Nitrate (NO3-) is often the dominant nitrogen species in aquatic ecosystems, but tracing its sources and transformations is always challenging. Atmospheric deposition contributes to nitrogen in aquatic ecosystems both directly and indirectly, but the relative contributions of the two pathways remain unclear. Furthermore, triple isotopes (S15N-S18O-Q17O) have recently been promoted as a more reliable way to quantify source contributions to NO3-in water than dual isotopes (S15N-S18O), but their differences in results remain unknown. In this study, we measured the concentration, isotopes of NO3-and water in Dongting Lake, investigated the nitrogen transformation process, and quantified the source contributions based on the dual and triple isotopes of NO3-using the SIAR model. Our results showed that seasonal differences in ion concentrations and isotopes were generally found in Dongting Lake. The seasonal variations of nitrogen and oxygen isotopes revealed the role of nitrification process and changes in source proportional contributions. The source contributions quantified based on S15N-S18O and S15N-S18O-Q17O are consistent, showing that soil nitrogen sources contribute the most, followed by agricultural fertilizer application and manure and sewage sources. Atmospheric deposition contributes the least to NO3-in Dongting Lake, with an average fraction of less than 9 %. Source contributions quantified on the basis of triple isotopes can effectively reduce uncertainties in the proportional contribution of atmospheric deposition. In Dongting Lake, most of the atmospheric deposition of NO3-is indirect, accounting for about 95.2 % and 89.9 % of the total atmospheric deposition to water NO3-fluxes in wet and dry seasons, respectively. The study reveals the source contribution and transformation process to NO3-in Dongting Lake, and provides an example for quantifying direct and indirect atmospheric deposition to inland lakes. The results provide valuable observational data for modelling the nitrogen cycle in aquatic ecosystems and help to better constrain the nitrogen budget in inland waters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant morbidity and mortality despite the end of its pandemic phase. The emergence of highly mutated SARS-CoV-2 variants of concern highlights the requirement of broad-spectrum antiviral countermeasures which possess both prophylactic and therapeutic efficacies. Here, we obtain a macrocyclic peptide, 6L3-3P11K, that effectively inhibits a wide range of SARS-CoV-2 variants and subvariants. Structural studies show that 6L3-3P11K forms homotrimers that lock the spike protein (S) trimer into a “closed” conformation by engaging a conserved non-receptor binding motif (non-RBM) of S. This interaction disrupts the binding between S and ACE2 receptor. Structure-guided modifications result in a thermostable and trypsin-resistant macrocyclic peptide, 6L3-1F3P11hR, that exhibits prophylactic and therapeutic effects against SARS-CoV-2 infection in a male hACE2 transgenic mouse model after intranasal administration. Our results provide a drug candidate for the control and prevention of COVID-19 and may stimulate further research on macrocyclic broad-spectrum anti-coronavirus drug development. Newly emerging SARS-CoV-2 variants underscore the need for broad-spectrum antiviral solutions. This study shows a macrocyclic peptide inhibitor that locks the SARS-CoV-2 spike trimer into a “closed” conformation by engaging a conserved region, and demonstrates that intranasal administration of the peptide inhibitor protects against Omicron variants.

Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (Viperin), an interferon-stimulated gene (ISG) product, restricts the replication of a broad spectrum of viruses through its radical S-adenosyl methionine (SAM) enzymatic activity, which converts cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). This conversion leads to premature termination of RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of certain RNA viruses. Coronaviruses, being RNA viruses, can be suppressed by viperin; certain strains, such as porcine epidemic diarrhea virus (PEDV), are directly influenced by ddhCTP, while others, such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), are inhibited by different pathways. In this study, we describe a previously unrecognized anti-coronavirus mechanism of viperin. Using Porcine Deltacoronavirus (PDCoV) as a model, we observed that viperin is strongly induced upon PDCoV infection and significantly inhibits viral replication. Furthermore, we found that viperin directly interacts with the viral non-structural protein 8 (nsp8) protein, disrupting the formation of the replication-transcription complex (RTC) and reducing RdRp activity. Our results further indicate that the central domain (residues 43-184) of viperin and the lysine 82 (K82) residue in the N-terminal domain of nsp8 are critical for this interaction and its antiviral function. We illustrate that the viperin-nsp8 interaction is preserved across all genera of α-, β-, γ-, and δ-coronaviruses. These findings reveal a unique anti-coronavirus mechanism of viperin and offer new insights into its potential as a target for inhibiting viral nsp8 function.

