Plastic waste in marine environments provides novel habitats for diverse organisms, forming distinct microbial ecosystems known as the ‘plastisphere’. Although bacteria of the plastisphere have been widely studied, the role of fungi in plastisphere formation and plastic degradation remains largely unexplored. Thus, we investigated temporal changes in culturable fungal community composition on three common plastic types—high-density polyethylene, low-density polyethylene, and polypropylene—across the early (seven days) and mature (30 days) plastisphere developmental stages through a marine mesocosm experiment. In total, 436 fungal strains were isolated and identified as belonging to 179 taxa, with Penicillium, Cladosporium, Trichoderma, Aspergillus, and Fusarium as the dominant genera. Temporal shifts in the species richness of the dominant genera were observed: Cladosporium showed higher species richness at the early stage, whereas that of Trichoderma increased at the mature stage. Plastic degradation assays revealed that 54.6% of the strains exhibited measurable degradation capacity, with patterns varying by plastic type rather than fungal developmental stage. Scanning electron microscope observations revealed surface damage patterns including cracks, pitting, and erosion on the plastic surfaces. These findings provide novel insights into the composition and functional heterogeneity of culturable fungal communities in the marine plastisphere and suggest that plastisphere fungi play diverse ecological roles beyond direct plastic degradation.
Pathogenic Escherichia coli is a major cause of foodborne illness worldwide and includes strains capable of causing severe disease. To establish a genome-informed framework for foodborne outbreak surveillance, we analyzed 1,029 E. coli isolates from clinical, food, livestock, and environmental sources using whole-genome sequencing. Pathogenic isolates obtained from human clinical cases or linked to documented outbreaks were classified as epidemiologically defined high-risk (EpiHR), whereas the remaining pathogenic isolates were classified as non-EpiHR. Virulence-associated genomic features were extracted using a bioinformatics pipeline, and four machine learning (ML) algorithms, including gradient boosting machine, random forest (RF), and support vector machines with linear and radial basis function kernels, were evaluated. Among them, the RF model showed the best performance, achieving an area under the curve (AUC) of 0.98 and accuracy of 0.93 in 10-fold cross-validation. Additional leave-one-group-out validation showed retained discrimination across held-out sequence types and serotypes, although performance was reduced when isolates were grouped by isolation source. Evaluation using an independent test dataset of 1,908 publicly available pathogenic E. coli genomes showed an AUC of 0.97 and a sensitivity of 0.98. Feature importance analysis using Shapley additive explanations identified influential predictive features, including traT, etpB, and enterotoxin-associated genes. A reduced 10-feature model achieved an AUC of 0.79 in the independent test dataset, supporting its exploratory use for future simplified screening approaches. These results indicate that genome-based ML provides a sensitive framework for surveillance-oriented prioritization of EpiHR pathogenic E. coli isolates, with model predictions interpreted together with epidemiological information.
Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathogen that causes severe community-acquired infections; however, the immune mechanisms controlling intracellular hvKp have yet to be clearly defined. In this study, we investigated the therapeutic potential of berberine against hvKp infection and elucidated the underlying molecular mechanisms using macrophage cell models and in vivo zebrafish models. Berberine significantly reduced intracellular hvKp survival in macrophages and improved survival in hvKp-infected zebrafish. Berberine markedly attenuated proinflammatory cytokine production and inhibited the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Notably, we found that hvKp exploited host lipid droplets (LD) biosynthesis to support its intracellular survival, and berberine effectively suppressed LD accumulation. Mechanistically, berberine promoted the nuclear translocation of transcription factor EB (TFEB), thereby enhancing lipolysis. Although berberine upregulated autophagy-related gene expression during hvKp infection, it did not induce lipophagy, the selective autophagic degradation of LD. Collectively, these findings indicate that berberine has potential as a therapeutic agent against hvKp infection by modulating host lipid metabolism to restrict bacterial intracellular survival.
