Allergic diseases are a class of important immune imbalance diseases that lack effective cures. Helminth-derived serine protease inhibitors (serpins) exert immunoregulatory effects similar to those of helminth, are controllable, have few side effects, and have great application potential in correcting unbalanced immune responses. However, the immune regulatory effects and mechanisms mediated by the enzyme inhibitory activity of helminth-derived serpins have not been investigated or clarified in previous studies. We obtained mutant serpin proteins with significantly reduced enzyme inhibitory activity by predicting and mutating the single key amino acid. The therapeutic effect of immunoregulation mediated by enzyme inhibitory activity of serpin on allergic inflammation was investigated based on an acute allergy model constructed in OVA-specific T-cell receptor transgenic C57BL/6 mice. Unlike the Trichinella spiralis-derived serpin (Ts-serpin), the mutant proteins showed significantly reduced inhibitory activity against the chymotrypsin and elastase. Meanwhile, the immunosuppressive ability of the mutant proteins was weakened and mutant protein intervention groups showed more severe allergic inflammation in the lungs. Additionally, enzyme inhibitory activity regulates the differentiation of macrophages and Treg cells to establish immune tolerance, which is the key to rapidly improving lung injury at the challenge and treatment stages of allergy. These findings suggest that the rapid regulatory properties of enzymatic reactions have great potential in the prevention and treatment of allergic inflammation.
Aspergillus fumigatus is a major opportunistic fungal pathogen, and increasing azole resistance poses a challenge for aspergillosis treatment. Squalene is an upstream precursor of ergosterol biosynthesis and may also be utilized by SHC-like triterpene cyclases, suggesting a potential link between squalene-associated metabolism, membrane adaptation, and azole response. However, the roles of SHC-like triterpene cyclase genes in A. fumigatus remain unclear. Here, we characterized three candidates, shc1, shc2, and shc3, using comparative bioinformatic analysis, gene deletion, phenotypic assays, azole susceptibility testing, transcriptomics, and host-interaction models. Sequence, genomic-context, phylogenetic, and structural analyses suggested divergence among the three candidates. Individual shc deletion caused limited effects on vegetative growth, whereas loss of shc1 mildly reduced susceptibility to voriconazole and posaconazole, as reflected by twofold MIC increases and lower inhibition rates. Transcriptomic analysis revealed distinct remodeling patterns, with Δshc3 showing the broadest transcriptional changes despite no detectable MIC shift. Targeted metabolite profiling and PI uptake analysis further supported an association between shc deletion, sterol/hopane-type triterpenoid balance, and membrane-associated properties. shc deletion also altered epithelial cell interaction phenotypes, while Δshc1 showed reduced lethality in Galleria mellonella. In clinical isolates, elevated shc transcription was associated with azole-resistant backgrounds. These findings suggest functional diversification among SHC-like triterpene cyclase genes and indicate that shc1 may contribute to azole-associated adaptation and virulence-related traits in A. fumigatus.
The link between diet and overall human health is well established, with certain diets known to promote better health and be associated with a reduced risk of developing chronic diseases. Previous research has also demonstrated the direct effects of diet on microbiome composition, diversity, and fitness, which can, in turn, alter colonization resistance and the immune response against enteric pathogens. However, much less is known regarding the direct effect of diet on the virulence of pathogenic bacteria. To examine the effect of diet on bacterial virulence while maintaining constant macronutrient composition, we used peptones from different sources (plant- and animal-based) to simulate various dietary protein sources. The peptones were examined for their effect on the virulence of enterohemorrhagic Escherichia coli (EHEC). We found that bacteria grown with peptone derived from casein - the main protein component of milk - exhibit a significant reduction in their type III secretion system (T3SS) activity. This effect manifested as a significant reduction in bacterial adherence to host cells and limited translocation activity of the T3SS effector Tir. We additionally found evidence suggesting that milk-derived peptone causes a shift in bacterial behavior from hyper-virulent, adherent bacteria to a more motile state. Finally, we observed that the inhibitory effects of milk-derived peptone extend beyond EHEC to other T3SS-possessing pathogens. While the specific inhibitory components in casein remain to be identified, our findings provide a foundation for developing casein-based, non-antibiotic therapies against bacterial diarrheagenic pathogens.
Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, reporter readouts that facilitate early discovery of cccDNA-modulating agents. Here, we developed two complementary hepatocyte HiBiT reporter models: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16), in which a secreted split-NanoLuc HiBiT signal is linked to cccDNA-associated expression, and a Cre/Lox-based recombinant cccDNA (rcccDNA) reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA with a matched HiBiT readout. Screening of 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. Palovarotene, a retinoic acid receptor-γ agonist, was selected as an exemplar concordant hit and reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems without overt cytotoxicity at the tested concentrations. Together, this dual-reporter strategy supports efficient cross-model triage of candidate cccDNA modulators for subsequent orthogonal validation.
Getah virus (GETV) is an emerging arthropod-borne zoonotic alphavirus that poses a growing threat to animal and public health, yet its virulence determinants remain poorly understood. Here, we report the first identification of a natural GETV variant isolated from the brain of diseased piglets. This variant harbors a 9-nucleotide deletion in the E1 glycoprotein stem region, resulting in the deletion of glutamine 397 (Q397) and valine 398 (V398), along with an asparagine‑to‑isoleucine substitution at position 396 (N396I) and the loss of phenylalanine 399 (F399). It also carries a threonine-to-methionine substitution at position 49 (T49M) in nonstructural protein 2 (NSP2). The NQVF motif (residues 396-399) in E1 protein is highly conserved among GETV strains and related alphaviruses, suggesting a shared functional role. Using reverse genetics, we demonstrated that the E1 deletion-but not the NSP2 mutation-drastically attenuates viral replication in vitro and completely abrogates lethality in neonatal mice by restricting systemic dissemination. Stepwise mutagenesis further revealed that the four‑residue motif functions synergistically as a virulence switch: single deletions partially reduce pathogenicity, while the quadruple deletion confers full attenuation, limiting tissue tropism and histopathology. Although both wild‑type and mutant viruses crossed the placental barrier in pregnant mice, the mutant exhibited reduced vertical transmission and maternal tissue replication. Our findings identify a highly conserved E1 stem motif as a critical regulator of GETV virulence, offering a strategic target for developing attenuated vaccines against GETV and other alphaviruses.
Gene therapy has emerged as a promising strategy for cancer treatment, yet challenges in efficient gene delivery remain a major barrier. Herpes simplex virus type 1 (HSV-1), as an oncolytic virus, has garnered attention for its potential in cancer therapy due to its replicative capacity, large genomic payload, and relatively low toxicity. Notably, syncytium-forming HSV-1 (SF-HSV-1) not only exhibits enhanced and sustained antitumor efficacy but also triggers profound immune responses. However, the exact molecular mechanisms orchestrating HSV-1-induced syncytium formation, its resulting cytotoxicity, and its precise role in immune modulation remain incompletely understood. This review aims to provide an in-depth exploration of the mechanisms underlying HSV-1 syncytium formation and its therapeutic implications in cancer gene therapy.
