The continuous evolution and drug-resistant mutations of influenza virus have placed higher demands on the study of its pathogenic mechanism, as well as the development of antiviral drugs and vaccines. As a key tool for real-time monitoring of viral infection, replication and transmission, viral visualization reporter systems have evolved into various strategies, including high-resolution microscopic imaging, exogenous nanomaterial labeling, recombinant reporter viruses, and virus infection-inducible reporter systems. This manuscript systematically reviewed the research progress of influenza virus visualization technologies, elaborated the principles, core breakthroughs, and application scenarios of each technique, analyzed the common limitations and specific problems existing in the current technical system, and prospected its development direction combined with cutting-edge research, providing a reference for technical selection and innovative applications in influenza virus visualization research.

[Objective] To efficiently express and purify wild-type and three active site-deleted variants (ENO1-M1, ENO1-M2, and ENO1-M3) of ENO1 through the insect cell/baculovirus expression system, thus providing a theoretical basis for subsequent functional research, antibody development, and inhibitor screening. [Methods] Wild-type and active site-deleted genes of ENO1 with a C-terminal 6×His tag were cloned into the pFastBac-HTB vector via molecular cloning and then transformed into DH10Bac competent cells to obtain recombinant Bacmid through screening. Recombinant baculovirus particles were transfected into ExpiSf9 insect cells to generate the P0 virus stock, which was then amplified to produce high-titer P1 virus for target protein expression. Expression products were purified by affinity chromatography (e.g., nickel column) and identified by SDS-PAGE, Coomassie Brilliant Blue staining, and Western blotting. The anti-ENO1 monoclonal antibody, single-chain antibody, and chimeric antibody were used for specific detection by Western blotting. On the basis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) under the catalysis by ENO1, the specific activity of ENO1 was measured by UV spectrophotometry at 240 nm. [Results] All recombinant plasmids and bacmids were successfully constructed, and high-level expression of recombinant proteins was achieved in insect cells. High-purity recombinant proteins were obtained, and Western blotting analysis confirmed their reactivity. The deletion of active sites of the enzyme did not affect the immunoreactivity. The specific activity of wild-type ENO1 was 691.28 U/mg, while those of the three deletion variants were significantly reduced. ENO1-M2 (with deletion of GSHAGNK at residues 156-162) and ENO1-M3 (with deletion of SPDPSRYI at residues 262-270) exhibited particularly pronounced losses of enzyme activity. [Conclusion] We successfully expressed and purified both wild-type and active site-deleted ENO1 proteins with retained reactivity by using the baculovirus expression vector system. The results confirm that GSHAGNK and SPDPSRYI are two key active sites of this enzyme, laying both theoretical and material foundations for further exploring the functions of ENO1 and developing its specific inhibitors.

Litopenaeus vannamei is one of the crustacean species with the highest production and economic value in the global aquaculture industry. Its health status is closely linked to the community succession and balance of the microbiota in the aquaculture system, and the stable supply of seeds is one of the fundamental and core components of the high-quality development of the shrimp aquaculture industry. Characterizing the pattern of bacterial community succession during the early developmental stages of shrimp is an essential prerequisite for achieving microbiome-based regulation in larviculture. [Objective] To establish a standardized bacterial community dataset covering the entire cycle of L. vannamei larviculture, thereby providing data support for systematic investigations on the microbial communities during shrimp larvae development. [Methods] Focusing on the complete developmental stages of L. vannamei larvae, larval shrimps and rearing water samples were collected from the larviculture system of the Zhejiang Mariculture Research Institute following standardized sampling and laboratory processing protocols. Bacterial community datasets were constructed by high-throughput 16S rRNA gene amplicon sequencing combined with the standard Dix-seq amplicon analysis pipeline. [Results] This dataset encompassed the raw paired-end sequencing reads from 102 samples (48 larval samples and 54 rearing water samples) across the entire cycle of shrimp larviculture, comprising a total of 204 fastq.gz files and 4 709 988 raw paired-end reads. After quality control, 4 255 994 effective sequences and 202 505 zero-radius operational taxonomic units (ZOTUs) were obtained. Data generation strictly followed standardized protocols, with multi-point composite sampling ensuring sample representativeness of microbial samples, and unified sample collection and nucleic acid extraction procedures were employed. All analytical parameters were publicly disclosed through the parameter card mechanism of the Dix-seq pipeline, guaranteeing the reproducibility and reliability of the data analyses. [Conclusion] The samples in this dataset cover the key developmental nodes of L. vannamei larvae, achieving synchronous monitoring of the bacterial communities throughout the entire larviculture period. It provides fundamental data for exploring bacterial community succession and host-environment interaction mechanisms during the early developmental stages of shrimp, thereby facilitating the development of green larviculture technologies based on microbiome regulation. Additionally, it serves as a benchmark for the standardized management and analysis of similar high-throughput sequencing data, which is of great significance for enhancing the standardization and operability of microbiome research in agricultural ecosystems.

