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Animal gastrointestinal coronaviruses such as porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) can cause oral infection despite being enveloped viruses, implying that they are capable of passing through the highly acidic gastric lumen. This study focused on the low pH acid resistance of coronaviruses, including PEDV, TGEV and equine coronavirus (ECoV) in fasted-state simulated gastric fluid (FaSSGF) at pH 1.6, which simulated gastric fluid. Interestingly, PEDV, TGEV and ECoV retained infectivity in FaSSGF, whereas SARS-CoV-2 was rapidly inactivated. Although FaSSGF containing surfactants did not affect the acid resistance of these gastrointestinal coronaviruses, FaSSGF including pepsin showed inactivation effects against PEDV, TGEV and ECoV. These findings suggest that animal gastrointestinal coronaviruses possess the potential ability to withstand low pH conditions in gastric acid environments.
West Nile virus (WNV) is a mosquito-borne pathogen of global concern that can cause fatal neuroinvasive disease, and no specific prophylaxis or treatment exists for infections by WNV and most related orthoflaviviruses. Here, we isolated and characterized antibodies from WNV convalescent individuals and report that neutralizing autoantibodies against type I interferons did not impair antiviral antibody development. Among the monoclonal antibodies with potent neutralizing activity against WNV that were identified, W010 targeted a distinct epitope within the envelope protein domain III (EDIII) and conferred both pre- and post-exposure protection in a murine WNV model, even when interferon signaling was impaired. A second protective antibody, W014, exhibited broad cross-neutralization of other pathogenic orthoflavivirus members, including Japanese encephalitis virus, Murray Valley encephalitis virus, Saint Louis encephalitis virus, and Usutu virus. These findings identify key neutralizing epitopes on WNV EDIII and provide candidates for the development of antibody-based interventions against encephalitic orthoflavivirus infections.
Objective: To construct a reverse genetic system for the genotype ON1 of human respiratory syncytial virus subtype A (HRSV-A) expressing fluorescent reporter genes. Methods: Recombinant plasmids encoding EGFP or mCherry were constructed based on the 2019 Beijing HRSV-A ON1 dominant strain (6914). Recombinant viruses, rescued by co-transfecting BSR/T7-9 cells with helper plasmids, were identified via indirect immunofluorescence, whole-genome sequencing, and Western blot. Biological properties were characterized through fluorescent quantitative RT-PCR (qRT-PCR), immunostaining plaque assay and fluorescent focus assays (FFA). Results: Two recombinant viruses expressing EGFP or mCherry (rRSVA6914-EGFP and rRSVA6914-mCherry) were successfully rescued. Western blot analysis confirmed that the expression levels of key structural proteins (G, F, and N) in the recombinant strains were consistent with the parental virus. Multistep growth curve analysis revealed that the replication kinetics of the two recombinant viruses in HEp-2 cells did not differ significantly from those of the parental strain. Two recombinant viruses exhibited substantial neutralizing activity against both palivizumab and nirsevimab used in clinical settings. Furthermore, the viral titer of rRSVA6914-mCherry in A549 cells [(1.19±0.05)×105 PFU/ml] was significantly higher than in HEp-2 cells [(7.60±0.79)×104 PFU/ml] (P<0.001). For rRSVA6914-EGFP, the viral titers determined by immunostaining plaque assay and FFA methods were (1.15±0.17)×105 PFU/ml and (1.36±0.19)×105 FFU/ml. For rRSVA6914-mCherry, the corresponding titers were (3.50±0.23)×104 PFU/ml and (3.37±0.07)×104 FFU/ml. There was no statistically significant difference between the immunostaining plaque assay and FFA methods (both P>0.05). Conclusion: The HRSV-A genotype ON1 reverse genetic system expressing fluorescent reporter genes has been successfully constructed and systematically verified, providing a scientific tool for investigating the pathogenic mechanism of genotype ON1 and for screening antiviral drugs. 