Zinc is an essential micronutrient with well-characterized immunomodulatory properties and has been widely investigated in viral infectious diseases, yet its specific functional role in neurotropic viral encephalitis remains poorly elucidated. In this study, we demonstrate that a zinc-supplemented (ZnS) dietary intervention confers protection against Japanese encephalitis virus (JEV) infection by suppressing macrophage-mediated inflammatory pathology in the central nervous system (CNS). Besides, zinc inhibited the phosphorylation of Bruton's tyrosine kinase (BTK) and NF-κB p65 in macrophages, thereby effectively curbing excessive neuroinflammation and alleviating JEV-induced neuronal damage in the murine brain. Our findings have identified a previously unrecognized mechanism by which zinc regulates immune responses in the central nervous system, and suggest zinc supplementation as a potential intervention for neurological complications arising from central nervous system infections.

Multiple studies have revealed a potential role for integrin α4 (ITGA4) in the pathogenesis of SARS-CoV-2/HIV/SIV infection, but its role in flavivirus infection remains elusive. Here, we used Japanese encephalitis virus (JEV) as a model, aiming to dissect the regulatory role of ITGA4 in flavivirus infection. We found that JEV infection activates vitamin A metabolism, which in turn induces the upregulation and activation of ITGA4. ITGA4 triggers cytoskeletal reorganization enhancing early viral replication in monocytes via the ITGA4-HSP70-pFAK-Rac1 signaling axis, and reinforces interactions between immune cells and brain microvascular endothelial cells, reducing the expression of tight junction proteins (ZO-1 and Occludin) and increasing blood-brain barrier permeability, thereby enabling viral neuroinvasion and inflammatory cell infiltration. Genetic knockdown of ITGA4 markedly reduces viral load, mitigates neuronal damage, and improves survival in JEV-infected mice. Moreover, ITGA4 enhances the replication of multiple flaviviruses (JEV, dengue virus serotype 2, Zika virus) in monocytes, highlighting a conserved pro-viral function. Collectively, this is the first report demonstrating that ITGA4 as a pivotal host factor orchestrating flavivirus pathogenesis, uncovering the novel dual mechanism through which ITGA4 links vitamin A metabolism, cytoskeletal remodeling, and blood-brain barrier (BBB) disruption to promote flaviviral neuroinvasion.

Toll-like receptors (TLRs) are a family of pattern recognition receptors that recognise both pathogen-associated and damage-associated molecular patterns. While their expression was initially believed to be restricted to immune cells, accumulating evidence now demonstrates their presence across multiple neural cell types. Due to their significant involvement in neuroinflammatory and neurodegenerative processes, TLRs have garnered growing attention for their potential contributions to neurocognitive disorders, including Alzheimer's disease, Parkinson's disease, stroke, amyotrophic lateral sclerosis, and other forms of dementia. Potential treatment targets for lowering neuroinflammation and slowing the evolution of neurocognitive diseases include TLR signalling pathways, namely the MYD88-dependent and TRIF-dependent cascades. To initiate signalling, Toll-like receptors (TLRs) recruit specific adaptor molecules that activate the transcription factors NF-κB and IRFs, which regulate the induction of innate immune responses. Over the past decade, a combination of genetic, biochemical, structural, cellular, and bioinformatics approaches has been utilised to elucidate the detailed molecular mechanisms underlying TLR signalling. These studies have clarified how TLRs interact with cytosolic innate immune sensors to orchestrate effective immunological reactions. The function of different TLRs expressed in various brain immune cells and their contribution to the pathophysiology of neuroinflammation are described. This paper discusses the involvement of TLRs in autoimmune and neuroinflammatory circumstances like multiple sclerosis (MS), bacterial meningitis, viral encephalitis, stroke, Alzheimer's disease, and Parkinson's disease. It is intended for TLR biologists and immunologists studying neuroinflammation, as well as neuroscientists delving into central nervous system processes mediated by TLRs.

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.

