Five IBDV strains—G61 (cIBDV, A1), 2340 and 2331 (nVarIBDV, A2d), 2341 (vvIBDV, A3), and NF8 (attIBDV, A8)—were isolated and preserved in our laboratory. NF8 is an attIBDV strain and kept in our laboratory. Leghorn male hepatoma (LMH) cells from ATCC were cultured in Dulbecco’s modified Eagle medium F12 (DMEM/F12) (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Lonsera, Shanghai, China), 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C and 5% CO2 atmosphere. SP2/0 myeloma cells from our laboratory were maintained in HT medium. The monoclonal antibodies mAb 6C5 against VP2 protein of IBDV and mAb 2G10 (37) against the Fiber-2 protein of Duck adenovirus 3 (DAdV-3) were generated and stored in our laboratory. Chicken sera against nVarIBDV, vvIBDV or cIBDV were prepared and stored in our laboratory.

All primers used in the experiments are listed in Table 1 . All eukaryotic recombinant plasmids were constructed into the pcDNA3.1 vector via homologous recombination, each fused with a C-terminal Flag tag. These recombinant plasmids include pc-nVarIBDV-VP2, pc-vvIBDV-VP2, pc-attIBDV-VP2, pc-cIBDV-VP2, pc-VP2-HVR, pc-201–347 aa, pc-201–327 aa, pc-201–307 aa, pc-201–287 aa, pc-201–267 aa, pc-201–337 aa, pc-225–337 aa, pc-VP2-delA, pc-VP2-delhep, pc-nVarIBDV-N213D, pc-nVarIBDV-K221Q, pc-nVarIBDV-K249Q, pc-nVarIBDV-I252V, pc-nVarIBDV-N254G, pc-nVarIBDV-D318G, pc-nVarIBDV-E323D, pc-nVarIBDV-K221Q, pc-vvIBDV-Q221K, pc-cIBDV-Q221K, pc-attIBDV-Q221K. BALB/c mice were purchased from experimental center for comparative medicine, Yangzhou University.

The VP2-HVR fragment was amplified from the nVarIBDV cDNA template using the primers pColdI-VP2-HVR-F/R and then cloned to pColdI vector. The correct recombinant plasmid named pColdI-VP2-HVR was transformed into E. coli Rosetta (DE3) cells and the expression of VP2-HVR was induced with 1.0 mM isopropyl β-D-thiogalactoside (IPTG) at 16°C for 15 h. Then cells were collected, resuspended in PBS, lysed by sonication and centrifuged. The supernatants and precipitates were collected for protein identification by SDS-PAGE and Western blot. The expressed VP2-HVR protein was then purified by Ni-Sepharose high-performance affinity gel (GE Healthcare Life sciences, Freiburg, Germany) according to manufacturer’s instructions and identified by SDS-PAGE and Western blot analysis.

The purified VP2-HVR protein was mixed with Freund’s complete adjuvant (Sigma-Aldrich, Missoula, MO, USA), and then was intraperitoneally injected into BALB/c mice at a dose of 50 μg/mouse for the first immunization. The second and third immunization were performed with 50 μg purified VP2-HVR protein mixed with Freund’s incomplete adjuvant (Sigma-Aldrich, Missoula, MO, USA) every two weeks. Mouse with the highest serum titers against VP2 was selected and immunized with 50 μg of VP2-HVR protein by intraperitoneal injection without adjuvant. Three days later, the spleen cells from the immunized mice were fused with SP2/0 cells according to the procedure previously described (38). To screen positive hybridoma cells, LMH cells transfected with the pc-VP2-HVR were used as antigen in IFA. Then the positive hybridomas were subcloned for three times by limiting dilution method. The ascites of the corresponding positive hybridomas were prepared using paraffin-primed Balb/c mice, and purified by the NAb™ Spin Columns (Thermo scientific™). The subclass of mAbs was identified using the mouse monoclonal antibody subtype identification Kit (Biodragon, Beijing, China).

LMH cells transfected with serial truncations of VP2 protein (pc-201–347 aa/201–327 aa/201–307 aa/201–287 aa/201–267 aa/201–337 aa/225–337 aa, pc-VP2-delA and pc-VP2-delhep) or VP2 proteins from IBDV strains of different virulence (pc-nVarIBDV-VP2, pc-vvIBDV-VP2, pc-attIBDV-VP2 and pc-cIBDV-VP2) were fixed with chilled acetone: ethanol solution (3:2) for 5 min. Then the solution was discarded and the fixed LMH cells were dried for 1 h. Subsequently, mAbs 5B5 or Flag, and IBDV antisera were used as the primary antibody, while the FITC-labeled goat anti-mouse or anti-chicken IgG (Sigma-Aldrich, USA) served as the secondary antibody. Both were diluted with PBS and then incubated with LMH cells at 37°C for 45 minutes. After three washes with PBS, LMH cells were observed under an inverted fluorescence microscope. The fluorescence intensity was analyzed using ImageJ (NIH). Briefly, the images were converted to grayscale, followed by setting the threshold and measurement area (with fixed threshold and range). The relative fluorescence intensity was quantified using the formula: (Mean fluorescence intensity of test group [IntDen/Area])/(Mean fluorescence intensity of positive group).