Human cytomegalovirus (HCMV) profoundly reprograms host transcription and RNA metabolism, yet its impact on transcription start site (TSS) regulation of host genes remains poorly understood. Here, we employed NanoCap Analysis of Gene Expression sequencing (NanoCAGE-seq) to investigate HCMV-driven changes in alternative TSS usage across the host transcriptome. We identified widespread TSS switching, with ribosomal protein genes (RPGs) emerging as a highly enriched category. Alternative TSS usage produced isoforms with distinct 5’untranslated regions (UTRs), thereby altering cis-regulatory elements that shape translational efficiency. Integrative transcriptomic and proteomic analyses revealed a paradoxical accumulation of RPG proteins despite transcriptional downregulation during infection. Using 5’ Rapid Amplification of cDNA Ends (5’RACE), we characterized four RPGs of RPL4, RPS11, RPS23, and RPS24 that generated 5’UTR variants through alternative TSS usage. Notably, isoforms containing a 5’terminal oligopyrimidine (5’TOP) motif were significantly enriched, correlating with mTOR activation induced by HCMV. Functional assays with bicistronic reporter constructs in HEK293 cells and infection models in human embryonic lung fibroblasts demonstrated that the RPL4 5’TOP isoform exhibited enhanced mTORC1-driven translation compared with non-5’TOP counterparts. Importantly, RPL4 upregulation facilitated viral protein synthesis and boosted production of infectious virions. Together, our findings reveal that dynamic TSS switching of RPGs provides a simple, yet effective, mechanism for fine-tuning mTORC1-responsive translation. By co-opting host transcriptional and translational programs, HCMV enhances ribosome function to optimize the cellular environment for productive viral replication.
Chinese cordyceps consists of fruiting body and sclerotia (larvae part) formed through the parasitism of the insect Hepialidae by the fungus Ophiocordyceps sinensis, but artificial cultivation is hindered by fruiting body abortion, severely impacting yield and quality. The interactions between the fungus, its host insects, and soil are critical for its development. Multi-omics analyses were conducted to compare normally and abnormally developing samples, examining microbial communities and metabolites in fungus-colonized host larvae and the mycosphere soil. Abnormal fruiting body development was associated with profound shifts in microbial ecology: fungal diversity increased significantly in both endophytic larvae and mycosphere soil, whereas bacterial diversity decreased within host larvae. The microbial composition in and around abnormal samples was markedly altered, characterized by a high enrichment of Penicillium fungi and a depletion of Bacillus bacteria. Cross-kingdom microbial network analysis showed fewer connections and lower stability in abnormally developing samples. Untargeted metabolomic profiling revealed significant accumulation of the antibiotics N1-hydroxy-roquefortine C and glandicoline A, both roquefortine C derivatives characteristic of Penicillium metabolism, along with enriched pathways involved in antibiotic biosynthesis. Exploratory Partial Least Squares Structural Equation Modeling (PLS-SEM) analysis supported an associative pathway in which Penicillium-mediated toxicity is correlated with microbial dysbiosis, which in turn is associated with fruiting body abortion. Conversely, Bacillus may play a critical role in suppressing Penicillium overgrowth and maintaining microbial homeostasis, representing a promising target for biocontrol strategies. This study is the first to reveal potential links between Penicillium‑mediated toxicity, microbial imbalance, and developmental disorders of Chinese cordyceps.
The synergistic interaction between Staphylococcus aureus and Candida albicans exacerbates polymicrobial infection severity and mortality compared to monomicrobial infections. C. albicans metabolism is known to enhance S. aureus virulence, whereas the role of S. aureus metabolic reprogramming in this cross-species synergy remains poorly defined. In this study, we demonstrate that C. albicans remodels S. aureus glycolytic pathways, thereby increasing its virulence in glucose-rich environments. C. albicans triggers metabolic reprogramming in S. aureus, characterized by a reduction of lactate and accumulation of upstream glycolytic intermediates, including 3-phosphoglycerate, 2-phospho-D-glycerate, and phosphoenolpyruvate. The metabolic reprogramming phenotype is glycolysis-dependent, which is evidenced by 2-deoxy-D-glucose-mediated inhibition of virulence enhancement. Addition of exogenous lactate lowers extracellular pH and reduces the hemolytic activity of S. aureus-C. albicans co-cultures. Inhibition of lactate dehydrogenase by oxamate or extracellular alkalinization with sodium hydroxide significantly increases hemolysis in S. aureus mono-cultures. S. aureus-C. albicans co-infections promote inflammatory cell infiltration, but do not affect the quantity of S. aureus, indicating that increased toxin levels, rather than bacterial quantity, drive the enhanced virulence. Collectively, these findings indicate that C. albicans enhances S. aureus virulence through metabolic reprogramming. Targeting lactate metabolism or glycolytic intermediates may disrupt this cooperative interaction, offering a novel therapeutic strategy against these polymicrobial infections.
