Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), is a highly contagious pathogen that primarily affects young chickens between three and six weeks of age. The disease primarily targets the bursa of Fabricius and is highly immunosuppressive, increasing susceptibility of affected birds to secondary infections [1, 2]. The disease remains to be a problem to poultry health in China and undergone significant evolution since its detection in the country [2]. Although vaccination has played a critical role in controlling the disease, evolutionary pressures from both the environment and vaccination have driven the emergence and spread of novel variant strains. These variants now predominate in China and present significant challenges to current vaccination efforts [3–5].

IBDV belongs to the genus Avibirnavirus within the family Birnaviridae and possesses a bi-segmented double-stranded RNA genome [6]. Segment A encodes a polyprotein that undergoes co- and post-translational cleavage to yield VP2, VP3, VP4, and a small non-structural protein VP5, while segment B encodes VP1, the viral RNA-dependent RNA polymerase [6]. Among these, VP2 forms the major structural and antigenic protein on the viral capsid surface [7–9]. The hypervariable region of VP2 (VP2_HVR), spanning amino acids 206 to 350, is pivotal in determining the antigenic type and virulence of IBDV strains [10]. Mutations, insertions, or deletions within this region drive antigenic drift and facilitate the emergence of immune escape variants [11–13].

Since its initial emergence in the early 1960s, two distinct serotypes of IBDV have been identified. Serotype-I is pathogenic to chickens, while serotype-II infects other avian species but is non-pathogenic to chickens [7]. Within serotype-I, multiple antigenic and pathogenic variants exist, including classical, variant, very virulent, attenuated, and recombinant strains [8]. Particularly, very virulent IBDV strains have caused widespread and severe outbreaks across Europe, Asia, and Africa, becoming predominant in China since the 1990s [14]. Simultaneously, novel antigenic variants distinct from strains circulating in the United States have emerged in Chinese poultry farms [15, 16]. These variants exhibit altered antigenicity, reduced susceptibility to neutralization by antibodies induced by classical or vaccine strains, and enhanced environmental persistence, complicating disease control efforts [16–18].

Vaccination remains the primary tool for controlling the disease, with both live attenuated and inactivated vaccines widely implemented [19]. However, the emergence and spread of immune-escape variants and recombinant viruses has made disease control and prevention programs increasingly challenging [14, 20–22]. These variants undermine the protective efficacy of current vaccines, particularly in regions where multiple IBDV genotypes co-circulate [23]. Genetic reassortment and recombination between vaccine and field strains, along with co-infections, contribute to the virus’s extensive genetic diversity and rapid evolution of IBDV in China [13, 24, 25]. These dynamics foster the periodic emergence of highly virulent or antigenically novel strains capable of evading host immunity. In the past couple of years, there have been increasing reports of very virulent and novel immune-escape variant strains of IBDV from different provinces in China [17]. Despite widespread vaccination, Shandong Province, one of China’s largest poultry-producing regions, has reported a growing number of IBD outbreaks affecting both commercial and backyard poultry flocks [14]. This rise suggests possible antigenic mismatch and the circulation of novel immune-escape variants. Moreover, backyard flocks may serve as potential reservoirs facilitating viral transmission to commercial operations. However, genetic and serological data on circulating field strains in this region remain limited, particularly regarding the VP2_HVR, a critical determinant of antigenicity and pathogenicity. To address these gaps, the present study aimed to detect and molecularly characterize IBDV strains from commercial and backyard poultry farms in Binzhou, Shandong Province. By analyzing the VP2 region and assessing serological responses alongside immunosuppression indicators such as bursa-to-body weight ratios, we aim to better understand the genetic diversity and antigenic variation of local strains and contribute to improved region-specific IBD control strategies.

Serum samples were collected from clinically healthy broiler chickens at the six designated farms (Farm A-F). Samples were randomly obtained at 18 days of age (n = 20) and 35 days of age (n = 20) from each farm. The samples were centrifuged at 3,000 rpm for 10 minutes to separate the serum, which was then aliquoted and stored at − 20°C until further analysis. Prior to testing, serum samples were diluted 1:2 and analyzed using an indirect ELISA with the IDEXX IBD Ab Test kit (# 99-09260, IDEXX Laboratories, Inc., Westbrook, ME, USA), following the manufacturer’s instructions. Absorbance was measured using a microplate reader, and antibody titers were calculated accordingly. A cut-off value of 396 was applied to determine seropositivity.

Approximately 100 mg of each tissue sample was homogenized using a sterile mortar and pestle in 1 mL of phosphate-buffered saline (PBS, pH 7.4) containing penicillin and streptomycin at a final concentration of 700 µg/mL. Homogenization was carried out on ice to minimize RNA degradation. The homogenates were then centrifuged at 8000 rpm for 5 minutes at 4°C, and 200 µL of the clarified supernatant was collected for RNA extraction. Total viral RNA was extracted using the Simply P Virus DNA/RNA Co-Extraction Kit (Cat. No. BSC67, Bioer Technology Co., Hangzhou, China) according to the manufacturer’s protocol. All procedures were performed under RNase-free conditions to maintain RNA integrity. The quality and concentration of extracted RNA were evaluated using both agarose gel electrophoresis and spectrophotometry. High-quality RNA samples were stored at − 80°C until further analysis.

