Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is an acute and highly contagious avian infection that primarily causes severe immunosuppression in chickens. Since the 1980s, very virulent IBDV (vvIBDV) has triggered a global crisis in the poultry industry due to its high mortality. Although IBD has become largely sporadic in recent years following widespread vaccination, the persistent threat of vvIBDV underscores the critical need for novel vaccines that are both safer and more effective. In this study, a lipid nanoparticle (LNP)-encapsulated DNA vaccine encoding the VP2 antigen of vvIBDV (pCAHLJVP2-LNP) was developed. Following in vitro characterization-including assessment of protein expression, particle size, polydispersity index, and encapsulation efficiency; we evaluated the immunogenicity and protective efficacy of the vaccine in specific pathogen-free (SPF) chickens using two immunization doses (10 or 20 μg) and two regimens (single or double immunization). The 20 μg dose elicited superior immune responses compared to the 10 μg dose. Notably, even a single immunization with 20 μg of pCAHLJVP2-LNP elicited specific neutralizing antibodies and conferred complete protection, preventing both bursal lesions and mortality in chickens after vvIBDV challenge. These findings demonstrate that pCAHLJVP2-LNP is a promising vaccine candidate capable of inducing comprehensive immune protection, highlighting its potential for clinical application in vvIBDV prevention.

Avian reticuloendotheliosis (RE) caused by reticuloendotheliosis virus (REV) is an important tumor and immunosuppressive disease posing a serious threat to poultry development. The REV envelope protein, glycoprotein (gp)-90, not only participates in cell receptor binding and viral assembly and release but also induces neutralizing antibody production. However, the antigenic epitope structure of gp90 has not yet been systematically understood. Therefore, in this study, the gp90 envelope protein of a predominant REV strain was prepared using a prokaryotic expression system, and a hybridoma cell line stably secreting the REV gp90 monoclonal antibody was developed via cell fusion and flow cytometry. Notably, a novel linear B-cell epitope, 195REESVRERL203, was identified for the first time in the gp90 of REV using peptide scanning. This epitope was located on the outer side of the gp90 midpiece and was conserved across REV strains. Overall, this study is of great significance for the systematic understanding of REV antigen structure and the development of virus detection methods.

The novel variant infectious bursal disease virus (nVarIBDV) has emerged as a significant threat to the poultry industry because it causes severe damage to the bursa of Fabricius. Moreover, due to its antigenic variation, currently available commercial IBDV vaccines are unable to provide complete protection against it. To address this challenge, we constructed a recombinant virus rMDV-varVP2, which expresses the VP2 gene of nVarIBDV using the Marek's disease virus type 1 (MDV1) vaccine strain as the backbone for its persistence, with the aim of achieving effective control against nVarIBDV. The recombinant virus was successfully rescued and confirmed by cytopathic effect observation, electron microscopy, PCR, and sequencing. Western blotting and immunofluorescence assays verified expression of the exogenous varVP2 protein. rMDV-varVP2 maintained stable VP2 insertion and expression through at least 20 serial passages and showed replication kinetics comparable to those of the parental rMSΔMeq strain in vitro. In specific-pathogen-free chickens, rMDV-varVP2 induced both humoral and cellular immune responses, including ELISA-detectable anti-VP2 antibodies, nVarIBDV-neutralizing antibodies, increased IFN-γ-secreting cells, and upregulated cytokine expression. After challenge with nVarIBDV SHG19 strain, vaccinated chickens were protected from bursal atrophy and histopathological damage, with significantly reduced viral loads in the bursa of Fabricius. These results demonstrate that rMDV-varVP2 provides effective protection against nVarIBDV challenge and represents a promising MDV1-vectored vaccine strategy for controlling nVarIBDV in poultry.

[This corrects the article DOI: 10.3389/fmicb.2022.1046832.].