Scedosporium is the second most prevalent fungal colonizer of the airways of cystic fibrosis (CF) patients after Aspergillus fumigatus. Chronic bacterial and fungal colonization drives persistent inflammation via damage-associated molecular patterns, contributing to lung injury. Owing to their distinct structural and biological properties, S. apiospermum and A. fumigatus may trigger different immune responses and exhibit different susceptibilities to macrophage mediated killing. We compared the transcriptomic and inflammatory profiles of human macrophages challenged with S. apiospermum (Sap) or A. fumigatus (Afu) for 4 h and 12 h, and assessed fungal survival. Sap activated multiple pro-inflammatory pathways, overlapping with those induced by Afu. At 4 h, 15 inflammation-related pathways were differentially regulated in Sap-infected macrophages (FDR 4 × 10-2 to 7 × 10-13), 12 of which were also upregulated in Afu-infected cells. The most highly dysregulated pathways involved TNF, IL-17, NF-κB signaling, and cytokine-cytokine receptor interactions. Sap induced a stronger, earlier inflammatory response, with three times more genes upregulated at 4 h than for Afu (671 vs. 200), and significantly higher levels of IL-6, IL-1β, TNF-α, IL-23, IL-10 (p <0.001), and IFN-α2 (p <0.05) secretion. The proportions of macrophages infected at 4 h were similar for the two fungi, but Sap conidia were more efficiently killed after 6 h (46.6% vs. 34.5%, p <0.05). Thus, S. apiospermum elicits a faster and stronger inflammatory macrophage response than A. fumigatus, potentially enhancing fungal clearance but also exacerbating airway inflammation in CF.
Porcine epidemic diarrhea virus (PEDV) causes substantial economic losses in the swine industry globally. Host factors regulating the intracellular replication of PEDV, particularly early RNA synthesis and structural protein production, are not well understood, limiting antiviral strategies. We report that glycoprotein non‑metastatic melanoma protein B (GPNMB) is a key host factor promoting PEDV infection. Initially identified as a PEDV S1-binding partner, GPNMB was confirmed to enhance infection via loss-and gain-of-function experiments in Vero and IPEC-J2 cells. Genetic knockout of GPNMB inhibited PEDV replication without affecting viral attachment or internalization. We show that GPNMB is necessary for the accumulation of double-membrane vesicles (DMVs) and promotes early viral RNA synthesis. Notably, GPNMB directly interacts with the PEDV spike (S) and nucleocapsid (N) proteins, increases their abundance, and facilitates their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, implicating it in structural protein maturation. Our work reveals a pivotal role for GPNMB in PEDV replication and nominates it as a target for host-directed antiviral intervention.
Fusobacterium necrophorum is a major opportunistic pathogen of ruminants that causes foot rot and liver abscesses and contributes to substantial economic losses. Current vaccines provide limited protection. Here, we prepared and evaluated outer membrane vesicles (OMVs)-based vaccine candidate derived from F. necrophorum. OMVs were purified by density-gradient centrifugation and were characterized for morphology, composition, and immunological activity. The purified OMVs showed intact vesicle structure and a uniform particle size distribution and contained virulence-associated antigens and pathogen-associated molecular patterns. OMVs were rapidly taken up by dendritic cells and promoted dendritic cell maturation, as indicated by activation of PI3K/AKT signaling and increased expression of MHC I, MHC II, CD80, and CD86. In vivo imaging showed persistent fluorescence at the injection site and signal accumulation in draining lymph nodes and the spleen. In mice, OMVs immunization induced high titers of antigen-specific IgG and elicited a Th1-biased cellular immune response. In a lethal challenge, OMVs immunization achieved 100% survival, significantly reduced liver bacterial loads, and alleviated inflammatory and necrotic liver lesions. In sheep, OMVs immunization also induced progressively increased antigen-specific IgG and a predominantly Th1 type cellular immune response, supporting strong immunogenicity in a natural host. Together, these results support F. necrophorum OMVs as a promising vaccine candidate containing multiple native antigens for the prevention and control of F. necrophorum - associated diseases in ruminants.