Complementary DNA (cDNA) synthesis was performed using random hexamer primers and the SuperScript™ Reverse Transcriptase Kit (TransGen Biotech Co., Ltd., Beijing, China). The reverse transcription reaction was performed in a 20 µL reaction volume following the manufacturer’s instructions: RNA and primers were initially denatured at 65°C for 5 minutes, followed by reverse transcription at 42°C for 50 minutes, and final inactivation at 70°C for 15 minutes. The synthesized cDNA was stored at − 20°C until further use. To amplify the HVR of the VP2 gene of IBDV, a pair of specific primers was employed: IBDP1 (forward: 5′-TCACCGTCCTCAGCTTAC-3′) and IBDP2 (reverse: 5′-TCAGGATTTGGGATCAGC-3′), targeting a 643 bp fragment. The PCR was conducted in a 25 µL reaction mixture containing 2 µL of cDNA, 0.4 µM of each primer, 200 µM dNTPs, 2.5 mM MgCl₂, 1× PCR buffer, and 1 U of Taq DNA polymerase (TransTaq, TransGen Biotech, China). Thermal cycling conditions were: 94°C for 5 minutes (initial denaturation); 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds; with a final extension at 72°C for 10 minutes. PCR products were resolved by electrophoresis on 1% agarose gels stained with ethidium bromide (0.5 µg/mL) and visualized using a UV transilluminator. A 100 bp DNA ladder was used as a molecular weight marker to confirm the expected product size.

Based on the availability of tissues after conducting the PCR assay, the IHC assay was performed on 17 tissue samples, as listed in Supplementary Table S3. Briefly, Bursa of Fabricius samples were excised into 1–2 cm³ sections, rinsed with physiological saline, and blotted dry using filter paper. Tissues were fixed in 10% neutral buffered formalin, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at a thickness of 0.6 µm. Sections were stained with hematoxylin and eosin (H&E), cleared with xylene, and mounted with neutral resin. Histological slides were examined and scanned using a microscope (Olympus VS200, Olympus Corporation), and the resulting images were processed and analyzed with Olyvia software.

The HVR of the VP2 gene was sequenced from sixty-eight randomly selected IBDV-positive bursal samples with characteristics summarized in Supplementary Table S1, including reference number, bird type, age, farm source, and vaccination status, as well as from the VIRGO7 vaccine strain (Vigoly Bio. Tech Co. Ltd., Beijing, China). Quality control, sequence assembly, and analysis were conducted using Geneious Prime software (Dotmatics). The obtained viral sequences were aligned and analyzed alongside 50-VP2 gene reference sequences retrieved from GenBank, representing various genotypes and phenotypes of IBDV reported in China and other regions globally. Multiple sequence alignment was performed using the Clustal Omega program. A phylogenetic tree was constructed, and evolutionary relationships were inferred using the maximum likelihood method with the Jukes-Cantor (JC69) genetic distance model, implemented in the PhyML program within Geneious Prime. The phylogenetic tree was visualized and annotated using the Interactive Tree of Life (iTOL) v5 online tool [26]. Predicted amino acid sequences of the VP2 proteins were aligned, and variability at each residue was analyzed to determine viral phenotypes, following previously described methods [27].

This study provides valuable insights into the dynamics of maternal immunity and the serological response to IBDV in poultry farms in Binzhou, Shandong Province, China. Our data reveal two distinct antibody response profiles across the farms. In Farms A and F, birds exhibited relatively high IBDV-specific antibody titers at 18 days of age, suggesting the presence of maternally derived antibodies. However, these titers declined significantly by 35 days, falling below the protective threshold. This rapid decline likely indicates suboptimal maternal immunity transfer from breeder flocks, possibly due to factors such as improper vaccine storage, low-quality vaccines, incorrect administration, or non-adherence to vaccination schedules [21, 28, 29]. Effective maternal antibody transfer is essential for early protection against IBDV, and inadequate levels are associated with increased susceptibility to infection in progeny [30]. Of note, Farm A showed a significant increase in PCR positivity at 35 days, highlighting the susceptibility of birds following the decay of maternal antibodies In contrast, Farm F showed early PCR positivity at 18 days, but both antibody titers and bursal integrity declined by 35 days, suggesting initial protection followed by waning immunity. These observations are consistent with prior studies indicating the protective role of maternal antibodies against IBDV during early life [31–33]. Conversely, Farms B, C, D, and E displayed low or undetectable antibody levels at 18 days, suggesting that maternal immunity was either absent or insufficient. These findings could reflect a lack of vaccination or insufficient immunity in breeder flocks, which may have been free of prior infection or had implemented effective biosecurity protocols. However, the significant increase in antibody titers by 35 days suggests that these birds were naturally exposed to IBDV, leading to an active immune response. This natural seroconversion was corroborated by a marked reduction in bursa-to-body weight ratios, confirming IBDV-induced bursal atrophy and immunosuppression. Although live IBDV vaccines have been shown to cause some degree of bursal damage, the amino acid signatures of circulating strains in our study were distinct from those of vaccine strains, further supporting the conclusion that field strain exposure caused the observed pathology.

