Emerging Microbes & Infections

MERS-CoV poses a constant pandemic risk, as its viral lineages continue evolving, and zoonotic spillover events could lead to random viral polymorphisms that might lead to human adapted variants. Currently, no small animal model reliably recapitulates both disease progression and transmission dynamics, which are critical aspects for counter-viral measures like vaccine development. Although the Syrian hamster is an optimal animal model for SARS-CoV-2 infection and transmission, it is naturally resistant to MERS-CoV infection. Dipeptidyl peptidase-4 (DPP4) is the functional receptor for MERS-CoV infection, and is highly expressed in human kidney, intestine, liver, and lung tissues. Here, we evaluated the suitability of a human DPP4 (hDPP4) transgenic Syrian hamster model for MERS-CoV research. We used two different MERS-CoV strains (EMC/2012 and D10540/2023) for intranasal inoculation of hamsters. Both strains replicated efficiently, led to comparable severe clinical outcomes, and had similar viral transmission efficiencies. MERS-CoV RNA and nucleoprotein antigen were mainly detected in the brain and the respiratory tract. In summary, we validated a novel hDPP4-transgenic hamster as a suitable model for MERS-CoV infection enabling vaccine and transmission research.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a global health concern due to a high fatality rate associated with human infections and no approved vaccines or therapeutics. While Syrian hamsters are a value animal model for coronavirus research, including SARS-CoV-2, MERS-CoV does not infect wild-type hamsters. Here, we generated transgenic Syrian hamsters expressing human dipeptidyl peptidase-4 (hDPP4), the cellular receptor for MERS-CoV., MERS-CoV replicated efficiently in the respiratory tract tissues of hDPP4 hamsters, causing lethal disease. Treatment with the 3CLpro inhibitor nirmatrelvir significantly reduced viral titers in the lower respiratory tract of infected hDPP4 hamsters. While airborne transmission was not observed, direct contact transmission was observed in all contact hDPP4 hamsters cohoused with infected cage mates. Immunization with purified MERS receptor-binding domain protein reduced virus replication and disease severity but did not prevent direct contact transmission. Collectively, our findings demonstrate that hDPP4 transgenic Syrian hamsters are useful for studying MERS-CoV pathogenesis, transmission, and countermeasure efficacy.

Influenza A virus (IAV) circulates widely in European pig populations and continues to diversify through frequent introductions from humans, followed by reassortment within swine. Spain represents a particularly dynamic ecological setting due to the coexistence of intensive whitepig production, extensive Iberianpig systems, and abundant wild boar populations. This study provides an integrated analysis of IAV evolution and genomic diversity in swine in Spain between 2019 and 2022, expanding on previous surveillance from 2016 to 2019. Sampling across 24 provinces yielded 66 new wholegenome sequences from Iberian and white pigs. We identified 18 genotypes, including 11 novel reassortants not detected in our previous survey. Several genotypes, such as H1huN2 G21 and G22, H3N2 G23, and the unusual H3N1 G12, were exclusive to the country. Some genotypes were detected across white pigs, Iberian pigs, and wild boar in Toledo and Badajoz, suggesting viral flow among swine populations. Phylogenetic analyses revealed ongoing introductions of H1N1pdm09 from humans into pigs, generating at least five reassortant genotypes (G10, G16-G19). These lineages incorporated pandemic internal cassettes and, in some cases, humanseasonal N2 segments, highlighting the continued role of humans as a source of viral incursions. Conversely, four zoonotic infections (H1N1v) detected in Spain between 2022 and 2026 were linked to genotypes circulating in white pigs, underscoring the bidirectional nature of IAV transmission at the human swine interface. Overall, this study demonstrates that Spain provides ecological conditions conducive to IAV diversification, reassortment, and zoonotic risk. The findings reinforce the need for sustained One Health surveillance. HighlightsO_LINovel swine influenza virus (SIV) genotypes exclusive to Spain C_LIO_LIPhylogenetic analysis of genomic segments of zoonotic variants of swine origin detected in Spain since 2022 C_LIO_LIShared circulation of influenza A compatible with interbreed transmission among domestic pigs and wild boar C_LI