While shotgun metagenomics is often used to profile antibiotic resistome in gut microbial communities, few studies have investigated if the choice of sequencing platform and assembly strategy affect what mobile genetic elements and antimicrobial resistance genes are recovered. In this study, we compared three platforms (Illumina, Oxford Nanopore, and PacBio HiFi) and seven assembly strategies on gut metagenomes from cattle, pig, and human as case studies. Long-read assemblies recovered 5- to 7-fold more plasmid sequence than Illumina in cattle and pig (mean 17.0 Mb vs. 3.1 Mb), while Illumina performed comparably in the less diverse human gut where high per-species coverage enabled effective short-read plasmid assembly. Long reads also detected more resistance genes on plasmid contigs. Hybrid assembly results depended on the algorithm: scaffolding-based OPERA-MS preserved long-read contiguity and recovered more plasmid-borne resistance genes, while the short-read-centric metaSPAdes hybrid mode produced fragmented assemblies. After collapsing haplotype redundancy, PacBio HiFi identified 2 and 49 unique multi-drug resistance plasmid lineages in cattle and pig, respectively. On the other hand, only 2 and 4 were identified from Illumina. Long reads also placed far more ARGs in a putative mobilization context (50-73%) compared to 14-21% for short reads. Platform and assembly strategy are thus key variables in mobilome and resistome characterization and should be accounted for in antimicrobial resistance surveillance.
Mycobacterium tuberculosis (Mtb) encounters diverse and fluctuating microenvironments during infection, including changes in nutrient availability and pH. While adaptation to individual host-associated conditions has been extensively studied, the impact of repeated environmental fluctuations on bacterial physiology and antibiotic survival remains unclear. In this study, we investigated how long-term adaptation to stable or fluctuating environments influences Mtb growth and drug responses. Mtb populations were serially passaged for six consecutive passages under combinations of different carbon sources and pH conditions that were either maintained consistently or altered across passages. Environmental history significantly affected bacterial growth dynamics and antibiotic survival. Notably, under identical final condition with cholesterol as a sole carbon source, populations adapted to a stable environment exhibited higher survival following bedaquiline and rifampicin treatment than populations exposed to fluctuating environments. These findings suggest that stable environments promote the optimization of growth and stress-response programs, whereas environmental fluctuations limit such optimization despite potentially increasing phenotypic heterogeneity and a possibility of survival. Together, our results identify environmental history as an important determinant of antibiotic survival in Mtb and highlight the need to consider dynamic host-like environments when investigating tuberculosis physiology and drug responses.
Antimicrobial resistance poses a major global health challenge, and infections caused by multidrug-resistant Gram-negative bacteria are associated with substantial morbidity and mortality. In contrast to many Gram-positive pathogens, Gram-negative bacteria combine intrinsic barriers with acquired determinants, including enzymatic drug inactivation, reduced outer membrane permeability, active efflux, and target modifications, which collectively compromise the efficacy of multiple antibiotic classes. Previous reviews have largely catalogued resistant pathogens or antimicrobial agents. This review provides a mechanism-focused overview of antimicrobial resistance in clinically important Gram-negative bacteria and explains how dominant resistance determinants translate into clinically relevant failure modes, such as delayed effective therapy, limited treatment options, and increased reliance on toxic last-line agents. Current and emerging therapeutic strategies are discussed through a mechanism-based lens, emphasizing newer β-lactam/β-lactamase inhibitor combinations and nontraditional approaches, including phages, antivirulence, and microbiome-based interventions. This review highlights the conceptual links between resistance mechanisms, clinical impact, and rational therapeutic choices and identifies priorities for future research aimed at mitigating antimicrobial-resistant Gram-negative infections.
The global rise of multidrug-resistant bacteria poses a critical threat to public health, and bacteriophage-derived endolysins have emerged as promising alternatives to conventional antibiotics. The engineered endolysin LNT113, derived from the Escherichia coli phage PBEC131 endolysin EC340, exhibits potent lytic activity against Gram-negative bacteria. This study investigated the transcriptomic responses of E. coli to sublethal LNT113 stress and identified genetic determinants required for bacterial adaptation to endolysin-induced stress. Transcriptomic analysis identified 552 differentially expressed genes (DEGs) following sublethal LNT113 exposure. Thirteen DEGs associated with stress response and envelope maintenance were individually deleted to generate thirteen mutant strains and to functionally evaluate their roles in bacterial stress tolerance. Among these, the ΔfabB and Δ(prmB-yfcL) mutants exhibited significantly reduced survival under sublethal LNT113 exposure, indicating increased susceptibility to the endolysin. Regarding the prmB-yfcL operon, individual genes were deleted to determine the gene critical for bacterial tolerance. Deletion of aroC and mepA rendered E. coli more susceptible to LNT113. Furthermore, 1-N-phenylnaphthylamine uptake assays demonstrated increased membrane permeability in the ΔfabB, ΔaroC, and ΔmepA mutants. Complementation with pWSK129::fabB, pWSK129::aroC, and pWSK129::mepA restored membrane integrity in the respective mutant strains. These findings suggest that fabB-mediated unsaturated fatty acid biosynthesis and mepA-dependent peptidoglycan remodeling are critical for maintaining envelope integrity under endolysin stress, whereas aroC may indirectly support bacterial tolerance to LNT113 via metabolic adaptation. This study provides insights into bacterial responses to LNT113 and offers a foundation for optimizing endolysin-based therapeutic strategies.
