Glycosylation plays a pivotal role in modulating the structure and immunogenicity of viral antigens. Three glycosylation sites on the receptor-binding domain (RBD) of SARS-CoV-2, including N331, N343 (N-linked), and T323 (O-linked), are highly conserved and remain unchanged across multiple variant strains. To investigate their functional relevance, a series of site-directed glycosylation-deficient mutants were generated on the basis of the RBD-dimer antigen of the ZF2001 vaccine (developed by ZFSW Biologics), including N-N331-NA, N-N343-NA, O-T323-NA, and an RBD-NA control. Comparative analyses of glycosylation-deficient RBD variants revealed a site-specific hierarchy in immune modulation. In particular, disruption of the N343 site led to a pronounced reduction in antigen-specific antibody titers and T helper cell-related cytokine responses in immunized mice, whereas mutations at N331 and T323 had more limited effects. Furthermore, glycan profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS), secondary structure assessment using microfluidic modulation spectroscopy (MMS), and molecular dynamics simulations revealed that the N343 glycan contributes to local structural stability and preserves key antigenic features of the RBD. Together, these results identify N343 as a critical glycosylation hotspot that governs RBD immunogenicity and antigenicity, providing a mechanistic foundation for the structure-based optimization of SARS-CoV-2 vaccines targeting glycan-regulated epitopes.

Respiratory syncytial virus (RSV) causes severe infant morbidity, yet the mechanisms underlying their suboptimal immunity and the failure of prefusion F-stabilized adult vaccines in infants remain unclear. Here we integrate deep mutational scanning of 761 preF-binding antibodies, repertoire profiling of 102 repeatedly exposed pediatricians and 61 RSV-experienced toddlers, structural analysis, and >40,000 viral genomes to decode RSV F immunity. We resolved 12 immunologically distinct functional antibody subclasses and uncovered a hierarchical activation threshold: apical epitope-targeting antibodies with superior neutralizing potency demand extensive somatic hypermutation and cooperative CDR networks, whereas central-to-basal epitope-directed, marginal or non-neutralizing antibodies engage germline-encoded antibodies through minimal mutations (e.g., S31G). Toddler repertoires are confined to low-threshold, non-productive sites; adult repertoires enrich for apical elite neutralizers whose immune pressure drives contemporary RSV-B evolution. These reframe pediatric RSV vulnerability as a threshold-gated repertoire deficit and prescribe vaccine strategies that actively redirect immunodominance from permissive epitopes toward high-barrier apical targets.

Respiratory syncytial virus (RSV) prefusion F (preF) vaccines have transformed adult prophylaxis, yet unmet needs in antigen stability, pediatric safety, and mucosal protection persist. Here, we develop an integrated structure-guided RSV vaccine design platform that couples allosteric stabilization, epitope-focused immunogen engineering, and route-specific mRNA delivery for systemic and mucosal immune activations. By mapping prefusion F "breathing" motions and applying a ThermoNet- and Rosetta-guided screening funnel, we identified R296, a stabilized prefusion F immunogen that reinforces the α1-α5 hinge and interprotomer interfaces while preserving key neutralizing epitopes. Cryo-EM confirmed that R296 retains a native-like prefusion architecture. And mRNA-LNP vaccination elicited potent, durable, and broadly protective neutralizing responses in mice, rats, and cotton rats, with clearance of detectable infectious virus and no evidence of Th2-skewed enhanced respiratory disease. To address pediatric safety, we designed a stalkless nanoparticle immunogen, Head38-50AB-3, which enriches high-potency apical epitopes while excluding stalk regions associated with low-potency or non-protective responses, conferring protection without VAERD-like pathology. Finally, we engineered an intranasal-delivered LNP that enables intranasal R296 mRNA delivery, inducing systemic neutralization together with robust nasal and bronchoalveolar secretory IgA (sIgA). R296 has now advanced to Phase 1 clinical trials. These results establish a modular framework for next-generation RSV vaccines.