West Nile virus (WNV) and Usutu virus (USUV) are neurotropic Orthoflaviviruses sharing a similar enzootic transmission cycle primarily involving Culex pipiens mosquitoes as vectors and birds as amplifying hosts. First identified in Africa, both viruses established endemicity across Europe over the past two decades, most likely introduced and spread by migratory bird species along Mediterranean flyways. In avian species, infection outcomes range from subclinical to fatal neuroinvasive disease, varying by viral strain, host immunity, and species susceptibility. Southern France emerges as a key hotspot for the circulation of these viruses, supported by diverse avian habitats conducive to year-round viral maintenance. This study investigated the prevalence of WNV and USUV in more than 2500 sedentary and migratory wild birds from these regions during 2024-2025 using molecular surveillance. Samples were collected using mist net and bird boxes, across multiple passerine and non-passerine taxa, spanning wetlands, urban fringes, and agricultural zones. Our analyses revealed widespread viral circulation across diverse species, mainly among passerines such as great tits, house sparrows, and barn swallows with USUV detected at higher rates than WNV in both study years. Overall prevalence was markedly higher in 2024 than in 2025, potentially reflecting climatic or ecological drivers. Migratory individuals likely seed viral introductions during seasonal passages, whereas resident populations sustain local enzootic cycles, facilitating overwintering persistence. These results highlight the pivotal role of mixed avifauna in arbovirus dynamics within Mediterranean Europe and emphasize the necessity for integrated, year-round surveillance targeting high-risk species and habitats. Enhanced monitoring will aid in predicting spillover risks and informing vector control strategies to mitigate zoonotic threats.

Pigs serve as reservoirs of former human influenza A virus (IAV) H1N1 and H3N2 lineages and act as mixing vessels for diverse strains, facilitating the emergence of novel IAVs. Understanding the spread and evolution of swine IAVs (swIAVs) is therefore crucial to assess the risk of strains with zoonotic potential emerging. This study uses a phylogeographic framework to investigate the predictors of swIAV dispersal across Europe. All publicly available swIAV genomic sequences were retrieved and subsampled for the ten largest European pig-producing countries. Discrete phylogeographic reconstructions were conducted for H1, H3, N1, N2 encoding genes and all internal gene segments. Our analyses indicate that viral dispersal predominantly occurred from north-western to southern and eastern Europe, with frequent long-distance transitions between non-adjacent countries. We also extended the discrete phylogeographical analyses with generalized linear models to test the association between viral movement and potential predictors, such as live pig trade, pork trade, pig densities, farm sizes, or the geographic distance between key pig production zones. We find that breeding pig trade is the only consistently well-supported predictor of between-country transition events, whereas pork trade and geographic distance were not supported. This highlights that farms importing breeding pigs from multiple countries could act as hotspots for reassortment of diverse swIAV strains. Strengthening external biosecurity on farms with emphasis on quarantining breeding pigs, limiting long-distance transport, and implementing a One Health surveillance system for earlier detection of emerging strains, could help curb the rapid spread and evolution of swIAV in Europe. Author summaryInfluenza A viruses that originally came from humans are now circulating in pigs. This is a public health concern because these viruses continue to evolve in pigs and may eventually return to humans in new forms that could cause more severe disease. To better prevent this risk, we need to understand the dispersal dynamics of influenza viruses infecting pigs. For this purpose, we retrieved all publicly available European swine influenza A virus genomic sequences. Using this genomic dataset, we reconstructed how the virus has spread across Europe and investigated the external factors that may have driven the spread. Our results indicate that swine influenza A viruses frequently spread between European countries, in particular from north-western to southern and eastern Europe, and that the trade of pigs intended for breeding is strongly associated with these patterns of spread. Consequently, measures such as quarantining of breeding pigs, could help preventing swine influenza variants from spreading between and becoming established in new regions. We also find major gaps in available genomic data from several European countries. We therefore recommend stronger European surveillance programs that include monitoring influenza viruses in both animals and humans to improve early detection of new emerging viral variants.

Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms (SNPs) in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.