目的： 构建携带荧光报告基因的人呼吸道合胞病毒A亚型（HRSV-A）ON1基因型反向遗传学系统。 方法： 利用2019年北京地区分离的HRSV-A优势流行株ON1基因型6914株，构建携带增强型绿色荧光蛋白（EGFP）或红色荧光蛋白（mCherry）报告基因的重组质粒。通过与辅助质粒共转染至BSR/T7-9细胞拯救重组病毒；利用间接免疫荧光、全基因组测序、Western blot等方法鉴定病毒；基于荧光定量RT-PCR、免疫染色空斑试验及荧光灶形成试验（FFA）评价其生物学特性。 结果： 成功拯救出携带EGFP或mCherry报告基因的两株重组病毒（rRSVA6914-EGFP与rRSVA6914-mCherry）。Western blot证实重组病毒关键结构蛋白（G、F、N）表达水平与亲本株一致。多步生长曲线分析显示，两株重组病毒在HEp-2细胞中的复制动力学与亲本株基本一致。重组病毒对临床使用的帕丽珠单抗和尼塞韦单抗均表现出显著的中和活性。rRSVA6914-mCherry在A549细胞［（1.19±0.05）×105 PFU/ml］中的病毒滴度高于HEp-2细胞［（7.60±0.79）×104 PFU/ml］（P<0.001）。rRSVA6914-EGFP采用免疫染色空斑试验法和FFA法测得的病毒滴度为（1.15±0.17）×105 PFU/ml和（1.36±0.19）×105 FFU/ml，rRSVA6914-mCherry为（3.50±0.23）×104 PFU/ml和（3.37±0.07）×104 FFU/ml，两种方法测得的病毒滴度差异均无统计学意义（均P>0.05）。 结论： 成功构建并系统验证了携带荧光报告基因的HRSV-A ON1基因型反向遗传系统，可为ON1基因型致病机制研究及抗病毒药物筛选提供科学工具。.
Rodents, which account for over 40% of mammalian species and occupy diverse terrestrial ecosystems, are major reservoirs for zoonotic viruses, including Coronaviridae, Hantaviridae, and Arenaviridae. However, the rodent species acting as central hubs for viral diversity and transmission remain poorly defined. To address this, we conducted systematic sampling across 18 counties and cities on Hainan Island between 2023 and 2024. A total of 2550 animals were collected, representing 14 Rodentia species, one Erinaceomorpha species, and one Scandentian species. Based on a statistical sampling framework, 1284 individuals were selected to generate 125 pooled gut samples for virome analysis. We identified 527 viral RNA species and recovered 175 complete or near-complete viral genomes. Remarkably, Rattus norvegicus and Rattus tanezumi accounted for 83.9% (442/527) of detected viral species, 68.6% (72/105) of novel viral sequences, and 88.6% (93/105) of cross-species viruses. Eight novel viruses showed recombination signals, seven originating from these two species. Among 13 human pathogenic viruses identified, 12 were detected in Rattus norvegicus and Rattus tanezumi. These findings suggest that these species may function as hub hosts for viral maintenance and spread, emphasizing the value of targeted surveillance to reduce future potential spillover risks.
In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway.
Hepatitis B virus (HBV), a major human pathogen, replicates its DNA genome by protein-primed reverse transcription of a pregenomic RNA (pgRNA). This process is directed by the pgRNA-borne epsilon (ε) element, which provides the origin for minus-strand DNA synthesis and mediates coencapsidation of pgRNA with the viral polymerase (P protein) into nucleocapsids. ε adopts a thermodynamically stable hairpin structure that is remodeled upon formation of functional ε-P complexes, but the nature of the rearranged RNA structure and its implication for pgRNA encapsidation has remained elusive. Guided by in silico analyses of ε-like elements from distantly related nackednaviruses, we identify a distinct conformation of HBVε whose defining feature is a cryptic stem-loop (cSL), masked within the upper stem of ε. The P-dependent cSL conformation reorganizes key sequences into a compact structural unit that enables initiation of DNA synthesis and packaging of the viral pgRNA-P complex. RNAs engineered to favor cSL formation exhibit increased P protein affinity and strongly enhanced priming activity in vitro while maintaining replication competence in cells. Mutational analyses identify the cSL and its immediate vicinity, but not the remaining upper stem sequence, as the dominant determinants of ε function. Genetic variation in cSL-forming potential across hepadnaviruses links in vitro priming competence to the energetic accessibility of this alternative fold. Together, our findings reveal ε as a P protein-dependent RNA switch that tightly couples pregenome encapsidation to reverse transcription competence. This regulatory mechanism advances our understanding of HBV replication and could be exploited for antiviral intervention.
