Yellow fever virus (YFV) is an arbovirus causing substantial human morbidity and mortality. The live-attenuated 17D strain serves as vaccine and is one of the most successful vaccines so far. Receptor usage between attenuated and pathogenic YFV strains remains unclear. Here we performed a barcoded, genome-wide human open-reading frame library screen and identified LRP8 (also named APOER2) as a receptor for YFV. We show that LRP8 expression increases YFV infection (17D and two clinical strains, BJ01 and Asibi) in cell lines by promoting entry. Adeno-associated virus-mediated expression of human LRP8 in mouse liver aggravates infection and pathology of the clinical strain BJ01. LRP8 knockdown decreases YFV infection in brain cells, primary human hepatocytes and mosquitoes. LRP8 directly interacts with YFV 17D particles via the viral envelope protein. A soluble LRP8 decoy protein can block YFV 17D and BJ01 infection. Our findings provide insights for understanding YFV entry, tropism and pathogenesis.

ABSTRACT A non-replicating Tiantan strain-based vaccinia virus (NTV) holds significant application prospects for vaccination and gene therapy and has recently entered clinical trials as a novel and safer vaccine candidate against monkeypox. However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-ΔF1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-ΔF1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (10 3 PFU) or a single dose (10⁵ PFU) of NTV-ΔF1L-C7L, comparable to that conferred by VTT. These findings demonstrate that NTV-ΔF1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak. IMPORTANCE A highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.

In early 2025, the first cluster of imported clade Ib mpox cases in China was reported. Here we reported the laboratory confirmation of these mpox cases and the isolation of replication-competent MPXV from environmental samples. Seven clinical specimens and six environmental samples (from trimmers, shoe inserts, cloth, bedsheets, carpets, pillows, and bedside tables) were collected for molecular and serological detection. These mpox cases were confirmed to be MPXV clade Ib-positive based on qPCR and MPXV-specific seroconversion. Furthermore, clinical and environmental swabs were selected for virus isolation using Vero cells. Replication-competent viruses were isolated from two mpox clinical samples and an environmental sample (designated as MPXV-Ib-CCDC-Tan-TJ04). MPXV clade Ib strains exhibited smaller plaques than those of clade IIb strains. Neutralization assay indicated a strong correlation between clades Ib and IIb. This study highlights the importance of sustained surveillance in laboratory investigations by combining molecular detection with serological testing. The isolation of infectious MPXV clade Ib from environmental samples emphasizes the importance of environmental monitoring, disinfection practices, and health education for the prevention and control of mpox outbreaks, which could provide critical scientific evidence for formulating public health interventions.

Climate change and human activities have increased the risk of virus spillover from wildlife, posing a threat to human health. A human ocular disease called persistent ocular hypertensive viral anterior uveitis (POH-VAU) has recently emerged; however, the cause is unclear. Here we report that POH-VAU is associated with covert mortality nodavirus (CMNV) of aquatic origin. CMNV is prevalent in farmed and wild aquatic animals worldwide. We confirmed CMNV infection in ocular tissues and seroconversion in 70 patients with POH-VAU. An exposure survey and analysis of logistic regression models revealed that CMNV exposure frequency, number of severe exposures and exposure severity were associated with an increased risk of POH-VAU. Epidemiological data indicate that frequent unprotected processing of aquatic animals and consumption of raw aquatic animals were commonly reported exposure events, collectively accounting for 71.4% of investigated cases. Challenge tests revealed that CMNV can cause elevated intraocular pressure and pathological damage to ocular tissues in mice and can infect mammalian cells in vitro. This study reveals that an aquatic animal virus is associated with an emerging human disease.

Summary Gene transcription within the viral core is a unique feature of poxvirus replication. Following entry into the cytoplasm, the poxvirus core dissociates from the lateral bodies and undergoes expansion, functioning as a compartment for early transcription. However, the mechanisms governing molecular exchange between the viral core and the host cytoplasm remain poorly understood. Here, we determine the structures of the portal complex and its pore on the poxvirus core at 7.1 Å and 4.9 Å resolution, respectively, using cryo-electron tomography and sub-tomogram averaging. The pore is assembled from three viral proteins, E8, E6, and L3, for which we constructed an atomic model. Structural and channel analyses reveal that the pore satisfies the geometric and electrostatic requirements for the transport of RNA and smaller negatively charged molecules, while excluding double-stranded DNA and cytosolic DNA sensors. Together, our findings establish a structural framework for understanding the assembly and function of the poxvirus portal complex and identify potential targets for antiviral intervention.

