Bifunctional agents that integrate solvent alignment and chiral differentiation could streamline NMR-based structural and stereochemical analysis. Yet alignment media that simultaneously induce enantiodifferentiation remain rare, particularly in protic solvents and across chemically diverse molecules. In this study, we demonstrate that the amphiphilic oligopeptide (FK)4 self-assembles into β-sheet fibrils that serve as such a bifunctional medium. Upon assembly, (FK)4 weakly aligns the solvent environment while providing sufficient intermolecular interactions for robust enantiomeric differentiation. (FK)4 supports the enantiodifferentiation of diverse chiral compounds (amino acids, carbohydrates, organic acids, and pharmaceuticals) across multiple nuclei (1H, 13C, and 19F), yielding well-resolved resonance bifurcations for accurate enantiopurity quantification through integration. The bifunction of (FK)4 extends to water and protic organic solvents, allowing both residual chemical shift anisotropies (RCSAs) measurements and enantiodifferentiation in the same sample. This dual functionality enables a streamlined postsynthesis analysis workflow for simultaneous determination of enantiomeric composition and relative configuration in racemic reaction products. Integrating structural elucidation and chiral analysis, (FK)4 offers a perspective on extending anisotropic NMR techniques to stereochemical differentiation with the potential to facilitate absolute configuration elucidation.

Ebolaviruses, including EBOV, SUDV, and BDBV, cause severe hemorrhagic fever, yet currently licensed monoclonal antibody (mAb) therapies against EBOV lack cross-species efficacy. While mAbs offer high specificity, favourable safety profiles, and durable serum persistence, their susceptibility to viral escape highlights the need for broader, more resilient antibody strategies. Bispecific antibodies (bsAbs), which concurrently target non-overlapping epitopes, have the potential to enhance neutralization potency, expand strain coverage, and mitigate mutation-driven resistance. In this study, we engineered three bsAb formats-CrossMab®, DVD-IgG, and IgG-ScFv-directed against distinct ebolavirus epitopes and systematically characterized their antiviral activities. All bsAbs exhibited potent neutralizing activity and conferred substantial protection in mouse challenge models. Notably, the IgG-ScFv format demonstrated the greatest improvements in neutralization potency and in vivo efficacy. These findings provide a framework for rational bsAb design and underscore their promise as next-generation immunotherapeutics capable of broad and durable protection against diverse ebolaviruses.

BACKGROUND:Crimean-Congo hemorrhagic fever (CCHF) is a highly pathogenic tick-borne disease with mortality rates of 5% to 82%. Although cynomolgus macaques models exist for Hoti and Afghanistan strains, Chinese CCHFV isolates remain insufficiently defined. METHODS:We evaluated the pathogenicity of 2 Chinese CCHFV strains in cynomolgus macaques: BA04032 (tick-derived) and 75024 (non-lethal human clinical isolate). Animals were monitored for clinical parameters, viremia, hematologic and biochemical changes, viral RNA loads in blood, swabs, and tissues, and histopathological alterations. RESULTS:Infection with strain 75024 caused mild disease, whereas BA04032 produced more severe illness with fever, viremia, thrombocytopenia, leukopenia, and extensive viral replication in multiple organs. Despite belonging to the Asian lineage, the two viruses fall into different genotypes with distinct clinical outcomes in this model. CONCLUSIONS:Chinese isolates exhibit distinct disease phenotypes in cynomolgus macaques, underscoring the value of this model for pathogenesis studies and evaluation of medical countermeasures.