Infectious bursal disease (IBD) is an important immunosuppressive disease of chicken caused by infectious bursal disease virus (IBDV). At present, the newly emerging novel variant IBDV (varIBDV) and the persistently prevalent very virulent IBDV (vvIBDV) are two major threats, while the non-var/vvIBDV, such as classic IBDV (cIBDV) and attenuated IBDV (attIBDV), also increases the complexity of clinical detection. In this study, a multiplex real-time quantitative fluorescence RT-PCR (qRT-PCR) was developed. Based on sequence analysis of different pathogenic IBDV strains, three probes with different fluorescent signals (FAM, VIC, CY5) and two pairs of primers were designed. Specifically, varIBDV exhibits three fluorescent signals (FAM, VIC, CY5), vvIBDV shows two signals (FAM, VIC), and non-var/vvIBDV displays one signal (FAM). The method possesses excellent specificity: no cross-reactivity was observed between different pathogenic IBDV types, nor with other common avian pathogens. This method has good reproducibility and high sensitivity, with a minimum detection limit of about 10 copies. Furthermore, in the detection of laboratory or clinical samples, the consistency rate of this method with the conventional sequencing analysis method reached 100%. In conclusion, this study developed for the first time a multiplex qRT-PCR that can universally detect IBDV and simultaneously distinguish between vvIBDV and varIBDV, which is of great significance for high-throughput emergency detection and comprehensive prevention and control of new IBDV epidemics.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive condition in chickens, caused by the infectious bursal disease virus (IBDV). The recent emergence of novel variant IBDV (nVarIBDV) poses a significant threat to the global poultry industry. However, currently available vaccines provide only limited protection against nVarIBDV strains. In this study, a recombinant avian metapneumovirus subtype B (aMPV/B) expressing the nVarIBDV VP2 gene (rLN16A-nVarVP2) was successfully rescued by inserting the gene between the G and L genes of the attenuated aMPV/B strain LN16-A. Immunofluorescence and Western blotting analyses confirmed stable VP2 expression in vitro. Further evaluation showed that VP2 insertion did not alter the growth kinetics of the parental virus, and the expression remained stable after 20 serial passages. A single immunization with rLN16A-nVarVP2 elicited robust humoral and cellular immune responses in specific pathogen-free chickens, inducing high levels of neutralizing antibodies against both nVarIBDV and aMPV/B, as well as Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-6) cytokines. Moreover, rLN16A-nVarVP2 conferred complete (100

Background/Objective: Infectious bursal disease (IBD) is an acute and highly contagious immunosuppressive disease in chickens caused by infectious bursal disease virus (IBDV). In recent years, a novel variant IBDV (nVarIBDV) has emerged and spread widely, inducing severe immunosuppression and posing a substantial threat to the poultry industry. More importantly, owing to antigenic variations, nVarIBDV can escape the immune protection of the existing vaccines. Therefore, it is imperative to develop a new vaccine that is antigenically matched to nVarIBDV. Methods: The major protective antigen gene VP2 of the representative nVarIBDV strain SHG19 was inserted into the eukaryotic expression plasmid pCAGGS to construct the recombinant plasmid pCASHGVP2. Subsequently, pCASHGVP2 was encapsulated in lipid nanoparticles (LNPs) to form pCASHGVP2-LNP nanoparticles. Finally, using the SPF chicken model, the immune efficacy of pCASHGVP2-LNP was preliminarily assessed by administering two vaccine doses (10 and 20 μg) and two immunization regimens (single or double immunization). Results: Efficient VP2 protein expression from pCASHGVP2 was confirmed by in vitro transfection experiments. The prepared pCASHGVP2-LNP nanoparticles exhibited an optimal particle size distribution and acceptable polydispersity index, indicating a homogeneous formulation. Furthermore, animal experiments showed that the candidate DNA vaccine elicited specific neutralizing antibodies after double immunization and protected immunized chickens from disease induced by nVarIBDV challenge. Conclusions: This study reports the first development of an LNP-encapsulated VP2 DNA vaccine (pCASHGVP2-LNP) against nVarIBDV, highlighting its potential application for the prevention of nVarIBDV.

Marek's disease (MD) continues to cause significant economic losses in the global poultry industry. During routine MD surveillance in China, MD virus type 2 (MDV-2), which has the potential for widespread dissemination, was detected. In this study, 2842 suspected MD samples were collected from 237 poultry flocks vaccinated against MD across 21 provinces and four municipalities in China. Among these, 534 samples (18.79%) from 95/150 flocks (63.29%) tested positive for MDV-2, with detections reported in all provinces and municipalities. A total of 54 MDV-2 strains were isolated and further characterized. The genomes of four isolates, SW20, JLWK1501, LB3, and ZH4, were sequenced and analyzed. Comparative analysis with reference MDV-2 strains (SB-1, 301B/1, and HPRS24) showed that 33 genes in the Chinese isolates differed in length due to insertions or deletions, whereas 27 genes exhibited single-nucleotide polymorphisms (SNPs) without length variation. Homology analysis demonstrated that the four Chinese isolates were more closely related to the US strain 301B/1 than to the US strain SB-1 or the UK strain HPRS24. Point mutations resulted in SNP differences in 18 genes between strain 301B/1 and the Chinese isolates. Additionally, variations in the lengths of four genes (ORF3, ORF413, R-LORF1, and R-LORF9) were observed among the four Chinese isolates, indicating marked variability in these genomic regions. These findings align with alterations reported in circulating MDV-2 strains in China. Pathogenicity assessment of strain SW20 in specific-pathogen-free chickens showed that the strain induced inflammatory liver nodules, thymic atrophy, and splenic enlargement. This study highlights the expanding distribution of MDV-2 in China and confirms its ability to induce inflammatory responses, representing the first systematic epidemiological investigation of MDV-2 in the country. The findings provide an important reference for MD control efforts and emphasize the ongoing need for active surveillance.