Exopolysaccharides (EPS) are carbohydrate polymers produced by various bacteria, including species of the genus Lactobacillus. Until now, the link between the functional properties of these bacteria and their EPS remains poorly understood. Lactobacilli are used in a wide range of applications due to their protective effects against inflammation and their role in maintaining intestinal homeostasis. However, the functional properties of their EPS remain weakly characterized. This study investigated the EPS produced by Lactobacillus gasseri (Lg-EPS), a human-derived probiotic, focusing on their immunomodulatory activity and inhibitory effects on the growth and virulence of Candida albicans, an opportunistic fungal pathogen associated with dysbiosis and immune dysfunction. The results show that Lg-EPS are composed of 59.1% glucose, 26.0% galactose, 14.6% rhamnose, and 0.3% N-acetyl-glucosamine. Functionally, Lg-EPS reduced the expression of pro-inflammatory cytokines (IL-6, IL-8, CCL-2) and IL-6 protein levels, while increasing IL-10 expression and secretion. These effects were associated with downregulation of TLRs, MyD88 and NF-κB, alongside upregulation of the aryl hydrocarbon receptor (AhR), an intracellular transcription factor involved in immune modulation. Additionally, Lg-EPS increase induction of AhR activity in HT29-Lucia™ AhR cells. Furthermore, Lg-EPS inhibited C. albicans biofilm formation and fungal growth. These findings suggest that Lg-EPS contribute to immune regulation and antifungal defense via AhR activation. This receptor, known for sensing microbial metabolites, mediates anti-inflammatory effects by suppressing NF-κB signaling, enhancing IL-10 production, and reinforcing epithelial barrier function. This is the first report to demonstrate the AhR-dependent anti-inflammatory effect of bacterial EPS.
The type IV secretion system (T4SS) acts as the central virulence determinant of Brucella, facilitating intracellular survival via the secretion of effector proteins. In this study, we combined bioinformatic prediction with translocation assays to identify seven novel VirB-dependent effectors. Functional characterization revealed distinct roles for these proteins in both bacterial physiology and host-pathogen interactions. We identified BT4E19 and BT4E43 as critical determinants of cell envelope integrity: BT4E19 is required for core oligosaccharide maintenance and nitrosative stress tolerance, whereas BT4E43 is essential for O-antigen biosynthesis and oxidative stress resistance. Furthermore, BT4E43 is required for the efficient avoidance of lysosomal trafficking during intracellular infection. Notably, BT4E4 displays dual functions under tested conditions, being essential for oxidative stress resistance while simultaneously functioning to inhibit Caspase-5-mediated pyroptosis. In vivo assays further demonstrated that both BT4E19 and BT4E43 are indispensable for establishing chronic infection in mice. Collectively, these findings expand the Brucella effector repertoire and uncover the dual functions of specific effectors in maintaining bacterial structural integrity and orchestrating immune evasion, highlighting them as potential targets for anti-virulence therapies.