Molecular analysis of the VP2_HVR revealed that most isolates belonged to genogroup A3, corresponding to the vvIBDV phenotype, while a smaller proportion clustered within genogroup A2, representing variant strains. The high sequence identity within genogroup A3 suggests close genetic relatedness; however, notable intraspecies variability and the presence of distinct sub-lineages highlight ongoing viral evolution, consistent with reports from other regions in China and globally [32, 34, 35]. Importantly, our phylogenetic data showed evidence of viral exchange between commercial and backyard poultry, with several isolates sharing > 97% identity across production systems. This suggests cross-transmission between different poultry sectors in the region. The continuous evolution of field strains is likely driven by immune selection pressure from vaccination and natural infection [3, 36, 37]. The co-circulation of multiple IBDV strains in both commercial and backyard poultry, including genetically identical strains, suggests cross-transmission between production systems. This finding underscores the need for integrated control strategies and coordinated surveillance across the poultry sector to prevent the spread of IBDV [38, 39]. The VP2 protein plays a pivotal role in IBDV virulence and antigenicity, and the mutations identified in this study provide important insights into viral evolution and immune escape. Most A3 isolates retained key amino acid residues at positions 222, 253, 279, and 284, which are well-established markers of vvIBDV strains and are critical for pathogenicity and antigenic recognition [40, 41]. However, the detection of novel mutations, particularly the A222V substitution, is noteworthy. Although this mutation has not been previously reported in Chinese strains, it has been documented in Indian isolates, is associated with immune escape mechanisms, and reduced binding affinity of neutralizing antibodies, thereby potentially undermining vaccine efficacy [15, 42]. The presence of A222V in circulating strains suggests ongoing antigenic drift and the emergence of new variants that could contribute to vaccine failure. Additionally, the identification of a D279H substitution in several isolates raises questions regarding its functional impact. While substitutions at position 279, including D279N, contribute to attenuation of virulence, the impact of D279H remains uncharacterized and requires in vivo studies to determine its role in viral pathogenicity and immune evasion. [34, 43]. Mutations at position 253, including Q253H, have also been implicated in enhanced virulence and resistance to neutralization by vaccine-induced antibodies, reinforcing the notion that these changes facilitate viral adaptation and immune escape [43]. Collectively, these findings underscore the dynamic nature of IBDV evolution in the field and highlight the challenges posed to current vaccination strategies, emphasizing the need for continuous molecular surveillance and vaccine updates to maintain effective disease control.

Our study also highlights the significant divergence between circulating field strains and commercial vaccine strains such as B87 and Virgo7, both of which possess attenuation-associated residues at key VP2 positions that are absent in the field isolates. Furthermore, the consistent identification of vvIBDV residues in backyard poultry isolates, without evidence of vaccine-associated amino acid markers, confirms that these strains are field-derived and not vaccine escape variants. This divergence underscores the potential limitations of current vaccines in providing effective protection against circulating vvIBDV strains. Experimental and field studies have demonstrated that vaccines including viral vector, immune-complex, and recombinant subunit formulations often exhibit reduced protective efficacy when challenged with contemporary vvIBDV field strains [44]. The genetic and phenotypic differences identified in our study further corroborate these findings and highlight the urgent need for vaccine reformulation to address the ongoing evolution of viral populations. Given the observed antigenic divergence, vaccination strategies require updating to include antigens more relevant to regional variants. This approach would enhance concordance with circulating field strains and may improve vaccine efficacy. Furthermore, optimizing breeder vaccination protocols is essential to ensure effective maternal antibody transfer to offspring. Ongoing molecular surveillance and regular updates to vaccine formulations are critical to prevent vaccine failure and to achieve sustained control of IBDV in poultry production [45, 46].

Our study demonstrated the emergence and predominant circulation of unique vvIBDV variants clustered in genogroup A3 in Binzhou, Shandong, China. These viruses primarily contain amino acid substitutions at residue positions 222 and/or 279 in the HVR of the VP2 protein, which are different from the vaccine strains. The novel A222V substitution, consistently observed in most A3 isolates, may represent an emerging molecular signature in Chinese vvIBDV populations. The observed unique mutations can affect virulence and modify the confirmation of dominant epitopes in the P domain of VP2 protein changing the antigenic properties of the emerging vvIBDV. The viruses could break through existing immunity and cause disease. Strict biosecurity measures are essential to restrict the spread of emerging viruses. In addition, continuous surveillance and animal challenge experimental studies are required to evaluate the pathogenicity and antigenicity of these viruses to select for candidates for the development of antigenically representative vaccines to circulating strains.