The development of genetically engineered multivalent subunit vaccines is needed to provide protection against infections caused by Clostridium septicum and multiple types of Clostridium perfringens. In this study, a pET-32a(+) plasmid encoding the C. septicum α-toxin gene and the C. perfringens β-toxin gene was constructed; a positive clone was designated pET-32a-α-β1. This plasmid, along with pET-32a-α-β2-ε-previously engineered to express a C. perfringens α-β2-ε trivalent recombinant chimeric fusion protein-was transformed into Escherichia coli DE3 for recombinant protein expression after isopropyl-β-D-thiogalactopyranoside induction. Recombinant proteins α-β1 and α-β2-ε were purified, mixed at a protein concentration ratio of 1:1, and subsequently freeze-dried. The freeze-dried proteins were reconstituted with an aluminum hydroxide adjuvant and administered to sheep at various doses. Based on antibody titers and serum neutralization titers in immunized sheep, 0.2 mg of mixed protein was identified as the minimum immunization dose for the subunit vaccine. Toxin neutralization tests performed 21 days post-vaccination showed that sera from immunized sheep neutralized 10, 10, 27, and nine times the mouse lethal doses of C. perfringens types B, C, and D, and C. septicum toxins, respectively. All immunized sheep were protected against C. perfringens types B, C, and D, and C. septicum toxins, with a survival rate of 100%; the negative control group showed a survival rate of 0%. These encouraging findings provide a foundation for the development of novel subunit vaccines.

The treatment of produced water from enhanced oil recovery is hindered by the formation of hazardous, polymer-containing oily sludge caused by the electrostatic complexation between cationic reverse demulsifiers and residual partially hydrolyzed polyacrylamide (HPAM). To address this, we designed a series of nonionic polyether demulsifiers with controlled ethylene oxide (EO)-propylene oxide (PO) diblock and EO-PO-EO triblock architectures using tetraethylenepentamine as initiator. The optimized triblock demulsifier T3-H-2 reduces oil-water interfacial tension to 2.4 ± 0.1 mN/m at 1 mg/L and decreases the dilational modulus from 34.1 ± 1.3 to 16.2 ± 0.8 mN/m (at 0.1 Hz). Bottle tests confirm that T3-H-2 (300 mg/L) achieves 99.8 ± 2.5% water separation from water-in-oil emulsions within 90 min and an oil removal efficiency of 95.2 ± 3.8% from oil-in-water emulsions after 24 h. The nonionic polyethers operate via film disruption, not charge neutralization, avoiding HPAM complexation. This yields 91.5 ± 3.6% HPAM retention in the aqueous phase vs. 0.7 ± 0.1% with a cationic demulsifier. The retained HPAM is well-suited for recycling in the preparation of fresh polymer flooding fluids. This work provides a sustainable pathway for treating polymer-containing produced fluids, achieving efficient oil-water separation while preventing hazardous oil sludge formation during produced water treatment.

