The Innate Immune Response to Mycobacterium tuberculosis Infection.

Infection with Mycobacterium tuberculosis causes >1.5 million deaths worldwide annually. Innate immune cells are the first to encounter M. tuberculosis, and their response dictates the course of infection. Dendritic cells (DCs) activate the adaptive response and determine its characteristics. Macrophages are responsible both for exerting cell-intrinsic antimicrobial control and for initiating and maintaining inflammation. The inflammatory response to M. tuberculosis infection is a double-edged sword. While cytokines such as TNF-α and IL-1 are important for protection, either excessive or insufficient cytokine production results in progressive disease. Furthermore, neutrophils-cells normally associated with control of bacterial infection-are emerging as key drivers of a hyperinflammatory response that results in host mortality. The roles of other innate cells, including natural killer cells and innate-like T cells, remain enigmatic. Understanding the nuances of both cell-intrinsic control of infection and regulation of inflammation will be crucial for the successful development of host-targeted therapeutics and vaccines.

Decoding Toll-like receptors: Recent insights and perspectives in innate immunity.

Toll-like receptors (TLRs) are an evolutionarily conserved family in the innate immune system and are the first line of host defense against microbial pathogens by recognizing pathogen-associated molecular patterns (PAMPs). TLRs, categorized into cell surface and endosomal subfamilies, recognize diverse PAMPs, and structural elucidation of TLRs and PAMP complexes has revealed their intricate mechanisms. TLRs activate common and specific signaling pathways to shape immune responses. Recent studies have shown the importance of post-transcriptional regulation in TLR-mediated inflammatory responses. Despite their protective functions, aberrant responses of TLRs contribute to inflammatory and autoimmune disorders. Understanding the delicate balance between TLR activation and regulatory mechanisms is crucial for deciphering their dual role in immune defense and disease pathogenesis. This review provides an overview of recent insights into the history of TLR discovery, elucidation of TLR ligands and signaling pathways, and their relevance to various diseases.

Host immune responses in the central nervous system during fungal infections.

Fungal infections in the central nervous system (CNS) cause high morbidity and mortality. The frequency of CNS mycosis has increased over the last two decades as more individuals go through immunocompromised conditions for various reasons. Nevertheless, options for clinical interventions for CNS mycoses are still limited. Thus, there is an urgent need to understand the host-pathogen interaction mechanisms in CNS mycoses for developing novel treatments. Although the CNS has been regarded as an immune-privileged site, recent studies demonstrate the critical involvement of immune responses elicited by CNS-resident and CNS-infiltrated cells during fungal infections. In this review, we discuss mechanisms of fungal invasion in the CNS, fungal pathogen detection by CNS-resident cells (microglia, astrocytes, oligodendrocytes, neurons), roles of CNS-infiltrated leukocytes, and host immune responses. We consider that understanding host immune responses in the CNS is crucial for endeavors to develop treatments for CNS mycosis.

ADAR RNA Modifications, the Epitranscriptome and Innate Immunity.

Modified bases act as marks on cellular RNAs so that they can be distinguished from foreign RNAs, reducing innate immune responses to endogenous RNA. In humans, mutations giving reduced levels of one base modification, adenosine-to-inosine deamination, cause a viral infection mimic syndrome, a congenital encephalitis with aberrant interferon induction. These Aicardi-Goutières syndrome 6 mutations affect adenosine deaminase acting on RNA 1 (ADAR1), which generates inosines in endogenous double-stranded (ds)RNA. The inosine base alters dsRNA structure to prevent aberrant activation of antiviral cytosolic helicase RIG-I-like receptors. We review how effects of inosines, ADARs, and other modified bases have been shown to be important in innate immunity and cancer.

The battle between host antiviral innate immunity and immune evasion by cytomegalovirus.