The soil-borne Gram-negative beta-proteobacterium Ralstonia solanacearum species complex (RSSC) causes bacterial wilt, a devastating plant disease that threatens crop production and food security worldwide. In this review, we first summarize current knowledge of RSSC pathogenicity and virulence, focusing on the factors that determine its ability to infect and cause disease, including advances in resistance breeding and the genetic basis of host resistance to bacterial wilt. Next, we highlight the questions provided from the previous studies and describe our recent studies, which revealed that the phc QS network is a highly complex regulatory system that dominates global gene expression and finely tunes RSSC virulence throughout the infection process, from root epidermis invasion to colonization of xylem vessels. Finally, we identify key knowledge gaps and discuss future research directions and practical strategies for the effective management of bacterial wilt.
Getah virus (GETV) is an emerging mosquito-borne pathogen with a broad host range, posing a significant threat to livestock and public health. Although putative N-linked glycosylation sites on its envelope proteins have been reported, their precise functional roles remain uncharacterized. In this study, tunicamycin inhibition assays demonstrated that GETV replication depends on the host glycosylation machinery. Enzymatic dissection revealed that the envelope proteins E1 and E2 carry complex-type N-linked glycans, which are critical for infectivity. By constructing envelope protein N-linked glycosylation site mutants, it was revealed that the E1-N141 site influences viral entry, whereas the E2-N262 site was critical for both viral adsorption and internalization. Mutations at all envelope protein N-linked glycosylation sites impaired the infectivity of progeny virions, resulting in delayed replication kinetics and significantly reduced viral titers. In ICR mice model, all mutants showed attenuated pathogenicity, with the E2-N200 mutation conferring the most substantial reduction in virulence. Notably, despite reduced virulence, some mutants (E1-N141A and E2-N200A/N262A) elicited neutralizing antibody responses stronger than those induced by wild-type virus and provided complete protection against subsequent challenge with wild-type GETV. These findings highlight the critical role of envelope protein glycosylation in GETV infectivity and pathogenesis, providing a molecular basis for rational vaccine design.
Streptococcus agalactiae, also known as Group B Streptococcus (GBS), colonizes the intestinal tract, where it must overcome bile salt-mediated membrane disruption to establish infection. However, the specific molecular mechanisms underlying this resistance remain unclear. In this study, a Himar1 transposon screen identified a bile salt hypersensitive spxA2 (SAHN016_RS09865) mutant. To elucidate how SpxA2 mediates bile salt stress adaptation and promotes GBS pathogenesis, we constructed the spxA2 deletion mutant ΔspxA2 by homologous recombination. We found that deletion of spxA2 significantly compromises membrane stability, as evidenced by a markedly increased negative surface charge and decreased hydrophobicity in the ΔspxA2 mutant. Under bile salt stress, the ΔspxA2 mutant exhibited severe membrane depolarization and compromised membrane integrity. Genetic and transcriptional analyses further revealed that the LiaFSR two-component system as the upstream regulator that significantly upregulated spxA2 transcription in response to bile salt stress, defining a novel LiaFSR-SpxA2 regulatory pathway playing a role in bile salt resistance in GBS. Importantly, this LiaFSR-SpxA2 regulatory axis plays a role in colonization and virulence in a tilapia infection model. Collectively, our work defines the LiaFSR-SpxA2 axis as a central regulator of membrane homeostasis that is critical for GBS to overcome innate host defenses, thereby facilitating intestinal colonization and the progression to systemic disease.
