Population structure and antibiotic resistance of swine extraintestinal pathogenic Escherichia coli from China.

Extraintestinal Pathogenic Escherichia coli (ExPEC) pose a significant threat to human and animal health. However, the diversity and antibiotic resistance of animal ExPEC, and their connection to human infections, remain largely unexplored. The study performs large-scale genome sequencing and antibiotic resistance testing of 499 swine-derived ExPEC isolates from China. Results show swine ExPEC are phylogenetically diverse, with over 80% belonging to phylogroups B1 and A. Importantly, 15 swine ExPEC isolates exhibit genetic relatedness to human-origin E. coli strains. Additionally, 49 strains harbor toxins typical of enteric E. coli pathotypes, implying hybrid pathotypes. Notably, 97% of the total strains are multidrug resistant, including resistance to critical human drugs like third- and fourth-generation cephalosporins. Correspondingly, genomic analysis unveils prevalent antibiotic resistance genes (ARGs), often associated with co-transfer mechanisms. Furthermore, analysis of 20 complete genomes illuminates the transmission pathways of ARGs within swine ExPEC and to human pathogens. For example, the transmission of plasmids co-harboring fosA3, blaCTX-M-14, and mcr-1 genes between swine ExPEC and human-origin Salmonella enterica is observed. These findings underscore the importance of monitoring and controlling ExPEC infections in animals, as they can serve as a reservoir of ARGs with the potential to affect human health or even be the origin of pathogens infecting humans.

The Barrier Disruption and Pyroptosis of Intestinal Epithelial Cells Caused by Perfringolysin O (PFO) from Clostridium perfringens.

Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.

Protection against the H1N1 influenza virus using self-assembled nanoparticles formed by lumazine synthase and bearing the M2e peptide.

There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.

The recombinant vaccine of Lactobacillus plantarum elicits immune protection against H1N1 and H9N2 influenza virus infection.

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, resulting in significant economic losses and numerous fatalities. Current vaccines, typically administered through injection, provide limited protection due to the frequent antigenic shift and drift of IAV strains. Therefore, the development of alternative broad-spectrum vaccine strategies is imperative. Lactic acid bacteria (LAB) represent promising candidates for vaccine engineering due to their low cost, high safety profile, and suitability for oral administration. In this study, we identified a strain of Lactobacillus plantarum (Lp) that is resistant to acid and bile salts and capable of colonizing the intestines of mice. Subsequently, we employed the RecE/T gene editing system to integrate headless hemagglutinins (mini-HA) into the genome of Lp, generating Lp-mini-HA-SP. Remarkably, immunization with Lp-mini-HA-SP elicited serum IgG antibody responses and conferred immune protection against H9N2 and H1N1 influenza virus challenges. Collectively, our findings offer a novel approach for the development of orally administered IAV vaccines and hold significant potential for future drug development endeavors.

Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Astrocytes-Secreted WNT5B Disrupts the Blood-Brain Barrier Via ROR1/JNK/c-JUN Cascade During Meningitic Escherichia Coli Infection.

The blood-brain barrier (BBB) is a complex structure that separates the central nervous system (CNS) from the peripheral blood circulation. Effective communication between different cell types within the BBB is crucial for its proper functioning and maintenance of homeostasis. In this study, we demonstrate that meningitic Escherichia coli (E. coli)-induced WNT5B plays a role in facilitating intercellular communication between astrocytes and brain microvascular endothelial cells (BMECs). We discovered that astrocytes-derived WNT5B activates the non-canonical WNT signaling pathway JNK/c-JUN in BMECs through its receptor ROR1, leading to inhibition of ZO-1 expression and impairment of the tight junction integrity in BMECs. Notably, our findings reveal that c-JUN, a transcription factor, directly regulates ZO-1 expression. By employing a dual luciferase reporting system and chromatin immunoprecipitation techniques, we identified specific binding sites of c-JUN on the ZO-1 promoter region. Overall, our study highlights the involvement of WNT5B in mediating intercellular communication between astrocytes and BMECs, provides insights into the role of WNT5B in meningitic E. coli-induced disruption of BBB integrity, and suggests potential therapeutic targeting of WNT5B as a strategy to address BBB dysfunction.

Mycobacterium tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Potential therapeutic strategies to halt viral neuroinvasion.

The viral infection of the central nervous system is a significant public health concern. So far, most clinical cases of viral neuroinvasion are dealt with supportive and/or symptomatic treatments due to the unavailability of specific treatments. Thus, developing specific therapies is required to alleviate neurological symptoms and disorders. In this review, we shed light on molecular aspects of viruses' entry into the brain which upon targeting with specific drugs have shown promising efficacy in vitro and in preclinical in vivo model systems. Further assessing the therapeutic potential of these drugs in clinical trials may offer opportunities to halt viral neuroinvasion in humans.

Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Discovery of Salifungin as a Repurposed Antibiotic against Methicillin-Resistant Staphylococcus aureus with Limited Resistance Development.