Cytomegalovirus (CMV) has successfully established a long-lasting latent infection in humans due to its ability to counteract the host antiviral innate immune response. During coevolution with the host, the virus has evolved various evasion techniques to evade the host's innate immune surveillance. At present, there is still no vaccine available for the prevention and treatment of CMV infection, and the interaction between CMV infection and host antiviral innate immunity is still not well understood. However, ongoing studies will offer new insights into how to treat and prevent CMV infection and its related diseases. Here, we update recent studies on how CMV evades antiviral innate immunity, with a focus on how CMV proteins target and disrupt critical adaptors of antiviral innate immune signaling pathways. This review also discusses some classic intrinsic cellular defences that are crucial to the fight against viral invasion. A comprehensive review of the evasion mechanisms of antiviral innate immunity by CMV will help investigators identify new therapeutic targets and develop vaccines against CMV infection.

Modeling suggests gene editing combined with vaccination could eliminate a persistent disease in livestock.

Recent breakthroughs in gene-editing technologies that can render individual animals fully resistant to infections may offer unprecedented opportunities for controlling future epidemics in farm animals. Yet, their potential for reducing disease spread is poorly understood as the necessary theoretical framework for estimating epidemiological effects arising from gene-editing applications is currently lacking. Here, we develop semistochastic modeling approaches to investigate how the adoption of gene editing may affect infectious disease prevalence in farmed animal populations and the prospects and time scale for disease elimination. We apply our models to the porcine reproductive and respiratory syndrome (PRRS), one of the most persistent global livestock diseases to date. Whereas extensive control efforts have shown limited success, recent production of gene-edited pigs that are fully resistant to the PRRS virus have raised expectations for eliminating this deadly disease. Our models predict that disease elimination on a national scale would be difficult to achieve if gene editing was used as the only disease control. However, from a purely epidemiological perspective, disease elimination may be achievable within 3 to 6 y, if gene editing were complemented with widespread and sufficiently effective vaccination. Besides strategic distribution of genetically resistant animals, several other key determinants underpinning the epidemiological impact of gene editing were identified.

Arabidopsis immune responses triggered by cellulose- and mixed-linked glucan-derived oligosaccharides require a group of leucine-rich repeat malectin receptor kinases.

The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked ß-1,3/ß-1,4-glucans (MLGs; e.g. ß-1,4-D-(Glc)2 -ß-1,3-D-Glc, MLG43) and cellulose (e.g. ß-1,4-D-(Glc)3 , CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. ß-1,4-d-(GlcNAc)6 , CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1-igp4 are altered in three RKs - AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) - with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD-RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD  = 1.19 ± 0.03 µm) and cellopentaose (KD  = 1.40 ± 0.01 µM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis.

Evolution of the Major Components of Innate Immunity in Animals.

Innate immunity is present in all animals. In this review, we explore the main conserved mechanisms of recognition and innate immune responses among animals. In this sense, we discuss the receptors, critical for binding to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs); the downstream signaling proteins; and transcription factors that govern immune responses. We also highlight conserved inflammatory mediators that are induced after the recognition of DAMPs and PAMPs. At last, we discuss the mechanisms that are involved in the regulation and/or generation of reactive oxygen species (ROS), influencing immune responses, like heme-oxygenases (HOs).

PTI-ETI synergistic signal mechanisms in plant immunity.

Plants face a relentless onslaught from a diverse array of pathogens in their natural environment, to which they have evolved a myriad of strategies that unfold across various temporal scales. Cell surface pattern recognition receptors (PRRs) detect conserved elicitors from pathogens or endogenous molecules released during pathogen invasion, initiating the first line of defence in plants, known as pattern-triggered immunity (PTI), which imparts a baseline level of disease resistance. Inside host cells, pathogen effectors are sensed by the nucleotide-binding/leucine-rich repeat (NLR) receptors, which then activate the second line of defence: effector-triggered immunity (ETI), offering a more potent and enduring defence mechanism. Moreover, PTI and ETI collaborate synergistically to bolster disease resistance and collectively trigger a cascade of downstream defence responses. This article provides a comprehensive review of plant defence responses, offering an overview of the stepwise activation of plant immunity and the interactions between PTI-ETI synergistic signal transduction.

Protective and stochastic correlation between infectious diseases and autoimmune disorders.

