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Kawaoka, Y.

Publications and source records attributed to Kawaoka, Y..

At least 19 recordsLinked to original sources
bioRxiv2026

NGS-RFA/FRA: A High Throughput Experimental and Computational Pipeline for Selective Mutational Scanning in Parallel

Influenza viruses evade vaccine and infection mediated immunity by accumulating mutations in their hemagglutinin (HA) protein. Predicting this evolution might be possible via selective mutational scanning (SMS) - the generation of many specific mutants of interest from currently circulating viruses and characterizing their escape potential and fitness with high accuracy (Mogling 2016). However, this task is challenging, even when focusing on a reduced set of key HA positions (Koel et al. 2013). Here we describe a high-throughput SMS method to address this challenge. Our approach consists of a three-stage pipeline: (1) a parallel optimized virus rescue process that generates balanced target mutant virus libraries (2) an assay to assess replicative fitness and neutralisation of these variants as a mixture, and (3) a bespoke statistical model to quantify statistically significant differences between these observables. We tested the pipeline on libraries of up to 134 variants finding excellent correlation to classical hemagglutination inhibition (HI) and plaque growth assays used to assess antigenic phenotype and replicative fitness respectively, as well as remarkable repeatability overall. Notably, the method reduces the timeline required to carry out such assessments with classical methods from about a year to several weeks. By enabling rapid and efficient characterization of influenza virus variants, this approach has the potential to greatly enhance surveillance efforts, transforming reactive monitoring into proactive forecasting.

evolutionary biology↗
bioRxiv2026

A rapidly deployable CRISPR-Cas3 diagnostic platform for emerging RNA viruses

Rapidly converting viral genome information into deployable molecular tests remains a major challenge in outbreak preparedness. We developed CONAN-SWIFT (Simple Workflow for Isothermal Field Testing), a sequence-to-test platform that integrates computational assay design, reverse-transcription loop-mediated isothermal amplification, CRISPR-Cas3 detection, reagent lyophilization and lateral-flow readout. Sequence-guided assays for Andes virus and Bundibugyo virus were established within approximately three weeks and extended to four additional filoviruses. A web-based designer supported crRNA selection, and systematic RT-LAMP primer optimization improved amplification performance. Recombinant Escherichia coli-expressed Cascade enabled standardized preparation of lyophilized Cas3-detection reagents, which were combined with a battery-operated isothermal device. The portable system detected as few as 10 input RNA copies per reaction within approximately 40 min. It also detected viral RNA and biologically contained, replication-incompetent Ebola virus in spiked human blood and concentrated wastewater. These findings establish the analytical feasibility of a rapidly adaptable CRISPR-Cas3 engineering framework for decentralized detection of emerging RNA viruses.

bioengineering↗
bioRxiv2026

Evolutionary constraints limit additional mammalian adaptation of bovine-derived H5N1 influenza viruses in ferrets

The outbreak of clade 2.3.4.4b H5N1 viruses among U.S. dairy cattle has raised concerns that sustained circulation among agricultural mammals could facilitate viral adaptation toward efficient human transmission. However, the evolutionary dynamics governing such adaptation remain poorly understood. Here we investigated the evolution of two bovine-derived H5N1 B3.13 genotype viruses during infection and airborne transmission in ferrets, building on prior characterization of their robust replication and inefficient airborne transmission. Within hosts, viral genetic diversity was limited and viruses were subject to genetic drift and weak purifying selection. Transmission, when it occurred, was characterized by stringent bottlenecks that sharply reduced viral genetic diversity. We found no evidence of mammalian adaptation during infection or transmission. Together, these findings indicate that bovine-derived H5N1 viruses face evolutionary constraints during acute mammalian infection and transmission, limiting movement toward enhanced airborne spread. These constraints may help explain why efficient mammalian replication does not necessarily coincide with efficient transmission. Continued circulation of HA clade 2.3.4.4b viruses nevertheless creates repeated opportunities for rare but consequential evolutionary events, underscoring the importance of sustained surveillance and risk mitigation.

microbiology↗
bioRxiv2025

A divergent betacoronavirus with a functional furin cleavage site in South American bats

