Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that causes substantial economic losses in the swine industry worldwide. The PDCoV NS6 protein is an accessory protein that plays a pivotal role in the viral life cycle and immune evasion. However, the functions of NS6 and its role in PDCoV pathogenesis remain largely unknown. In this study, we prepared a monoclonal antibody (mAb) 5-A11 that specifically recognizes the PDCoV NS6 protein. The mAb 5-A11 exhibited high specificity for PDCoV, with no cross-reactivity with several major porcine pathogenic viruses. Furthermore, the epitope recognized by mAb 5-A11 was precisely mapped to residues 70EYGSIYGKDFI80 of the NS6 protein using Western blot analysis. Notably, this epitope is highly conserved among different PDCoV isolates. Substantial variations were observed when comparing this epitope with the corresponding regions in the NS6 proteins of other δ coronaviruses, suggesting potential differences in the structure, function, and antigenicity of their NS6 proteins. Our findings provide valuable tools and insights for further elucidating the functions of the NS6 protein and its role in PDCoV pathogenesis, as well as for developing diagnostic and therapeutic strategies against PDCoV infection.

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47-49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens.

Membranous nephropathy (MN), an autoimmune disease, can manifest at any age and is among the most common causes of nephrotic syndrome in adults. In 80% of cases, the specific etiology of MN remains unknown, while the remaining cases are linked to drug use or underlying conditions like systemic lupus erythematosus, hepatitis B virus, or malignancy. Although about one-third of patients may achieve spontaneous complete or partial remission with conservative management, another third face an elevated risk of disease progression, potentially leading to end-stage renal disease within 10 years. The identification of phospholipase A2 receptor as the primary target antigen in MN has brought about a significant shift in disease management and monitoring. This review explores recent advancements in the pathophysiology of MN, encompassing pathogenesis, clinical presentations, diagnostic criteria, treatment options, and prognosis, with a focus on emerging developments in pathogenesis and therapeutic strategies aimed at halting disease progression. By synthesizing the latest research findings and clinical insights, this review seeks to contribute to the ongoing efforts to enhance our understanding and management of this challenging autoimmune disorder.

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66. Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.

OBJECTIVE: Since the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal antibodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli.
RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein.
CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion assembly and virus-host interactions. We selected the hydrophilic region of M protein for prokaryotic expression, purification, and recombinant protein production. Utilizing hybridoma technology, we prepared the monoclonal antibody (mAb) 24-A6 against M protein. The mAb 24-A6 was shown to be suitable for use in immunofluorescence assays, western blotting, and immunoprecipitation, with specificity for PDCoV and no cross-reactivity with other five porcine viruses. The M protein was observed to be expressed as early as 3 h after PDCoV infection, increasing its expression over the duration of infection. Notably, the antigenic epitope of the M protein identified as 103SPESRL108 recognized by mAb 24-A6 was found within a conserved structural domain (SWWSFNPETNNL) of the coronavirus M protein, indicating a crucial overlap between a functionally important viral assembly region and a region recognized by the immune system. Our findings provide valuable insights into mAb 24-A6 targeting the antigenic epitope of M protein and may contribute to the development of diagnostic tools for PDCoV infection and fundamental research into the function of PDCoV M protein.

Egg is one of the eight major food allergens, and ovalbumin (OVA) is the most abundant allergenic protein in eggs. In this study, the effects of pulsed electric field (PEF)-assisted Alcalase hydrolysis on the spatial conformation and potential allergenicity of OVA were studied, and the mechanism of its inhibiting allergic reactions effect was revealed. PEF-assisted Alcalase hydrolysis increased the degree of hydrolysis, surface hydrophobicity, and free sulfhydryl group content. Moreover, the reduction in the α-helix content, fluorescence intensity, and disulfide bond content suggested that PEF promoted the OVA hydrolysis by Alcalase. Additionally, enzyme-linked immunosorbent assay data indicated that PEF-assisted Alcalase hydrolysis hindered OVA binding to immunoglobulins E and G1. Finally, based on bioinformatics combined with mass spectrometry, PEF-assisted Alcalase reduced OVA-induced allergic reactions by destroying epitopes in OVA. Overall, PEF technology further destroyed the epitopes of allergens by targeting the binding sites of substrates and enzymes to improve the affinity of enzymes and substrates, reducing allergic reactions.