The development of therapeutic vaccines against human papillomavirus (HPV)–associated malignancies remains challenging due to the immunosuppressive tumour microenvironment and the limited efficacy of existing delivery platforms. In this study, we designed and systematically compared two mRNA vaccine strategies based on a core–shell structured lipopolyplex (LPP) delivery system: a codon- and untranslated region-optimised non-replicating mRNA (nr-mRNA) and a self-amplifying mRNA (sa-mRNA). In the TC-1 murine model of HPV-driven cancer, both vaccine formulations effectively activated systemic antitumour immune responses, significantly enhancing the infiltration of functional CD8⁺ T cells and natural killer cells into tumours and promoting the repolarisation of tumour-associated macrophages towards an M1 phenotype. Notably, the sa-mRNA–LPP platform achieved comparable therapeutic efficacy at only one-fifth the dose of nr-mRNA–LPP, highlighting its superior potency and dose-sparing potential. Further analysis revealed that immune response induced by sa-mRNA–LPP was predominantly localised to the tumour site, with no significant immune activation detected in peripheral lymphoid organs such as lymph nodes and spleen, suggesting a stronger capacity for localised immunomodulation within the tumour. In summary, this study validates the high efficiency of the LPP platform in delivering different mRNA vaccines and elucidates the unique mechanism by which sa-mRNA exerts potent antitumour effects at low doses through localised immune remodelling. These findings provide important experimental support for the clinical translation of LPP-based mRNA vaccines targeting HPV-associated cancers.

Human adenovirus type C (HAdV-C) causes upper respiratory infections in children and may lead to severe pneumonia. During the implementation and subsequent relaxation of non-pharmaceutical interventions, HAdV-C emerged as a transiently dominant circulating strain in Beijing. However, the fine-scale genomic architecture and the evolutionary trajectories governing its recombination remains insufficiently characterized. Between March 2023 and August 2024, respiratory samples from Beijing patients were collected and screened by quantitative PCR. In conjunction with high-throughput whole-genome sequencing, five complete HAdV-C genomes were characterized, predominantly identified as genotypes C1 and C108, maintaining over 98% intra-typic sequence identity. Phylogenetic analysis based on whole genomic sequences revealed at least five evolutionary branches within C108. Phylogenetic reconstruction revealed a complex diversification within C108, delineating at least five distinct evolutionary clades. Specifically, the four C108 strains partitioned into two divergent sub-lineages, exhibiting close phylogenetic affinities with sequences from China and the United States. Furthermore, recombination analysis identified six discrete recombination patterns. Selection pressure analysis further demonstrated heterogenous evolutionary constraints across the genome; notably, immune-relevant early genes such as E1a_26KD exhibited elevated dN/dS ratios, harbouring multiple positive selection sites. These adaptive mutations were distributed across 23 of 33 annotated genes (69.7%), suggesting extensive diversifying selection. These findings elucidate that HAdV-C evolution is synergistically driven by frequent recombination events and potent selective pressures. This study provides critical evidence for the spatiotemporal dynamics and genomic surveillance of the emerging HAdV-C variants.

