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Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) has recently emerged in Europe, causing widespread outbreaks in cattle and wild ruminant populations. The lack of commercial vaccines at the time of emergence highlighted the urgent need to develop effective vaccines for disease control. Therefore, we developed and evaluated the safety and efficacy of a novel subunit candidate vaccine against EHDV-8, based on the recombinant VP2 protein (rVP2) from EHDV-8 expressed in a baculovirus system. Ten Holstein-Friesian calves were randomly assigned to vaccinated (n = 5) or control (n = 5) groups. Animals in the vaccinated group received two doses of 200 μg of rVP2 protein 21 days apart, while control animals were sham-vaccinated. Fourteen days after the booster dose, both groups were challenged with a field strain of EHDV-8. Clinical signs were recorded daily, and blood samples were collected at regular intervals to assess viremia and immune response. The rVP2 vaccine candidate was well tolerated, with only transient, low-grade fever and mild local reactions. Vaccinated calves developed neutralizing antibodies by day 28 post-vaccination and, upon EHDV-8 challenge on day 35 post-vaccination, showed no clinical signs or viremia. In contrast, all control calves developed viremia, mild clinical signs, and EHDV-specific antibodies after challenge. Notably, vaccinated animals remained negative by VP7 ELISA even post-challenge, indicating that the rVP2-based vaccine afforded protection from viral replication and disease and enabled DIVA (Differentiating Infected from Vaccinated Animals) capability. In conclusion, these results demonstrate that the rVP2-based subunit vaccine confers complete protection from EHDV-8 under experimental conditions in cattle, indicating the possibility of its use for disease control strategies.

Background Infectious bursal disease virus (IBDV) remains a major threat to the global poultry industry, causing significant economic losses. Recent surveillance data indicate that genetically diverse strains of IBDV are circulating in different regions of China. Methods In this study, both commercial and backyard chicken flocks from Shandong Province were investigated for evidence of IBDV circulation using a combination of serological assays and molecular detection techniques. Results Despite routine vaccination, antibody responses were highly variable, with some flocks showing reduced maternal immunity while others exhibiting active seroconversion by 35 days of age. Many birds showed an abnormally low bursa-to-body weight ratio, with a significant decline observed between 18 and 35 days of age in most farms, indicating immunosuppression. RT-PCR detected IBDV in 68 bursal samples, most of which were classified as very virulent strains (vvIBDV) belonging to genogroup A3, while a smaller subset was identified as variant strains within genogroup A2 based on phylogenetic analysis of the VP2 gene. Comparison of the VP2 protein sequence with other Chinese strains revealed variations in key hypervariable region residues critical for antibody binding. Conserved markers of very virulent strains persisted, while the novel substitutions A222V and D279H emerged, neither of which is present in commonly used vaccine strains. Conclusions Our findings highlight emerging IBDV variants with altered VP2 antigenicity, potentially causing vaccine escape and increasing poultry health and economic risks. Graphical abstract

Infectious bursal disease (IBD) is a severe immunosuppressive disease caused by the infection of infectious bursal disease virus (IBDV) in chicken. Recently, an emerging mutant named novel variant IBDV (nVarIBDV) has rapidly spread in China and become a prevalent strain. However, little is known about the unique antigenic sites of nVarIBDV escaped from current IBDV vaccines. Here, the expressed hypervariable region (HVR) of VP2 (VP2-HVR) of nVarIBDV was used as an immunogen and a novel monoclonal antibody (mAb) against VP2 (mAb 5B5) was generated. Immunofluorescence assay (IFA) and ELISA demonstrated that mAb 5B5 specifically reacted with nVarIBDV and its VP2 protein, but not with classical IBDV (cIBDV), very virulent IBDV (vvIBDV), or attenuated IBDV (attIBDV) strains. Epitope mapping and site mutagenesis assay revealed that mAb 5B5 recognized the conformational epitope in peak A (212-224 aa) and heptapeptide (326-332 aa) regions, and identified residue 221K in VP2 as the key antigenic site, which is conserved exclusively in nVarIBDV strains. Notably, K221Q mutation in VP2 of nVarIBDV significantly altered the reaction profile for sera against vvIBDV or cIBDV. Neutralization assays revealed that mAb 5B5 could inhibit replication of an engineered attIBDV carrying 221K in Leghorn male hepatoma (LMH) cells. Structural analysis further found that 221K is surface-exposed and alters local electrostatic potential, possibly facilitating immune evasion. All these demonstrated that 221K is a unique antigenic site in VP2 of nVarIBDV associated with immune escape, providing novel insights into the antigenicity of nVarIBDV and novel targets for efficient diagnostics, vaccine design, and molecular surveillance of IBDV.