Foot-and-mouth disease virus serotype O remains a global threat, particularly the O/CATHAY topotype in East and Southeast Asia. Because timely access to emerging field isolates can be limited, reverse genetics provides a flexible approach for generating viruses representing selected circulating strains for vaccine matching and efficacy evaluation. Here, we generated O/CATH/APQA-V18 and evaluated its suitability for vaccine assessment in pigs. A contemporary Vietnamese O/CATHAY strain was selected based on viral protein-1 phylogenetic analysis, and its P1 capsid-coding region was incorporated into an O1 Manisa backbone using reverse genetics. The recombinant virus was characterized by real-time reverse transcription polymerase chain reaction, transmission electron microscopy, and sequence analysis. Two-dimensional virus neutralization tests using sera against O/PanAsia-2 and O/Primorsky yielded antigenic relationship values of 0.04 and 0.11, respectively, indicating poor antigenic matching. In vivo, O/CATH/APQA-V18 induced clinical signs in unvaccinated pigs, confirming its infectivity and pathogenicity. Vaccinated pigs had low neutralizing antibody titers against O/CATH/APQA-V18 before challenge, and only 25% were protected. These findings support O/CATH/APQA-V18 as a genetically defined and reproducible experimental virus for vaccine matching and protective efficacy evaluation against contemporary O/CATHAY viruses.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that seriously threatens livestock health. Protective immunity induced by vaccination is primarily associated with the generation of neutralizing antibodies; however, conventional virus neutralization tests are time-consuming and require the handling of live virus, limiting their suitability for rapid and repeated evaluation in routine settings. In this study, we established a surrogate neutralization assay for the functional evaluation of neutralizing antibody activity against FMD virus (FMDV) serotype O. The assay measures changes in luciferase-based luminescent signals generated following exposure of LgBiT-expressing cells to HiBiT-tagged virus-like particle (VLP) preparations. FMDV serotype O-derived VLPs exhibited stable capsid protein expression, assembly characteristics, and morphological integrity similar to those of virus particles, as confirmed by sucrose gradient fractionation and electron microscopy. In the presence of neutralizing antibodies, a reduction in luminescent signals was observed, enabling functional discrimination of antibody activity. The surrogate assay results showed a strong correlation with those of the conventional virus neutralization test for FMDV serotype O (R[2] = 0.9068). This study demonstrates the feasibility of the surrogate neutralization assay for assessing the functional activity of neutralizing antibodies and its potential applicability to vaccine immunogenicity assessment and functional antibody analysis.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne bunyavirus with widespread and growing geographic distribution that causes severe hemorrhagic fever and death. Vaccine candidates targeting the viral nucleoprotein (NP) have shown efficacy in both mouse and non-human primate models but the mechanism of protection is unclear. Here we employ the closely related Hazara virus (HAZV) to investigate how the intracellular antibody receptor TRIM21 uses anti-NP antibodies to neutralize infection. We show that TRIM21 can detect incoming NP particles within hours of infection and that this results in a potent block to infection. Electroporated-antibody-dependent neutralization assay (EDNA) experiments reveal that TRIM21 inhibits viral transcription, protein expression and genome synthesis and reduces the production of infectious virions. Mutations and domain deletions within TRIM21 reveal that neutralization requires antibody-binding by the PRYSPRY domain but is only partially dependent on the E3 ubiquitin ligase RING domain. The data suggest a dual restriction mechanism in which NP cross-linking by TRIM21 physically interferes with NP function whilst parallel ubiquitination labels the protein for degradation. This dual mechanism is similar to that used by TRIM5 against retroviruses and suggests that antiviral TRIMs may utilize their capacity for self-assembly both for catalytic activation and viral caging.

The Hendra virus (HeV; Henipavirus hendraense) and Nipah virus (NiV; Henipavirus nipahense), members of the genus Henipavirus, are high-consequence pathogens that have caused multiple zoonotic outbreaks with high mortality. Frequently, pigs or horses have acted as bridging hosts between bats, the reservoir hosts, and people. We used pseudotyped viruses with the fusion (F) and glycoprotein (G) envelope proteins of the NiV and Ghana virus (GhV; Henipavirus ghanaense) in neutralization tests (PVNTs) to screen serum samples from 138 apparently healthy horses in northwest Nigeria. No antibodies to GhV were detected in the 138 samples tested. Two serum samples were positive for antibodies to NiV on an initial screen using a single serum dilution, giving a prevalence of 1.45% (95% CI: 0.4-5.13%). Both positive samples were from female, non-local breeds of horses that were used for polo and were 15 and 20 years old. The only significant risk factor found through Chi-squared analysis was the older age category (p = 0.028). On titration in a PVNT, a half-maximal inhibitory concentration was calculated for both samples (IC50 = 11.6 and 16.8). These data suggest the limited exposure of horses to NiV in the sample region but confirms that horses can act as a sentinel population for the circulation of henipaviruses.