This study compared the rhizosphere microbial communities of two closely related Monotropastrum species (M. humile, Mh; and M. humile var. glaberrima, Mhg) and identified key soil factors associated with their assembly. Bacterial and fungal communities were profiled by Illumina high-throughput sequencing, and soil physicochemical properties were assessed across multiple sites in Zhejiang Province, China. The bacterial communities of both species were dominated by Proteobacteria and Acidobacteriota at the phylum level, while the dominant fungal groups belonged to Ascomycota and Basidiomycota. The two plants shared several dominant bacterial genera, including Serratia, Burkholderia-Caballeronia-Paraburkholderia, and Bradyrhizobium, as well as common dominant fungal genera such as Saitozyma and Podila. Despite these similarities, species-specific enrichment patterns were observed. The rhizosphere of Mhg contained higher abundances of Acidothermus and Lactarius, whereas Mh preferentially enriched Cedecea, Klebsiella, and Russula. Bacterial communities were shaped by pH, soil organic matter (SOM), available potassium (AK), and available phosphorus (AP), whereas fungal communities were primarily influenced by pH, alkali-hydrolyzable nitrogen (AN), and SOM (p < 0.05). These results suggest that both host identity and soil properties contribute to rhizosphere microbial assembly, with clear host-associated differentiation in microbial communities. Notably, the identified host-associated microbial taxa, particularly key mycorrhizal fungi, may serve as potential microbial inoculants, providing new opportunities for the conservation and cultivation of mycoheterotrophic plants.
Fungal extracellular vesicles (EVs) have emerged as critical mediators of fungal physiology, virulence, and host-pathogen interactions. Since their first description in Cryptococcus neoformans, EVs have been identified in several fungal species and shown to transport a broad repertoire of bioactive cargo. Increasing evidence indicates that fungal EVs participate in multiple biological processes, including cell wall remodeling, stress adaptation, biofilm formation, antifungal resistance, and modulation of host immune responses. Recent advances in cryo-electron microscopy, multi-omics approaches, and functional genetics have substantially expanded our understanding of the molecular mechanisms governing EV biogenesis, cargo selection, and extracellular trafficking. These studies have further revealed that EV cargo loading is a highly regulated process linked to intracellular proteostasis, glycosylation, lipid homeostasis, and environmental adaptation. In parallel, the intrinsic immunogenicity and structural stability of fungal EVs have highlighted their translational potential as diagnostic biomarkers, vaccine platforms, therapeutic targets, and nanoscale delivery systems. Given the increasing global burden of invasive fungal infections, this review focuses on EVs derived from clinically relevant human fungal pathogens. We summarize recent advances in EV biogenesis, cargo regulation, their roles in pathogenesis, highlight emerging translational applications, and discuss key unresolved questions and future research directions in the field.
Dual specificity phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases that regulate diverse cellular processes through dephosphorylation of phosphorylated substrates. DUSPs are commonly found in eukaryotes, bacteria, archaea, and viruses. However, structural and biochemical characterization of bacterial DUSP remains limited, as only one bacterial DUSP has been identified thus far. In this study, we investigated a novel putative bacterial DUSP from Candidatus Chlorohelix allophototropha, referred to as CCaDUSP. The crystal structure of CCaDUSP showed the presence of a well-conserved catalytic motif with a characteristic phosphate-binding loop. Biochemical analyses further confirmed that CCaDUSP exhibits phosphatase activity and contains dual general acid/base residues, both of which contribute to its enzymatic activity. These findings not only represent the first characterization of a novel bacterial DUSP with dual general acid/base residues but also provide a foundation for understanding the diversity of DUSP proteins in bacteria.