The novel variant infectious bursal disease virus (nVarIBDV), which has been widely prevalent since 2017, threatens the poultry industry by inducing severe bursal atrophy and intense inflammatory responses. Understanding the inflammatory mechanism underlying nVarIBDV infection is critical for preventing virus-induced damage. Pyroptosis, an inflammatory type of programmed cell death, may contribute to the inflammation and bursa of Fabricius damage induced by nVarIBDV. Here, we found that the nVarIBDV infection induced severe inflammatory responses both in vivo and in vitro , which were associated with pyroptosis. Further research revealed that the viral protein VP3 drove pyroptosis induced by nVarIBDV infection by indirectly activating the Caspase-3-GSDME pathway, leading to GSDME cleavage. Mechanistically, VP3 directly interacted with and activated Caspase-9, thereby initiating Caspase-9-Caspase-3-GSDME pathway. More importantly, the Serine 33 of VP3 was identified as the key amino acid for interacting with and activating Caspase-9. Mutation of this residue significantly weakened the ability of VP3 to interact with Caspase-9 and activate the Caspase-9-mediated pyroptosis pathway, altered the binding mode of the Caspase-9-VP3 protein complex, and ultimately reduced the ability of VP3 to induce pyroptosis. In conclusion, our results elucidated a novel mechanism by which nVarIBDV infection induced inflammatory responses whereby viral protein VP3 triggered pyroptosis by targeting the Caspase-9-Caspase-3-GSDME pathway.

Background: Chicken infectious anemia virus (CIAV) is a globally significant immunosuppressive pathogen that causes substantial economic losses to the poultry industry, with particularly severe outbreaks in China in recent years. Given the limitations of existing vaccines, especially the residual virulence associated with live attenuated vaccines, there is an urgent need to develop novel, safer, and more effective vaccine strategies. Methods: In this study, the VP1 and VP2 genes of CIAV were cloned and expressed in Escherichia coli to develop a cost-effective subunit vaccine. Since VP1 primarily formed inclusion bodies, a “VP2-assisted co-refolding” strategy was employed. This involved denaturing VP1 and refolding it via gradient dialysis in the presence of soluble VP2, thereby leveraging VP2’s natural chaperone-like function to restore conformational epitopes. The refolded VP1/VP2 protein complexes, emulsified at different ratios, were used to immunize 3-day-old specific pathogen-free (SPF) chickens, followed by challenge with a virulent CIAV strain. Results: The vaccine formulation with a VP1:VP2 ratio of 1:1 provided the best protection, achieving 71.4% (5/7) protective efficacy, as evidenced by significantly reduced thymic atrophy and a higher thymus index. Conclusions: These findings validate the feasibility of using an economical prokaryotic expression system combined with a rational protein refolding strategy to produce a protective subunit vaccine candidate against CIAV, offering a promising alternative for disease control.