LMH cells transfected with indicated plasmids or infected with attIBDV were lysed with RIPA buffer (CWbio, Beijing, China) containing protease and phosphatase inhibitors (CST, MA, USA) for 20 min on the ice. The supernatant of the lysate collected by centrifuging and mixed with SDS loading buffer was boiled and then used for SDS-PAGE analysis. Subsequently, the proteins were transferred onto nitrocellulose membrane (GE Healthcare Life sciences, Freiburg, Germany). The membrane was then blocked with 5% skim milk in PBST and incubated with mAbs 5B5 or 6C5 at room temperature for 2 h. After being washed three times with PBST, the membrane was incubated with HRP-labeled goat anti-mouse IgG, for 1 h. Following additional three washes with PBST, the membrane was developed with luminol and peroxide (CWbio, Beijing, China) and imaged using an automatic imaging system (Tanon 5200). Densitometric analysis of Western blot bands was conducted using ImageJ (NIH). The analytical procedure included background subtraction correction, where identically sized measurement frames were applied to capture both target band signals and adjacent background signals in band-free regions, with background values subsequently deducted from target values. All data were normalized to GAPDH as an internal control, with relative expression levels calculated as the ratio of corrected target protein density to corresponding GAPDH density values.

The sandwich ELISA was used to verify the cross-reactivity of monoclonal antibodies with different types of IBDV. Briefly, the purified mAb 6C5 was diluted with a coating buffer (0.159 g Na₂CO₃, 0.293 g NaHCO₃, 100 mL ddH₂O, pH 9.6) to a final concentration of 15 μg/mL, and coated in ELISA plate with 100 μL per well at 4°C overnight. After three times washing with PBST buffer (PBS containing 0.05% Tween-20, pH 7.4), the wells were blocked with 5% skim milk at 37°C for 2 h. Washing three times as above, 100 μL dilution of different types of IBDV (nVarIBDV, vvIBDV, cIBDV and attIBDV) with the same viral copy number was added and incubated at 37°C for 1 h. After washing, 100 μL HRP conjugated mAbs 5B5, 6C5 and 2G10, diluted 1:300, were added and incubated for 1 h. Following another three washes, the 100 μL volume of 3, 3’,5,5’-Tetramethylbenzidine (TMB) ELISA substrate solution (Solarbio, Beijing, China) was added and incubated at 37°C for 15 min in the dark. Finally, 50 μL of 2 M H₂SO₄ was added to terminate the reaction and the values of OD₄₅₀ nm were measured using a Microplate Spectrophotometer.

To map the epitope recognized by mAb 5B5, a series of truncated VP2-HVR variants were cloned into the pcDNA3.1 vector. During the first screening round, five truncated constructs spanning residues 201–347 aa (V1), 201–327 aa (V2), 201–307 aa (V3), 201–287 aa (V4), and 201–267 aa (V5) were expressed. In the second round, V1 was further dissected into two overlapping peptides (201–337 aa and 225–337 aa). Additionally, using pc nVarIBDV-VP2 as the template, deletion mutants lacking either the peak A region (Δ212–224 aa) or the heptapeptide motif (Δ326–332 aa) were generated, respectively. The truncation strategy is schematically illustrated in Figure 1A . Furthermore, site-directed mutagenesis was performed to introduce single amino acid substitutions (N213D, K221Q, K249Q, I252V, N254G, D318G, E323D) into pc-nVarIBDV-VP2, and reciprocal mutations (Q221K) were engineered into pc-cIBDV-VP2, pc-vvIBDV-VP2, and pc-attIBDV-VP2. Epitope mapping was subsequently conducted using the IFA described above.