Postbiotics derived from lactic acid bacteria (LAB) have attracted growing interest as stable and potentially safer alternatives to probiotics for use in foods and health-related products. Comprehensive safety evaluation remains essential before their broader application. In this study, we assessed the safety profiles of RHT3201, a postbiotic preparation derived from Lacticaseibacillus rhamnosus IDCC 3201, through genomic, genotoxic, acute oral, and subchronic oral toxicity studies. Whole-genome analysis showed that IDCC 3201 lacks antimicrobial resistance genes and exhibits no hemolytic activity, supporting the genomic safety of the source strain. RHT3201 showed no genotoxic potential in either in vitro or in vivo assays, as evidenced by no structural or numerical chromosomal aberrations at concentrations up to 5,000 μg/ml in CHL/IU cells and no increase in micronucleated polychromatic erythrocytes, with no suppression of bone marrow erythropoiesis by oral administration of RHT3201 at doses up to 15,000 mg/kg/day using a mouse model. In rats, single oral doses of up to 15,000 mg/kg caused no mortality, treatment-related clinical signs, or gross pathological abnormalities, indicating an approximate lethal dose greater than 15,000 mg/kg. In a 90-day repeated-dose oral toxicity study, no adverse treatment-related effects were observed at doses up to 5,000 mg/kg/day. Mild liver and thyroid histopathological findings were considered adaptive and reversible. Accordingly, the no-observed-adverse-effect level was determined to be 5,000 mg/kg/day. Taken together, these findings support the safety of RHT3201 as a LAB-derived postbiotic ingredient.
Marine dinoflagellates are gaining attention as sustainable bioresource for polyunsaturated fatty acids (PUFAs), particularly omega-3 such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In the present study, we analyzed the FAs and transcriptomic profiles of marine dinoflagellates Amphidinium carterae (D-044) and Prorocentrum minimum (D-127) to evaluate their potential as FAs producers. Gas chromatography-FA methyl ester (GC-FAME) analysis showed that A. carterae is a superior omega-3 producer, yielding a total FA content of 67.6 mg/g DW. DHA accounted for 26.7% of the total FAME profile, which is significantly higher than that of P. minimum (18.1 mg/g DW; DHA 13.1%). Gene Ontology (GO) annotation revealed genes related to FAs and lipid metabolism in A. carterae (1,217 genes) and in P. minimum (2,317 genes), which provide a molecular basis for dinoflagellates with high lipid productivity. Notably, three lipid droplet-associated hydrolase (LDAH) genes with diverse evolutionary origins were identified from A. carterae. These findings suggest a potential expansion of the genetic repertoire related to lipid storage and metabolism, highlighting A. carterae and LDAH as candidates for future biotechnological applications and microalgal metabolic engineering.
Three Gram-stain-negative, strictly aerobic, motile bacterial strains, designated IMCC44359T, IMCC44632T, and IMCC44653, were isolated from coastal surface seawater collected near Jangbong Island in the Yellow Sea. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences assigned the isolates to the genus Aliikangiella. Strains IMCC44632T and IMCC44653 shared identical 16S rRNA gene sequences and exhibited high genomic relatedness (99.0% average nucleotide identity and 92.0% digital DNA-DNA hybridization), indicating that they represent a single species. In contrast, strain IMCC44359T showed low genomic relatedness to these strains and to previously validly published Aliikangiella species, supporting its recognition as a distinct species. The genome of IMCC44359T (5.95 Mbp; 36.5 mol% G + C) is substantially larger than those of IMCC44632T and IMCC44653 (~3.75 Mbp; 40.1-40.2 mol% G + C), and all genomes encode aerobic chemoorganotrophic metabolism and biochemical capacities consistent with adaptation to marine environments. The isolates grew under mesophilic and moderately halophilic conditions typical of coastal seawater bacteria, with growth occurring at ranges at 10-40℃, pH 6.0-9.0, and 0.5-7.5% NaCl (optimum, 30℃, pH 7.0-8.0, and 2.0-3.0% NaCl). All strains contained ubiquinone-8 (Q-8) as the sole respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were the major polar lipids. The dominant cellular fatty acids were iso-C15:0 and summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl). Integrated phylogenetic, genomic, phenotypic evidence supported the recognition of two novel species within the genus Aliikangiella, for which the names Aliikangiella litoralis sp. nov. (type strain IMCC44359T = KCTC 18089T = JCM 37879T = HNIBRBA19635T) and Aliikangiella aequoris sp. nov. (type strain IMCC44632T = KCTC 18090T = JCM 37880T = HNIBRBA19636T) are proposed.