Trichoderma reesei is recognized as the microorganism with the most potent protein secretion capability known to date. It is extensively utilized for cellulase production and has garnered significant attention in heterologous protein synthesis. Although notable progress has been achieved in the screening of expression elements, chassis engineering, and fermentation process optimization, critical challenges persist, and the large-scale industrial application of heterologous protein expression has not yet been fully realized. This paper reviews the strategies and recent research advances regarding the high-efficiency expression of heterologous proteins in T. reesei. Furthermore, this paper discusses the limiting factors for its industrial-scale application and proposes novel recommendations and strategies to enhance heterologous protein yields.

[Objective] The heat-labile enterotoxin (LT) is widely recognized as a potent mucosal immunoadjuvant. However, the mechanisms underlying its interaction with host epithelial cells remain incompletely understood, which makes it difficult to separate its toxicity from its adjuvant activity. This limitation has severely hampered its clinical application. Therefore, this study aims to elucidate the differential regulatory effects of LT and its A (LTA) and B (LTB) subunits on epithelial cells, as well as the mechanism underlying the initiation of their initial adjuvant activity. [Methods] The biologically active LTB, LTA, and its mutant LTA(R192G) were prepared via prokaryotic expression. Using the human small intestinal epithelial cell line FHs 74 Int and a mouse jejunal ex vivo intestinal segment model, we examined cell viability and apoptosis, analyzed inflammatory cytokine expression, and investigated changes in the NF-κB and NLRP3 inflammasome pathways. [Results] The three prepared proteins all possessed biological activity and could be effectively internalized by cells. LTB primarily induced early apoptosis, significantly up-regulated the expression of IL-6, IL-8, IL-1β, and TNF-α, and activated the NF-κB pathway. LTA triggered significant pyroptosis, specifically up-regulated the expression of IL-18 and IL-1β, and activated the NLRP3 inflammasome pathway. The mutant LTA(R192G) exerted weakened effects but could still activate the NLRP3 pathway. [Conclusion] The adjuvant activity of LT stems from its subunits activating immune responses in epithelial cells through different mechanisms: LTB primarily promotes pro-inflammatory cytokine production via the NF-κB pathway, while LTA mainly relies on the NLRP3 pathway to induce IL-1β/IL-18 secretion and pyroptosis. This discovery not only reveals the initial molecular events of LT-initiated mucosal immunity, but more importantly, lays a solid theoretical foundation for developing novel, safe, and efficient mucosal vaccine adjuvants through targeted modification of LTA and LTB subunits.