Heteromeric amino acid transporters (HATs), including y+LAT1-4F2hc complex, are responsible for transporting amino acids across membranes, and mutations in y+LAT1 cause lysinuric protein intolerance (LPI), a hereditary disorder characterized by defective cationic amino acid transport. The relationship between LPI and specific mutations in y+LAT1 has yet to be fully understood. In this study, we characterized the function of y+LAT1-4F2hc complex in mammalian cells and determined the cryo-EM structures of the human y+LAT1-4F2hc complex in two distinct conformations: the apo state in an inward-open conformation and the native substrate-bound state in an outward-open conformation. Structural analysis suggests that Asp243 in y+LAT1 plays a crucial role in coordination with sodium ion and substrate selectivity. Molecular dynamic (MD) simulations further revealed the different transport mechanism of cationic amino acids and neutral amino acids. These results provide important insights into the mechanisms of the substrate binding and working cycle of HATs.

BACKGROUND:Linezolid shows therapeutic potential for pediatric gram-positive bacterial central nervous system infections (CNSIs). However, its efficacy, safety profile, and cerebrospinal fluid (CSF) pharmacokinetics require detailed evaluation. METHODS:This prospective 2-center observational study enrolled children with confirmed or suspected gram-positive CNSIs. Clinical outcomes and adverse events were compared between linezolid-treated patients and a matched vancomycin cohort. Population pharmacokinetic (PopPK) modeling with nonlinear mixed-effects analysis quantified linezolid exposure in plasma and CSF. RESULTS:Among 45 matched pediatric CNSIs patients per group, linezolid demonstrated a 91.1% clinical response rate and 68.9% cure rate (vancomycin cure rate, 68.9%). However, noninferiority to vancomycin was not established for the primary end point, possibly influenced by intergroup baseline variability and extended treatment duration. Adverse events occurred more frequently with linezolid, including gastrointestinal (48.9% vs 24.4%, P = .02) and hematologic effects (73.3% vs 53.3%, P = .05). Plasma trough concentrations >7 µg/mL were correlated with elevated risk of leukopenia and neutropenia (odds ratio [OR], 9.38; 95% confidence interval [CI], 1.21-72.6 and OR, 40.2; 95% CI, 2.15-748.50). However, no treatment discontinuations occurred due to adverse events. The PopPK model analyzed 135 linezolid concentrations (90 plasma/45 CSF), identifying body weight as the primary covariate influencing distribution. Plasma and CSF trough concentrations showed a strong correlation (r = 0.87; 95% CI, .75-.98). CONCLUSIONS:Linezolid demonstrated favorable clinical efficacy and tolerability in pediatric CNSIs, with CSF concentrations that correlated with plasma levels and exhibited predictable pharmacokinetics.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic henipaviruses without approved human vaccines or therapies. Here, we report on a highly potent bispecific therapeutic that combines an anti-fusion (F) nanobody with an anti-receptor binding protein (RBP) antibody to deliver a dual-targeting biologic that is resistant to viral escape. We show that the nanobody, DS90, engages a unique, conserved site within prefusion F of NiV and HeV, and provides neutralization and complete protection from NiV disease. Bispecific engineering of DS90 with the anti-RBP mAb m102.4 results in neutralization, elimination of viral escape and superior protection from NiV disease compared to leading monovalent approaches. These findings carry implications for the development of cross-neutralizing immunotherapies that limit the emergence of henipaviral escape mutants. ### Competing Interest Statement The authors have declared no competing interest.