A priori, early exposure to a wide range of bacteria, viruses, and parasites appears to fortify and regulate the immune system, potentially reducing the risk of autoimmune diseases. However, improving hygiene conditions in numerous societies has led to a reduction in these microbial exposures, which, according to certain theories, could contribute to an increase in autoimmune diseases. Indeed, molecular mimicry is a key factor triggering immune system reactions; while it seeks pathogens, it can bind to self-molecules, leading to autoimmune diseases associated with microbial infections. On the other hand, a hygiene-based approach aimed at reducing the load of infectious agents through better personal hygiene can be beneficial for such pathologies. This review sheds light on how the evolution of the innate immune system, following the evolution of molecular patterns associated with microbes, contributes to our protection but may also trigger autoimmune diseases linked to microbes. Furthermore, it addresses how hygiene conditions shield us against autoimmune diseases related to microbes but may lead to autoimmune pathologies not associated with microbes.

Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusanaga.

Lectins are versatile proteins that specifically recognize and interact with sugar moieties expressed on the cell surface. The potential of lectin in drug targeting and delivery has instigated interest to identify natural lectins. Crabs have been identified as a rich source of lectin because the innate immune system is activated on encounter of pathogens and helps in the production of lectin. Although the presence of lectins in crab's hemolymph is well documented, little information about lectin in hepatopancreas, a vital organ for immunity and digestion in crustaceans, is currently available. A calcium dependent lectin (75 kDa) was purified from the hepatopancreas of the freshwater crab Oziotelphusa naga by bioadsorption and fetuin linked Sepharose 4B affinity chromatography technique. The isolated hepatopancreas lectin is calcium dependent and maximum agglutination was observed with rabbit erythrocytes. The hemagglutinating activity of the hepatopancreas lectin was effectively inhibited by sugars, such as α-lactose, GlcNAc, trehalose and NeuAc. Compared to sialylated N-glycosylated proteins including transferrin and apo transferrin, sialylated O-glycosylated proteins like fetuin exhibited stronger inhibitory effect. The ability of erythrocytes to bind hepatopancreas lectin has been diminished by desialylation of the potent inhibitor, indicating the significance of sialic acid in lectin-ligand interactions. The purified hepatopancreas lectin showed a broad spectrum of antimicrobial activity against bacteria Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, E. coli and fungi Candida albicans and Aspergillus niger. The findings of this study demonstrate the significance of hepatopancreas lectin as a multifunctional defense protein that inhibits the growth of bacteria and fungi.

A chimeric strain of porcine reproductive and respiratory syndrome virus 2 derived from HP-PRRSV and NADC30-like PRRSV confers cross-protection against both strains.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor.

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Effect of vaccination route (intradermal vs. intramuscular) against porcine reproductive and respiratory syndrome using a modified live vaccine on systemic and mucosal immune response and virus transmission in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with worldwide distribution and an enormous economic impact. To control PRRS virus (PRRSV) infection, modified live vaccines (MLVs) are widely used in the field, mainly administered via an intramuscular (IM) route. Currently, some MLVs are authorized for intradermal (ID) administration, which has many practical and welfare advantages. The objectives of the study were to compare the immune responses (systemic in blood and mucosal in lungs) and vaccine efficacy in preventing challenge strain transmission after IM or needle-free ID immunization of piglets with an MLV against PRRSV-1 (MLV1). METHODS: Groups of sixteen 5-week-old specific pathogen-free piglets were vaccinated with Porcilis PRRS® (MSD) either by an IM (V+ IM) or ID route (V+ ID) using an IDAL®3G device or kept unvaccinated (V-). Four weeks after vaccination, in each group, 8 out of the 16 piglets were challenged intranasally with a PRRSV-1 field strain, and one day later, the inoculated pigs were mingled by direct contact with the remaining 8 sentinel noninoculated pigs to evaluate PRRSV transmission. Thus, after the challenge, each group (V+ IM, V+ ID or V-) included 8 inoculated and 8 contact piglets. During the postvaccination and postchallenge phases, PRRSV replication (RT-PCR), PRRSV-specific antibodies (ELISA IgG and IgA, virus neutralization tests) and cell-mediated immunity (ELISPOT Interferon gamma) were monitored in blood and bronchoalveolar lavages (BALs). RESULTS: Postvaccination, vaccine viremia was lower in V+ ID pigs than in V+ IM pigs, whereas the cell-mediated immune response was detected earlier in the V+ ID group at 2 weeks postvaccination. In the BAL fluid, a very low mucosal immune response (humoral and cellular) was detected. Postchallenge, the vaccine efficacy was similar in inoculated animals with partial control of PRRSV viremia in V+ ID and V+ IM animals. In vaccinated sentinel pigs, vaccination drastically reduced PRRSV transmission with similar estimated transmission rates and latency durations for the V+ IM and V+ ID groups. CONCLUSIONS: Our results show that the tested MLV1 induced a faster cell-mediated immune response after ID immunization two weeks after vaccination but was equally efficacious after IM or ID immunization towards a challenge four weeks later. Considering the practical and welfare benefits of ID vaccination, these data further support the use of this route for PRRS MLVs.