One major aftermath of COVID-19 pandemic is cardiovascular consequences. SARS-CoV-2 binds to ACE2 and downregulates vasodilation. Dengue favors hypotension by weakening endothelial glycocalyx leading to plasma leakage. C1q levels, immune complexes (ICs), and proteomic profiles in serum samples from 52 COVID-19 and 19 pre-pandemic Dengue cases were studied. Unlike Dengue, COVID-19 serums showed elevated coagulation proteins promoting vaso-occlusion and peripheral artery diseases. The stress-induced chaperone and atherosclerosis marker, GRP78 (gene/ protein) was found upregulated upon SARS-CoV-2 spike expression in cardiac/ lung cell lines. Elevated GRP78 levels were also observed in serum samples from COVID-19-diagnosed individuals and subjects with myocardial infarction (MI) in post COVID-era. Surprisingly, spike antibodies (Abs) showed cross-binding to GRP78 and possibly contributed to the observed higher-level ICs in COVID-19 serums (cardiovascular embolism?). Co-localization studies showed that spike Abs (analogous to pro-atherosclerotic GRP78 auto-Abs) could directly bind to upregulated cellular GRP78 (type II hypersensitivity?). Both pathways could worsen vascular injury and atherosclerosis, leading to cardiac complications in COVID-19 cases with narrowed vessels.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

Global efforts to prevent and mitigate the impacts of high pathogenicity avian influenza (HPAI) H5 on domestic animals, humans, and wildlife rely on timely and transparent information that is both accurate and interpretable across countries and sectors. International epidemiological and genomic databases, such as the World Animal Health Information System (WAHIS), the Global Animal Disease Information System (EMPRES-i+), the Global Initiative on Sharing All Influenza Data (GISAID), and the National Center for Technological Bioinformation Virus Portal (NCBI) provide essential information for surveillance, research, and decision-making. To evaluate how well these resources capture recent wildlife impacts, we consolidated information from these databases and complementary public sources including government reports, scientific literature, and news articles, on wildlife mortality associated with HPAI H5 in the Americas from November 2021 to July 2024. The consolidated dataset comprised 615,883 wild birds (287 spp.) and 63,409 wild mammals (39 spp.). In comparison, WAHIS represented 16,902 wild birds (261 spp.) and 6,323 wild mammals (31 spp.) while EMPRES-i+ captured a substantially smaller portion of affected host diversity for both wild birds (105 spp.) and wild mammals (27 spp.). Genomic databases (GISAID and NCBI) represented 7,027 whole genome equivalents of H5 viruses from wild birds (175 spp.) and 371 from wild mammals (26 spp.). These discrepancies indicate that international databases, while essential, provide an incomplete picture of HPAI impacts on wildlife, with significant geographic and taxonomic asymmetries attributable to differences in surveillance capacity, reporting practices, sequencing effort, and data-sharing pathways. Studies and management strategies relying on these resources without complementary validation may therefore mistake data gaps for real-world epidemiological patterns. Strengthening data reporting standards, improving validation procedures, and integrating international databases with national reports, scientific publications, and other sources will enhance the reliability of epidemiological analyses and support more effective One Health surveillance, risk assessment, and conservation action. Author summaryHigh pathogenicity avian influenza (HPAI) H5 viruses, often called bird flu viruses, can cause severe disease in birds and mammals, including humans. Because of their relevance for human health, livestock production, and wildlife conservation, international databases were established to share information on when and where these viruses are detected, which species are affected, and what virus strains are found. These databases are essential tools for governments, scientists, and conservation practitioners working to track outbreaks, understand how these viruses spread and evolve, and guide surveillance and response. In this study, we compiled and compared information on recent HPAI H5 events in wildlife in the Americas available in international databases with information from other public sources, including reports from governments, scientific literature, and news articles. We found important discrepancies in how countries and species affected were represented across sources. As a result, international databases might not fully capture the actual distribution or conservation impact of HPAI H5 on wildlife. Our findings also show why decision-makers and scientists should interpret database-derived patterns carefully. We provide recommendations to improve international databases to address these gaps and better inform One Health risk assessment and wildlife conservation actions.