Antibodies have emerged as central components of therapeutic strategies against viral infectious diseases, functioning as key effectors in both prevention and treatment. While traditional antibody discovery has relied heavily on high-throughput screening, the field is now shifting toward rational antibody design, which requires integrative insights into sequence-structure-function relationships. However, existing resources provide a valuable foundation but remain limited in scope, highlighting the need for a standardized and well-annotated antibody database that integrates multidimensional features to further support systematic exploration, cross-pathogen comparison, and rational antibody design. Here, we introduce the Multidimensional Antiviral Antibody Database (MAAD; raabmd.org/raab/index), a curated platform dedicated to antibody, nanobody and single-chain variable fragment targeting three high-impact RNA virus families, Coronaviridae (SARS-CoV-1, SARS-CoV-2, MERS-CoV), Orthomyxoviridae (influenza virus), and Pneumoviridae (respiratory syncytial virus, human metapneumovirus), which were selected due to the large, high-quality datasets accumulated in recent years. MAAD further incorporates a suite of interactive analysis modules, including CDR and germline annotation, similarity-based sequence analysis, sequence-based clustering and structure-guided identification of antigen-antibody interface residues, complemented by per-site entropy and mutation rate profiling. These features enable in-depth exploration of antibody sequence characteristics, thereby facilitating functional and structural insights for rational antibody design. Together, by bridging antibody sequence, structure, and function, MAAD offers an open and standardized platform that advances comparative antiviral research and supports therapeutic antibody discovery.

SARS-CoV-2 continuously accumulates mutations in the spike receptor-binding domain (RBD), affecting both viral infectivity and antibody evasion. Systematic characterization of RBD mutations is therefore essential for understanding viral adaptation under immune pressure and predicting evolutionary trajectories. In this study, we employed a two-step, non-replicating pseudovirus deep mutational scanning (DMS) platform to measure the effects of all single amino acid substitutions in the RBD of Omicron variant JN.1 and its descendant lineage XEC within a full-length spike background. To identify representative antibodies for escape profiling, we first evaluated six RBD-targeting monoclonal antibodies against JN.1 and XEC pseudoviruses. Only BD55-1205 and 719-14 sIgA retained substantial neutralizing activity and were selected for subsequent escape mapping. The results showed that most single RBD amino acid mutations did not significantly enhance pseudovirus cellular invasion. Among mutations that are functionally retaining and confer marked escape from either antibody, most high escape substitutions cluster within the receptor-binding motif (RBM) and receptor-binding ridge. Furthermore, BD55-1205 and 719-14 sIgA each exhibited distinct, antibody-specific escape sites, demonstrating that different epitope preferences exert unique selective pressures within the same viral lineage. Overall, this pseudovirus-based DMS analysis elucidates the molecular mechanisms of immune escape and fitness for the JN.1 and XEC lineages. Our findings provide critical insights for forecasting SARS-CoV-2 evolution under population immunity and offer guidance for assessing emerging variants, selecting vaccine strains, and optimizing therapeutic antibodies.

SUMMARY Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) exhibit distinct seasonal epidemiology, with RSV circulating in early autumn and hMPV peaking in midwinter, yet the structural basis for this niche partitioning remains undefined. Here, we integrate in situ cryo-electron tomography and functional virology to decode the architectural logic governing their entry dynamics. RSV employs a matrix (M)-regulated prefusion F (pre-F) organization, partitioning trimers into stabilizing hexagonal superlattices and fusion-competent pools to maintain superior thermotolerance. By contrast, hMPV compensates for its intrinsically unstable, monomeric pre-F with extreme ribonucleoprotein polyploidy, packaging ∼4-fold more genome equivalents to ensure productive infection. Fusion events localize exclusively to M-depleted, non-arrayed membrane regions, establishing a spatial checkpoint for activation. These findings reveal a conserved trade-off between environmental resilience and genomic redundancy that dictates divergent pneumoviral entry strategies, explaining their distinct seasonal ecological niches.

Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are major causes of severe respiratory infections in young children, older adults, and immunocompromised individuals. Here, we isolated RSV fusion (F) protein-specific B cells from pediatricians who are routinely exposed to these viruses. We then derived monoclonal antibodies (mAbs) from those B cells to characterize their binding and neutralization profiles. Among the isolated mAbs, we found that CNR2056 and CNR2053 (targeting site Ø of the pre-F protein) potently neutralized diverse RSV A and B strains; another mAb, CNR2047 (targeting site III), uniquely exhibited cross-neutralization capacity against both RSV and hMPV variants. In vivo, prophylactic administration of CNR2056 and CNR2053 controlled lung viral loads and pathology in RSV A2- and B9320-challenged cotton rats. Moreover, a prophylactic dose of 0.5 milligrams per kilogram of CNR2047 resulted in complete protection against hMPV in the lungs of BALB/c mice. Structural analysis revealed unique binding modes for the three mAbs, supporting the potential for rational mAb cocktail design. Deep mutational scanning for RSV F further demonstrated that mutations required to evade CNR2053 and CNR2056 were primarily in evolutionarily constrained sites, suggesting a fitness cost to immune escape. Rationally combining site Ø- and site III-directed mAbs (e.g., CNR2056-CNR2047) into cocktails conferred additive effects, expanding coverage to hMPV and minimizing risk of escape variants. Thus, rationally designed cocktails of CNR2056, CNR2053, and CNR2047 may offer a versatile immunoprophylactic agent against a range of pneumoviruses with potential to protect against both current and future variants.

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for various EriCoVs. Analysis of APN orthologs from 139 species reveals that EriCoVs exhibit a restricted host range, primarily utilizing APN from hedgehogs and, to a lesser extent, felids and some other species, shaped by specific molecular determinants at the virus–receptor interface. Cryo-EM analysis of EriCoV–APN complex reveals a novel APN-binding mode that is distinct from those used by alpha- and deltacoronaviruses. Functional assays further demonstrate that transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes plasma membrane fusion. Neutralizing antibodies targeting the EriCoV RBD and hedgehog APN were generated and effectively blocked EriCoV pseudovirus entry or amplification in hedgehog-APN expressing human cells or primary hedgehog cells. The structural basis underlying the pan-EriCoV neutralization mediated by the RBD-targeting antibody BD23-0177 was further elucidated by cryo-EM analysis. Together, these findings uncover an unexpected convergent evolution of APN utilization among merbecoviruses, establishing a foundation for risk assessment and the development of targeted countermeasures.

Hepatitis A virus (HAV) is an unusual picornavirus with two types of extracellular virions: nonenveloped particles (nHAV) shed in feces and quasi-enveloped particles (eHAV) circulating in blood. Both enter cells by clathrin-dependent endocytic pathways merging in late endolysosomes with capsid binding to ganglioside receptors. Phosphatidylserine receptors facilitate eHAV endocytosis, but no protein receptor has been identified for nHAV. Here, we show low-density lipoprotein receptor (LDLR) is such a receptor. LDLR knockout did not alter viral attachment to cells, but restricted cellular uptake of nHAV (not eHAV). Soluble LDLR ectodomain blocked nHAV entry, as did antibody to LDLR. Recombinant LDLR-related protein-associated protein 1, a pan-LDLR family chaperone, also inhibited nHAV entry, including residual entry into knockout cells, suggesting other LDLR family members may similarly facilitate endocytosis. Reconstituting full-length LDLR expression restored nHAV entry in knockout cells, whereas LDLR mutants lacking LA repeats 4 to 7 or the EGF-like/propeller domain did not. ELISAs confirmed LDLR binds nHAV, optimally above pH7, without destabilizing the capsid. A 1.7Å resolution cryoelectron microscopy (cryo-EM) structure revealed LDLR interacts with VP1 at the fivefold vertex of the capsid. Extreme blurring of the LDLR density prevented detailed identification of LDLR interactions, and suggested binding does not follow particle symmetry, being either flexible or to multiple LDLR regions. Additional cryo-EM studies show ganglioside GD1a binds to a similar region of the capsid. Collectively, these data reveal the LDLR to be an important entry factor, shuttling nHAV from the extracellular environment to endolysosomes where it is likely released at low pH to bind gangliosides.