Background Senecavirus A (SVA) is a pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical signs are similar to those of foot-and-mouth disease, porcine vesicular disease, and vesicular stomatitis. Therefore, identification of SVA as a cause of PIVD is important to eliminate this emerging pathogen. Methods In this study, an indirect ELISA based on the VP2 epitope (VP2-epitp-ELISA) was developed to detect antibodies directed against SVA. Results A novel linear epitope ( 271 GLRNRFTTGTDEEQ 284 ) was first identified at the C-terminus of the VP2 protein by epitope mapping. The diagnostic performance of VP2-epitp-ELISA was estimated by testing a panel of known background sera from swine. Under the optimum test conditions, when the cutoff value was 37%, the diagnostic sensitivity (Dn) and diagnostic specificity (Dp) of the assay were 91.13% and 91.17%, respectively. The accuracy of VP2-epitp-ELISA was validated and further compared with that of commercial diagnostic kits. The diagnostic results showed that VP2-epitp-ELISA did not cross-react with serum positive for other idiopathic vesicular diseases and had a concordance rate of 90.41% with the Swinecheck ® SVA bELISA. Conclusions These results indicate that VP2-epitp-ELISA is suitable for specific detection of antibodies against SVA in swine.

The feline panleukopenia virus (FPV) is a highly contagious virus that affects cats worldwide, characterized by leukopenia, high temperature and diarrhea. Recently, the continuous prevalence and variation of FPV have attracted widespread concern. The aim of this study was to investigate the isolation, genetic evolution, molecular characterization and epidemiological analysis of FPV strains among cats and dogs in China from 2019 to 2024. The 41 FPV strains, including 38 feline strains and 3 canine strains, were isolated from rectal swab samples by inoculating monolayer FK81 cells and performing a plaque purification assay. The viral and hemagglutination titers of these 41 FPV strains were 104.33~106.33 TCID50/0.1 mL and 7.0 log2~9.7 log2, respectively. Based on the complete VP2 gene, the nucleotide homology of these FPV strains was 98.91~100%, and the homology with 24 reference FPV strains from different countries and hosts was 98.85~100%. The phylogenetic analysis revealed that 41 FPV strains were more closely related to the FPV strains of Asian origin (Asian FPV strain group) than those of European and American origin (European and American FPV strain group). Furthermore, 12 mutation sites of the VP2 protein were found in these FPV strains, of which 91 and 232 amino acid sites were previously reported. Moreover, the 91 amino acid site was found to be a positive selection site with the highest dN/dS value in the selection pressure analysis. Importantly, 35 FPV strains with 91S substitution in the VP2 protein (FPV-VP2-91S strains) had formed obvious evolutionary branches in the Asian FPV strain group. The analysis of all available VP2 protein sequences of Chinese FPV strains in the GenBank database showed that the occurrence rate of FPV-VP2-91S strains had been increasing from 15.63% to 100% during 2017~2024, indicating that the FPV-VP2-91S substitution in the VP2 protein was a noteworthy molecular characteristic of the dominant FPV strains in China. These results contribute to a better understanding of their genetic evolution and renew the knowledge of FPV molecular epidemiology.

Background Canine parvovirus (CPV) poses an emerging threat to endangered felids, yet its epidemiology and evolutionary dynamics in tigers remain poorly characterized. Methods Pathogen detection was performed through necropsy, colloidal gold assays, and PCR on a deceased Bengal tiger, followed by genetic and molecular characterization of the identified CPV. Results This study isolated a CPV-2c strain (HNSY-031) from captive Bengal tigers. Genetic analysis revealed its closest relationship to Vietnamese canine parvoviruses, with 100% identity to CPV strains circulating in China and Vietnam since 2020. Molecular characterization identified 12 amino acid mutations within the VP2 protein, including key residues associated with host adaptation and viral evolution. Conclusions The isolated CPV-2c strain may have originated from multiple potential sources, including contact with domestic or wild animals or contaminated environments. These findings highlight the necessity of implementing comprehensive biosecurity protocols around wildlife facilities, expanding vaccination programs for animals in surrounding regions, and establishing ongoing surveillance systems to monitor potential sources of pathogen exposure.