BackgroundSyphilis is a sexually transmitted disease (STD) caused by Treponema pallidum (TP) which could progress to neurosyphilis affecting the nervous system. However, the pathogenesis of neurosyphilis remain unknown. In addition, current diagnosis of neurosyphilis is mainly based on serological tests and cerebrospinal fluid (CSF) analysis, with limitations in terms of sensitivity and specificity.MethodsFrom April 2021 to April 2023, 129 patients with syphilis were enrolled and divided into the non-neurosyphilis group (common syphilis, CS, n=19), asymptomatic neurosyphilis group (AN, n=77), symptomatic neurosyphilis group (NS, n=33) and a control group (n=15). Forty-eight cytokines/chemokines in the CSF samples were measured and analysed in combination with clinical indices. Feature selections were further analysed for the using different machine learning model for the diagnosis and progression predicting of neurosyphilis.ResultsMachine learning-based feature selection identified a three-cytokine panel (RANTES, MIP-1β, and IL-3) that effectively distinguished syphilis patients from controls (AUC = 0.869, 95% CI: 0.821–0.917). For prediction of symptomatic neurosyphilis progression, IL-2Rα emerged as the optimal standalone biomarker, achieving an AUROC of 0.843 (95% CI: 0.784–0.902) for discriminating asymptomatic from symptomatic disease. Multivariable analysis confirmed IL-2Rα retained independent discriminatory power after adjusting for age (multivariable AUC = 0.876, 95% CI: 0.827–0.925). Age-stratified ROC analysis revealed IL-2Rα maintains robust diagnostic utility across all adult age groups, with optimal Youden-derived cut-offs of 21.82 U/mL (sensitivity 100.0%, specificity 85.7%, AUC 0.939) for 35–45 years; 25.48 U/mL (sensitivity 83.3%, specificity 88.9%, AUC 0.889) for 45–55 years; and 24.54 U/mL (sensitivity 73.7%, specificity 84.6%, AUC 0.789) for >55 years. The combination of IL-2Rα and IP-10 significantly improved predictive accuracy within specific age strata, achieving AUCs of 0.949 (35–45 years), 1.000 (45–55 years), and 0.814 (>55 years) when distinguishing symptomatic from asymptomatic neurosyphilis.ConclusionIn conclusion, IL-2Rα and IP-10 represent promising predictive biomarkers for neurosyphilis progression, with their combined application achieving high diagnostic accuracy for distinguishing symptomatic from asymptomatic disease. These findings establish a foundation for CSF cytokine-guided risk stratification in syphilis patients, although prospective validation in larger, age-matched cohorts is warranted to optimize clinical cut-offs and confirm generalizability across diverse populations.

Nipah virus (NiV) is a highly lethal zoonotic pathogen, causing disease that can spread between animals and people with a mortality rate about 40% to 70%. No commercially approved vaccines or therapeutics are available. In this study, novel NiV vaccines targeting the attachment glycoprotein (G) or fusion glycoprotein (F) of NiV were developed utilizing bacteriophage T4 as a carrier platform. The immune effects induced in mice by intranasal immunization (i.n.) and intramuscular injection (i.m.) show that i.n. immunization with bacteriophage T4 coupled with Nipah virus G or F protein can not only stimulate humoral immunity and cellular immunity, but also induce excellent mucosal immune responses. Complete protection against lethal NiV challenge was achieved in Syrian hamsters after intranasal immunization with either T4-G, T4-F, or the mixture of T4-G and T4-F (1:1). Due to its safety and modular versatility, the bacteriophage T4 carrier system presents a promising strategy for developing vaccines against NiV and other viruses.

BACKGROUND:The COVID-19 pandemic drastically altered human behaviors alongside meteorological and environmental conditions, yet how these shifts differentially impacted respiratory versus intestinal infectious diseases remains unclear. This study investigates the evolving relationships between environmental exposures and disease transmission across pre-pandemic, pandemic, and post-pandemic phases. METHODS:We analyzed surveillance data for notifiable respiratory and intestinal diseases in Sichuan Province (2006-2023), integrating meteorological and environmental pollution data via principal component analysis (PCA). A modeling framework combining PCA with generalized additive distributed lag non-linear models (PCA-GAM-DLNM) was applied to quantify the non-linear and lagged effects of composite environmental exposures on disease risks. RESULTS:A significant divergence emerged during the pandemic: respiratory diseases declined sharply due to non-pharmaceutical interventions (NPIs), whereas intestinal diseases (e.g., infectious diarrhea) remained stable or increased, correlating with worsened water pollution indicators despite improved air quality. We identified four distinct exposure-lag patterns, ranging from immediate effects (acute hemorrhagic conjunctivitis) to protection-risk reversal effects (mumps). Simulations indicated that using medium-to-high exposure levels as baselines yields more reliable early warnings than average levels. CONCLUSIONS:Respiratory and intestinal diseases exhibit differential environmental sensitivities. The persistence of intestinal diseases during the pandemic suggests that NPIs targeting airborne transmission are insufficient for waterborne risks. Future public health strategies require integrated management coordinating air and water quality control, with targeted interventions accounting for specific disease lag mechanisms and extreme weather risks.