Metagenomic next-generation sequencing (mNGS) emerging as a standout in the clinical setting. In this study, we harnessed the power of mNGS to explore the pathogenic spectrum and temporal variations in respiratory tract specimens collected from adult patients with severe pneumonia who were admitted to the Intensive Care Units (ICUs) of two hospitals in Guangxi, China. From December 2021 to July 2022, 44 respiratory tract samples (including sputum and bronchoalveolar lavage fluid) from 25 adult patients (comprising 18 males and 7 females) diagnosed with severe pneumonia and admitted to the ICUs of two hospitals in Guangxi. A customized mNGS detection protocol was developed and applied for analyzing the composition and temporal variations of pathogens within the respiratory tract samples. Among these patients, the bacteria, fungi, and viruses were markedly higher detected by mNGS compared to conventional microbial culture methods (P < 0.001). The most prevalent bacteria detected were Stenotrophomonas maltophilia (61.36

Human sapovirus (HuSaV) has long occupied a peripheral position in etiological research on acute gastroenteritis. However, in the context of widespread rotavirus vaccination, the pathogen spectrum of childhood diarrhoea has been reshaped; molecular diagnostics have continued to evolve; and evidence from birth cohorts and environmental surveillance has accumulated. Together, these developments have prompted a systematic reassessment of the public health significance of HuSaV in childhood diarrhoea, outbreaks in childcare facilities and schools, asymptomatic infection, and persistent infection in immunocompromised hosts. Here, we review key advances in HuSaV research across disease burden and epidemiological patterns, genome organization and evolution, natural history and immune responses, experimental models and host interactions, complex clinical phenotypes, and surveillance-to-public-health translation. We further propose priority directions for the next 5 years, including the establishment of standardized antigen panels and reference strain repositories, integration of serological and genotyping data within longitudinal cohorts, elucidation of viral receptor recognition, cell entry and immune-evasion mechanisms, and the development of closed-loop linkages between experimental-model research and population-level surveillance systems. Overall, HuSaV has evolved from a historically underestimated pathogen associated with acute gastroenteritis in infants and young children into a key enteric viral pathogen that requires dedicated surveillance, mechanistic investigation and translational intervention strategies in the post-rotavirus vaccine era. The research paradigm is likewise shifting from descriptive epidemiology towards an integrated framework that combines disease burden, host immunity, experimental systems and transmission surveillance.

Langya virus (LayV), classified in the species Parahenipavirus langyaense, is a newly identified zoonotic paramyxovirus discovered in febrile patients in eastern China in recent years. The discovery of LayV has expanded the field beyond the prevailing paradigm of highly pathogenic, bat-borne henipaviruses represented by Nipah virus (NiV) and Hendra virus (HeV), toward a parahenipavirus spillover model shaped by shrews, rodents, and agricultural ecological interfaces. Reported cases have mainly presented with fever, fatigue, cough, and vomiting, and may be accompanied by leukopenia, thrombocytopenia, and abnormalities in hepatic and renal function. However, owing to the limited number of cases, restricted surveillance coverage, incomplete follow-up data, and the lack of standardized serological tools, the true burden of infection, full spectrum of natural hosts, role of intermediate hosts, and animal-to-human transmission pathways remain unresolved. Sustained human-to-human transmission has not been confirmed. In recent years, accumulating evidence from viromic analyses of shrew lung tissues along the eastern coast of China, positive detection in the Ussuri white-toothed shrew (Crocidura lasiura) in South Korea, characterization of the LayV genome architecture, P-gene RNA editing, ELISA/multiplex RT-qPCR/CRISPR-Cas12a diagnostic platforms, and structure-based F/G antigen engineering and antibody development has begun to move LayV research from pathogen discovery toward mechanistic dissection and the construction of countermeasure platforms. In this Review, we systematically summarize the discovery of LayV, clinical features and epidemiological evidence, taxonomy and molecular evolution, animal host ecology, noncanonical receptor-mediated entry mechanisms, diagnostic approaches, and immunological intervention strategies. We further propose key research priorities centered on One Health surveillance, identification of the unknown receptor, structure-guided F/G antigen design, and integrated sampling across animal, environmental, and human populations.