Exploring novel antimicrobial drugs and strategies has become essential to the fight MRSA-associated infections. Herein, we found that membrane-disrupted repurposed antibiotic salifungin had excellent bactericidal activity against MRSA, with limited development of drug resistance. Furthermore, adding salifungin effectively decreased the minimum inhibitory concentrations of clinical antibiotics against Staphylococcus aureus. Evaluations of the mechanism demonstrated that salifungin disrupted the level of H+ and K+ ions using hydrophilic and lipophilic groups to interact with bacterial membranes, causing the disruption of bacterial proton motive force followed by impacting on bacterial the function of the respiratory chain and adenosine 5'-triphosphate, thereby inhibiting phosphatidic acid biosynthesis. Moreover, salifungin also significantly inhibited the formation of bacterial biofilms and eliminated established bacterial biofilms by interfering with bacterial membrane potential and inhibiting biofilm-associated gene expression, which was even better than clinical antibiotics. Finally, salifungin exhibited efficacy comparable to or even better than that of vancomycin in the MRSA-infected animal models. In conclusion, these results indicate that salifungin can be a potential drug for treating MRSA-associated infections.

Antimicrobial Resistance and Genomic Characteristics of Escherichia coli Strains Isolated from the Poultry Industry in Henan Province, China.

Escherichia coli (E. coli) is an important foodborne pathogen and a biomarker for monitoring antimicrobial resistance. Investigating the prevalence of E. coli in the poultry industry holds great importance, particularly in Henan province, a major poultry-producing region in China. Here, we investigated the antimicrobial resistance (AMR) phenotypes of E. coli strains obtained from the poultry industry in Henan, China. A total of 344 E. coli strains were isolated from 638 samples collected from seven farms, three slaughterhouses, and ten terminal markets. Approximately 96.4%, 81.7%, and 52.5% of the isolates from the farms, slaughterhouses, and terminal markets exhibited multidrug resistance. Whole-genome sequencing was performed on 169 strains to reveal their genomic characteristics. The sequence type (ST) analysis revealed that ST10 and ST156 were the most frequent types within the poultry supply chain, whereas ST10 and ST162 were commonly found across the farms, slaughterhouses, and terminal markets. Fourteen ST10 E. coli strains belonged to phylogenetic group A, while fifteen ST165 and six ST162 E. coli strains belonged to phylogenetic group B1. In addition, several antimicrobial resistance genes and virulence factor genes were identified. The blaNDM-5 gene mediated carbapenem resistance in two E. coli strains, while mcr-1-mediated colistin resistance was detected in nine E. coli strains. Phylogenetic group A exhibited fewer virulence genes compared to other groups of E. coli. Plasmid replicons, such as IncFIB (AP001918), IncX1, IncFIC (FII), and IncFII (pHN7A8), were frequently observed. These findings provide valuable insights into the current AMR profiles of E. coli strains isolated from the poultry industry in Central China and highlight the need to implement good manufacturing practices and reduce antibiotic usage to mitigate potential risks associated with E. coli.

ESKtides: a comprehensive database and mining method for ESKAPE phage-derived antimicrobial peptides.

'Superbugs' have received increasing attention from researchers, such as ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), which directly led to about 1 270 000 death cases in 2019. Recently, phage peptidoglycan hydrolases (PGHs)-derived antimicrobial peptides were proposed as new antibacterial agents against multidrug-resistant bacteria. However, there is still a lack of methods for mining antimicrobial peptides based on phages or phage PGHs. Here, by using a collection of 6809 genomes of ESKAPE isolates and corresponding phages in public databases, based on a unified annotation process of all the genomes, PGHs were systematically identified, from which peptides were mined. As a result, a total of 12 067 248 peptides with high antibacterial activities were respectively determined. A user-friendly tool was developed to predict the phage PGHs-derived antimicrobial peptides from customized genomes, which also allows the calculation of peptide phylogeny, physicochemical properties, and secondary structure. Finally, a user-friendly and intuitive database, ESKtides (http://www.phageonehealth.cn:9000/ESKtides), was designed for data browsing, searching and downloading, which provides a rich peptide library based on ESKAPE prophages and phages. Database URL:  10.1093/database/baae022.

EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

Application of Recombinant Lactic Acid Bacteria (LAB) Live Vector Oral Vaccine in the Prevention of F4+ Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a neurotropic pathogen that causes lethal encephalitis. The high susceptibility and massive proliferation of JEV in neurons lead to extensive neuronal damage and inflammation within the central nervous system. Despite extensive research on JEV pathogenesis, the effect of JEV on the cellular composition and viral tropism towards distinct neuronal subtypes in the brain is still not well comprehended. To address these issues, we performed single-cell RNA sequencing (scRNA-seq) on cells isolated from the JEV-highly infected regions of mouse brain. We obtained 88,000 single cells and identified 34 clusters representing 10 major cell types. The scRNA-seq results revealed an increasing amount of activated microglia cells and infiltrating immune cells, including monocytes & macrophages, T cells, and natural killer cells, which were associated with the severity of symptoms. Additionally, we observed enhanced communication between individual cells and significant ligand-receptor pairs related to tight junctions, chemokines and antigen-presenting molecules upon JEV infection, suggesting an upregulation of endothelial permeability, inflammation and antiviral response. Moreover, we identified that Baiap2-positive neurons were highly susceptible to JEV. Our findings provide valuable clues for understanding the mechanism of JEV induced neuro-damage and inflammation as well as developing therapies for Japanese encephalitis.