Reticuloendotheliosis virus (REV) is an immunosuppressive virus in poultry that can cause acute reticular neoplasms, chronic lymphoid tumors, stunting syndrome, and secondary infections. In many countries, the lack of effective vaccines has resulted in a high prevalence of REV infections and substantial economic losses. Enzyme-linked immunosorbent assay (ELISA)-based antibody detection is an important tool for monitoring the REV prevalence in poultry farms. ELISA coating antigens generally consist of either whole virus or viral protein; however, most commercially available REV antibody ELISA detection kits use whole virus as the coating antigen, which limits their applicability in certain diagnostic and research settings. In this study, the gp90 protein from a dominant REV strain was expressed and purified using 293F suspension cell eukaryotic expression system. Using recombinant gp90 protein as the coating antigen, an indirect ELISA for detecting gp90 antibodies (gp90-ELISA) was developed. After optimization, the optimal conditions were as follows: coating antigen concentration of 4 µg/mL with overnight incubation at 4 °C; blocking with 5% skim milk at 37 °C for 1.5 h; serum dilution of 1:200 with incubation at 37 °C for 45 min; secondary antibody dilution of 1:1000 with incubation at 37 °C for 30 min; and color development using TMB substrate at room temperature in the dark for 10 min. The cut-off value was defined as an OD450 ≥ 0.22 for positive samples and <0.22 for negative samples. The developed gp90-ELISA specifically detected REV-positive sera at a maximum serum dilution ratio of 1:3200. Intra- and inter-assay variation coefficients were ≤10%, indicating that the gp90-ELISA had good specificity, sensitivity, and reproducibility. Laboratory serum testing showed that the gp90-ELISA successfully detected sera from chickens immunized with the gp90 protein or infected with REV. Furthermore, analysis of clinical serum samples demonstrated 100% concordance between the gp90-ELISA results and a commercial whole-virus-coated ELISA kit. These results indicate that the gp90-ELISA is a reliable supplementary method to whole-virus-coated ELISA and has potential utility in disease surveillance and evaluation of immune responses.

Avian metapneumovirus subtype B (aMPV/B) infections significantly affect the global poultry industry. However, the virulence determinants and attenuation mechanism remain unknown. Here, a series of chimeric and mutant viruses was constructed, and their pathogenicity was evaluated in a specific-pathogen-free (SPF) chicken model. First, substitutions in different genes (N, P, F, SH, G, and L) and amino acid sites (323rd, 396th, and 522nd residues in F protein) in virulent (LN16-V) and attenuated (LN16-A) viruses revealed that the residue 396th in F protein is closely related to replication ability in vitro and in vivo and is a critical determinant of aMPV/B virulence in chickens. Further studies revealed that the K396R mutation in the F protein decreases the adsorption of aMPV/B to DF-1 cells, which reduces the binding of the F protein with αVβ1 integrin. Structural and surface plasmon resonance analysis indicated that the K396R mutation primarily reduced the electrostatic potential of the RDD motif of the F protein that binds with αVβ1 and decreased the binding between the F protein and αVβ1, which is a critical determinant of the replication ability of aMPV/B. Collectively, these findings not only contribute to a better understanding of the aMPV attenuation mechanism but also offer novel strategies for developing Metapneumovirus members live vaccines.IMPORTANCEBoth aMPV and hMPV belong to the Metapneumovirus family and cause the most acute respiratory diseases in poultry and humans, respectively. Recently, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the USA, largely attributed to aMPV/B infections. Live-attenuated vaccines developed by the blind passage of virulent strains in tissue culture have been widely used to prevent aMPV/B infection. However, the mechanism of aMPV/B attenuation remains unclear. Here, we identified the F gene as a key determinant of the virulence of aMPV/B and confirmed that residue 396 in the F protein plays an important role in attenuating the virulence of aMPV/B. Importantly, we found that the K396R mutation decreased the binding affinity between the F protein and αVβ1 and reduced the replication ability of aMPV/B. This is the first study to identify the key virulence genes and amino acid residues of aMPV/B and elucidate the molecular mechanisms underlying the attenuation of virulence. Our work provides fundamental insights into aMPV/B pathogenicity and offers direction for guiding the rational design of novel and more effective vaccines against aMPV/B and, by extension, related pathogens, such as hMPV.

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in innate immunity by inducing antiviral proteins in response to interferon signals. Marek's disease virus (MDV), a member of the alphaherpesvirus family, exerts potent tumorigenic and immunosuppressive effects. Recent studies have primarily focused on the tumorigenic mechanisms of MDV, and the mechanism of immune evasion has not been fully understood. In this study, we showed that MDV reduced the production of interferon-stimulated genes (ISGs) by inhibiting the phosphorylation and nuclear translocation of STAT1. Using a dual-luciferase reporter system, we screened for viral proteins that significantly suppress interferon-stimulated response element (ISRE) promoter activity. Meq overexpression markedly reduced ISRE promoter activity and ISG expression, whereas infection with Meq-deficient MDV induced higher ISG production in vitro and in vivo than infection with wild-type MDV. Meq also inhibited the phosphorylation and nuclear translocation of STAT1. Further experiments showed that Meq interacted with JAK1 and tyrosine kinase 2 (TYK2) and thereby inhibited JAK1-STAT1 interactions. Meq degraded TYK2 via a caspase-mediated pathway. The Meq-deficient MDV mutant replicated less efficiently than the wild-type MDV, both in vitro and in vivo. Collectively, these findings demonstrate that Meq played an immunosuppressive role in MDV by attenuating the JAK-STAT signaling pathway, which facilitated escape from innate immune surveillance mechanisms.