Ergosteryl-β-glucosidase (Egh1) catalyzes the hydrolysis of ergosteryl-β-glucoside for producing free ergosterol and glucose. This enzyme plays a crucial role in maintaining intracellular membrane lipid homeostasis and regulating the morphological stability of organelles, including vacuoles. However, the biological functions of this enzyme class in plant pathogenic fungi remain poorly understood. [Objective] To elucidate the role of MoEGH1, an Egh1 homolog in Magnaporthe oryzae, in appressorium development, sterol metabolism, autophagy regulation, and pathogenicity. [Methods] MoEGH1, homologous to yeast EGH1, was identified by sequence homology analysis. A deletion mutant ΔMoegh1 was constructed through targeted gene replacement. The biological functions of MoEgh1 were systematically examined through phenotypic characterization, genetic complementation, autophagic flux assays, and assessments of appressorium formation and turgor pressure. [Results] The deletion of MoEGH1 markedly impaired the growth, development, and pathogenicity of M. oryzae. ΔMoegh1 displayed significantly reduced radial growth and conidiation, an increased proportion of malformed conidia, a substantial decrease in appressorial turgor pressure, and defective appressorium formation. These developmental defects led to an almost complete loss of pathogenicity. Furthermore, compared with the wild‑type strain, ΔMoegh1 demonstrated heightened sensitivity to rapamycin and sterol‑targeting antifungal agents, including natamycin, amphotericin B, and itraconazole. Additionally, the mutant displayed abnormally elevated activity of the target of rapamycin (TOR) signaling pathway concomitant with reduced levels of autophagy. [Conclusion] MoEgh1 is essential for vegetative growth, conidiation, and appressorium formation in M. oryzae. MoEgh1 regulates the autophagy process and sterol homeostasis by modulating TOR activity, thereby playing a critical role in the pathogenicity of this fungus.

[Objective] To investigate the specific role and molecular mechanism of the host protein protein disulfide isomerase A4 (PDIA4) during bovine viral diarrhea virus (BVDV) infection, thus providing a theoretical basis for elucidating the role of this protein in viral replication and developing novel prevention and control strategies. [Methods] Western blotting and qPCR were employed to analyze PDIA4 expression in Madin-Darby bovine kidney (MDBK) cells following BVDV infection. The expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62) were measured by Western blotting and laser confocal microscopy. MDBK cell lines with pdia4 knockdown and overexpression were constructed. The effects of pdia4 on LC3 and p62 expression were examined, and the autophagic flux was evaluated by a tandem GFP-mRFP-LC3 reporter system. Cells were subjected to starvation or treated with bafilomycin A1 (BafA1), followed by BVDV infection. Viral replication was assessed by measuring BVDV mRNA levels through qPCR and double-stranded RNA (dsRNA) levels through immunofluorescence staining. [Results] BVDV infection significantly upregulated the endogenous expression of PDIA4. The LC3 level increased progressively with infection time, whereas the p62 level showed an initial increase followed by a decrease. The cell lines with pdia4 knockdown and overexpression were successfully established. pdia4 knockdown significantly increased both LC3 and p62 levels, whereas pdia4 overexpression increased the LC3 level and decreased the p62 level. Under starvation or BafA1 treatment, pdia4 knockdown inhibited BVDV replication, whereas pdia4 overexpression promoted BVDV replication. [Conclusion] BVDV infection upregulates host PDIA4 expression. PDIA4 promotes BVDV replication by activating the autophagic flux through regulation of LC3 and p62. These findings provide new insights into the pathogenic mechanism of BVDV and offer a theoretical basis for developing targeted antiviral strategies.

[Objective] To investigate the mechanism by which Camphora kanahirae leaves alleviate alcoholic liver disease through the regulation of the gut microbiota-short-chain fatty acid-intestinal barrier pathway. [Methods] Sixty mice were randomly allocated into six groups [normal, model, silymarin (100 mg/kg), and low-, medium-, and high-dose (100, 200, and 400 mg/kg, respectively) C. kanahirae leaves], with 10 mice in each group. After 14 consecutive days of administration, a mouse model of alcoholic liver disease was established. The liver index, alanine aminotransferase (ALT) and aspartate transferase (AST) levels, colon histopathological changes, short-chain fatty acid content, and alterations in gut microbiota structure were measured. [Results] Compared with the model group, medium- and high-dose C. kanahirae leaves reduced ALT and AST levels (P<0.05). Pathological evaluation showed that C. kanahirae leaves at all doses alleviated alcohol-induced colon mucosal damage and inflammatory cell infiltration to varying degrees. Alcohol damage resulted in decreases in levels of short-chain fatty acids, such as acetic acid, propionic acid, butyric acid, and valeric acid (P<0.05). C. kanahirae leaves promoted the proliferation of beneficial bacteria, increased short-chain fatty acid levels, and enhanced intestinal barrier function. Furthermore, 16S rRNA gene analysis of the gut microbiota indicated that alcohol intake led to dysbiosis, and C. kanahirae leaves effectively reversed this microbial imbalance, restoring the composition and diversity of the gut microbiota. [Conclusion] C. kanahirae leaves exert hepatoprotective effects by regulating the gut microbiota balance, promoting short-chain fatty acid production, and maintaining intestinal barrier integrity.