Self-amplifying RNA (saRNA) derived from alphavirus replicons enables robust intracellular RNA amplification and high-level protein expression at substantially lower doses than nonreplicating messenger RNA (mRNA) platforms. Venezuelan equine encephalitis virus (VEEV)-based replicons lacking the viral structural genes are among the most extensively characterized saRNA backbones, combining efficient cytoplasmic replication with a favorable safety profile. However, incorporating chemical nucleoside modifications such as N1-methylpseudouridine (m1ψ), now standard in conventional mRNA workflows, can directly impact replicase activity and hinder RNA amplification, necessitating backbone-specific optimization. Recent studies have shown that incorporation of 5-methylcytidine (m5C) preserves VEEV saRNA replication while reducing innate immune activation and improving expression durability. In an independent study, phosphatase treatment to remove residual 5'-triphosphates was shown to enhance saRNA functionality. Building on these findings, here we describe a streamlined protocol for generating m5C-modified VEEV-based saRNA using a single-step in vitro transcription (IVT) strategy. This protocol utilizes PCR-generated DNA templates with an encoded poly(A) tail, CleanCap AU for co-transcriptional capping, and post-transcriptional phosphatase treatment to minimize immunostimulatory RNA species. Finally, the RNA was purified using the phenol-chloroform-isoamyl alcohol method and functionally evaluated by transfection into HEK293T cells. This protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA suitable for downstream functional and translational studies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: IVT and purification of saRNA with modified nucleotides Basic Protocol 2: Functional assessment of IVT-generated saRNA in HEK293T Cells.

Nasu-Hakola disease is a rare autosomal recessive disorder characterized by progressive cognitive decline and bone cyst formation and is commonly associated with triggering receptor expressed on myeloid cells 2 (TREM2) variants. Herein, we report a novel TREM2 frameshift variant in a middle-aged man from a consanguineous Chinese family who presented with early-onset dementia and right ankle pain. Neuroimaging and skeletal examinations revealed cerebral atrophy and bone cystic lesions. Whole-exome sequencing followed by Sanger confirmation identified a homozygous TREM2 c.257del (p.D86Afs*103) variant. The patient was diagnosed with Nasu-Hakola disease, and his cognitive deterioration continued despite treatment with donepezil and memantine. Functional assays in human embryonic kidney 293T cells demonstrated preserved mRNA expression of the mutant construct but markedly reduced protein levels compared with that of wild-type. We also conducted a descriptive literature review of 54 previously reported cases of homozygous or compound heterozygous TREM2 variants to highlight the variability in neurodegenerative and skeletal phenotypes. To the best of our knowledge, this is the first report of a TREM2 c.257del variant in a Chinese family. Our findings expand the mutational spectrum of Nasu-Hakola disease and highlight substantial phenotypic heterogeneity, emphasizing the importance of early genetic testing in patients with unexplained early-onset dementia, even in the absence of bone lesions.