Background/Objectives: Bovine ephemeral fever virus (BEFV) causes an economically important arthropod-borne cattle disease. In 2023, a lineage I "Mayotte-like" (ML) strain emerged in Israel during an outbreak in vaccinated herds, raising concerns about its antigenic match with the live-attenuated vaccine based on Australian strain 919. We compared G-protein neutralizing sites and cross-neutralization among the vaccine strain, Israeli lineage IIIa strains from 2000 and 2021, and ML. Methods: G1-G3 neutralizing-site sequences were compared. Serum-neutralization assays tested all four viruses using sera from 87 cattle across five vaccination/exposure groups. Log2-transformed titers were compared within animals. Results: ML differed from the vaccine strain at eight amino acid positions within G1-G3, whereas the lineage IIIa strains differed from the vaccine strain at three or four positions. In vaccinated, unexposed calves, neutralizing titers against the vaccine and 2021 strains were 4.0- and 2.7-fold higher, respectively, than against ML (both p < 0.01). In unvaccinated 2021-exposed cattle, titers against the 2021 strain were 6.5-fold higher than against ML (p < 0.01). In unvaccinated 2023-exposed cattle, titers against ML were 4.76-fold higher than against the vaccine strain (p < 0.01). Between-strain differences were smaller in vaccinated 2023-exposed cattle. Conclusions: Sequence divergence and cross-neutralization indicate a markedly reduced antigenic match between the Australian 919 vaccine strain and the ML strain. Given the continued circulation and emergence of antigenically divergent BEFV strains and their potential for geographic spread, challenge and field-effectiveness studies are urgently needed to determine the clinical protection afforded by the current vaccine. Lineage-matched or multivalent vaccines should therefore be prioritized to broaden protection against circulating and newly emerging strains.

Hantaan virus (HTNV) is the predominant causative agent of hemorrhagic fever with renal syndrome (HFRS) in China, yet no specific antiviral therapy is currently available. Neutralizing antibodies (NAbs) represent a promising strategy, but most existing anti-HTNV NAbs are heterologous and carry immunogenicity risks. Here, two fully human antibodies from a previously established human anti-HTNV phage display library were generated and characterized. KJJ3, a VL-VL tandem antibody derived from clone 3-12, showed weak binding to inactivated HTNV and minimal neutralizing activity (IC50 = 27.12 μg/mL). However, KJJ4, an engineered IgG4 antibody derived from clone 4-19 and carrying the S108P hinge mutation to prevent Fab-arm exchange, bound inactivated HTNV antigen and recombinant glycoprotein Gn (residues 19-371) in a dose-dependent manner, with only weak binding to Gc. Surface plasmon resonance yielded an association rate constant of 4.70 × 10[3] M[-1]s[-1], a dissociation rate constant of 3.06 × 10[-3] s[-1], and an equilibrium dissociation constant of 650 nM for monomeric Gn19-371. In a Vero E6 focus-reduction microneutralization assay, KJJ4 neutralized HTNV in vitro with an IC50 of 2.879 μg/mL. These data establish KJJ4 as a fully human anti-HTNV antibody with experimentally defined in vitro binding and neutralizing activity, warranting further evaluation in animal models of HTNV infection.

