Blood-tissue barriers are specialized interfaces that safeguard organ homeostasis by restricting pathogen dissemination. Zika virus (ZIKV), an emerging flavivirus of global concern, exhibits an exceptional ability to breach multiple barriers—including the blood-brain, blood-placental, blood-testis, and blood-retinal barriers—enabling neuroinvasion, vertical and sexual transmission, and ocular disease. ZIKV employs diverse strategies to cross these barriers: receptor-mediated entry, disruption of tight junctions, and hijacking immune cells or extracellular vesicles as viral carriers. Adaptive mutations further refine tissue tropism and enhance barrier traversal efficiency. Insights from cell culture, organoid, animal, and ex vivo tissue models reveal not only the conserved and tissue-specific mechanisms of barrier penetration but also the downstream pathological consequences in the affected organs. Understanding how ZIKV breaches these interfaces and induces organ-specific pathology deepens our knowledge of host-pathogen interactions and provides a framework for designing barrier-protective and disease-mitigating strategies against ZIKV and other pathogens that breach blood-tissue barriers.

Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs1-3. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE)4,5 and transposase-mediated (CAST)6 insertion systems, particularly for larger payloads, showing a 11-fold, 61-fold and 12-fold improvement for a 9.5 kb insertion, respectively. Notably, QuadPE was effective in both dividing and non-dividing primary cells such as human primary T cells and post-mitotic neurons, establishing QuadPE as a powerful and precise platform for large-fragment gene insertion without the need for double-stranded breaks or recombinases.

Background Adolescent depressive disorders (ADD) pose a critical global public health challenge, yet comprehensive assessments of their long-term burden, key risk factors, and future trajectories remain limited. Objectives This study aimed to quantify the global incidence, disability-adjusted life years (DALYs), and attributable risk factors for ADD from 1991 to 2021, and to project future burden trends to 2039. Method We conducted a population-based analysis using data from the Global Burden of Disease Study 2021 across 204 countries and territories. Analyses included joinpoint regression for trend analysis, comparative risk assessment for risk factors (bullying victimization, intimate partner violence, childhood sexual abuse), and Bayesian age-period-cohort modeling for burden prediction. Results From 1991 to 2021, global ADD incidence increased significantly. The rise was more pronounced in males and the 10–14-year age group. East Asia saw a decline, while high-income North America experienced a sharp increase. DALYs attributable to violence and bullying rose, particularly in high-SDI regions. Projections indicate a continued global increase and a widening gender gap, with females disproportionately affected. Conclusions The global burden of ADD is escalating, with distinct patterns by sex, age, and region. Violence and bullying are key drivers. These findings underscore an urgent need for targeted, gender-sensitive, and region-specific mental health interventions for adolescents.

ZIKV infection is associated with testicular damage and abnormal spermatogenesis. However, the molecular mechanisms underlying these pathogenic processes remain unclear. Here, we demonstrate that ZIKV disrupts Leydig cells' ability to produce testosterone, leading to decreased sperm counts and motility. Specifically, the non-structural protein NS2A of ZIKV downregulates testosterone production by directly binding to mRNA of CYP17A1, a key enzyme in testosterone synthesis, thereby inhibiting its translation. Notably, the sole membrane-traversing segment and its flanking loops of NS2A are crucial for this interaction with CYP17A1 mRNA. Scanning mutagenesis studies within this sequence identified amino acid residues critical for NS2A binding and the suppression of CYP17A1 mRNA translation. Testicular inoculation of adeno-associated virus (AAV) delivering ZIKV-NS2A or its mutant showed that ZIKV-NS2A alone is sufficient to affect steroidogenesis and spermatogenesis in vivo. Moreover, a mutant virus generated by reverse genetics, containing a single amino acid mutation that abolishes NS2A's binding to CYP17A1 mRNA, exhibited significantly lower inhibition of steroidogenesis and spermatogenesis compared to the wild-type virus in mouse models. These findings enhance our understanding of how ZIKV impacts male reproductive health and provide crucial insights for future preventive and therapeutic strategies.