Positive-sense RNA viruses that constitute a large class of human pathogens employ various strategies to suppress and evade host immune defenses. Understanding the dynamic interaction between the viral life cycle and immune signaling is crucial to designing effective antiviral strategies. Although significant progress has been made, quantitative models that can accurately capture the intricate interactions and the intertwined dynamics during viral infection of cells remain missing. In this study, we develop a comprehensive mathematical model that integrates the intracellular viral life cycle with key cellular innate immune pathways, including RIG-I-mediated detection and JAK-STAT signaling. The model provides mechanistic insights into long-standing observations, capturing both virus-specific dynamics and innate immune response, and the key components driving their coupled dynamics. For example, a comparison of viruses shows how the Japanese Encephalitis virus undergoes a dramatic reduction in viral load in cells, due to its rapid replication that robustly activates the RIG-I pathway, in contrast to the poor immune control of Hepatitis C virus. More importantly, our model demonstrates how virus-host interactions exhibit a sharp transition boundary behavior, where minor differences in immune strength or viral suppression capacity can determine whether infections resolve or persist. We propose that ISG mRNA translation and viral replication predominantly dictate these bimodal infection outcomes. Additionally, the model not only recapitulates IFN desensitization but also identifies the molecular players involved. We demonstrate how our model's ability to capture IFN dynamics allows us to predict optimal timing and dosing strategies for interferon-based prophylactic therapies. Together, our approach reveals fundamental features that govern the delicate balance between the establishment of infection and immune control in RNA virus infections.
Indoor air pollution caused by particulate matter, VOCs, and pathogenic microorganisms has become more and more serious. The use of numerous petroleum-based polymer filters also causes environmental pollution. Therefore, it is urgent to develop green, degradable, and long-term-stable indoor air filters with synergistic purification of multiple pollutants. Polylactic acid nonwovens are considered candidates for the next generation of air filters because of their green and degradable properties. Herein, we developed a multistage S-scheme heterojunction (Ag3PO4/MXene/Bi2WO6, AMB)-modified PLA electrospun nanofiber membrane (AMB/PLA ENM) using continuous electrospinning equipment. The AMB/PLA ENMs not only exhibit high-efficiency air-filtration performance but also demonstrate robust photocatalytic activity for formaldehyde, bacteria, and viruses. The filtration performance of AMB/PLA ENMs for PM0.3 achieved 99.99%/77.21 Pa at 32 L/min. Moreover, AMB/PLA ENMs exhibit excellent visible-light-driven photocatalytic activities for gaseous formaldehyde, reaching 76.77% within 200 min, and for liquid ciprofloxacin at 82.25% within 240 min. The establishment of the built-in electric field and the increased oxygen vacancies by MXene in the AMB heterojunction significantly enhance the photocatalytic performance of AMB/PLA ENMs. It also shows a significant killing rate of 99.99% against Escherichia coli, Staphylococcus aureus, and the H1N1 virus. This synergistic purification of multiple pollutants is attributed to a dual-functional strategy that integrates multistage S-scheme heterojunction photocatalysts with a PLA electrospun nanofiber membrane to achieve highly efficient and low-pressure air filtration and long-term stable photocatalytic performance. In addition, the AMB/PLA ENMs maintain self-cleaning, good mechanical properties, and degradability, even after long-term utilization.
Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling.In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.