Bats are natural reservoirs for a wide range of RNA viruses. Members of the genus Betacoronavirus, including Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome virus (MERS-CoV), have attracted particular attention due to their recent zoonotic emergence. However, much of the known diversity of betacoronaviruses is based on data from Asia, Africa, and the Middle East, with limited genomic information available from the Americas. Herein, we report the complete genome of a novel bat betacoronavirus identified from a Pteronotus parnellii bat sampled in Brazil. Phylogenetic analysis revealed that this virus is sufficiently distinct from the five recognized Betacoronavirus subgenera to represent a new subgenus. Of note, the spike protein of this novel bat coronavirus possesses a functional furin cleavage site at the S1/S2 junction with a unique amino acid sequence motif (RDAR) that differs from that found in SARS-CoV-2 (RRAR) by only one amino acid. Comparative structural analysis identified other betacoronaviruses in bats with furin cleavage sites at the S1/S2 junction, suggesting that this region is a structurally permissive "hotspot" for cleavage site incorporation. Our study provides a broader understanding of the phylogenetic and functional diversity of bat coronaviruses as well as their zoonotic potential.

microbiology↗
bioRxiv2025

A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4+ and CD8+ T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.

microbiology↗
bioRxiv2025

In Vivo Evolution of Monoclonal Antibody CR3022 to Achieve Cross-Neutralization of SARS-CoV-2 and Implications for Vaccine Strategies Against SARS-Related Viruses

The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We developed a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. SARS-CoV/SARS-CoV-2 sequential immunization led to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM) that resembled the affinity-matured CR3022 from a human, but now also adapted to key variants and divergent sarbecoviruses. While simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol elicited dominant responses to other epitopes. X-ray crystal structures revealed that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.

immunology↗
bioRxiv2024

Structural basis of broad protection against influenza virus by a human antibody targeting the neuraminidase active site via a recurring motif in CDR H3

Influenza viruses evolve rapidly, driving seasonal epidemics and posing global pandemic threats. While neuraminidase (NA) has emerged as a vaccine target, shared molecular features of NA antibody responses are still not well understood. Here, we describe cryo-electron microscopy structures of the broadly protective human antibody DA03E17, which was previously identified from an H1N1-infected donor, in complex with NA from A/H1N1, A/H3N2, and B/Victoria-lineage viruses. DA03E17 targets the highly conserved NA active site using its long CDR H3, which features a DR (Asp-Arg) motif that engages catalytic residues and mimics sialic acid interactions. We further demonstrate that this motif is conserved among several NA active site-targeting antibodies, indicating a common receptor mimicry strategy. We also identified potential antibody precursors containing this DR motif in all donors of a healthy human donor BCR database, highlighting the prevalence of this motif and its potential as vaccine targeting. Our findings reveal shared molecular features in NA active site-targeting antibodies, offering insights for NA-based universal influenza vaccine design.

immunology↗
bioRxiv2024

Influenza A virus exploits the motility of membrane cytoskeletal actomyosin filaments for its genome packaging in the host cell

Influenza A virus encodes its genome in eight segments of viral ribonucleoproteins (vRNPs) replicated in the host cell nucleus. Our understanding of host factors involved in driving vRNP selective packaging remains incomplete. To address this, we used advanced immuno-freeze-etch electron microscopy to visualise the vRNP packaging process and atomic force live-cell imaging (AFM) to examine the motility of membrane cytoskeletal actin filaments. In the cytoplasm, vRNPs were mainly localised on mottled membrane-like structures, suggesting intracellular trafficking through such structures. After reaching the cytoplasmic side surface of the plasma membrane, vRNPs formed many aggregates while associating with actin filaments. Antibody labelling also detected myosin along actin filaments entangled in vRNPs. Blocking myosin activity with blebbistatin prevented the active movement of membrane cytoskeletal actin filaments just below the plasma membrane visualised by AFM and abrogated proper aggregation of vRNPs. Thus, actomyosin motility appears to be crucial for the selective packaging of vRNPs.

cell biology↗
bioRxiv2024

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E. coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.