Chemical and Pharmacological Prospection of the Ascidian Cystodytes dellechiajei.

Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.

cGLRs Join Their Cousins of Pattern Recognition Receptor Family to Regulate Immune Homeostasis.

Pattern recognition receptors (PRRs) recognize danger signals such as PAMPs/MAMPs and DAMPs to initiate a protective immune response. TLRs, NLRs, CLRs, and RLRs are well-characterized PRRs of the host immune system. cGLRs have been recently identified as PRRs. In humans, the cGAS/STING signaling pathway is a part of cGLRs. cGAS recognizes cytosolic dsDNA as a PAMP or DAMP to initiate the STING-dependent immune response comprising type 1 IFN release, NF-κB activation, autophagy, and cellular senescence. The present article discusses the emergence of cGLRs as critical PRRs and how they regulate immune responses. We examined the role of cGAS/STING signaling, a well-studied cGLR system, in the activation of the immune system. The following sections discuss the role of cGAS/STING dysregulation in disease and how immune cross-talk with other PRRs maintains immune homeostasis. This understanding will lead to the design of better vaccines and immunotherapeutics for various diseases, including infections, autoimmunity, and cancers.

NLRP3 Negative Regulation Mechanisms in the Resting State and Its Implications for Therapeutic Development.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular sensor of the innate immune system that detects various pathogen- and danger-associated molecular patterns, leading to the assembly of the NLRP3 inflammasome and release of interleukin (IL) 1ß and IL-18. However, the abnormal activation of the NLRP3 inflammasome has been implicated in the pathogenesis of autoinflammatory diseases such as cryopyrin-associated autoinflammatory syndromes (CAPS) and common diseases such as Alzheimer's disease and asthma. Recent studies have revealed that pyrin functions as an indirect sensor, similar to the plant guard system, and is regulated by binding to inhibitory 14-3-3 proteins. Upon activation, pyrin transitions to its active form. NLRP3 is predicted to follow a similar regulatory mechanism and maintain its inactive form in the cage model, as it also acts as an indirect sensor. Additionally, newly developed NLRP3 inhibitors have been found to inhibit NLRP3 activity by stabilizing its inactive form. Most studies and reviews on NLRP3 have focused on the activation of the NLRP3 inflammasome. This review highlights the molecular mechanisms that regulate NLRP3 in its resting state, and discusses how targeting this inhibitory mechanism can lead to novel therapeutic strategies for NLRP3-related diseases.

Structural and Phylogenetic In Silico Characterization of Vitis vinifera PRR Protein as Potential Target for Plasmopara viticola Infection.

Fungi infection, especially derived from Plasmopara viticola, causes severe grapevine economic losses worldwide. Despite the availability of chemical treatments, looking for eco-friendly ways to control Vitis vinifera infection is gaining much more attention. When a plant is infected, multiple disease-control molecular mechanisms are activated. PRRs (Pattern Recognition Receptors) and particularly RLKs (receptor-like kinases) take part in the first barrier of the immune system, and, as a consequence, the kinase signaling cascade is activated, resulting in an immune response. In this context, discovering new lectin-RLK (LecRLK) membrane-bounded proteins has emerged as a promising strategy. The genome-wide localization of potential LecRLKs involved in disease defense was reported in two grapevine varieties of great economic impact: Chardonnay and Pinot Noir. A total of 23 potential amino acid sequences were identified, exhibiting high-sequence homology and evolution related to tandem events. Based on the domain architecture, a carbohydrate specificity ligand assay was conducted with docking, revealing two sequences as candidates for specific Vitis vinifera-Plasmopara viticola host-pathogen interaction. This study confers a starting point for designing new effective antifungal treatments directed at LecRLK targets in Vitis vinifera.