Despite the application of H-1 parvovirus as an anticancer drug, the relationship between its specific tropism and oncolytic activity has been unknown. H-1 viral infection induced cytopathic effects in HeLa cells, whereas Kilham rat virus (KRV), similar to H-1 virus, did not. To explore which segments of the viral protein 2 (VP2) capsid protein in the H-1 virus determine susceptibility to human cancer cells, chimeric H-1 viruses with specific gene segments of H-1 VP2 were constructed. Delineation of the VP2 capsid protein revealed a minimum domain (K208-L435 in the H-1 VP2 protein) to determine infectivity in human cancer cells; however, this domain was not sufficient to maintain infectivity. To solve this problem, further construction of chimeric H-1 viruses illustrated the necessity of segments covering both M1-N87 and D104-P206 in the H-1 VP2 protein, based on chimeric H-1 viruses designated as YCH44, YCH45, and YCH46. Both the variable region 4b (VR4b) domains from KRV VP2 and VR8 from H-1 VP2 were required for the same purpose, based on chimeric H-1 viruses designated as YCH-HK8, YCH16, YCH17, YCH18, and YCH19. We confirmed that chimeric viruses carrying these segments infected human lung adenocarcinoma A549 and pancreatic cancer Panc-1 cells, whereas the parental KRV did not. Taken together, these findings indicate that specific domains of the H-1 virus VP2 capsid protein determine infectivity toward human cancer cells.
16S rRNA gene amplicon sequencing is the most widely used approach for characterizing microbial communities, yet analyzing such data requires navigating a fragmented landscape of bioinformatics tools with distinct installation requirements, parameter settings, and data formats. Here we present 16S-Pipeline, an open-source, web-based platform that provides a complete workflow from raw FASTQ files to publication-ready statistical analyses. 16S-Pipeline automatically detects sequencing type (paired-end, single-end, long-read), variable region, and sequencing platform (Illumina, PacBio HiFi, Nanopore), then performs quality filtering, primer trimming, amplicon sequence variant (ASV) inference via DADA2, taxonomy assignment against SILVA v138.1, phylogenetic tree construction, and optional functional prediction via PICRUSt2. Downstream analyses include alpha and beta diversity, taxonomic composition visualization, differential abundance testing using five complementary methods (ALDEx2, DESeq2, ANCOM-BC2, LinDA, MaAsLin2) with consensus reporting, and KEGG pathway mapping. Built-in NCBI SRA integration enables downloading public datasets for re-analysis and generates submission metadata spreadsheets for data deposition. The interactive web interface built on FastAPI and Plotly Dash enables researchers to perform complex microbiome analyses without command-line expertise. 16S-Pipeline is freely available at https://github.com/tatsu1207/16S-Pipeline under the MIT License.
Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine pleuropneumoniae (PP), a high contagious respiratory disease with significant impact on the swine industry in both clinically and economically. Despite of the several attempts to control APP, the emergence of novel serotypes and antimicrobial resistance (AMR) strains highlights the importance of monitoring the genetic characteristics of APP at single nucleotide level. Despite the importance of genomic surveillance of APP to develop effective control strategies, genetic information on the recent Korean isolates of APP is not available at whole genome level. Therefore, in this study, six APP strains were isolated from porcine lungs with characteristic lesions of PP from 2022 to 2024. And their whole genomic sequences, serotypes, virulence factors, and AMR traits were investigated using combined short-and long-read sequencing methods. In silico PCR serotyping identified the isolates as serotype 1, 7, and 15, while one isolate was non-typeable. Multiple AMR genes including Hinf_PBP3_BLA, Ecol_ EFTu_PLV, tet(B), tet(O), tetR, sul2, aph(3'')-Ib, aph(6)-Id, and aph(3')-Ia were detected. Also, these genes were located with adjacent to mobile genetic elements, suggesting the possibility of horizontal gene transfer. Phylogenetic comparison with 40 global APP complete genomes, presented that Korean isolates were closely related with China and Switzerland strains. This study provides the whole genome sequences based genetic characterization on the recent Korean isolates of APP, and this study emphasizes that continuous monitoring of APP genomic variation to support effective control of porcine pleuropneumoniae.