The workshop on 'Reactive and therapy induced BM changes linked to systemic infectious and non-infectious disorders including MAS/HLH' of the 22nd meeting of the European Association for Haematopathology held in Dubrovnik, 2024, included 58 cases. These encompassed a broad range of infections, autoimmune disorders, malignancies and therapy-effects, or a combination of these factors, of which 28 had an associated Hemophagocytic Lymphohistiocytosis (HLH) / Macrophage Activation Syndrome (MAS). Histoplasmosis, the infection mostly associated with HLH, showed a wide variability of BM changes, with or without focal lesions. Leishmaniasis, less often associated with HLH, induced BM changes that mimic myelodysplastic syndrome. BM changes after COVID-19 infection included myeloid and megakaryocytic hypoplasia, erythroid hyperplasia, dyserythropoiesis, hemophagocytosis, and possibly ring granulomas. Other infectious causes included viruses (HHV-8, EBV, Parvovirus B19), mycobacterial infections, and human granulocytic anaplasmosis. HLH may arise in association with the full spectrum of EBV-related disorders, including acute infection, systemic chronic active EBV disease, viral reactivation, and EBV-associated malignancies. BM changes associated with autoimmune diseases included plasmacytosis, myeloid hyperplasia and hemophagocytosis, with or without meeting the criteria of MAS/HLH, the latter often triggered by a secondary infection or exacerbation of the disease. Haematologic malignancies (EBV-positive and negative) with HLH encompassed B-cell, T-/NK-cell, and myeloid neoplasms. In addition, the workshop included therapy-induced BM changes, such as differentiation syndrome, lenalidomide-associated B-ALL, therapy-related dysplasia, gelatinous transformation, CAR-T-induced BM hypoplasia, and CAR-T-associated HLH. Finally, the workshop demonstrated the presence of T-cell expansions in a variety of conditions, which should not be misinterpreted as T-cell malignancy.
Cytochrome P450 (CYP450) enzymes represent a critical class of detoxification enzymes that play essential roles in antioxidant defense, xenobiotic metabolism, and innate immune responses in animals. However, knowledge regarding the gene sequences and functional characteristics of CYP450 genes in mollusks remains limited. In this study, a novel CYP450 gene, designated Cf-CYP450, was cloned from the Zhikong scallop (Chlamys farreri). The open reading frame (ORF) of Cf-CYP450 spans 1497 bp and encodes a polypeptide of 498 amino acids. The N-terminus of the Cf-CYP450 protein contains a transmembrane domain, whereas the C-terminus harbors a conserved P450 domain. Phylogenetic analysis indicates that Cf-CYP450 is a newly identified member of the molluscan CYP450 family. Quantitative real-time PCR analysis revealed that Cf-CYP450 mRNA is ubiquitously expressed across various scallop tissues, with the highest expression observed in gill tissue. Furthermore, the expression of Cf-CYP450 is inducible by multiple stimuli, including pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), and poly(I:C). As anticipated, infections with bacteria (including various Vibrio species) and viruses (e.g., acute viral necrosis virus) significantly alter Cf-CYP450 expression levels, suggesting its involvement in scallop innate immunity. Dual-luciferase reporter (DLR) assays further demonstrated that overexpression of Cf-CYP450 in HEK293T cells markedly suppresses the activity of multiple immune-related reporter genes. Additionally, Cf-CYP450 dose-dependently inhibits the activation of ISRE (Interferon stimulated response element) reporter genes induced by poly(I:C) or scallop IRF1 (Interferon regulatory factor 1). Notably, Cf-CYP450 expression in HEK293T cells also induces significant phosphorylation of MAPK (Mitogen-activated protein kinase) signaling proteins, such as JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular regulated protein kinases 1/2). These findings collectively indicate that Cf-CYP450 plays a pivotal role in scallop defense against exogenous pathogens and may mediate diverse immune responses through participation in distinct signaling pathways or regulation of specific effector genes.