The infectious bursal disease virus (IBDV) can cause severe immunosuppression and high mortality in chickens, posing a significant threat to the poultry farming industry. Pyroptosis mediated by gasdermin E (GSDME) may be closely related to the tissue damage caused by IBDV. In this study, 3-week-old specific-pathogen-free (SPF) White Leghorns chickens were inoculated intranasally with 1000 copies/200 μL of very virulent IBDV (vvIBDV) Gx strain, and we analyzed GSDME expression in chicken tissues and the cleavage site of GSDME by Caspase-3. Tissue distribution results showed that GSDME and IL-1β transcription in the bursa of Fabricius and kidneys were significantly upregulated by more than five-fold (p < 0.01) following vvIBDV infection, indicating a close association with vvIBDV-induced tissue lesions. Further studies demonstrated that Caspase-3 could cleave GSDME at conserved sites (D270), releasing the active N-terminal fragment (GSDME-N) to induce pyroptosis. Furthermore, although IBDV proteins cannot directly cleave GSDME, the viral protein VP3 enhances Caspase-3-mediated GSDME cleavage, thereby triggering pyroptosis. The above results reveal the role of GSDME-dependent pyroptosis in the pathogenesis of IBDV and provide new ideas for the prevention and control strategies against IBDV.

Chicken infectious anemia (CIA) is a highly contagious disease caused by the chicken infectious anemia virus (CIAV), and it poses a serious threat to the poultry industry. However, effective control measures and strategies have not been identified. In this study, a recombinant Marek’s disease virus (rMDV) expressing the VP1 and VP2 proteins of CIAV was successfully constructed using CRISPR/Cas9, and a commercial Marek’s disease virus (MDV) vaccine strain was used as the vector. VP1 and VP2 expression by rMDV was confirmed by immunofluorescence assay and western blot analysis, which revealed robust in vitro expression. Further analysis showed that the VP1 and VP2 genes integrated into the MDV genome did not alter the growth kinetics of the virus and remained stable even after 20 passages, indicating the genetic stability of the recombinant virus. In animal studies, vaccination of one-day-old specific-pathogen-free chickens with rMDV induced high levels of CIAV-specific antibodies (1 × 105) and neutralizing antibodies (1:25) and a potent cellular immune response. Moreover, rMDV vaccination conferred an 85% protective index against challenge with a highly virulent strain of CIAV, significantly reducing the occurrence of anemia and thymic atrophy caused by CIAV infection and dramatically suppressing CIAV replication in the thymus. Collectively, these results highlight the potential of rMDV as a vaccine candidate for preventing and controlling CIAV infection, thus offering a new avenue for mitigating the impact of CIA on the poultry industry.

Inflammation is key important pathogenic response that occurs in several poultry diseases. NOD-like receptor protein 3 inflammasome plays a central role in initiating inflammation. However, the lack of antibodies against chicken-derived inflammatory factors presents a major bottleneck in related studies. In this study, we developed a monoclonal antibody (mAb) against chicken NLRP3 using hybridoma technology. This antibody can recognize both eukaryotic and prokaryotic NLRP3 proteins as well as the upregulation of endogenous NLRP3 in HD11 cells induced by lipopolysaccharide or infectious bursal disease virus. Furthermore, a novel antigenic epitope, 42DELEKVTHPSS52, located in the PYD domain of chicken NLRP3 and specifically recognized by this mAb was identified. This epitope is unique to chickens and valuable for distinguishing chicken NLRP3 from orthologs in other species. The developed mAb provides an important tool for detecting chicken NLRP3 and facilitates further study of its antigenic structure and biochemical characteristics.