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in infants, young children, and elderly people worldwide. Developing a safe and broadly effective vaccine remains a critical and unmet public health need. This study comparatively evaluated the immunogenicity and protective efficacy of respiratory syncytial virus (RSV) mRNA vaccines expressing a modified G protein extracellular domain (Gecto), delivered via either lipid nanoparticle (LNP) or lipid-polymer hybrid (LPP) platforms. Both RSV Gecto-mRNA elicited robust humoral and cellular immunity in mice. Although the humoral immunity in both groups showed a Th2 bias, the advantage of the LNP platform lies in its ability to simultaneously trigger potent humoral and Th1-type cellular immunity. Comparing the two groups, The LNP group elicited a stronger Th1-biased cellular response, manifested by high expression of cytokines such as IL-2 and TNF-α in CD4+T cells, and the production of higher neutralizing antibody titers against A/B subtypes of RSV strains than the LPP group,which elicited slightly higher Th2 bias IgG antibody titers. Viral challenge confirmed that both groups provided effective cross-protection against RSV A2 and B9320, significantly reducing lung viral load and histopathology, with comparable protective efficacy.These findings demonstrate that both platforms enabled robust delivery of the RSV Gecto-mRNA vaccine and generated cross-protective immunity, albeit with divergent response characteristics. These results inform the selection of RSV vaccine delivery platforms aimed at distinct immune correlates of protection.

The persistent emergence of SARS-CoV-2 variants continues to compromise current vaccine efficacy, driving the development of broad-spectrum coronavirus vaccines to address variant evasion and future outbreaks. To develop a pan-coronavirus vaccine, we identified some conserved T/B epitopes across spike proteins of human-infecting coronaviruses, focusing on two conserved long peptides, VV and VS, which demonstrated broad immunogenicity in PBMCs from COVID-19 convalescent patients. By structurally fusing the VV and VS long peptides with heptad repeat 1/2 (HR1/2) domains from the S2 subunit, we engineered a trimeric immunogen HR1-VV-HR2-VS. This design induced superior cellular and humoral immune responses compared to individual peptide components in immunized mice. The vaccine also significantly reduced viral loads and attenuated lung pathology in mice challenged with HCoV-229E, SARS-CoV-2 prototype strain, and the KP.2 variant, demonstrating cross-protective immunity. Therefore, these results indicated that HR1-VV-HR2-VS vaccine elicits cross-protective immunity, highlighting its potential as a universal coronavirus vaccine. In addition, we developed an innovative peptide vaccine platform based on the HR1-HR2 trimeric structural protein, which serves as a potent polypeptide fusion scaffold to significantly enhance peptide immunogenicity.

In 2022, a global outbreak of mpox was anticipated, with several cases reported in non-endemic countries in early May. Given the challenge of distinguishing the mpox virus (MPXV) from other pathogens based solely on symptoms, there is an urgent need for prompt and reliable MPXV detection methods. In this study, we developed assays using recombinase-aided amplification (RAA) to identify MPXV and evaluated their applicability with clinical samples. The assays were designed to detect the N4R gene of MPXV. All assays demonstrated detection limits of 1 copy/µL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus, confirming their high specificity. Our established assay provides results in less than 50 min. Furthermore, we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings, especially in areas with limited laboratory resources. Overall, three RAA-based nucleic acid assays for MPXV were established, providing a powerful tool for efficient, rapid, and specific detection of MPXV infection.

Yellow fever virus (YFV), an arbovirus causing substantial human morbidity and mortality, was the first human virus discovered over a century ago. The live-attenuated 17D vaccine is among the most successful vaccines in medicine. Despite the importance of YFV, its receptor has remained unknown. Here, we performed a barcoded, genome-wide human ORF library screen and identified LRP8 (also named APOER2) as a receptor for YFV. We show that LRP8 expression specifically boosts YFV infection in cell lines by promoting entry. AAV-mediated expression of human LRP8 in mouse liver aggravates infection and pathology. LRP8 knockdown abolishes YFV infection in brain cells, primary human hepatocytes, and notably in mosquitoes. Biochemically, LRP8 directly interacts with YFV particles via the viral envelope protein. This function of LRP8 is conserved across species, particularly in mosquitoes and primates. A soluble LRP8 decoy protein can block YFV infection in vitro and in mice, providing a potential therapeutic or prophylactic strategy. Our findings provide groundwork for understanding YFV entry, tropism, and pathogenesis, and may enable development of novel therapeutics to treat YFV infection. ### Competing Interest Statement The authors have declared no competing interest.