Engineering the genetic code-by reassigning multiple of the 64 natural codons-enables making organisms resistant to all viruses, preventing genetic information exchange, and allowing the biosynthesis of genetically encoded unnatural polymers. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using genome synthesis, directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple synthetic Escherichia coli genomes. We construct six partially recoded E. coli strains bearing up to 45.8% of a synthetic genome with a deleterious 57-codon genetic code. As our analyses revealed widespread defects-including unassigned codons in Syn61 and Syn57-we apply multi-omics to revise our genome design and mitigate defects. Using multi-omics, we show that recoding induces transcriptional and translational changes leading to fitness defects under hundreds of conditions. Finally, we develop a multi-omics-guided evolution strategy that rapidly restores fitness, enabling genome synthesis with radical changes.
Susceptibility to infection after solid organ transplantation (SOT) reflects interactions among epidemiologic exposures, allograft-specific factors, pharmacologic immunosuppression and host innate and adaptive immune functions. Substantial variability in susceptibility to viral, bacterial, and fungal infections occurs among recipients despite comparable immunosuppressive regimens. Accumulating evidence demonstrates that allelic variation, including single nucleotide polymorphisms, occurs in genes involved in multiple aspects of innate immune function and influence infectious risk after SOT. These genes encode pattern recognition receptors, complement components, phagocyte and natural killer cells functions, soluble opsonins, and cytokines. These variants are associated with altered susceptibility to bacterial, invasive fungal, and viral infections including cytomegalovirus, Epstein-Barr virus, BK polyomavirus, and hepatitis viruses. The contribution of allelic variation to infectious susceptibility remains incompletely defined. Identification of high-risk genotypes may enable individualized infectious surveillance, tailored antimicrobial prophylaxis, and host-directed interventions to advance precision transplantation.
In insects, two lysozyme types have been described: c-type lysozymes, which are well characterized at both the genetic and functional levels, and i-type lysozymes, whose functions remain comparatively less understood. Although traditionally recognized for their antibacterial activity, insect lysozymes have been implicated in a broad range of biological processes, including defense against fungi, viruses, and parasites, as well as digestion, microbiota regulation, nutrient recycling, and developmental transitions. In this review, we synthesize current knowledge on the diversity, structure, and functions of insect lysozymes across different taxa and feeding strategies. We propose that the multifunctionality of these enzymes positions them at the interface of immunity, metabolism, and development. Finally, we highlight major knowledge gaps, including the limited understanding of i-type lysozymes and the scarcity of studies in hemimetabolous insects, which constrain our understanding of lysozyme evolution and functional diversification.
Rabies continues to pose a significant public health challenge in China. To offer a scientific foundation for formulating rabies prevention and control strategies, this study conducted a national survey to comprehend the current status of rabies post-exposure prophylaxis (PEP) clinics construction and the surveillance of rabies-exposed individuals in China. The Chinese Center for Disease Control and Prevention (CCDC) developed a national rabies prevention and control questionnaire, and conducted a survey across 31 provinces in Mainland China. The questionnaire was completed by the person in charge of rabies prevention and control in each provincial disease prevention and control institution. By the end of 2022, 24,304 PEP clinics were reported in Mainland China. The number of PEP clinics per capita in China is 1.75 per 100,000. Nationwide, 47% of PEP clinics were capable of handling category III exposures. A total of 10,770,543 PEP clinic visits were reported. East China reported the highest number of rabies exposures (4,022,265). Among category III exposures, only 35% reported using rabies immunoglobulin (RIG). 97% of rabies exposures were attacked by dogs or cats. In South China, exposures caused by cats were nearly as frequent as those caused by dogs (47% and 48% respectively). The distribution of PEP clinics across China is uneven. This pattern appears broadly consistent with the historical geographic distribution of rabies risk, which may reflect a risk-informed allocation of resources. We recommended implementing tiered prevention and control strategies, with emphasis on enhancing the stockpile of RIG, dogs and cats population management, and health education in regions with higher rabies exposure risks. In provinces with a high incidence of rabies outbreaks, it is essential to actively advocate for the inclusion of rabies vaccines and RIG in the reimbursement scope of the New Rural Cooperative Medical Insurance Scheme, and raise the reimbursement rate and limit. The "One Health" approach should be adopted, including mandatory immunization and surveillance management systems for dogs and cats.