Vaccination is the most effective preventative measure against economically devastating poultry diseases, such as infectious bursal disease (IBD), H9 subtype avian influenza (AI), and Marek's disease. In recent decades, various vaccination strategies have been investigated and developed. Among these, multivalent and combination vaccines, which confer protection against multiple diseases in a single dose, have emerged as a significant advancement in veterinary medicine. This study demonstrates that the attenuated Marek's disease virus serotype 1 (MDV-1) vaccine strain, rMSΔMeq, known for its safety and robust carrier immunogenicity, can serve as a vector for the heterologous expression of multiple antigenic proteins. We constructed the recombinant virus rMDV-VP2-HA by inserting the H9 subtype avian influenza virus (AIV) hemagglutinin (HA) expression cassette into the UL41 region and the infectious bursal disease virus (IBDV) VP2 expression cassette into the US2 region. The growth characteristics of this recombinant virus were consistent with those of the parental virus, and it stably expressed the HA and VP2 genes. Vaccination with rMDV-VP2-HA induces high titers of IBDV-neutralizing antibodies and hemagglutinin inhibition (HI) antibodies and elicits strong cellular immune responses. Specifically, vaccination with rMDV-VP2-HA enhances interferon-gamma (IFN-γ) expression in response to stimulation with HA and VP2 proteins. Meanwhile, cytokines associated with both Th1 and Th2 responses were also upregulated. Remarkably, chickens vaccinated with rMDV-VP2-HA achieved complete protection against very virulent IBDV, H9 subtype AIV, and very virulent MDV. These findings underscore the exceptional practical potential of rMDV-VP2-HA as the first MDV-1 vector-based multivalent vaccine candidate for combating these poultry diseases.IMPORTANCECommercial vaccines for infectious bursal disease (IBD) and H9 subtype avian influenza (AI) require multiple doses, increasing costs and causing stress in chicken flocks. Additionally, their efficacy is frequently compromised by maternal antibody interference. This underscores the urgent need for a multivalent, multi-component, and single-dose vaccines capable of streamlining immunization protocols, overcoming maternal antibody interference, and providing lifelong immunity. Our research demonstrates, for the first time, serotype 1 Marek's disease virus (MDV-1) can stably express multiple exogenous genes. More significantly, the rMDV-VP2-HA elicits robust humoral and cellular immune responses, achieving complete protection against H9 subtype AIV, very virulent IBDV, and very virulent MDV with a single immunization. These findings contribute to enhancing the efficiency of disease prevention and confirm that the MDV-1 is an ideal vector for developing multivalent vaccines, achieving the goal of "multiple protections with a single shot." This advancement represents a significant progression in the prevention and control of economically important poultry diseases.

Chicken infectious bursal disease (IBD) is an acute, highly contagious, lethal, and immunosuppressive disease caused by the infectious bursal disease virus (IBDV). The newly emerged novel variant IBDV (nVarIBDV) and the continuously circulating very virulent IBDV (vvIBDV) are the two predominant epidemic strains that pose a threat to the poultry industry in several countries, including China. However, on-site rapid detection methods for distinguishing nVarIBDV from vvIBDV remain lacking. In this study, a neutralizing monoclonal antibody (mAb), 2F10, capable of differentiating nVarIBDV from vvIBDV was successfully developed for the first time. The antigenic epitope recognized by mAb 2F10 is conformation-dependent, with residue 318 of VP2 being the key determinant of its specificity. Significant differences in hydrogen bonding, hydrophobic interactions, and salt bridges at the VP2-mAb interface-caused by the consistent substitution of aspartic acid (in nVarIBDV) and glycine (in vvIBDV) at residue 318-constitute the core mechanism by which mAb 2F10 selectively recognizes nVarIBDV but not vvIBDV. Further data confirm that mAb 2F10 can be utilized to develop a competitive ELISA for the specific detection of nVarIBDV antibodies. This study not only contributes to a deeper understanding of the antigen structure and immune evasion mechanism of nVarIBDV but also provides a valuable tool for molecular tracing and differential detection of this novel variant.

Pro-IL-1β is an important inflammatory factor and is also a biomarker for detecting early pro-inflammatory immune responses. However, commercially available antibodies against chicken inflammatory factors are lacking, which prohibits an in-depth exploration of the mechanism of chicken inflammation. This study cloned and expressed chicken pro-IL-1β, and developed a hybridoma cell line 1E12 capable of stably secreting chicken pro-IL-1β monoclonal antibody (mAb). The secreted mAb 1E12 can recognize exogenous or endogenous chicken pro-IL-1β by Western blot and IFA techniques, and for the first time, a novel antigen epitope 13SSLSEETFY21 of chicken pro-IL-1β recognized by this mAb was identified. This study not only provides an important tool for the detection and research of chicken pro-IL-1β, but also has significant implications for understanding the antigenic structure and biochemical characteristics of chicken pro-IL-1β.