Dear Editor, African swine fever(ASF),caused by the African swine fever virus(ASFV),is a highly contagious swine disease with nearly 100％mortality in severe hemorrhagic cases(Dixon et al.,2019).As the swine industry is vital to agriculture,recent ASF outbreaks have raised concerns about global economic stability and food security(Wang et al.,2019).Controlling ASF is challenging due to the virus's stability,ability to evade immunity,and lack of effective vaccines or treatments.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections among infants and older adults, posing a significant threat to global public health. The prophylactic use of neutralizing antibodies (nAbs) underscores the need to understand elite RSV antibody neutralization mechanisms, which is fundamental for developing next-generation therapies with enhanced potency and broader activity. In this study, we utilized H2L2 transgenic mice encoding human immunoglobulin variable regions for immunization and successfully screened multiple antibodies with significant neutralizing activity using the Beacon Optofluidic system. One of these antibodies, PR306007, exhibited significantly superior broad-spectrum neutralization against both RSV-A and B subgroups. Cryo-electron microscopy (Cryo-EM) structural analysis revealed that PR306007 binds to a unique epitope that overlaps with antigenic sites II and V of the F protein, with its primary binding regions located at the base of the α6 and α7 helices of site II, and residues S173 and N175 of site V. This binding mode offers valuable insights into enhanced neutralization activity and potentially reduces the risk of emerging immune evasive mutants. Furthermore, PR306007 showed potent in vivo antiviral activity against RSV infection and demonstrated good efficacy against both lower and upper respiratory tract infections, making it a promising prophylactic candidate for broad prevention. These findings provide new insights for the future development of RSV vaccines or nAbs.

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus–receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstract Convergent utilization of hedgehog APN by EriCoVs ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. National Key R&D Program of China, 2024YFC2607300, 2023YFC2605500 the National Natural Science Foundation of China, 82322041, 32270164, 323B2006 Scientific Research Innovation Capability Support Project for Young Faculty, ZYGXONJSKYCXNLZCXM-H16 Natural Science Foundation of Hubei Province, 2023AFA015 Strategic Priority Research Program, XDB1310000 National Science Foundation Grants, 32325004, T2394482, 12034006 Basic Research Program Based on Major Scientific Infrastructures, CAS-JZHKYPT-2021-05, CAS-YSBR-010 Ministry of Science and Technology of China, CPL-1233 Changping Laboratory, 2025D-04-01 [1]: pending:yes

Several human papillomavirus (HPV) L1-based virus-like particle (VLP) vaccines are in development to meet future global vaccination needs. Type-specific monoclonal antibodies with good reactivity to all types of vaccines are urgently needed to evaluate vaccine potency. In this study, binding activity, neutralizing activity, conformational sensitivity, immunodominance in human serum, and versatility were compared among antibodies. A broad-spectrum binding antibody (C4-F5-127) was selected as the capture antibody; four type-specific neutralizing antibodies (6-F5-77, 11-F5-187, 16-F5-196, and 18-F5-203) were selected as detection antibodies for HPV6, 11, 16, and 18, respectively. These antibodies formed a standardized and universal in vitro relative potency (IVRP) assay kit. High-resolution cryo-electron microscopy (cryo-EM) structures of HPV6-6-F5-77, HPV11-11-F5-187, HPV16-16-F5-196 and HPV18-18-F5-203 complexes define the location and nature of epitopes, revealing serotype specific binding modes and neutralization mechanisms. The IVRP results were correlated with potency data from mouse models, offering an efficient alternative to in vivo potency experiments.