CoronaVac, an inactivated SARS-CoV-2 vaccine, was one of the first deployed during the COVID-19 pandemic. Given the limited vaccine availability and the urgent need to assess effectiveness in target populations, a risk-based allocation clinical trial was designed to generate evidence under the ethical and logistical constraints at the beginning of the COVID-19 vaccination. In Manaus, Brazil, participants working in public service (education and public safety) aged 18-49 years were assessed regarding the risk of severe COVID-19 disease. Participants with one or more comorbidities, who were considered at higher risk of severe COVID-19 outcomes if infected, were allocated to early vaccination, while participants without comorbidities were enrolled as an unvaccinated comparison group. Blood samples were collected before each vaccine dose (D0 and D28) and during in-person follow-up visits (D90 and D180). Additional information was obtained through phone calls. Clinical cases of COVID-19, hospitalizations, deaths, and antibody titrations were the evaluated endpoints, considered after the second week following the second dose of the vaccine. A total of 6,226 participants were included: 1,139 in the low-risk group, and 5,087 in the high-risk vaccinated group. COVID-19 incidence was statistically significantly lower (p < 0.001) in the vaccinated group at D28 (5.2% vs. 40.9%) and D90 (10.3% vs 31.6%). Hospitalization and death rates were low, with no difference observed between the groups. There was a decline in highly reactive titers at D180 in the vaccinated group. This pioneer risk-based allocation clinical trial provided evidence that CoronaVac reduced the risk of severe COVID-19 outcomes among individuals with comorbidities, effectively aligning their risk with that of lower-risk, unvaccinated individuals. Beyond its clinical implications, the study underscores the importance of adaptive, real-world research designs in rapidly generating actionable evidence in response to emerging public health threats. ClinicalTrials.gov Registration: NCT04789356.
Rabies encephalitis, caused by rabies virus, is an almost uniformly fatal neurotropic infection once clinical disease develops. Classical presentations of furious rabies, typically associated with a definitive history of dog bite and characteristic features such as hydrophobia and aerophobia, progress rapidly to death. In contrast, atypical presentations, particularly paralytic rabies, often lack classical clinical features posing significant diagnostic challenges. Diagnostic confirmation may be further complicated by recent post-exposure vaccination, confounding antibody assays, while molecular detection remains technically demanding and limited in availability. With advances in intensive care, rare instances of survival have been reported, especially in children. The authors report the case of a 2-y-old toddler with history of dog bite, who presented with rapidly progressive quadriparesis, where the diagnosis of paralytic rabies was supported by rising CSF and serum rabies virus neutralising antibodies. The child survived following comprehensive intensive care and was successfully discharged after a six-week hospital stay.
Dengue fever, a mosquito-borne disease caused by dengue virus (DENV), has become a global health problem, and no FDA-approved drug is currently available. Qingwen Baidu Decoction (QBD) is used to treat the critical phase of dengue fever in China, but its mechanism of action remains unclear. In this work, we integrated bioinformatics analysis, machine learning, and network pharmacology to investigate the possible molecular targets and potential active chemical components of QBD. Common targets between differentially expressed genes from DENV infected samples and predicted targets of QBD were identified by bioinformatics analysis and refined by machine learning algorithms including LASSO, random forest and SVM-RFE. Three core genes, CXCL10, EZH2 and EPHB2 were significantly overexpressed in dengue fever patients, indicating their potential diagnostic and therapeutic value. Single cell transcriptome analysis further revealed that QBD primarily targets dendritic cells, monocytes and macrophages. Immune infiltration analysis using ssGSEA showed that these three core genes were significantly associated with CD4+ and CD8+ T cell subtypes, suggesting their involvement in host immune regulation. Molecular docking and molecular dynamics simulations identified eight chemical components of QBD as potential active ingredients. Based on these computational predictions, we hypothesize that QBD may exert its therapeutic effects through dual mechanisms, including directly binding to DENV proteins to inhibit viral replication, while also regulating the function of CXCL10 and EZH2 to alleviate DENV-induced inflammatory responses and modulate host immunity. These results provide a theoretical reference for future experimental validation and drug development.