Flaviviruses are zoonotic pathogens transmitted by infected mosquitoes and ticks,posing a persistent threat to global health.These infections lead to a diverse spectrum of diseases,broadly classified into two phenotypes:systemic hemorrhagic conditions,such as dengue and yellow fever,and neurological complications,exempli-fied by West Nile virus(WNV)and Zika virus(ZIKV)infections.The interactions between flaviviruses and human host cells are pivotal to the viral lifecycle and the activation of host immunity.Investigating the molec-ular mechanisms of flavivirus-host interactions is essential for understanding viral pathogenesis,managing epi-demics,optimizing therapeutic strategies,and enhancing public health security.Flaviviruses utilize various strategies to evade the host immune system,and understanding these mechanisms is critical for the develop-ment of antiviral therapeutics.This study employs bibliometric methods,leveraging CiteSpace and VOSviewer software,to analyze literature on flavivirus-host interactions and the associated immune responses.By exam-ining developmental trends and pivotal research areas,this analysis provides insights and guidance for future studies in this field.

BACKGROUND:Accurate and timely diagnosis of tuberculosis (TB) and drug-resistant TB is crucial; however, current methods have limitations in sensitivity and scope, especially for detecting drug resistance and differentiating other respiratory pathogens. METHODS:We developed a multiplex PCR-based targeted next-generation sequencing (tNGS) assay and validated its analytical performance, including limit of detection (LoD), precision, and resistance to interference. A single-centre prospective study was conducted with 181 suspected TB patients to evaluate the assay's clinical performance using bronchoalveolar lavage or sputum samples, compared against microbiological culture and Xpert MTB/RIF. RESULTS:The tNGS assay demonstrated a low LoD of 10 copies/mL for TB and other respiratory pathogens, with high precision and resistance to interference. In clinical validation, it achieved 94.94 % sensitivity in confirmed TB cases and showed a 92.86 % positive percent agreement with Xpert MTB/RIF for rifampicin resistance, while also identifying additional mutations. The assay accurately detected nontuberculous mycobacteria (NTM) and other respiratory pathogens, aiding differential diagnosis. CONCLUSION:The tNGS assay provides reliable detection of TB, drug-resistant mutations, and respiratory pathogens, including NTM, thereby enhancing differential diagnosis and supporting effective treatment strategies in the management of patients with suspected TB.

Foxes are susceptible to SARS-CoV-2 in laboratory settings, and there have also been reports of natural infections of both SARS-CoV and SARS-CoV-2 in foxes. In this study, we assessed the binding capacities of fox ACE2 to important sarbecoviruses, including SARS-CoV, SARS-CoV-2, and animal-origin SARS-CoV-2 related viruses. Our findings demonstrated that fox ACE2 exhibits broad binding capabilities to receptor-binding domains (RBDs) of sarbecoviruses. We further determined the cryo-EM structures of fox ACE2 complexed with RBDs of SARS-CoV, SARS-CoV-2 prototype (PT), and Omicron BF.7. Through structural analysis, we identified that the K417 mutation can weaken the ability of SARS-CoV-2 sub-variants to bind to fox ACE2, thereby reducing the susceptibility of foxes to SARS-CoV-2 sub-variants. In addition, the Y498 residue in the SARS-CoV RBD plays a crucial role in forming a vital cation-π interaction with K353 in the fox ACE2 receptor. This interaction is the primary determinant for the higher affinity of the SARS-CoV RBD compared to that of the SARS-CoV-2 PT RBD. These results indicate that foxes serve as potential hosts for numerous sarbecoviruses, highlighting the critical importance of surveillance efforts.

Enterovirus 71 (EV71), a prominent pathogen associated with hand, foot, and mouth disease (HFMD), has been reported worldwide. To date, the advancement of effective drugs targeting EV71 remains in the preliminary experimental stage. In this study, magnolol demonstrated a significant dose-dependent inhibition of EV71 replication in vitro. It upregulated the overall expression level of nuclear factor erythroid 2 - related factor 2 (Nrf2) and facilitated its nucleus translocation, resulting in the increased expression of various ferroptosis inhibitory genes. This process led to a reduction in reactive oxygen species (ROS) accumulation induced by viral infection. Additionally, magnolol exhibited a broad-spectrum antiviral effect against enteroviruses. Notably, treatment with magnolol substantially enhanced the survival rate of EV71-infected mice, attenuated viral load in heart, liver, brain, and limb tissues, and mitigated tissue inflammation. Taken together, magnolol emerges as a promising candidate for the development of anti-EV71 drugs.