Background:New clades and lineages emerged with the globally prevalent of Mpox virus (MPXV), accompanied by changing clinical symptoms, pathogenesis and transmission dynamics in associated with specific clades and lineages. Methods:Here, we developed a two tube multiplex real-time fluorescent quantitative PCR (mrt-qPCR) assay for simultaneous differentiation of MPXV clades Ia, Ib, II, and innovative binding lock nucleic acid (LNA) probes to detect A.1, B.1 and C.1 lineages within the clade IIb. Results:The assay demonstrated high sensitivity (33-69 copies/reaction) and specificity with expected linearity and stability. The intra-assay and intre-assay coefficients of variations (CV) were below the acceptable threshold of 5%, and the mrt-qPCR method has good stability and reproducibility. Clinical validation using 109 qPCR positive, 1 clade IIb B.1 virus strain and 15 negative specimens revealed 100% concordance for the differentiation of the three clade II and 97.60% for the differentiation the three lineages. The two tube multi-test system streamlined workflows, enabling efficient screening of diverse clinical samples (swabs from skin lessions, oropharynx and rectum, saliva and plasma). Conclusions:We have established a two-tube multiplex qPCR method for detecting different clades and lineages of the MPXV. This method addresses the issue of false-negative detection of MPXV clade Ib caused by gene fragment deletion, and has also enabled the development of a rapid detection approach for the predominantly circulating clade IIb (including lineages A.1, B.1, and C.1). This cost-effective assay provides an important tool for accurate diagnosis, typing and epidemiological surveillance of MPXV.

Filoviruses, including Ebola and Marburg viruses, present significant global health challenges due to their high mortality rates. Despite the availability of several Ebola virus vaccines, none provide cross-protection against multiple filovirus species. In this study, we developed recombinant live attenuated measles virus-based vaccine candidates designed to express Ebola or Marburg virus glycoproteins and generate virus-like particles for pan-filovirus immunization. The administration of these candidates by intraperitoneal injection into IFNAR-/- mice elicited robust antibody and cellular immune responses specific to Ebola and Marburg virus glycoproteins, with virus-like particles expressing candidates inducing the strongest immunity. Importantly, all vaccines containing Ebola virus glycoproteins afforded complete protection against mouse-adapted Ebola virus; meanwhile, those with Marburg glycoproteins provided protection against lethal replication competent vesicular stomatitis virus-Marburg virus challenges. These findings support the potential of measles virus-based vectors and virus-like particles as promising platforms for the development of vaccines targeting multiple filoviruses.

Nipah virus (NiV) infection is highly lethal in humans, and the development of vaccines that provide rapid protection is critical for addressing NiV outbreaks. In this study, we demonstrate that a single intranasal immunization with the chimpanzee adenoviral-vectored NiV vaccine, AdC68-F, induced robust and sustained cellular and humoral responses in BALB/c mice, and provided complete protection against challenge with the NiV-Malaysia strain (NiV-M) in Syrian hamsters. Notably, AdC68-F, administered at a dose of 5 × 109 viral particles, offered a complete prophylactic protection window as few as 7 days before exposure to a lethal NiV-M challenge. Furthermore, passive transfer of sera from AdC68-F or AdC68-G immunized animals conferred complete protection against NiV-M infection in naive hamsters. These findings underscore the pivotal role of antigen-specific immunity in controlling NiV infection and highlight the potential of single-dose intranasal AdC68-based NiV vaccines for rapid protection during outbreaks. By providing rapid and effective protection, these vaccines could help reduce human-to-human transmission and aid in curbing NiV outbreaks.

Nipah virus (NiV) is a zoonotic pathogen that causes severe encephalitis and respiratory disease in humans and multiple mammalian species. However, no licensed vaccines or therapeutics are currently available against NiV infection. In this study, we developed three mRNA vaccine candidates using a lipid nanoparticle (LNP) delivery platform: mRNA-F-LNP, comprising mRNA encoding the fusion protein (F); mRNA-G-LNP, containing mRNA encoding the attachment glycoprotein (G); and mRNA-GF-LNP, in which mRNAs encoding both F and G proteins were co-encapsulated at a 1:1 molar ratio. All three mRNA-LNPs induced robust and sustained immune responses in both mice and Syrian hamsters. Sera from immunized Syrian hamster showed high levels of cross-neutralizing antibodies against both NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B) strains. Notably, all three mRNA-LNPs conferred complete protection against a lethal challenge with NiV-M in Syrian hamsters. These findings demonstrate that these mRNA-based vaccines are highly immunogenic and efficacious, highlighting their potential as promising candidates for NiV vaccine development.