The infectious clone of the NF8 strain (attIBDV) was constructed as previously described (39). All primers involved below are from the Table 2 . The infectious clone of the NF8 strain was constructed as follows. For segment A assembly, two overlapping fragments were generated through multi-step overlap PCR. The first fragment underwent three successive amplifications using primer pairs A1/A4, A2/A4, and A3/A4. The second fragment was amplified through four sequential PCR steps with primer pairs A5/A6, A5/A7, A5/A8, and A5/A9. Following purification, the full-length segment A flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences was generated via overlap PCR using primers A3/A9. The engineered fragment was digested with NotI/NheI and directionally cloned into pcDNA3.1 to generate infectious clone pc-NF8A. Based on the plasmid pc-NF8A, primer pairs A3/attIBDV-Q221K-R and attIBDV-Q221K-F/A9 were introduced Q221K mutation and the mutated plasmid was named pc-NF8A-221K. For segment B construction, two overlapping fragments were similarly amplified: the first using three-step PCR (B1/B4, B2/B4, B3/B4) and the second through four-step PCR (B5/B6, B5/B7, B5/B8, B5/B9). Full-length segment B was assembled using primers B3/B9. The fragment was digested with NotI/NheI and cloned into pcDNA3.1 to generate pc-NF8B. The infectious viruses rNF8 and its variant rNF8-221K were generated through co-transfection of LMH cells with either pc-NF8A or pc-NF8A-221K alongside pc-NF8B. At 5 days post-transfection (dpt), transfected cells underwent three freeze-thaw cycles to release viral progeny. The cell lysate was subsequently subcultured in fresh LMH cells for viral amplification. At 5 days post-inoculation (dpi), the infected cells were harvested for rescue virus confirmation via IFA and sequencing validation.

LMH cells transfected with pc-nVarIBDV-VP2 or infected with pc-rNF8-221K were separately lysed in NP-40 lysis buffer for 30 min on ice. Following centrifugation at 12,000 × g for 10 min at 4°C, the resultant supernatant was incubated with the specified antibodies overnight and followed by an addition of Protein A/G magnetic beads for 3 h at 4°C. Upon centrifugation at 1,000 × g for 5 min at 4°C, the supernatant was discarded, and the beads were washed five times using ice-cold PBS. Then, the beads were lysed in SDS loading buffer and subjected to Western blot assays.

MAbs 5B5 and 6C5 were diluted to 60 µg/mL each. The mutant rNF8-221K was diluted to 1000 TCID50 per 500 µL and mixed with equal volumes of the diluted mAbs or nVarIBDV antiserum. After incubation at 37°C for 1 h, 1 mL of the mixture was added to each well of a 6-well plate containing a 90% confluent LMH cell monolayer. At 2 hours post-infection (hpi), the cells were divided into two experimental groups: one group was maintained in DMEM/F12 maintenance medium containing 1% FBS, while the other group received additional supplementation with the mAbs 5B5 or 6C5 (6 µg/mL) or nVarIBDV antiserum in the maintenance medium. Cells from both groups were harvested at 4 days post-infection (dpi) for Western blot analysis to assess viral replication. In a parallel experiment, LMH cells were first infected with rNF8-221K for 2 h and subsequently cultured in DMEM/F12 medium supplemented with 1% FBS and mAbs 5B5 or 6C5 concentration gradients. Supernatants were collected at 24-hour intervals, and at 4 dpi, cells were collected for Western blot analysis, while the corresponding supernatants were subjected to TCID50 assays.

The VP2-HVR amino acid sequences of IBDVs circulating in China, including nVarIBDV, vvIBDV, cIBDV, varIBDV and attIBDV, were retrieved from GenBank (listed in Table 3 ), and were aligned in DNAstar MegAlign software. The tertiary structure of VP2 protein was gathered from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/). The PyMOL software (Version 2.5.2, Schrödinger, LLC) was used to visualize the spatial distribution of epitopes recognized mAb 5B5.

All data were shown as the means ± SD of three independent experiments. Statistical differences were assessed using Student’s t- test and an unpaired two- tailed Student’s t- test using GraphPad Prism V.7.0 software. For all experiments, p < 0.05 was considered statistically significant (*p<0.05, **p<0.01).

To obtain the VP2-HVR protein, the recombinant plasmid pColdI-VP2-HVR was first constructed, and then transformed into the E. coli Rosetta cells and expressed as His-tagged fusion protein. As shown in Figure 2A , The recombinant VP2-HVR protein was successfully expressed in a soluble form in the Rosetta (DE3) expression system. SDS-PAGE analysis revealed a specific band of approximately 18 kDa in the supernatant, which corresponded to the expected molecular weight. In contrast, no corresponding band was detected at this molecular weight range in the empty pColdI vector control group. This expression was further confirmed by an anti-His monoclonal antibody ( Figure 2B ). Next, the recombinant VP2-HVR protein was purified using a Ni Sepharose™ affinity chromatography column. As shown in Figure 2C , SDS-PAGE revealed a specific band corresponding to the recombinant VP2-HVR protein. Furthermore, Western blot assay indicated that the recombinant VP2-HVR protein could be effectively recognized by chicken sera against nVarIBDV ( Figure 2D ). Collectively, these results demonstrated that the prokaryotic recombinant VP2-HVR protein of nVarIBDV was successfully expressed with good antigenicity.