synthetic biology↗
bioRxiv2023

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

bioinformatics↗
bioRxiv2023

Characterization of an EG.5.1 clinical isolate in vitro and in vivo

EG.5.1 is a subvariant of the SARS-CoV-2 Omicron XBB variant that is rapidly increasing in prevalence worldwide. EG.5.1 has additional substitutions in its spike protein (namely, Q52H and F456L) compared with XBB.1.5. However, the pathogenicity, transmissibility, and immune evasion properties of clinical isolates of EG.5.1 are largely unknown. In this study, we used wild-type Syrian hamsters to investigate the replicative ability, pathogenicity, and transmissibility of a clinical EG.5.1 isolate. Our data show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5, and both EG.5.1 and XBB.1.5 are attenuated compared to a Delta variant isolate. We also found that EG.5.1 is transmitted more efficiently between hamsters compared with XBB.1.5. In addition, unlike XBB.1.5, we detected EG.5.1 virus in the lungs of four of six exposed hamsters, suggesting that the virus tropism of EG.5.1 is different from that of XBB.1.5 after airborne transmission. Finally, we assessed the neutralizing ability of plasma from convalescent individuals and found that the neutralizing activity against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. This suggests that EG.5.1 effectively evades humoral immunity and that the amino acid differences in the S protein of EG.5.1 compared with that of XBB.1.5 or XBB.1.9.2 (i.e., Q52H, R158G, and F456L) alter the antigenicity of EG.5.1. Our data suggest that the increased transmissibility and altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in the human population.

microbiology↗
bioRxiv2023

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

molecular biology↗
bioRxiv2022

Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody to antigenically distinct omicron SARS-CoV-2 subvariants

The rapid evolution of SARS-CoV-2 Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identify S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) and derived from an individual previously infected with SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrates broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1). Furthermore, it protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis reveals that this antibody targets a class 1 epitope via multiple hydrophobic and polar interactions with its CDR-H3, in addition to common class 1 motifs in CDR-H1/CDR-H2. Importantly, this epitope is more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.

immunology↗
bioRxiv2022

Host network-based discovery of critical regulators of innate immunity, virus growth, and pathogenesis in influenza virus infection

Innate immunity is protective against viruses, but also can facilitate pathological infection responses. Despite intensive research, our understanding of the mechanisms that regulate innate immunity in virus infection remains incomplete. Systems biology-based data-driven modeling approaches hold substantial promise toward discovery of crucial innate immune signaling regulators, yet model-derived predictions are almost completely unexplored. Here, we carried out systematic experimental validation of candidate regulators predicted by a transcriptional association network model of influenza virus-infected cells. We identified dozens of novel innate immune signaling regulators with potent effects on the replication of influenza and other viruses, and importantly, we established the biological relevance of a validated regulator in vivo. Collectively, these findings aid in clarifying mechanisms of influenza virus pathogenicity and might lead to innovative approaches for treating influenza virus disease. Similar data-driven modeling strategies may be applicable for the study of other pathogen systems or immunological disorders.

microbiology↗
bioRxiv2022

Can SARS-CoV-2 transmit from a dead body?

Although it has been 2.5 years since the COVID-19 pandemic began, the transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a dead infected body remains unclear, and often, in Japan bereaved family members are not allowed to view in-person a loved one who has died from COVID-19. In this study, we analyzed the possibility of SARS-CoV-2 transmission from a dead body by using the hamster model. We also analyzed the effect of Angel-care--in which the pharynx, nostril, and rectum are plugged--and embalming on reducing transmissibility from dead bodies. We found that SARS-CoV-2 could be transmitted from the body of animals that died within a few days of infection; however, Angel-care and embalming were effective in preventing transmission from the dead body. These results suggest that protection from infection is essential when in contact with a SARS-CoV-2-infected dead body, and that sealing the cavities of a dead body is an important infection control step if embalming is not done. ImportanceWe found that SARS-CoV-2 could be transmitted from a dead body presumably via postmortem gases. However, we also found that postmortem care, such as plugging the pharynx, nostrils, and rectum, or embalming could prevent transmission from the dead body. These results indicate that protection from infection is essential when handling infected corpses, and that appropriate care of SARS-CoV-2-infected corpses is important.

microbiology↗
bioRxiv2022

Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates

The prevalence of the Omicron subvariant BA.2.75 is rapidly increasing in India and Nepal. In addition, BA.2.75 has been detected in at least 34 other countries and is spreading globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs was higher than that of BA.2 and BA.5. Of note, BA.2.75 caused focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which was not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicated better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 and should be closely monitored.

microbiology↗
bioRxiv2022

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

microbiology↗