BackgroundThe rigorous non-pharmacological interventions (NPIs) during SARS-CoV-2 outbreak posed a deep impact on the etiology and epidemiology of acute respiratory tract infections (ARTIs). We aimed to elucidate the changing patterns during and post NPIs of SARS-CoV-2 in Shenzhen, China.MethodsA total of 4610 outpatients with ARTIs from the fever clinic of our hospital were enrolled between June 2022 and May 2024, and nasopharyngeal swabs were collected and tested for twenty-five common respiratory pathogens using well-established RT-qPCR. The two year’s period was further divided into three stages: Stage 1 with strict NPIs, Stage 2 with outbreak of SARS-CoV-2 and Stage 3 with regular epidemic of SARS-CoV-2. Demographic and clinical data were also collected and analyzed.ResultsOverall, 57.05% (2630/4610) of patients were positive for at least one of tested respiratory pathogens, with top five pathogens of IAV (17.09%), H.influenzae (13.97%), SARS-CoV-2 (10.11%), IBV (7.38%) and HAdV (5.66%). Except for SARS-CoV-2, IAV and H.influenzae dominated the three stages, while the other pathogens varied. Meanwhile, positivity rates of most viral pathogens have increased post NPIs. Moreover, HAdV and H.influenzae infections were more frequently found in males. and higher overall rates of viral and bacterial infections were found in both children and the elderly. Notably, the results indicate a higher positivity rate in summer and autumn, with the lowest rate observed in winter. The overall co-infection rate was 24.62%, and the most frequent co-infections were between IAV, SARS-CoV-2, HAdV and H.influenzae.ConclusionsIn conclusion, the etiology and epidemiological patterns of ARTIs during and post NPIs of SARS-CoV-2 in Shenzhen have changed overtime, and sex, age and seasonal patterns were also found. The findings could provide useful information for the public health measures and the clinical management of respiratory infections.

Zoonotic H7N9 avian influenza virus infection remains a global concern because of its pandemic potential. Therefore, developing effective antibodies and vaccines against H7N9 is vital for preventing and controlling major outbreaks. Here, we isolated a human VH3-30 gene-encoded antibody, designated 6Y13, from a survivor of H7N9 infection. This antibody recognized the hemagglutinins (HAs) of the representative H7 subtype zoonotic viruses spanning two decades of antigenic evolution and potently neutralized epidemic H7N9 viruses in vitro. Moreover, 6Y13 conferred complete protection in mice against lethal H7N9 challenge in both prophylactic and therapeutic experiments. Structural analysis by cryoelectron microscopy indicated that 6Y13 binds to a unique conserved site on the HA head, distinct from the receptor-binding site and lateral patch. Nevertheless, 6Y13 efficiently blocked viral receptor binding without interfering with HA receptor binding, independent of Fc-mediated steric hindrance. Our findings provide a promising therapeutic candidate against pan-H7 subtype viruses and are beneficial for the design of H7 subtype influenza vaccine immunogens.

The ongoing outbreak and emergence of monkeypox virus (MPXV) clade Ib and IIb lineage have heightened its transmissibility, morbidity, and mortality, underscoring the urgent need for safe and effective vaccines against MPXV infection. In this study, we developed a "two-in-one" nanoparticle vaccine, DAM-NP, which incorporates bivalently fused M1 and A35 antigens conjugated to a self-assembled 24-meric ferritin nanoparticle. Preclinical evaluations demonstrated that DAM-NP elicits robust humoral and cellular immune responses, providing complete protection against lethal vaccinia virus challenge. Importantly, pre-existing antibodies resulting from prior infection with the VACV Tiantan strain utilized for smallpox prevention did not diminish vaccine efficacy. A single dose was sufficient to prevent both morbidity and mortality following a lethal MPXV challenge. These findings establish DAM-NP as a highly immunogenic and promising single-dose vaccine candidate for prevention of MPXV and other orthopoxvirus infections.

The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.

Metabolite analysis is essential for understanding the biochemical processes and pathways that sustain life, providing insights into the complex interactions within cellular systems and clinical examinations. This review explores recent applications of nuclear magnetic resonance (NMR) spectroscopy in metabolite studies. Various methods enhancing analytical accuracy for metabolome profiling and metabolic pathway studies, including spectral simplification techniques, quantitative NMR, high-resolution MAS NMR, and isotopic labeling, are discussed. The application of NMR in in situ and in vivo studies is also covered, highlighting in-cell NMR and in vivo MRS techniques. Last but not least, we discuss recent advancements in NMR hyperpolarization, with a focus on dynamic nuclear polarization (DNP), chemically induced dynamic nuclear polarization (CIDNP), para-hydrogen-induced polarization (PHIP), and signal amplification by reversible exchange (SABRE). These advancements offer significant potential for enhancing the sensitivity and accuracy of metabolite studies and are expected to further deepen the study and understanding of metabolites and metabolic pathways.