In spite of well-established global immune landscape, SARS-CoV-2 is still able to further spread and continue causing infection waves. The current understanding about the reason behind is limited, and it is still difficult to predict the evolution or spreading tread of SARS-CoV-2. Therefore, it is necessary to investigate whether the establishment of population immunity has changed the virus evolution or spreading pattern. In this investigation, one overall analysis of the SARS-CoV-2 spreading in the past several years have been carried out through one thorough genomic epidemiology study, with Germany being chosen as one representative location in view of the systemic efforts for genomic surveillance. The growth advantage of a few predominant variants in its early spreading period has been evaluated through a logistic regression model. The results have revealed that the major circulating SARS-CoV-2 variants since 2023 are mainly derived from the Omicron BA.2 family. Since middle of 2024, most predominant variants were produced primarily through recombination, indicating that the evolution derived from recombination might be the major driving force for the continuous spread of SARS-CoV-2 despite the existence of population immunity. Furthermore, the lower growth advantage of recently emerged variants might possibly lead to a tread of reduction in the frequency of infection wave. The information revealed from this investigation suggests that although short-term spreading tread can be affected by specific virus feature as well as local immunity landscape, the long-term spreading tread is mainly decided by the genomic diversity of the viruses, and can be predicted through phylogenetic and genomic epidemiology investigation. The results have emphasized the importance of maintaining the efforts for genomic surveillance of SARS-CoV-2, which is essential from both medical and research perspectives.

SARS-CoV-2 continues to evolve from the Omicron serotype, with BA.2.86 sublineage JN.1 and descendants such as KP.2 predominating in 2025-26. By early 2026, the JN.1-derived NB.1.8.1 and XFG variants had largely replaced other variants globally, with a more recent emergence of the highly divergent BA.3.2 saltation variant. Elderly individuals continue to be at greatest risk of clinical complications from COVID-19, yet contemporary data on kinetics of immune potency and breadth following multiple vaccinations remain very limited in this group. We studied a cohort of forty-three healthy older adults (median age = 85 years, IQR 75-88, 40% female). Using both pseudotyped virus (PV) and surrogate virus neutralisation (SVNT) based assays, we demonstrate that JN.1 and KP.2 vaccinations six months apart elicit high potency neutralisation across all studied variants except BA.3.2.2, which escaped neutralisation almost completely in all individuals. Waning of neutralising activity in serum was observed to be modest in the [~]6 months between vaccine doses, suggesting sustained immunity following multiple vaccines. While absolute neutralisation titres remained highest against ancestral Wu-1 at all timepoints due to multiple historical exposures and accumulation, the recall responses revealed a shift in immunodominance. After the recent KP.2 vaccine dose, neutralisation against full-length Wu-1 spike was not boosted, whereas all tested JN.1 descendants and BA.3.2.2 showed significant boosts, indicating that immune imprinting against ancestral Wu-1 was partially overcome. Interestingly, RBD-specific neutralising responses experienced a boost following KP.2 vaccination, suggesting that RBD responses remain imprinted but that they constitute a small proportion in the overall Wu-1 neutralising response as immune imprinting is alleviated.

Background and AimsHepatitis E virus (HEV) infections are a growing threat to global public health. To obtain an in-depth understanding of HEV infections in untreated and ribavirin-treated rats, we characterized the early HEV viral kinetics using rat HEV (rHEV) as a surrogate model and using two routes of virus inoculation: intravenous (I.V.) or oral infection. Approach and ResultsWe frequently collected feces, serum, and tissue samples up to 60 days after infection in both infection models to characterize the rHEV viral kinetics. A ~2-week delay in quantifiable RNA levels in feces was observed in the oral versus the I.V. infection model. Early rHEV viral kinetics in feces were found to be multiphasic and showed good concordance with those in the various tissue compartments studied. Comparison of the viral kinetics in these samples also revealed that the liver may serve as the initial site of rHEV replication, followed by replication in the intestine and spleen. While a dosage of 60 mg/kg/day ribavirin was found optimal to maintain rHEV RNA levels at (nearly) undetectable in feces, levels were detectable in the liver and increased both in feces and liver after treatment discontinuation. ConclusionsWe found that the two rHEV infection models share similar multiphasic viral kinetics with the liver as the main site of viral replication. Additionally, the rHEV RNA load in feces could be used as a reliable proxy for that in the liver, spleen, and intestine. We also show that ribavirin at 60 mg/kg/day was partially effective in preventing viral rebound. These findings may aid in exploring the correlation between the infection phases and antiviral efficacy, ultimately guiding therapeutic decisions.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