Bovine coronavirus (BCoV) is an important pathogen associated with enteric disease in calves, contributing to significant economic losses worldwide. To provide an updated overview of BCoV epidemiology, we conducted a systematic review and meta-analysis of studies published up to October 2025. Seventy-two eligible studies comprising 29,045 samples from nine countries were included, yielding a pooled global prevalence of 20.39% (95% CI: 15.07-26.99). Substantial heterogeneity was observed, reflecting variations in geographic regions, detection methods, and study populations. To complement the epidemiological analysis, 298 fecal samples from diarrheic calves in northeastern China were screened by RT-PCR, identifying 36 BCoV-positive samples (12.08%). Co-infection analysis revealed that most positive samples contained additional enteric viruses, indicating complex viral interactions in calf diarrhea. A novel BCoV strain was isolated in MDBK cells and designated DDFX98. Viral identity was confirmed by PCR, transmission electron microscopy, and immunofluorescence assay. Complete genome sequencing demonstrated that DDFX98 belongs to the GIIb subtype and shares high nucleotide identity with contemporary circulating strains. Comparative analysis of the spike (S) protein revealed multiple amino acid substitutions predominantly located within the S1 subunit. Structural modeling suggested localized conformational variations, particularly in surface-exposed and flexible regions, while preserving the overall spike architecture. In silico predictions further indicated minor alterations in glycosylation potential and epitope-associated regions. Collectively, this study integrates global epidemiological evidence with molecular and structural characterization of a contemporary BCoV isolate, providing insights into S protein variability and its potential structural - functional implications.
Rotavirus (RV) replication occurs within viroplasms (VMs) and is initiated by two RV non-structural proteins NSP2 and NSP5. Viruses exploit host cellular components for their replication and assembly; however, information on the roles of host proteins in VM dynamics and RV replication is limited. Thus, in the current study, we used proteomics to identify host proteins that interact with NSP5 during RV infection to delineate their role in virus replication. A large number of host proteins (n = 128) were found to interact with NSP5, and Gene Ontology enrichment analysis revealed the enrichment of various metabolic processes during RV infection. One of the essential host proteins, ATP citrate lyase (ACLY), which is involved in the de novo lipid synthesis pathway, was found to interact with C-terminal region of RV-NSP5 and colocalize within VMs. Following RV infection, the serine 455 phosphorylation of ACLY was induced, suggesting increased enzymatic activation. This correlates with enhanced lipid droplet production via increased acetyl-coenzyme A expression levels, which in turn supports VM formation. The ACLY inhibitors SB204990 or hydroxycitric acid tripotassium hydrate significantly reduced RV-infection in vitro. This anti-rotaviral effect of drug was further validated in BALB/c suckling mice by measuring viral protein expression and viral titers. In the presence of ACLY inhibitor, reduced viral titers, reduced viral protein expression, and improved small intestinal histopathology were observed. These findings suggest a protective effect of drugs against RV infection in vivo and highlight ACLY as a potential anti-rotaviral target for the development of new therapeutics.
Peach shoot blight, caused by Diaporthe amygdali, is a serious challenge for China's peach industry, contributing to annual yield losses of 20-50%. Despite its economic significance, the molecular mechanisms underlying D. amygdali pathogenicity remain poorly characterized. Velvet proteins are fungal-specific regulatory factors that exhibit functional diversity across plant pathogenic fungi; however, their roles in D. amygdali remain uncharacterized. Here, we identified and systematically characterized four velvet proteins in D. amygdali, including DaVeA, DaVelB, DaVelC, and DaVosA. Expression analysis revealed that DaVEA, DaVELB, and DaVELC exhibited significantly higher expression during the asexual stage; notably, DaVEA and DaVELB maintained markedly upregulated expression at the invasive stages. We further obtained single-gene velvet protein deletion mutants and their complemented strains, finding that deletion of DaVEA or DaVELB significantly reduced vegetative growth, abolished asexual reproduction, weakened the pathogenicity, increased melanin accumulation, altered colony surface hydrophobicity, and compromised stress tolerance. In contrast, deletion of DaVELC or DaVOSA affected only asexual reproduction. We further observed that deletion of DaVEA or DaVELB led to significant downregulation of the genes encoding cell wall-degrading enzymes and the fusicoccin biosynthetic gene cluster-a phytotoxic diterpene metabolite implicated in fungal virulence. Moreover, the four velvet proteins form complex interactions in D. amygdali. Our findings expand the functional repertoire of velvet proteins in fungal phytopathogens and clarify DaVeA and DaVelB as potential molecular targets for future studies aimed at developing control strategies.