•In our study, we expressed the IBDV antigenic VP2 protein in the cytoplasm of food-grade L. lactis NZ3900 with pNZ8149.•To enhance antigen delivery, we targeted gut M cells for antigen presentation.•The results of this study indicate r-L. lactis expressing foreign protein is a highly specific antigen expression and delivery system that can be used to induce the high level of neutralizing antibodies in immunized animals. Infectious bursal disease virus (IBDV) is a highly contagious disease that results in enormous economic losses in the global poultry sector. Lactic acid bacteria are an appealing vehicle for the safe and effective delivery of heterologous protein antigens. Oral administration of the commensal bacterium Lactococcus lactis expressing recombinant fusion proteins has been used to elicit mucosal and systemic immune responses. In this study, a Lactococcus lactis NZ3900 strain co-expressing the outer membrane protein (Omp) H of the microfold (M) cell-targeting ligand and the viral capsid protein (VP)2 antigen of IBDV was genetically engineered, and its immunopotentiating capacity as an oral and injected vaccine in chickens was evaluated. Western blotting analysis demonstrated that VP2-OmpH was expressed in the cytoplasm of cells and had high immunoreactivity. An in vivo study showed that in the absence of any adjuvant, the recombinant L. lactis VP2-OmpH strain stimulated the immune response and protected against very virulent IBDV challenge in 100% and 80% of chickens immunized by injection and oral administration, respectively. Moreover, the antiviral neutralizing antibody titers induced by injection administration were higher than those induced by oral administration. Mucosal secretory IgA titers induced by oral administration were higher than those induced by injection administration. These results suggested that the recombinant L. lactis VP2-OmpH strain is a promising candidate vaccine to prevent IBDV infection.

Feline parvovirus (FPV), a single-stranded DNA virus, is accountable for causing feline panleukopenia, a highly contagious and often lethal disease that primarily affects cats. The epidemiology prevalence and pathogenicity of FPV in certain regions of China, however, remains unclear. The aim of this research was to investigate the epidemiology of FPV in different regions of China in 2021 and compare its infectivity and pathogenicity. In this research, a total of 36 FPV strains were obtained from diverse regions across China. Phylogenetic analysis was performed based on the VP2 and NS1 sequences, and two representative strains, FPV027 and FPV072, which belonged to different branches, were selected for comparative assessment of infectivity and pathogenicity. The results revealed that all strains were phylogenetically classified into two groups, G1 and G2, with a higher prevalence of G1 strains in China. Both and experiments demonstrated that FPV072 (G1 group) exhibited enhanced infectivity and pathogenicity compared to FPV027 (G2 Group). The structural alignment of the VP2 protein between the two viruses revealed mutations in residues 91, 232, and 300 that may contribute to differences in infectivity and pathogenicity. The findings from these observations will contribute significantly to the overall understanding of the molecular epidemiology of FPV in China and facilitate the development of an effective FPV vaccine.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout ( Oncorhynchus mykiss ) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM + B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

Infectious bursal disease virus (IBDV) is one of the most important immunosuppressive viruses in poultry, causing the global spread of infectious bursal disease (IBD). It poses a significant threat to the healthy development of the poultry industry. Vaccination is an effective approach for controlling IBDV infection. Therefore, reliable immune monitoring for IBDV is critical for maintaining poultry health. The enzyme-linked immunosorbent assay (ELISA) is a common technique used to detect specific antibodies in clinical serum testing and for the serological evaluation of IBDV vaccines. Among the currently available and under development IBDV vaccines, IBD VP2 subunit-based vaccines account for a considerable proportion. These vaccines stimulate the production of antibodies that are specific only to VP2. However, most IBDV antibody ELISA kits approved for use have applied the whole virus as the coating antigen, which does not adequately meet the diverse requirements for IBDV detection across different conditions. This study utilized a prokaryotic expression system to express the VP2 protein of the IBDV epidemic strain, assembling it into virus-like particles to be used as coating antigens. This approach enabled the establishment of an indirect ELISA method for detecting IBDV VP2 antibody (VP2-ELISA). The optimal coated antigen concentration was determined to be 2.5 μg/mL, with overnight coating at 4 °C; sealing with 5% skim milk at 37 °C for 4 h; serum dilution at 1:500 with incubation at 37 °C for 30 min; secondary antibody dilution at 1:4000 with incubation at 37 °C for 40 min; and then incubation with the substrate solution 3,3′,5,5′-tetramethylbenzidine at room temperature for 20 min. The criterion for interpreting the detection results was OD450nm ≥ 0.111 indicates IBDV antibody positivity, while OD450nm < 0.111 indicates negativity. The established VP2-ELISA can specifically detect IBDV-positive sera at the lowest serum dilution of 1:6400, with intra- and inter-batch coefficients of variation of <2%. This indicates that the VP2-ELISA exhibits good specificity, sensitivity, and stability. Detection experiments using 20 laboratory-immunized chicken serum samples and 273 clinical serum samples demonstrated that the results of VP2-ELISA were consistent with those of commercial ELISA kits coated with whole virus. In summary, the VP2-ELISA developed in this study offers advantages in immune response detection for IBD VP2 subunit-based vaccines and is appropriate for evaluating the efficacy of IBD vaccines and detecting clinical serum samples.