BackgroundInfluenza A virus (IAV) is a major public health burden, causing seasonal epidemics and occasional pandemics. Its transmission from avian species to mammals and subsequent spread requires adaptive changes in the viral genome. Understanding these molecular adaptations is essential for pandemic preparedness, and machine learning offers a powerful approach to uncover the evolution and biology of IAV. ResultsOur calibrated WaveSeekerNet model accurately predicted the host source of 8 IAV segments (Macro F1-score: 0.9728), significantly improving the reliability of predicted probabilities, with calibration errors approaching zero. Interpretation showed that avian-adapted IAVs consistently activated G/C content, whereas mammalian-adapted IAVs generally activated A/T content. This distinction was confirmed by codon-level analysis, in which G/C-rich codons were rewarded for the avian hosts and A/T-rich codons for the mammalian hosts. We defined host-adaptive distance to quantify species barriers and proposed it as a risk-assessment metric. We hypothesized the Mammalian Adaptation Zone (MAZ), a zone where the virus is expected to adjust its host-adaptive distance to reach, thereby helping it establish persistent mammalian lineages. The analysis also revealed the Hard Distance of avian-origin viruses (e.g., H5Nx, H9N2), indicating they have not yet established persistent mammalian lineages. Finally, analysis of human H7N9 (2013, China) and non-human mammalian H5Nx (North America) viruses showed that WaveSeekerNet accurately identified key mammalian-adaptive mutations, including PB2-E627K and PB2-D701N. ConclusionsWaveSeekerNet elucidated IAV host-adaptation mechanisms in silico, providing insights into the underlying mechanisms of host adaptation and informing improved surveillance and intervention strategies.

Introduction: Synthetic oligopeptides provide a rapid and cost-efficient approach to developing antibodies and diagnostics for emerging viral variants. Methods: This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC). Binding affinity was computationally estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), while experimental validation was performed using enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical diagnostic accuracy testing was done using plasma samples from RT-PCR-confirmed COVID-19 patients and pre-COVID-19 controls. Results: S621 demonstrated nanomolar binding affinity (Kdapp = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). Diagnostic evaluation yielded a sensitivity of 89.92% and specificity of 27.79%, corresponding to an overall accuracy of 71.79%. Discussion: These findings demonstrate that a single synthetic peptide derived from a conserved spike subdomain can function as a high-affinity surrogate for full-length antigens, supporting its potential application in rapid peptide-based immunodiagnostics.

While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line-dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air-liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line-derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.

Shigella sonnei is a leading cause of bacterial dysentery and a high priority WHO pathogen because of the spread of multidrug resistant strains. Understanding microbiome-Shigella-host interactions during colonization of the gastrointestinal tract, and the development of vaccines have been hampered by the lack of small animal models of shigellosis. Here, we developed a murine model of intestinal colonization with S. sonnei. Pre-treatment of mice with antibiotics disturbed the intestinal microbiome and rendered mice susceptible to high level, gastrointestinal colonization with S. sonnei for over one week. Infection with S. sonnei CS14 harbouring a stable virulence plasmid induced an initial inflammatory response in wild type mice, with weight loss and elevated levels of fecal lipocalin 2; the S. sonnei Type III Secretion System was responsible for this inflammatory response. Expression of O-antigen and Group IV capsule by S. sonnei promoted sustained intestinal colonization, with infected mice developing mucosal and systemic antibody responses predominantly directed at these glycans. Finally, infection with S. sonnei induced a degree of protection against subsequent re-challenge. Overall, this murine model successfully mimics aspects of S. sonnei colonization and should be helpful in understanding how S. sonnei successfully survives within the gastrointestinal tract and competes with the microbiota as well as the evaluation of vaccine candidates.