To generate monoclonal antibodies (mAbs) targeting the VP2 protein of nVarIBDV, BALB/c mice were immunized with purified recombinant VP2- HVR protein derived from nVarIBDV. Since only an attIBDV strain (NF8) - but not nVarIBDV, vvIBDV, or cIBDV - was adapted to cell culture in our study, the positive sera from immunized mice were verified by reacting with LMH cells transfected with pc-VP2-HVR or infected with NF8. IFA results revealed strong serum reactivity against VP2-HVR and NF8 ( Figure 3A ). Splenocytes from high-titer responders were subsequently fused with SP2/0 myeloma cells, yielding 16 positive hybridoma clones. Among these, mAb 5B5 demonstrated strain-specific recognition of nVarIBDV, showing no cross-reactivity with vvIBDV, cIBDV or attIBDV strains in an in-house established IBDV-specific sandwich ELISA assay ( Figure 3B ). The cutoff value was calculated as 0.26 using the formula: cutoff = OD450 mean + 3×SD based on 43 avian-derived viral samples (OD450 mean = 0.086, SD = 0.058). To further confirm the specificity of mAb 5B5 for nVarIBDV, LMH cells transfected with plasmids pc-nVarIBDV-VP2, pc-vvIBDV-VP2, pc-attIBDV-VP2 and pc-cIBDV-VP2 were used to IFA and Western blot analysis. As described in Figure 3C , mAb 5B5 specifically recognized VP2 protein of nVarIBDV, but not that of vvIBDV, cIBDV or attIBDV strains, which was consistent with the data of ELISA. However, 5B5 could not react with any denatured VP2 protein of different IBDV types including nVarIBDV, vvIBDV, cIBDV or attIBDV (data not shown), suggesting that mAb 5B5 recognized the conformational epitope of VP2. To validate the binding specificity of monoclonal antibody 5B5 to nVarIBDV VP2, immunoprecipitation assays were conducted in LMH cells transiently expressing VP2 proteins from nVarIBDV, vvIBDV, cIBDV, and attIBDV. As shown in Figure 3D , mAb 5B5 demonstrated strong binding affinity for nVarIBDV VP2, while showed no detectable binding for VP2 proteins of vvIBDV, cIBDV or attIBDV (data not shown). Collectively, these data demonstrate that mAb 5B5 specifically recognizes nVarIBDV-derived VP2 without cross-reactivity to VP2 from other IBDV phenotypes.

To localize the conformational B-cell epitope recognized by mAb 5B5, a series of truncated VP2-HVR constructs were designed ( Figure 1A ). In the first round, five truncated fragments of the VP2-HVR protein from nVarIBDV were generated and used to map the epitope recognized by mAb 5B5. As described in Figures 1B, C , mAb 5B5 reacted only with the V1 (201-347aa) fragment, but not with the other constructed fragments. In the second round, V1 was further truncated to precisely define the epitope recognized by mAb 5B5. As shown in Figures 1C, D , mAb 5B5 reacted only with V6 (201-337aa). Notably, the deletion of the peak A (DNYQFSSQYKTPGG, 212-225aa) or the heptapeptide region (WSASGSS, 326-332aa) of the VP2 protein from nVarIBDV abolished the reaction between mAb 5B5 and the VP2 protein. Since there are seven amino acid differences (including D213N, Q221K, Q249K, V252I, G254N, G318D, and D323E) in the peak A and the heptapeptide region of VP2-HVR between nVarIBDV and other IBDV strains, to further determine the key residues recognized by mAb 5B5, N213D, K221Q, K249Q, I252V, N254G, D318G, or E323D mutation was individually introduced into the VP2 of nVarIBDV. The corresponding plasmids, designated as pc-nVarIBDV-N213D, pc-nVarIBDV-K221Q, pc-nVarIBDV-K249Q, pc-nVarIBDV-I252V, pc-nVarIBDV-N254G, pc-nVarIBDV-D318G, and pc-nVarIBDV-E323D, were subsequently generated. As described in Figure 4A , IFA analysis revealed that mAb 5B5 could still react with the VP2 of nVarIBDV carrying N213D, K249Q, I252V, N254G, D318G or E323D, but not with the VP2 of nVarIBDV with K221Q. To further confirm this, a panel of VP2 mutant of vvIBDV, cIBDV, or attIBDV with Q221K were constructed. As shown in Figure 4B , mAb 5B5 could effectively recognize the VP2 of vvIBDV, cIBDV, or attIBDV with Q221K. All these data demonstrated that the epitope recognized by mAb 5B5 is located in the peak A and heptapeptide regions of VP2, and 221K in VP2 of nVarIBDV is the key antigenic site in the epitope for mAb 5B5.