Given the intimate relationship between humans and dogs, the H3N2 canine influenza viruses (CIVs) pose a threat to public health. In our study, we isolated four H3N2 CIVs from 3,758 dog nasal swabs in China between 2018 and 2020, followed by genetic and biological analysis. Phylogenetic analysis revealed 15 genotypes among all available H3N2 CIVs, with genotype 15 prevailing among dogs since around 2017, indicating the establishment of a stable virus lineage in dogs. Molecular characterization identified many mammalian adaptive substitutions, including HA-G146S, HA-N188D, PB2-I292T, PB2-G590S, PB2-S714I, PB1-D154G, and NP-R293K, present across the four isolates. Notably, analysis of HA sequences uncovered a newly emerged adaptive mutation, HA-V223I, which is predominantly found in human and swine H3N2 viruses, suggesting its role in mammalian adaptation. Receptor-binding analysis revealed that the four H3N2 viruses bind both avian and human-type receptors. However, HA-V223I decreases the H3N2 virus’s affinity for human-type receptors but enhances its thermal stability. Furthermore, attachment analysis confirmed the H3N2 virus binding to human tracheal tissues, albeit with reduced affinity when the virus carries HA-V223I. Antigenic analysis indicated that the current human H3N2 vaccines do not confer protection against H3N2 CIVs. Collectively, these findings underscore that the potential threat posed by H3N2 CIVs to human health still exists, emphasizing the necessity of close surveillance and monitoring of H3N2 CIVs in dogs.

As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.

Flaviviruses strategically utilize the endoplasmic reticulum (ER) in their replication cycles. However, the role of ER autophagy (ER-phagy) in viral replication process remains poorly understood. Here, we reveal that prolonged Zika virus (ZIKV) infection results from the degradation of ER-phagy receptor FAM134B, facilitated by viral NS2A protein. Mechanistically, ER-localized NS2A undergoes K48-linked polyubiquitination at lysine (K) 56 by E3 ligase AMFR. Ubiquitinated NS2A binds to FAM134B and AMFR orchestrates the degradation of NS2A-FAM134B complexes. AMFR-catalyzed NS2A ubiquitination not only targets FAM134B degradation but also hinders the FAM134B-AMFR axis. Notably, a recombinant ZIKV mutant (ZIKV-NS2AK56R), lacking ubiquitination and ER-phagy inhibition, exhibits attenuation in ZIKV-induced microcephalic phenotypes in human brain organoids and replicates less efficiently, resulting in weakened pathogenesis in mouse models. In this work, our mechanistic insights propose that flaviviruses manipulate ER-phagy to modulate ER turnover, driving viral infection. Furthermore, AMFR-mediated flavivirus NS2A ubiquitination emerges as a potential determinant of viral pathogenecity. Flaviviruses strategically utilize the endoplasmic reticulum (ER) in their replication cycles, but the role of ER-phagy in viral replication process remains poorly understood. Here, the authors show Zika virus targets key ER-phagy receptor to inhibit ER-phagy process and physiologically elucidate the basic mechanisms and viral pathogenicity by regulating ER-phagy.

Noncoding RNAs (ncRNAs) constitute a class of RNA molecules that lack protein-coding capacity. ncRNAs frequently modulate gene expression through specific interactions with target proteins or messenger RNAs, thereby playing integral roles in a wide array of cellular processes. The Flavivirus genus comprises several significant members, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV), which have caused global outbreaks, resulting in high morbidity and mortality in human populations. The life cycle of arthropod-borne flaviviruses encompasses their transmission between hematophagous insect vectors and mammalian hosts. During this process, a complex three-way interplay occurs among the pathogen, vector, and host, with ncRNAs exerting a critical regulatory influence. ncRNAs not only constitute a crucial regulatory mechanism that has emerged from the coevolution of viruses and their hosts but also hold potential as antiviral targets for controlling flavivirus epidemics. This review introduces the biogenesis of flavivirus-derived ncRNAs and summarizes the regulatory roles of ncRNAs in viral replication, vector-mediated viral transmission, antiviral innate immunity, and viral pathogenicity. A profound comprehension of the interplay between ncRNAs and flaviviruses will help formulate efficacious prophylactic and therapeutic strategies against flavivirus-related diseases.