Flagellin O-linked glycans are required for the interactions between Campylobacter jejuni and Acanthamoebae castellanii.

The predation and engulfment of bacteria by Acanthamoebae facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the physiology, ecology and evolution of pathogenic bacteria. Acanthamoebae species can be reservoirs for many important human pathogens including Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne enteritis worldwide, despite being a microaerophile that is incapable of withstanding atmospheric levels of oxygen long-term. The persistence and transmission of this major pathogen in the natural environment outside its avian and mammalian hosts is not fully understood. Recent evidence has provided insight into the relationship of C. jejuni and Acanthamoebae spp. where Acanthamoebae are a transient host for this pathogen. Mutations to the flagella components were shown to affect C. jejuni-A. castellanii interactions. Here, we show that the motility function of flagella is not a prerequisite for C. jejuni-A. castellanii interactions and that specific O-linked glycan modifications of the C. jejuni major flagellin, FlaA, are important for the recognition, interaction and phagocytosis by A. castellanii. Substitution of the O-linked glycosylated serine 415 and threonine 477 with alanine within FlaA abolished C. jejuni interactions with A. castellanii and these mutants were indistinguishable from a ΔflaA mutant. By contrast, mutation to serine 405 did not affect C. jejuni 11168H and A. castellanii interactions. Given the abundance of flagella glycosylation among clinically important pathogens, our observations may have a wider implication for understanding host-pathogen interactions.

Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity.

BACKGROUND: Japanese encephalitis virus (JEV) remains a predominant cause of Japanese encephalitis (JE) globally. Its infection is usually accompanied by disrupted blood‒brain barrier (BBB) integrity and central nervous system (CNS) inflammation in a poorly understood pathogenesis. Productive JEV infection in brain microvascular endothelial cells (BMECs) is considered the initial event of the virus in penetrating the BBB. Type I/III IFN and related factors have been described as negative regulators in CNS inflammation, whereas their role in JE remains ambiguous. METHODS: RNA-sequencing profiling (RNA-seq), real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze the gene and protein expression changes between mock- and JEV-infected hBMECs. Bioinformatic tools were used to cluster altered signaling pathway members during JEV infection. The shRNA-mediated immune factor-knockdown hBMECs and the in vitro transwell BBB model were utilized to explore the interrelation between immune factors, as well as between immune factors and BBB endothelial integrity. RESULTS: RNA-Seq data of JEV-infected hBMECs identified 417, 1256, and 2748 differentially expressed genes (DEGs) at 12, 36, and 72 h post-infection (hpi), respectively. The altered genes clustered into distinct pathways in gene ontology (GO) terms and KEGG pathway enrichment analysis, including host antiviral immune defense and endothelial cell leakage. Further investigation revealed that pattern-recognition receptors (PRRs, including TLR3, RIG-I, and MDA5) sensed JEV and initiated IRF/IFN signaling. IFNs triggered the expression of interferon-induced proteins with tetratricopeptide repeats (IFITs) via the JAK/STAT pathway. Distinct PRRs exert different functions in barrier homeostasis, while treatment with IFN (IFN-ß and IFN-λ1) in hBMECs stabilizes the endothelial barrier by alleviating exogenous destruction. Despite the complex interrelationship, IFITs are considered nonessential in the IFN-mediated maintenance of hBMEC barrier integrity. CONCLUSIONS: This research provided the first comprehensive description of the molecular mechanisms of host‒pathogen interplay in hBMECs responding to JEV invasion, in which type I/III IFN and related factors strongly correlated with regulating the hBMEC barrier and restricting JEV infection. This might help with developing an attractive therapeutic strategy in JE.