To further investigate the effect of the Q221K mutation on the antigenicity of VP2, LMH cells were transfected with wild-type VP2 of nVarIBDV, vvIBDV, cIBDV, and attIBDV, along with their corresponding mutants with Q221K or K221Q, and then were analyzed by IFA using chicken sera against different types of IBDV. As described in Figure 5 , although sera against nVarIBDV could recognize all the wild type VP2 proteins and their corresponding mutants, it had significantly enhanced reactivity with the wild type of VP2 of nVarIBDV compared to its mutant with K221Q. Notably, sera against vvIBDV or cIBDV demonstrated favorable affinity for the VP2 proteins of vvIBDV, cIBDV, and attIBDV, as well as their respective mutants. However, sera against vvIBDV or cIBDV could not recognize the wild type VP2 protein of nVarIBDV, but could react with the VP2 of nVarIBDV with K221Q ( Figure 5 ). All these demonstrated that Q221K mutation not only serves as key antigenic site recognized by mAb 5B5, but also significantly alters the antigenicity of VP2 of nVarIBDV, which might be associated with the immune escape of nVarIBDV from current IBDV vaccine.

To assess the inhibitory potential of monoclonal antibody 5B5 (mAb 5B5) on IBDV infectivity, we employed reverse genetics to rescue a point-mutated attenuated strain, designated rNF8-221K ( Figure 6A ), owing to the limited permissiveness of nVarIBDV in LMH, DF-1, and DT40 cell lines. The rescued strain was validated by IFA and Sanger sequencing (data not shown). Notably, mAb 5B5 exhibited specific binding to rNF8-221K but not to the parental strain rNF8 ( Figure 6B ). Moreover, a high-affinity interaction between mAb 5B5 and rNF8-221K was further confirmed by in immunoprecipitation ( Figure 6C ). Neutralization assays demonstrated that supplementation with 60 μg/mL mAb 5B5 in the culture medium significantly suppressed rNF8-221K infection/replication in LMH cells compared to untreated controls ( Figures 6D, E ). To investigate the potential inhibitory effect of mAb 5B5 on viral egress, rNF8-221K-infected LMH cells were treated with varying concentrations of mAb 5B5 or control mAb 6C5. Western blot and TCID₅₀ analyses demonstrated that mAb 5B5 significantly suppressed viral replication in a dose-dependent manner, with marked inhibition observed even at 7.5 μg/mL ( Figures 7A–C ). In contrast, the control mAb 6C5 showed no comparable inhibitory activity. These findings indicated that mAb 5B5 effectively restricts IBDV replication during the intracellular phase of infection.

To assess sequence conservation of the key 221K antigenic site within VP2 (recognized by mAb 5B5), we aligned representative VP2 sequences from major IBDV types circulating in China—including nVarIBDV, varIBDV, vvIBDV, cIBDV, and attIBDV—alongside five laboratory-isolated strains. The results identified 11 unique amino acid residues within the VP2-HVR: 213N, 221K, 222T, 249K, 252I, 254N, 284A, 286I, 299S, 318D, and 323E. Among these, 213N, 221K, 252I, 254N, 299S, 318D, and 323E served as significant molecular markers distinguishing Chinese nVarIBDV (A2d) from the Chinese isolates of A2a type variants BX (1996), GZ901 (1996) and early U.S. varIBDV strains Variant E. Furthermore, 221K and 252I were exclusively present in Chinese nVarIBDV (A2d), constituting distinguishing features from other IBDV genotypes ( Figure 8A ). The spatial structure of the peak A region and the heptapeptide region in the VP2 protein was visualized using PyMOL software. As described in Figure 8B , the peak A region and the heptapeptide region were exposed on the surface of the P domain of the VP2 protein and were spatially adjacent to each other. This distribution contributed to the formation of the conformational epitope recognized by mAb 5B5. Moreover, the key antigenic site 221K was situated at the outermost tip of the peak A, making it more spatially accessible for binding with antibodies. Notably, although the substitution of residue 221 from Q to K did not change the overall structure of VP2 ( Figures 8C, D ), it altered local electrostatic potential, possibly facilitating antigenicity and immune evasion.