Monoclonal antibodies (mAbs) targeting the SARS-CoV-2 receptor-binding domain are used to treat and prevent COVID-19. However, the rapid evolution of SARS-CoV-2 drives continuous escape from therapeutic mAbs. Therefore, the ability to identify broadly neutralizing antibodies (bnAbs) to future variants is needed. Here we use deep mutational scanning to predict viral receptor-binding domain evolution and to select for mAbs neutralizing both existing and prospective variants. A retrospective analysis of 1,103 SARS-CoV-2 wild-type-elicited mAbs shows that this method can increase the probability of identifying effective bnAbs to the XBB.1.5 strain from 1% to 40% in an early pandemic set-up. Among these bnAbs, BD55-1205 showed potent activity to all tested variants. Cryogenic electron microscopy structural analyses revealed the receptor mimicry of BD55-1205, explaining its broad reactivity. Delivery of mRNA-lipid nanoparticles encoding BD55-1205-IgG in mice resulted in serum half-maximal neutralizing antibody titre values of ~5,000 to XBB.1.5, HK.3.1 and JN.1 variants. Combining bnAb identification using viral evolution prediction with the versatility of mRNA delivery technology can enable rapid development of next-generation antibody-based countermeasures against SARS-CoV-2 and potentially other pathogens with pandemic potential.

Despite the discovery of plant viruses as a new class of pathogens over a century ago, the structure of plant virus replication machinery and antiviral pesticide remains lacking. Here we report five cryogenic electron microscopy structures of a ~330-kDa RNA-dependent RNA polymerase (RdRp) from a devastating plant bunyavirus, tomato spotted wilt orthotospovirus (TSWV), including the apo, viral-RNA-bound, base analogue ribavirin-bound and ribavirin-triphosphate-bound states. They reveal that a flexible loop of RdRp's motif F functions as 'sensor' to perceive viral RNA and further acts as an 'adaptor' to promote the formation of a complete catalytic centre. A ten-base RNA 'hook' structure is sufficient to trigger major conformational changes and activate RdRp. Chemical screening showed that ribavirin is effective against TSWV, and structural data revealed that ribavirin disrupts both hook-binding and catalytic core formation, locking polymerase in its inactive state. This work provides structural insights into the mechanisms of plant bunyavirus RdRp activation and its dual-targeted site inhibition, facilitating the development of pesticides against plant viruses.

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2. (SARS-CoV-2) and its variant strains, including Alpha, Beta, Gamma, Delta, and now Omicron, pose a significant challenge. With the constant evolution of the virus, Omicron and its subtypes BA.1, BA.2, BA.3, BA.4, and BA.5 have developed the capacity to evade neutralization induced by previous vaccination or infection. This evasion highlights the urgency in discovering new monoclonal antibodies (mAbs) with neutralizing activity, especially broadly neutralizing antibodies (bnAbs), to combat the virus.In the current study, we introduced a fully human neutralizing mAb, CR9, that targets Omicron variants. We demonstrated the mAb’s effectiveness in inhibiting Omicron replication both in vitro and in vivo. Structural analysis using cryo-electron microscopy (cryo-EM) revealed that CR9 binds to an epitope formed by RBD residues, providing a molecular understanding of its neutralization mechanism. Given its potency and specificity, CR9 holds promise as a potential adjunct therapy for treating Omicron infections. Our findings highlight the importance of continuous mAb discovery and characterization in addressing the evolving threat of COVID-19.

The tremendous success of mRNA vaccine during the COVID-19 pandemic has captured attention globally and highlighted the transformative potential of mRNA technology in addressing infectious diseases[1].In comparison to conventional protein antibody-based therapies,the delivery of mRNA-encoding antibod-ies presents a cost-effective and versatile approach with several advantages.