Background/Objectives: COVID-19, caused by SARS-CoV-2, has emerged as a global pandemic since its outbreak in 2019. As an increasing number of variants have emerged, especially concerning variants such as Omicron BA.1, BA.2, XBB.1, EG.5, which can escape the immune system and cause repeated infections, they have exerted significant pressure on monoclonal antibodies and the treatment approaches for COVID-19. Broad spectrum antiviral medication was urgently needed. In this study, we developed several bispecific antibodies based on the IgG-scFv format and one trispecific antibody containing Fab fragments with different anti-virus mechanisms studied previously. The Fab fragments are from h11B11, S2P6, and S309 respectively. Method: all recombinant antibodies were expressed by HEK 293. The pseudoviruses’ neutralization assay and the virus challenge to BALB/c mice were deployed to assess the efficiency of recombinant antibodies in vitro and in vivo. Results: the bispecific antibodies exhibited a favorable pseudoviruses neutralization activity, with IC50 values ranging from 8 to 591 ng/mL. The trispecific antibody performed even better, with IC50 values ranging from 5 to 27 ng/mL. Furthermore, the virus challenge to mice confirmed that the bispecific antibodies, including the trispecific antibody, had decent therapeutic efficacy. Conclusions: our study provided several supplements to the therapeutic measures of COVID-19 based on multispecific antibodies, supporting the great potential of the multispecific antibodies strategy in dealing with emerging pathogens.

Recently, the monkeypox virus (MPXV) outbreak was once again declared by the World Health Organization as a global health emergency, and currently, there is no specific drug against MPXV. During the replication cycle, MPXV produces two distinct forms of viral particles: extracellular enveloped virus (EEV), released via exocytosis, and intracellular mature virus (IMV), expelled through host cell lysis. A29 and B6 proteins are membrane proteins found on the IMV and EEV viral particles, respectively. This study designed two different bispecific antibodies (bsAbs) targeting specific antigens of the MPXV: the developed bsAb 9F8-3A1 targets two non-competitive binding epitopes on the MPXV protein A29, while bsAb 9F8-7C9 targets different antigen-binding epitopes on both A29 and B6. The in vitro and in vivo characterization assays demonstrated that the bsAbs provided complete protection against three poxvirus strains: vaccinia virus (VACV) Tiantan, VACV Western Reserve (VACV WR), and MPXV, surpassing the efficacy of all the parental monoclonal antibodies. Notably, the bsAb 9F8-7C9 exhibited the most effective antiviral activity. In vivo pharmacokinetic experiments showed that these two bsAbs have long half-lives in rhesus macaques. In conclusion, this study successfully developed two bispecific antibodies that target different epitopes, providing crucial insights for the development of decent antiviral drugs against MPXV and other orthopoxviruses.IMPORTANCEMpox is a viral zoonotic disease caused by MPXV infection. Since 2022, cases of mpox have been reported in non-endemic countries. The number of infections and deaths continues to rise, posing a serious threat to global health and safety. Currently, there are no specific treatments for mpox, making the development of effective therapeutic options urgent. In recent years, antibody-based drugs have been extensively studied for the treatment of various significant human viruses. However, there is a lack of research on therapeutic monoclonal antibodies for mpox, particularly in the development and application of bsAbs. In this context, we have designed effective bsAbs that demonstrate high antiviral activity both in vitro and in vivo. This research provides a theoretical foundation for the development of specific therapeutic agents for mpox and offers new approaches for clinical treatment, which is crucial for controlling the current outbreak.

Understanding of infection dynamics is important for public health measures against monkeypox virus (MPXV) infection. Herein, samples from multiple body sites and environmental fomites of 77 acute MPXV infections (HIV co-infection: N = 42) were collected every two to three days and used for detection of MPXV DNA, surface protein specific antibodies and neutralizing titers. Skin lesions show 100% positivity rate of MPXV DNA, followed by rectum (88.16%), saliva (83.78%) and oropharynx (78.95%). Positivity rate of oropharynx decreases rapidly after 7 days post symptom onset (d.p.o), while the rectum and saliva maintain a positivity rate similar to skin lesions. Viral dynamics are similar among skin lesions, saliva and oropharynx, with a peak at about 6 d.p.o. In contrast, viral levels in the rectum peak at the beginning of symptom onset and decrease rapidly thereafter. 52.66% of environmental fomite swabs are positive for MPXV DNA, with highest positivity rate (69.89%) from air-conditioning air outlets. High seropositivity against A29L (100%) and H3L (94.74%) are detected, while a correlation between IgG endpoint titers and neutralizing titers is only found for A29L. Most indexes are similar between HIV and Non-HIV participants, while HIV and rectitis are associated with higher viral loads in rectum. Here the authors measure viral load in samples from skin lesions, saliva, oropharynx, and rectum of 77 patients with acute monkeypox virus infection as well as from environmental fomite swabs and show a high seropositivity rate for antibodies against A29L and H3L.