The emergence of novel infectious disease has intensified demand for more advanced vaccine development and more potent adjuvants to enhance immunogenicity. Here we introduce a dynamic DNA supramolecular matrix assembled from five unmodified, short DNA single strands, serving as a safe, multifaceted adjuvant platform. This DNA matrix elicits a robust humoral response with minimal adverse effects, generating potent neutralizing antibodies and conferring robust protection against SARS-CoV-2 and Streptococcus pneumoniae infections. Its dynamic colloidal feature prolongs the in vivo retention of both DNA and antigen, facilitating lymphatic-targeted transportation and presentation. This process leads to a robust pro-inflammatory response in both the vaccinated site and draining lymph node, which, in turn, promotes the recruitment and activation of immune cells, leading to a rapid, effective antigen-specific antibody response. The enhanced function of DNA matrix depends on the canonical TLR9–MyD88 signalling axis in dendritic cells. In addition, only right-handed, not left-handed, chirality of the DNA strands forms d-DNA matrix and promotes immune activations. Thus, this DNA matrix functions as an all-in-one adjuvant platform, opening promising avenues for future vaccine design. A DNA matrix material potentiates humoral response through multiple administration routes, generating neutralizing antibodies and conferring robust protection against infection.

A safe and effective vaccine is urgently needed to prevent acute respiratory infections caused by respiratory syncytial virus (RSV). Oral administration offers several advantages, including ease of delivery, minimal stress for vaccine recipients, and greater safety than the systemic injection. In this study, we developed an oral vaccine candidate based on the human adenovirus serotype 5 (Ad5) vector, Ad5-PreF-DS2, encoding a prefusion protein of RSV with a dsRNA as an endogenous adjuvant. We evaluated the immunogenicity and protective efficacy of oral immunization against an RSV challenge in mice, comparing it with those of IM and IN immunizations. Subsequently, we performed an in-depth analysis of the B cell immune response to the oral vaccine. Our findings indicate that oral vaccines elicited a robust antibody response, T-cell response, and B-cell response, and provide effective protection against RSV infection in mice. Importantly, dsRNA adjuvants significantly enhanced T-cell immune responses and increased neutralizing antibody levels when administered via oral vaccination (P < 0.05). These preclinical data demonstrate the capacity of an oral vaccine to induce protective immunity against RSV and support further development of RSV vaccine.

Autophagy is a highly conserved intracellular degradation system that is crucial for nutrient recycling, thus regulating plant growth and development as well as in response to various stresses. Halophytic plant Lycium ruthenicum Murray (L. ruthenicum) is considered as a potential model plant for studying the physiological mechanisms of salt stress tolerance in plants. Although the genome sequence of L. ruthenicum is available, the characteristics and functions of the salt stress-related genes remain largely unknown. In the present study, a total of 36 AuTophaGy-related (ATG) genes were identified in L. ruthenicum and detailed characteristics of them were given. Quantitative real-time polymerase chain reaction analysis revealed that the expression of 25 LrATGs was significantly upregulated after salt stress treatments. Furthermore, the autophagic marker line pSuper:GFP-LrATG8g was generated and used to demonstrate the salt stress-induced autophagy, as revealed by measuring autophagic flux and observing autophagosome formation. The pSuper:LrATG5-GFP overexpression (OE) lines were also generated and further phenotypic analysis showed that OE-LrATG8g and OE-LrATG5 plants exhibited better salt tolerance than that of WT plants. To the best of our knowledge, this study firstly reports a detailed overview of LrATGs-mediated autophagy in L. ruthenicum response to salt stress. These findings contribute to a global understanding of the characteristics of ATG genes in L. ruthenicum and lay a foundation for future functional study.