Lymphocystis disease viruses (LCDVs), members of the Lymphocystivirus genus of the Iridoviridae family, infect various freshwater and marine fish species. They cause the chronic disease lymphocystis, which is non-fatal, but substantially reduces the commercial value of the infected fish. To date, four genotypes of LCDV (LCDV1-4) have been identified, all of which encode the viral homologue of B-cell lymphoma 2 (Bcl-2), a key inhibitor of apoptosis. In this study, we performed biochemical and structural analyses of LCDV2 Bcl-2. Binding assays revealed that LCDV2 Bcl-2 exhibits binding selectivity toward BH3 domain-containing zebrafish proteins. It interacted with zBaxA and zNoxa, but not with zBaxB, zBid, or zBeclin 1, distinguishing it from mammalian and herpesviral Bcl-2 proteins. Subsequent structural determination of LCDV2 Bcl-2 in complex with the BH3 domain of zBaxA demonstrated that they interact in a canonical manner, primarily mediated by the BH3 consensus motif residues of zBaxA. In addition, a subpocket formed by two phenylalanine residues in LCDV2 Bcl-2 plays a key role in determining binding selectivity.
Phytoplasmas are wall-less obligate parasites of plants and insects. Several phytoplasma strains within the Peanut Witches' Broom (PWB; 16SrII) group are associated with significant disease losses across diverse crops and weeds. We present complete, single contig genome assemblies for two Indian parthenium phyllody strains, 'Candidatus Phytoplasma asiaticum' PR34 and 'Ca. P. australasiaticum' PR08, generated through host DNA depletion and hybrid Illumina-Nanopore sequencing. Both genomes display characteristic features of reductive evolution (∼614 kb and 589 kb, respectively) but show notable differences from previously sequenced PWB phytoplasmas. In contrast to most of PMU-rich phytoplasma genomes, neither PR34 nor PR08 retains intact Potential Mobile Units. Instead, both harbor numerous open reading frames encoding group II intron reverse transcriptase/ maturase proteins, predominantly of the mitochondrial-like type, with PR34 containing 52 and PR08 28 such loci that together constitute > 4% of each genome. These observations support the hypothesis that intron-associated processes may contribute to genome variability in the absence of canonical PMUs. Comparative analyses support the classification of PR34 as a distinct species within the PWB complex and reveal both conserved Sec-dependent effectors (SAP05, SAP11, and SAP54/PHYL1) and lineage-specific secreted proteins with predicted nuclear localization. Additional retained features include functional sodA genes and multiple truncated HlyB-like transporters. Collectively, these high-quality genomes illustrate a genomic configuration in which extensive genome reduction and loss of PMUs coexist with the retention of core virulence factors and an expanded repertoire of group II introns, providing a framework for future investigation of genome plasticity in phytoplasmas.
This study presents the first investigation of acetyl-11-keto-β-boswellic acid (AKBA)'s anti-human cytomegalovirus (HCMV) activity in vitro and elucidates its underlying mechanisms. In HCMV Towne strain-infected WI-38 cells, AKBA (1-12 μM) exhibited negligible cytotoxicity while significantly suppressing virus-induced cytopathic effects (CPE) at 6-10 μM, with dose-dependent reduction of viral proteins (IE1/2 and p52) expression, viral DNA copy number (UL123, UL44, and UL32), and infectious viral progeny titer (TCID50). Time-of-addition experiments demonstrated the primary antiviral activity of AKBA during post-entry phase, along with direct virion inactivation. Transcriptome analysis revealed that AKBA significantly downregulated the expression of the host factor NR4A1 induced by HCMV, a finding further validated by Western blotting. Further gene knockdown experiments confirmed that silencing NR4A1 significantly reduced the expression of viral proteins IE1/2, thereby validating NR4A1 as a key host factor for HCMV infection. These findings indicate that AKBA has a potent and dose-dependent inhibitory effect on HCMV replication in WI-38 cells, and proves that this effect is mediated through two different mechanisms: one is the downregulation of the expression of the key host factor NR4A1, and the other is the direct inactivation of HCMV viral particles.