OBJECTIVE: To analyze the sequence characteristics of Rhipicephalus microplus Enolase gene, and to predict the secondary and tertiary structure and antigenic epitopes of the Enolase protein.
METHODS: Sixty-two engorged female R. microplus were sampled from a yellow cattle breeding farm in Zhijiang County, Huaihua City, Hunan Province in June 25, 2022. Genomic DNA was isolated from R. microplus, and the Enolase gene was amplified using PCR assay, followed by cloning, sequencing and expression of the amplification product. The sequence characteristics of the Enolase gene were analyzed using the software Clustal X, and the gene sequence was translated into amino acid sequences. The secondary and tertiary structures of the Enolase protein were deduced using the software PRABI, and the physicochemical properties of the Enolase protein were analyzed using the software PRABI. In addition, the B- and T-cell epitopes of the Enolase protein were predicted using the software ABCpred Prediction, Scratch, IEDB and NetCTL.
RESULTS: The R. microplus Enolase gene sequence was 1 323 bp in size, and the contents of A, T, G and C bases were 24.5%, 22.5%, 27.0% and 26.0%,with 47.0% of A + T content and 53.0% of G + C content. The R. microplus Enolase gene encoded 434 amino acids, and the Enolase protein had a molecular weight of 47.12 kDa. The secondary structure of the Enolase protein contained 186 α-helixes (42.86%), 32 β-turns (7.37%), 144 random coils (33.18%) and 72 extended strands (16.59%). The Enolase protein was most probably present in cytoplasm (76.7%), followed by in mitochondrion (39.1%) and nucleus (21.7%), and the Enolase protein had no signal peptide or transmembrane domain. In addition, the Enolase protein had 14 B-cell dominant epitopes and 8 T-cell dominant epitopes.
CONCLUSIONS: The R. microplus Enolase gene sequence exhibits a GC preference, and its encoding Enolase protein is an acidic and hydrophilic protein, with α-helixes and random coils as its primary structure, and presenting B- and T-cell dominant epitopes, which is a potential target for development of vaccines against R. microplus.
［摘要］ 目的 分析微小扇头蜱Enolase基因序列特征, 并预测其所编码Enolase蛋白二、三级结构及抗原表位。方法 2022年6月25日在湖南省怀化市芷江县某黄牛养殖场采集62只雌性饱血微小扇头蜱, 提取其DNA, PCR扩增其Enolase 基因, PCR扩增产物克隆、测序并表达。采用软件Clustal X分析Enolase 基因序列特征, 并将基因序列翻译成氨基酸 序列。采用PRABI软件推导出Enolase蛋白二、三级结构, 并对其理化性质进行分析; 采用ABCpred Prediction、Scratch、IEDB和NetCTL软件预测Enolase蛋白B、T细胞抗原表位。结果 微小扇头蜱Enolase基因序列全长1 323 bp, 碱基A、T、G、C含量分别为24.5%、22.5%、27.0%、26.0%, A + T含量为47.0%、G + C含量为53.0%。该基因共编码434个氨基酸; Enolase蛋白分子量大小为47.12 kDa, 其二级结构含186个 (42.86%) α-螺旋、32个 (7.37%) β-转角、144个 (33.18%) 无规卷 曲、72个 (16.59%) 扩展链。Enolase蛋白存在于细胞质中的概率最大 (76.7%), 其次是线粒体 (39.1%) 和细胞核 (21.7%), 该蛋白无信号肽和跨膜结构域。Enolase蛋白预计有14个B细胞优势抗原表位和8个T细胞优势抗原表位。结论 微小 扇头蜱Enolase 基因序列呈GC偏好; 其所编码的Enolase蛋白为酸性亲水性蛋白, 以α-螺旋和无规卷曲为主要结构, 且具 有B、T细胞优势抗原表位, 是微小扇头蜱疫苗研发的一种理想靶标。.