Background/Objectives: Antimicrobial peptides (AMPs) offer promising strategies for combating drug-resistant bacteria such as Escherichia coli (E. coli). This study evaluated the synergistic effects of the human-derived AMP LL37 and its fragments FK16 and FK13, in combination with polymyxin B, colistin, and vancomycin, against three E. coli strains in vitro. Methods: Because the outer membrane of E. coli acts as a barrier to antimicrobial agents, we assessed the membrane-permeabilizing activity of these AMPs. We further examined electrostatic interactions between the cationic AMPs and bacterial surfaces via zeta potential measurements. To simulate physiological conditions, we investigated how Mg[2+] and Ca[2+] ions, common in blood, stabilize the bacterial outer membrane and influence AMP activity. Additionally, we assessed cytoplasmic membrane permeabilization as a key mechanism of antibacterial action. Finally, to support clinical translation, we evaluated the cytotoxicity of the AMPs on human dermal fibroblasts. Results: Checkerboard assays revealed that the peptide-antibiotic combinations exerted synergistic effects against the tested E. coli strains in both MHB medium and cation-adjusted MHB medium, with FICI values ranging from 0.3125 to 0.5 and <0.375 to 0.5, respectively. All three AMPs (LL37, FK16, and FK13) exhibited strong outer membrane-permeabilizing activity. Zeta potential measurements revealed a correlation between membrane disruption and surface charge neutralization. The presence of Mg[2+] and Ca[2+] ions was found to stabilize the bacterial outer membrane. Cytoplasmic membrane permeabilization was confirmed as a key antibacterial mechanism. Cytotoxicity assays confirmed the safety profile of these AMPs on human cells. Conclusions: The study demonstrates that LL37 and its fragments enhance the activity of conventional antibiotics against E. coli through membrane disruption and charge neutralization, as evidenced by the FICI values (0.3125-0.5), membrane disruption and charge neutralization. While divalent cations in physiological conditions influence AMP efficacy, these peptides show potential for clinical application against E.coli.

Goose astrovirus genotype 1 (GAstV-1) is a major causative agent of visceral gout in goslings, leading to substantial economic losses across Chinese poultry farms. Currently, no commercial vaccine is available, rendering early serological surveillance essential for disease control. In this study, a sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a monoclonal antibody (mAb) targeting the ORF2 capsid protein of GAstV-1. Following optimization of antigen coating concentration, serum dilution, mAb concentration, and incubation time, the assay demonstrated a detection limit at a serum dilution of 1:160 and showed no cross-reactivity with antisera against other avian pathogens, including H9N2 avian influenza virus (H9N2-AIV), avian reovirus (ARV), goose parvovirus (GPV), goose astrovirus genotype 2 (GAstV-2), and Tembusu virus (TMUV). The cutoff percent inhibition (PI) value was established at 25%, based on 40 GAstV-negative sera. Intra- and inter-assay coefficients of variation (CV) ranged from 0.41 to 0.88% and 3.12-6.95%, respectively, confirming excellent reproducibility. Evaluation of 35 clinical serum samples revealed an 88.6% (31/35) concordance with virus neutralization tests (VNT), with a kappa value of 0.77. These findings indicate that the ORF2-based ic-ELISA is a reliable, cost-effective, and high-throughput tool for seroepidemiological investigation and post-vaccination monitoring of GAstV-1 in geese.

Fluoride contamination in water sources poses significant risks to human health and ecosystems, necessitating effective treatment strategies. This study investigates the influence of calcium chloride (CaCl2) and the basicity of polyaluminum chloride (PAC) on fluoride removal using PAC-PDMDAAC coagulation. Through systematic jar tests, the effects of varying PAC basicity (0.5, 1.0, and 2.0) and CaCl2 addition sequences were examined under different fluoride concentrations (1.0, 3.0, and 10.0 mg/L). Results indicate that PAC with lower basicity (B = 0.5) exhibits superior fluoride removal efficiency, showing an increase of 20%-40% compared to PAC (B = 2.0), primarily due to the electro-neutralization mechanism of monomeric aluminum species (Ala). The introduction of PDMDAAC reduced the coagulation performance of PAC, especially at an initial fluoride concentration of 1 mg/L. The fluoride removal rate decreased from 80% to 57% at B = 0.5, from 91% to 62% at B = 1.0, and from 72% to 25% at B = 2.0. The order of adding CaCl2 affects the removal rate of fluoride, with the CaCl2 + PAC sequence outperforming the reverse sequence. The study highlights the importance of optimizing coagulant basicity and CaCl2 dosing order to enhance fluoride removal efficiency, providing valuable insights for water treatment processes. These findings contribute to the development of more efficient and cost-effective coagulation strategies for fluoride removal in water treatment plants.