FOR TREATMENT OF HUMAN METAPNEUMOVIRUS INFECTIONS

Human metapneumovirus (HMPV) is a major cause of respiratory infections, particularly among vulnerable populations, yet effective therapeutics remain unavailable. Monoclonal antibodies (mAbs) offer a promising approach for both treatment and prevention. Here, we describe the discovery and characterization of 4F11, a highly potent and broadly neutralizing mAb with demonstrated in vitro and in vivo efficacy against HMPV. Using cryo-electron microscopy, we defined a unique mechanism of binding HMPV employed by 4F11, which distinguishes it from previously characterized RSV and HMPV mAbs. 4F11 targets an epitope located at the apex of the prefusion F protein (site O) with a 1:1 stoichiometry, distinct from the 3:1 stoichiometry observed with other HMPV site O antibodies. Unlike other site O antibodies, which penetrate the glycan shield between Asn57 and Asn172, 4F11 binds vertically and directly interacts with the Asn172 glycan, representing a unique glycan-dependent mode of recognition. In vitro, 4F11 displayed high potency and broad neutralization across diverse HMPV strains. It also showed a low propensity for resistance development, with only a single escape mutation (K179E) identified, a mutation not found in any published HMPV sequence to date. Viruses rescued with the K179E escape mutation had significantly decreased fitness in vitro compared to wild-type virus. In a hamster challenge model, 4F11 significantly reduced viral loads in both the lungs and nasal turbinates. These findings highlight 4F11 as a promising candidate for therapeutic development, particularly for immunocompromised individuals and other high-risk groups.

immunology↗

bioRxiv2025

A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 {Delta}3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. IMPORTANCESmall-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins ({Delta}3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

microbiology↗