[Objective] To elucidate the role of arginine methyltransferase in the growth, development, and pathogenicity of Botrytis cinerea. [Methods] Bioinformatics approaches were used to perform sequence alignment, phylogenetic analysis, and conserved domain prediction of the arginine methyltransferase HMT2 from eight fungal species, including B. cinerea, Saccharomyces cerevisiae, Fusarium graminearum, and Magnaporthe oryzae. The gene encoding this enzyme in B. cinerea was identified as BcHMT2. The BcHMT2-deleted mutant was constructed via homologous recombination, followed by phenotypic characterization and pathogenicity assays. [Results] The BcHMT2-deleted mutant exhibited a significantly reduced growth rate, significantly weakend cell wall-degrading enzyme activity, and decreased sclerotial production and conidiation. Meanwhile, the hyphal cells became smaller and the conidial morphology was abnormal. In addition, the mutant displayed markedly attenuated pathogenicity on tomato fruits and tobacco leaves. [Conclusion] This study reveals the regulatory role of BcHMT2 in the growth, development, and pathogenicity of B. cinerea, providing novel insights into the research on the prevention and control of gray mold.

[Objective] To investigate the ability of enterovirus A71 (EV-A71) to traverse the blood-brain barrier (BBB) and infect human cerebral organoids, as well as the impact of EV-A71 on BBB integrity following retrograde invasion into the central nervous system via neural routes, and to characterize the infection profiles of EV-A71 in the BBB and human cerebral organoids and the associated inflammatory responses. [Methods] An invitro BBB model was established by seeding human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes into a Transwell system. Human cerebral organoids were generated from human induced pluripotent stem cells (hiPSCs), and a BBB-human cerebral organoid co-culture model was subsequently established. EV-A71 at three titers (10², 10⁴, and 10⁶ TCID₅₀) was used to infect each cell type of the BBB to evaluate viral replication kinetics and cytopathic effects (CPE) across different cell types. Subsequently, the BBB-human cerebral organoid co-culture model was infected with 10⁴ TCID₅₀ EV-A71 using two distinct routes: (1) an apical inoculation strategy, in which EV-A71 was directly applied to the human brain microvascular endothelial cell layer; and (2) a retrograde infection strategy, in which human cerebral organoids were first infected and subsequently co-cultured with the BBB model. Viral RNA copy numbers in culture supernatants and cells were quantified by RT-qPCR. BBB integrity was assessed by measuring transendothelial electrical resistance, and inflammatory responses were evaluated by determining the expression levels of inflammatory cytokines in human cerebral organoids using RT-qPCR. [Results] EV-A71 efficiently replicated in human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes, inducing pronounced CPE. Among these cell types, astrocytes were the most susceptible to EV-A71 infection, showing the most rapid progression of cytopathic changes, followed by brain vascular pericytes. Human brain microvascular endothelial cells were relatively less susceptible, requiring higher viral titers to establish effective infection. Under apical inoculation conditions, EV-A71 replicated within the BBB, compromised BBB integrity, traversed the barrier to infect human cerebral organoids, and significantly activated inflammatory responses. In the retrograde infection model, EV-A71 replicated within human cerebral organoids, preferentially targeted neurons and astrocytes, triggered cellular inflammatory responses, and subsequently invaded the BBB in a retrograde manner, resulting in compromised structural integrity of the BBB. [Conclusion] EV-A71 can traverse the BBB to infect human cerebral organoids and activate inflammatory responses, and can also infect human cerebral organoids first and subsequently disrupt BBB integrity in a retrograde manner. This study systematically characterizes the infection dynamics of EV-A71 in an invitro BBB-human cerebral organoid model, provides a robust experimental model for in-depth analysis of the mechanisms underlying EV-A71 invasion of the central nervous system, and lays a research foundation for future investigations into viral pathogenesis and antiviral therapeutic development.