In 2018, two novel influenza-like virus genomes were first identified in basal vertebrates: the Asiatic toads (Bufo gargarizans) and spiny eels (Mastacembelus aculeatus). Their hemagglutinin (HA) proteins exhibit remarkably low amino acid sequences homology (23.0% and 42.8%, respectively) compared to influenza B virus (IBV), their closest canonical influenza virus relative. This study revealed that the Asiatic toad influenza-like virus HA (tHA) demonstrates dual receptor specificity, bound both α2-3 (avian-type) and α2-6 (human-type) sialic acid (SA) receptors, whereas the spiny eel influenza-like virus HA (eHA) lacks this capability. Biophysical characterization showed reduced thermal stability (lower Tm values) for both tHA and eHA compared to canonical influenza HA. Furthermore, we determined the cryo-EM structures of apo-tHA, tHA in complex with either α2-3 SA receptor or α2-6 SA receptor, as well as apo-eHA and eHA bound to GM2 complex. Our analysis revealed that tHA has a shorter length and looser HA trimer packing compared to canonical HA. These findings collectively indicate that influenza-like viruses in basal vertebrates have evolutionarily acquired dual SA receptor-binding capacity, a trait critical for cross-species transmission in influenza viruses. However, the observed thermolability of these HA proteins suggests that host physiological temperatures may impose a barrier to zoonotic spillover.

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 Mpox virus (MPXV) is an orthopoxvirus that caused a global outbreak in 2022. The poxvirus DNA polymerase complex is responsible for the replication and integrity of the viral genome; however, the molecular mechanisms underlying DNA replication fidelity are still unclear. In this study, we determined the cryoelectron microscopy (cryo-EM) structures of the MPXV F8-A22-E4 polymerase holoenzyme in its editing state, in complex with mismatched primer-template DNA and DNA containing uracil deoxynucleotide. We showed that the MPXV polymerase has a similar replication-to-edit transition mechanism to proofread the mismatched nucleotides like the B-family DNA polymerases of other species. The unique processivity cofactor A22-E4 undergoes conformational changes in different working states and might affect the proofreading process. Moreover, we elucidated the base excision repair (BER) function of E4 as a uracil-DNA glycosylase and the coupling mechanism of genome replication and BER, characteristic of poxviruses. Our findings greatly enhance our molecular understanding of DNA replication fidelity of orthopoxviruses and will stimulate the development of broad-spectrum antiviral drugs.

The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as fusion proteins in some organisms and viruses. However, the mechanism underlying DNA unwinding initiation by these helicase-primase fusion enzymes and the functional association between domains have not been elucidated. Herein, we report the cryo-EM structures of mpox virus E5, the founding member of these helicase-primase enzymes, in various enzymatic stages. Notably, E5 forms a head-to-head double hexamer encircling dsDNA, disrupted by the conformational rearrangement of primase domains upon nucleotide incorporation. Five E5-ssDNA-ATP structures further support an ATP cycle-driven non-classical escort model for E5 translocation. Finally, the helicase domain is found to enhance the primase function as a DNA scaffold. Together, our data shed light on the E5-mediated DNA unwinding model including dsDNA loading, DNA melting, ssDNA translocation, and provide a reasonable interpretation for evolutionary preservation of helicase-primase fusion from a functional perspective.

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and the elderly. However, natural infection fails to induce durable immune protection, and existing mRNA vaccines for older adults exhibit limited long-term efficacy. We developed an antigen engineering strategy inserting ESCRT/ALIX-binding region (EABR) into truncated RSV prefusion F (PreF) cytoplasmic tails to form enveloped virus-like particles (eVLPs). In murine models, PreF-EABR mRNA vaccines elicited higher, more persistent neutralizing antibodies than conventional PreF mRNA, correlating with enhanced germinal center B cell and memory B cell responses. A lower dose of PreF-EABR mRNA (1 μg) suppressed viral load and pathology comparable to higher-dose PreF mRNA (2.5 μg). Transcriptomic analysis showed PreF-EABR mRNA activated toll-like receptor and chemokine signaling pathways, enhancing antibody longevity via platelet-associated signatures. This study explores the development and possible mechanism of long-lasting RSV mRNA vaccines by eVLPs technology, which also suggest its potential application in other vaccines.