Kyasanur Forest Disease (KFD) is a tick-borne viral haemorrhagic fever endemic to forested regions of South India, with case fatality rates ranging from 3% to 10% and seasonal outbreaks causing recurrent illness among at-risk populations such as forest workers and villagers. The historically used formalin-inactivated KFD vaccine showed limited effectiveness in field studies, providing only about 62% protection. This moderate level of protection highlights the need for improved vaccines that offer higher efficacy and longer-lasting immunity for populations living in endemic areas. A major challenge in vaccine development is the limited understanding of immune responses that protect against KFD, along with the lack of preclinical models that fully reflect the disease as it occurs in humans. Existing animal models-including murine systems and limited non-human primate studies -have contributed to understanding viral replication, lethality, viraemia kinetics, and basic immunological responses. However, murine models often fail to reproduce the full spectrum of human immunopathology, particularly haemorrhagic manifestations and complex host immune responses, while non-human primate models remain limited by cost, accessibility, and incomplete characterisation. Furthermore, variability in experimental endpoints and the absence of standardized immunogenicity and neutralisation assays restrict cross-study comparability and hinder identification of immune correlates of protection. This review critically synthesises current knowledge on KFD animal models and host immune responses, identifying key gaps in translational relevance. We propose a prioritised roadmap that includes: (i) development and validation of advanced preclinical models that better mimic human disease progression and immune dysregulation; (ii) systematic identification of serological and cellular correlates of protection; and (iii) standardisation of virological, serological, and immunological assays to support regulatory evaluation and vaccine benchmarking. Addressing these challenges through coordinated interdisciplinary efforts will accelerate the development of next-generation KFD vaccines and therapeutics tailored to endemic populations.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that causes a substantial threat to human health and livestock; however, the epitranscriptomic mechanisms that support its replication remain poorly defined. Here, we identify a proviral host factor C2H2 zinc-finger protein ZNF33B that promotes JEV infection through coupling N[6]-methyladenosine (m[6]A) RNA modification to autophagy regulation. Mechanistically, ZNF33B recruits METTL14 to stabilize the METTL3-METTL14 methyltransferase complex, thereby increasing global m[6]A deposition. Multi-omics analyses reveal that ZNF33B selectively binds m[6]A-modified sites within the antiviral transcript Trim25 (c.1567 and c.1669 bp) to accelerate its decay. We further demonstrate that TRIM25 functions as an E3 ubiquitin ligase that catalyzes K48-linked ubiquitination of ATG7 at lysines 389 and 423, leading to its proteasomal degradation and ultimately suppressing autophagic flux. In contrast, ZNF33B-mediated Trim25 degradation counteracts its inhibitory effect on autophagy, creating a favorable environment for viral replication. In vivo, adeno-associated virus (AAV)-mediated ZNF33B delivery increases mouse brain m[6]A levels, decreases TRIM25 expression, elevates ATG7 abundance, exacerbates JEV-induced neuropathology, and accelerates mouse mortality. Together, these findings reveal a previously uncharacterized ZNF33B-m[6]A-TRIM25-autophagy axis that JEV hijacks to evade host antiviral responses, providing new insights into flaviviral pathogenesis and potential therapeutic targets.

The maturation and infectivity of orthoflaviviruses is driven by interactions and structural changes of the two envelope proteins, prM and E, which are controlled by protonation and deprotonation events during the viral life cycle when the virus encounters acidic and neutral pH environments inside and outside the cell. Histidine residues act as sensors for these pH-induced conformational transitions, which differ between mosquito-borne and tick-borne orthoflaviviruses in some aspects. We identified six histidine residues that are conserved among the envelope proteins of mammalian tick-borne orthoflaviviruses, but not among those of mosquito-borne orthoflaviviruses. These residues might account for some of the structural differences observed between these two orthoflavivirus groups. We therefore conducted a mutational analysis by replacing each of the conserved histidine residues with alanine and determined the effect of these mutations on the maturation and infectivity of tick-borne encephalitis virus (TBEV). One of the histidine residues (H208, located in an insert unique to mammalian tick-borne orthoflaviviruses at the prM-binding site in E) was shown to be an important determinant of efficient prM cleavage. In line with recent findings regarding the infectivity of immature tick-borne orthoflaviviruses, this mutant was only slightly less infectious than the mature wild-type virus. The presence of histidine residue 208 appears to be linked to the particular requirements of the complex ecological life cycle of TBEV and likely reflects an evolutionary adaptation to the specific interactions of the virus with mammalian hosts and/or tick vectors.

Brain [18F]FDG PET can reveal metabolic abnormalities that precede, exceed, or clarify structural MR imaging findings. Among inflammatory brain diseases, the strongest clinical rationale is currently in autoimmune encephalitis, where fluorodeoxyglucose (FDG) PET increases diagnostic sensitivity, supports syndrome-oriented metabolic pattern recognition, and may contribute to selected follow-up. In viral encephalitis, use is selective rather than routine. In post-coronavirus infectious disease (COVID) condition and related postinfectious syndromes, FDG PET may support biological stratification and differential diagnosis in a subset of patients. Interpretation remains highly dependent on clinical context and methods. Translocator protein (TSPO) PET adds mechanistic information on neuroimmune activation but belongs mainly to the research domain.