Mpox poses a heightened risk of severe disease and mortality among individuals with HIV, yet the molecular mechanisms and immunopathology underlying multi-organ damage caused by the mpox virus (MPXV), particularly in the context of HIV co-infection, remain poorly understood. Here, we observe increased MPXV replication, more extensive skin lesions, and impaired humoral and cellular immune responses in SIV-MPXV co-infected rhesus macaques compared to those infected with MPXV alone. Multi-organ proteomic and phosphoproteomic analyses reveals upregulation of proteins involved in immune and inflammatory pathways in skin lesions and across multiple organs, especially in immune-related tissues. Abnormal activation of DNA replication and cell cycle signaling pathways, which may contribute to enhanced viral replication, is evident in both MPXV and SIV-MPXV co-infected groups. CDK4/6 may present a potential therapeutic target to suppress MPXV replication. These comprehensive proteomic datasets offer valuable insights into the pathogenesis of MPXV in the context of SIV co-infection and support ongoing efforts to mitigate the impact of mpox.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and children. mRNA vaccines based on the lipopolyplex (LPP) platform have been previously reported, but they remain unapplied in RSV vaccine development. In this study, we developed a novel LPP-delivered mRNA vaccine that expresses the respiratory syncytial virus prefusion protein (RSV pre-F) to evaluate its immunogenicity and protective effect in a mouse model. We synthesized mRNAs with gene modification for RSV pre-F and prepared mRNA vaccines using the LPP delivery platform, referred to as RSV pre-F LPP-mRNA. RSV pre-F protein expression in mRNA vaccines was characterized in vitro. Then, we evaluated the effects of the immune response and protection of this mRNA vaccine in mice up to 24 weeks post-vaccination. Following booster immunization, robust and long-lasting RSV pre-F-specific IgG antibodies were detected in the serum of mice, which exhibited Th1/Th2 balanced IgG response and cross-neutralizing antibodies against different subtypes (RSV A2, B18537, and clinical isolate hRSV/C-Tan/BJ 202301), with a clear dose–response relationship observed. RSV pre-F-specific IgG antibodies were maintained in the mice for an extended period, lasting up to 18 weeks post-immunization. Concurrently, multifunctional RSV F-specific CD8+ T cells (IFN–γ, IL-2, and TNF-α) were detected in the mice. After RSV A2 challenge, the RSV pre-F LPP-mRNA vaccine led to a significant reduction in viral replication, while reduced pathological damage was observed in lung tissue. The LPP-delivered mRNA vaccine expressing RSV pre-F induces a robust and long-lasting immune response and protection, indicating good prospects for further development and application.

Human alphaherpesvirus 2 (HSV-2) is the main pathogen resulting human genital herpes, which poses a major threat to the socio-economic development, while there is no effective vaccine. In this study, we developed a novel lipopolyplex (LPP)-delivered mRNA vaccine expressing the HSV-2 envelope glycoprotein gD and evaluated its immunogenicity in mice. The mRNA vaccine was prepared from the genetically modified gD mRNA synthesized in vitro combined with the LPP delivery platform and it was named gD-ORI mRNA. The expression of gD antigen in the mRNA vaccine was validated in vitro by Western blotting and indirect immunofluorescence assay, then the immune responses induced by this mRNA vaccine in mice were evaluated. The immunization with gD mRNA alone induced strong humoral and cellular immune responses in mice. Robust and long-lasting gD-specific IgG antibodies were detected in the mouse serum after booster immunization with gD-ORI mRNA. The immunized mice exhibited a Th1/Th2 balanced IgG response and robust neutralizing antibodies against HSV-2, and a clear dose-response relationship was observed. The gD-specific IgG antibodies were maintained in mice for a long time, up to 18 weeks post-booster immunization. At the same time, multifunctional gD-specific CD4+ and CD8+ T cells in vaccinated mice were detected by intracellular cytokine staining (ICS). This novel gD-expressing mRNA vaccine delivered by LPP induces strong and long-lasting immune responses in mice post booster immunization and has a promising prospect for development and application. This study provides scientific evidence and reference for the development of a new mRNA vaccine for HSV-2.