The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a potential zoonotic threat, owing to its adaptation to the human angiotensin-converting enzyme 2 receptor, highlights the need for antiviral strategies to control emerging HKU5-CoVs. However, despite the importance of the main protease (Mpro) as a key antiviral target, structural and biochemical characterization of HKU5-CoV Mpro in the context of clinical inhibitors has remained limited. In this study, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro in its apo state and in complex with the clinical inhibitors nirmatrelvir and ensitrelvir. These structures served as a foundation for the characterization of HKU5-CoV-2 Mpro via modeling and molecular dynamics simulations. Biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 Mpro exhibited nearly identical kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 Mpro. Structural analysis revealed a highly conserved S1 subsite but distinct local environments in the S1', S2, and S4 substrate-binding sites relevant to inhibitor recognition. Despite these variations, nirmatrelvir and ensitrelvir showed potent inhibitory activity, with comparable double-digit nanomolar IC50 values across all three Mpro proteins. Integrated structural modeling and molecular dynamics simulations showed that HKU5-CoV-2 Mpro retains the ligand-induced active-site rearrangements observed in HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition. These findings provide a structural framework for understanding the susceptibility of emerging Merbecoviruses to clinical Mpro inhibitors and support the development of pan-Coronavirus antivirals. Author summaryAs coronaviruses continue to emerge from wildlife reservoirs, determining whether current clinical antivirals can inhibit divergent viral targets and how they engage these proteins is crucial. This study focuses on Pipistrellus bat coronavirus HKU5, particularly the newly identified lineage 2 (HKU5-CoV-2), which has recently attracted attention as a potential zoonotic coronavirus. We determined high-resolution crystal structures of the HKU5-CoV-1 main protease and used these structures to build and analyze models of the HKU5-CoV-2 protease. Our biochemical and structural analyses show that approved COVID-19 protease inhibitors, including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova), potently inhibit HKU5 Mpro and reveal conserved features of inhibitor recognition. These findings provide a structural foundation for designing coronavirus protease inhibitors with broader activity against emerging coronaviruses.

Foot-and-mouth disease (FMD) remains a major burden in endemic regions, where inactivated vaccines are constrained by cost, short duration of immunity, cold-chain dependence, and high-containment manufacturing. Here, we engineered a recombinant bovine papular stomatitis virus (rBPSV) expressing the FMDV A24 Cruzeiro capsid precursor P1-2A together with an attenuated 3C protease (3Cpro L127P). Infection of ovine fetal turbinate (OFTu) cells resulted in robust capsid protein expression and assembly of abundant 25-30 nm icosahedral virus-like particles (VLPs). Intramuscular immunization of two BPSV-seropositive calves with rBPSV (107 TCID50 on days 0, 21 and 35) induced strong anti-FMDV humoral responses. By day 28, liquid-phase blocking ELISA (LPBE) titers exceeded laboratory defined protective threshold ([&ge;]1.9 log10), and homologous neutralizing titers against A24 Cruzeiro surpassed the threshold associated with protection ([&ge;]1.36 log10). Intra-serotypic cross-neutralization was observed against A/Argentina/2001, whereas no neutralization was detected against heterologous serotype O1 Campos. Pre-existing anti-BPSV antibodies did not prevent induction of neutralizing responses. These findings establish first proof-of-concept that BPSV can serve as a cattle-adapted vector platform for delivery of FMDV VLPs and other heterologous antigens.

Gonorrhea is the second most common bacterial sexually transmitted infection and affects about 80 million people worldwide annually. The causative agent, Neisseria gonorrhoeae, has become resistant to almost every antibiotic used for its treatment. There is no licensed vaccine against gonorrhea. Therefore, there is an urgent need to develop novel prevention and treatment strategies to curb the spread of gonorrhea. The gonococcus has evolved several mechanisms to evade complement, a key arm of immune defenses against this pathogen, including binding of the human complement inhibitors Factor H (FH) and C4b-binding protein (C4BP). We previously showed that chimeric molecules fusing the gonococcal binding domains of FH and C4BP to IgG Fc and IgM Fc, respectively, mediate complement-dependent killing of gonococci in vitro and attenuate gonococcal colonization of mouse vaginas when administered topically. Here, we fused C4BP domains 1 and 2, which contain the gonococcal binding region, to IgG Fc bearing the IgM tail-piece to facilitate Fc hexamerization. This molecule, called C4BP-Hexa IgG Fc, showed [~]650-fold greater complement-dependent bactericidal activity on a molar basis than monomeric C4BP-IgG1 Fc. C4BP-Hexa IgG Fc enhanced association with and uptake by human neutrophils in a complement-independent manner. Despite off-target complement activation in solution, C4BP-Hexa IgG Fc reduced both the duration and the bacterial burden of gonococcal vaginal colonization in human FH and C4BP transgenic mice when administered intravaginally daily. In conclusion, we show proof-of-concept of the efficacy of a hexameric C4BP IgG Fc fusion molecule against N. gonorrhoeae, which could aid in the fight against this multidrug-resistant pathogen.