Influenza virus is a segmented, single-stranded, negative-sense RNA virus. Viral genome transcription (to make viral messenger RNA) and replication (to make more viral genome) of influenza virus are catalyzed by the influenza viral RNA-dependent RNA polymerase (FluPol) in the context of the viral ribonucleoprotein complexes in the nucleus of infected cells. The dynamics of the transcription and replication are tightly regulated throughout the viral life cycle, with a switch from transcription to replication in the later stages of infection being essential for efficient progeny virus production. The mechanism by which the virus achieves the switch has emerged recently through structural and functional studies. Here, we summarize the current hypotheses of the regulatory mechanisms governing the switch. Specifically, we highlight our recent findings showing that the late expression of the viral nonstructural protein NS2, which resulted from a suboptimal splicing site in the NS segment, functions as a molecular timer to mediate the transcription-to-replication switch.

Recent avian-origin H3N8 influenza A virus (IAV) that have infected humans pose a potential public health concern. Alterations in the viral surface glycoprotein, hemagglutinin (HA), are typically required for IAVs to cross the species barrier for adaptation to a new host, but whether H3N8 has adapted to infect humans remains elusive. The observation of a degenerative codon in position 228 of HA in human H3N8 A/Henan/4-10/2022 protein sequence, which could be residue G or S, suggests a dynamic viral adaptation for human infection. Previously, we found this human-isolated virus has shown the ability to transmit between ferrets via respiratory droplets, with the HA-G228S substitution mutation emerging as a critical determinant for the airborne transmission of the virus in ferrets. Here, we investigated the receptor-binding properties of these two H3N8 HAs. Our results showed H3N8 HAs have dual receptor-binding properties with a preference for avian receptor binding, and G228S slightly increased binding to human receptors. Cryo-electron microscopy structures of the two H3N8 HAs with avian and human receptor analogs revealed the basis for dual receptor binding. Mutagenesis studies reveal that the Q226L mutation shifts H3N8 HA's receptor preference from avian to human, while the G228S substitution enhances binding to both receptor types. H3N8 exhibits distinct antigenic sites compared to H3N2, prompting concerns regarding vaccine efficacy. These findings suggest that the current H3N8 human isolates are yet to adapt for efficient human-to-human transmission and further continuous surveillance should be implemented.IMPORTANCEInfluenza virus transmission remains a public health concern currently. H3N8 subtype influenza A viruses infect humans and their HAs acquire the ability to bind to both human and avian receptors, posing a threat to human health. We have solved and analyzed the structural basis of dual receptor binding of recently human-infecting H3N8 HA, and we demonstrate that the G228S enhances human receptor binding and adaptation. We also found that HN/4-10 H3N8 HA has distinct antigenic sites, which challenges vaccine efficacy. Taken together, our work is critical to the prevention and control of human H3 influenza virus infection.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic henipaviruses without approved human vaccines or therapies. Here, we report on a highly potent bispecific therapeutic that combines an anti-fusion glycoprotein nanobody with an anti-receptor-binding glycoprotein (RBP) antibody to deliver a dual-targeting biologic that is resistant to viral escape. We show that the nanobody, DS90, engages a unique, conserved site within the fusion glycoprotein of NiV and HeV and provides neutralization and complete protection from NiV disease. Bispecific engineering of DS90 with the anti-RBP monoclonal antibody m102.4 results in neutralization, elimination of viral escape and superior protection from NiV disease compared to leading monovalent approaches. These findings carry implications for the development of cross-neutralizing immunotherapies that limit the emergence of henipaviral escape mutants.