ObjectiveTo analyze the host factors affecting the drug resistance of Helicobacter pylori (Hp).MethodsPatients with Hp infection were consecutively recruited in the Affiliated Suzhou Hospital of Nanjing Medical University from November 2021 to October 2023. Endoscopic biopsy specimens were collected for pathological diagnosis, Hp strain culture and antimicrobial susceptibility test. Nineteen factors involving the basic information, lifestyle, dietary habits, and health status of the patients were collected through electronic medical records and questionnaires. Logistic regression was used to evaluate the association between the patients' factors and drug resistance to clarithromycin, levofloxacin, amoxicillin, furazolidone, tetracycline and metronidazole.ResultsA total of 115 patients (Hp strain 115) with Hp infection who met the inclusion and exclusion criteria were enrolled. There were 53 males (46.09%) and 62 females (53.91%), with an average age of (45.16±13.39) years. Gastroscopic pathology showed 86 cases (74.78%) of superficial gastritis, 6 cases (5.22%) of atrophic gastritis, 14 cases (12.17%) of intestinal metaplasia, 6 cases (5.22%) of low-grade intraepithelial neoplasia, and 3 cases (2.61%) of high-grade intraepithelial neoplasia/gastric cancer. The drug resistance rates of Hp strains to metronidazole, levofloxacin and clarithromycin were 91.30% (105/115), 53.04% (61/115) and 51.30% (59/115), respectively. Resistance to amoxicillin, furazolidone and tetracycline was not found. Dual drug resistance: levofloxacin + metronidazole dual resistance rate was 50.43% (58/115), clarithromycin + metronidazole dual resistance rate was 47.83% (55/115), clarithromycin + levofloxacin dual resistance rate was 36.52% (42/115). Multidrug resistance: clarithromycin + levofloxacin + metronidazole triple resistance rate was 34.78% (40/115). Multivariate Logistic regression analysis showed that (metronidazole was not included in the multivariate analysis due to the absence of sensitive strains), previous Hp eradication history (OR=74.782, 95% CI: 10.377-538.886, P < 0.001) and tap water (OR=4.919, 95% CI: 1.160-20.859, P=0.031) increased the risk of clarithromycin resistance, and age ≥50 years increased the risk of levofloxacin resistance (OR=4.261, 95% CI: 1.420-12.785, P=0.010), previous Hp eradication history (OR=5.855, 95% CI: 2.209-15.517, P < 0.001), 40-59 years old (OR= 3.269, 95% CI: 1.254-8.520, P=0.015) increased the risk of dual resistance to clarithromycin and levofloxacin.ConclusionsThe drug resistance rate of Hp strains isolated from patients in the Affiliated Suzhou Hospital of Nanjing Medical University to metronidazole, levofloxacin and clarithromycin were high, and dual drug resistance and multidrug resistance were prominent. Age, previous Hp eradication history and drinking water source may be associated with single or dual drug resistance to clarithromycin and levofloxacin. Comprehensive consideration, reasonable selection of antibiotics and individualized treatment should be taken into account during Hp eradication.

Sepsis, characterized as life-threatening organ dysfunction resulting from dysregulated host responses to infection, remains a significant challenge in clinical practice. Despite advancements in understanding host-bacterial interactions, molecular responses, and therapeutic approaches, the mortality rate associated with sepsis has consistently ranged between 10 and 16%. This elevated mortality highlights critical gaps in our comprehension of sepsis etiology. Traditionally linked to bacterial and fungal pathogens, recent outbreaks of acute viral infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), among other regional epidemics, have underscored the role of viral pathogenesis in sepsis, particularly when critically ill patients exhibit classic symptoms indicative of sepsis. However, many cases of viral-induced sepsis are frequently underdiagnosed because standard evaluations typically exclude viral panels. Moreover, these viruses not only activate conventional pattern recognition receptors (PRRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) but also initiate primary antiviral pathways such as cyclic guanosine monophosphate adenosine monophosphate (GMP-AMP) synthase (cGAS)-stimulator of interferon genes (STING) signaling and interferon response mechanisms. Such activations lead to cellular stress, metabolic disturbances, and extensive cell damage that exacerbate tissue injury while leading to a spectrum of clinical manifestations. This complexity poses substantial challenges for the clinical management of affected cases. In this review, we elucidate the definition and diagnosis criteria for viral sepsis while synthesizing current knowledge regarding its etiology, epidemiology, and pathophysiology, molecular mechanisms involved therein as well as their impact on immune-mediated organ damage. Additionally, we discuss clinical considerations related to both existing therapies and advanced treatment interventions, aiming to enhance the comprehensive understanding surrounding viral sepsis.

Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL10 protein (pUL10) is a homolog of glycoprotein M (gM), which is conserved in herpesviruses. pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners.

Coronavirus disease 2019 (COVID-19) was first reported three years ago, when a group of individuals were infected with the original SARS-CoV-2 strain, based on which vaccines were developed. Here, we develop six human monoclonal antibodies (mAbs) from two elite convalescents in Wuhan and show that these mAbs recognize diverse epitopes on the receptor binding domain (RBD) and can inhibit the infection of SARS-CoV-2 original strain and variants of concern (VOCs) to varying degrees, including Omicron strains XBB and XBB.1.5. Of these mAbs, the two most broadly and potently neutralizing mAbs (7B3 and 14B1) exhibit prophylactic activity against SARS-CoV-2 WT infection and therapeutic effects against SARS-CoV-2 Delta variant challenge in K18-hACE2 KI mice. Furthermore, post-exposure treatment with 7B3 protects mice from lethal Omicron variants infection. Cryo-EM analysis of the spike trimer complexed with 14B1 or 7B3 reveals that these two mAbs bind partially overlapped epitopes onto the RBD of the spike, and sterically disrupt the binding of human angiotensin-converting enzyme 2 (hACE2) to RBD. Our results suggest that mAbs with broadly neutralizing activity against different SARS-CoV-2 variants are present in COVID-19 convalescents infected by the ancestral SARS-CoV-2 strain, indicating that people can benefit from former infections or vaccines despite the extensive immune escape of SARS-CoV-2.

With advances in cancer biology and an ever-deepening understanding of molecular virology, oncolytic virus (OV)-driven therapies have developed rapidly and become a promising alternative to traditional cancer therapies. In recent years, satisfactory results for oncolytic virus therapy (OVT) are achieved at both the cellular and organismal levels, and efforts are being increasingly directed toward clinical trials. Unfortunately, OVT remains ineffective in these trials, especially when performed using only a single OV reagent. In contrast, integrated approaches, such as using immunotherapy, chemotherapy, or radiotherapy, alongside OVT have demonstrated considerable efficacy. The challenges of OVT in clinical efficacy include the restricted scope of intratumoral injections and poor targeting of intravenous administration. Further optimization of OVT delivery is needed before OVs become a viable therapy for tumor treatment. In this review, the development process and antitumor mechanisms of OVs are introduced. The advances in OVT delivery routes to provide perspectives and directions for the improvement of OVT delivery are highlighted. This review also discusses the advantages and limitations of OVT monotherapy and combination therapy through the lens of recent clinical trials and aims to chart a course toward safer and more effective OVT strategies.

Supplementary Methods and Materials, Figure Legend

•The first global exploration of alternative splicing changes in COVID-19 and relevant respiratory diseases.•The specificities and similarities between alternative splicing events in different respiratory diseases.•Identification of regulatory network of RBP and alternative splicing in respiratory diseases.•Defining the interactions of alternative splicing and cell abundance in respiratory diseases.

Development of biologically relevant and clinically relevant human cerebral cortex models is demanded by mechanistic studies of human cerebral cortex‐associated neurological diseases and discovery of preclinical neurological drug candidates. Here, rational design of human–sourced brain‐like cortical tissue models is demonstrated by reverse engineering and bionic design. To implement this design, the acoustic assembly technique is employed to assemble hiPSC‐derived neural progenitors and neurons separately in a label‐free and contact‐free manner followed by subsequent neural differentiation and culture. The generated microtissues encapsulate the neuronal microanatomy of human cerebral‐cortex tissue that contains six‐layered neuronal architecture, a 400‐µm interlayer distance, synaptic connections between interlayers, and neuroelectrophysiological transmission. Furthermore, these microtissues are infected with herpes simplex virus type I (HSV‐1) virus, and the HSV‐induced pathogenesis associated with Alzheimer's disease is determined, including neuron loss and the expression of A β . Overall, a high‐fidelity human‐relevant in vitro histotypic model is provided for the cerebral cortex, which will facilitate wide applications in probing the mechanisms of neurodegenerative diseases and screening the candidates for neuroprotective agents.