Canine morbillivirus (CDV) vaccine quality control requires rapid and reliable analytical methods to ensure vaccine consistency and compliance with regulatory standards. In this study, a gold nanoparticle-based lateral flow immunoassay (LFA-CDV) was developed and evaluated as a rapid antigen detection platform for modified live virus (MLV) vaccine assessment. A total of 100 CDV vaccine batches were analyzed and compared with multiplex real-time RT-PCR targeting the H gene, which served as an analytical reference method. Gold nanoparticles with a mean diameter of approximately 20 ± 5 nm were synthesized and conjugated with monoclonal antibodies specific to the CDV envelope protein to enable visual immunochromatographic detection. The assay demonstrated high analytical specificity, with no cross-reactivity against tested heterologous viral or bacterial agents and no interference from common substances. Analytical sensitivity testing established a limit of detection of 10 ³ TCID₅₀/0.1 mL, which is compatible with regulatory potency requirements for CDV vaccines. Comparative evaluation showed a sensitivity of 98.84% (95% CI: 93.7-99.9), specificity of 85.71% (95% CI: 57.2-98.2), and overall accuracy of 97.0% (95% CI: 91.5-99.4). Positive and negative predictive values were 97.70% and 92.31%, respectively. Because RT-qPCR served as an analytical comparator, positive percent agreement (PPA) and negative percent agreement (NPA) were also calculated and reached 98.84% and 85.71%, respectively. Overall agreement between methods was 97%, with a Cohen's kappa coefficient of 0.90 (95% CI: 0.78-1.00), indicating almost perfect agreement. These findings demonstrate that the developed LFA-CDV assay is a rapid, specific, and analytically robust tool for vaccine quality assessment. Integration of LFA with molecular methods may provide an efficient complementary approach for vaccine evaluation and batch-release testing.
Hepatitis C virus self-testing (HCVST) has emerged as a potential strategy to expand testing among key populations. We assessed the feasibility of HCVST in cisgender men-who-have-sex-with-men (cis-MSM) and transgender women (TGW) on pre-exposure prophylaxis (PrEP). This cross-sectional study included cis-MSM or TGW attending a PrEP consultation in Rio de Janeiro (Brazil). Participants performed HCVST using blood-based and oral-fluid kits on the same day under observation. Difficulties, errors and assistance during HCVST were recorded. Re-reading and re-testing concordance [Kappa(k)] and values/preferences were assessed. A total of 250 participants (88% cis-MSM, age = 34 [IQR,28-41] years, 42% with high education level) were included. The main steps where participants requested assistance (95%CI) for blood-based HCVST were to add buffer [35.6%(29.9-41.8)] and to collect blood sample with the dropper [34.0%(28.4-40.1)]. The main error during oral fluid HCVST was incorrect collection of oral fluid [29.6% (95%CI,24.2-35.6)]. A total of 62.4% (95%CI,56.2-68.2) and 28.8% (95%CI,23.5-34.8) participants needed assistance in at least one step of blood-based and oral fluid HCVST, respectively. Lower education level was associated with higher odds of needing assistance for blood-based HCVST [aOR = 2.07 (95%CI,1.99-3.59),p = 0.009]. Re-reading and re-testing k-indexes were 0.92 and 0.89 for blood-based, and 1.00 and 0.75 for oral fluid HCVST, respectively. More than 95% of people felt safe; would repeat or would recommend HCVST. A total of 46.4% (95%CI,40.3-52.6) preferred oral fluid versus 36.4% (95%CI,30.6-42.6) who preferred blood-based. A relatively high proportion of participants needed assistance, especially for blood-based HCVST. Despite these challenges, high re-reading and re-testing agreements were observed and HCVST was well-accepted.