Riemerella anatipestifer (RA) is the primary causative agent of infectious serositis in ducks, causing significant economic losses. In this study, a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the conserved region of the ompA gene was developed. Specific primers and a FAM-labeled probe were designed, and amplification products were visualized using phenol red-based colorimetric detection and a lateral flow dipstick (LFD) system. Among the five candidate primer sets, primer set 2 was selected because it showed the highest amplification efficiency and specificity, with no cross-reactivity detected against 12 common waterfowl pathogens. Under optimal conditions, the phenol red-based LAMP assay yielded visible results after incubation at 65 °C for 30 min, while the LAMP-LFD assay required an additional 3~5 min probe hybridization step, with detection limits of 7.76 × 102 copies/μL for the phenol red-based method and 7.76 × 100 copies/μL for the LAMP-LFD method. Thirty clinical samples suspected of RA infection were analyzed using conventional PCR and the developed visual LAMP assays. The positive detection rates obtained with the LAMP-LFD and phenol red-based LAMP methods were 63.3% and 60%, respectively, showing high concordance with conventional PCR (56.7%). In conclusion, the LAMP assay integrating phenol red visualization and lateral flow dipstick detection is rapid, sensitive, and easy to perform, and both detection formats show potential for point-of-care or on-site applications, and can be used for the early diagnosis and detection of RA.

Fowl adenovirus serotype 4 (FAdV-4) is the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS) and is associated with high mortality rates (20–80%) in 3–6-week-old chickens. This study aimed to develop a rapid and specific method for detecting FAdV-4. Two monoclonal antibodies (mAbs 6B3 and 8G11) against the FAdV-4 penton protein were successfully generated using hybridoma technology, both of which exhibited high titers (1:100,000) and strong serotype specificity. Specificity analysis confirmed that these mAbs recognized 12 FAdV-4 isolates from diverse origins without cross-reactivity to other FAdV serotypes or common avian pathogens. On this basis, a colloidal gold immunochromatographic assay was developed; systematic optimization of its key parameters yielded an optimal labeling pH of 8.3, an antibody labeling concentration of 7.2 μg/mL, and optimal coating concentrations for the test line (T-line) and control line (C-line) of 2.5 mg/mL and 1.5 mg/mL, respectively. Performance evaluation of the method demonstrated that it achieved a detection sensitivity of 7.81 × 104 TCID₅₀/0.1 mL with a detection time of 15 min. Clinical sample validation revealed a positive concordance rate of 91.6%, a negative concordance rate of 100%, and an overall concordance rate of 98.6% relative to those of the ELISAs. The proposed colloidal gold immunochromatographic method offers advantages such as ease of operability, rapid visualization and high specificity and serves as a practical technical tool for onsite rapid diagnosis and epidemiological surveillance of FAdV-4 infection, thus it has high potential in the prevention and control of HHS.

IntroductionGoose adenovirus type 4 (GoAdV-4) is an emerging pathogen that causes inclusion body hepatitis and hepatic necrosis in goslings, with mortality rates reaching up to 80% in severe outbreaks. Since its first identification in China in 2022, GoAdV-4 has spread rapidly across major goose-producing provinces, posing a serious threat to the domestic goose industry. However, no sensitive and specific quantitative assay has been available for the rapid diagnosis and viral load monitoring of GoAdV-4.MethodsWe developed a TaqMan-based real-time quantitative PCR (qPCR) assay targeting the hexon gene of GoAdV-4 and systematically validated its analytical sensitivity, specificity, and repeatability. The assay was then applied to 582 clinical samples of five specimen types (tissues, blood, goose embryos, and cloacal swabs), and its diagnostic performance was compared with conventional PCR.ResultsThe assay demonstrated a linear detection range of 6.4 × 101 to 6.4 × 107 copies/μL (R2 = 0.9968) with an amplification efficiency of 97.5%, and a limit of detection (LOD) as low as 6.4 × 101 copies/μL. No cross-reactivity was observed with fowl adenovirus serotype 4, duck adenovirus type 3, egg drop syndrome virus, goose circovirus, goose astrovirus, goose parvovirus, or nuclease-free water. Intra-assay and inter-assay coefficients of variation ranged from 0.87% to 1.17% and 1.06% to 1.48%, respectively, indicating high reproducibility. The assay identified 55 GoAdV-4-positive samples (9.45%), compared with 48 positives (8.25%) detected by conventional PCR, with an overall concordance rate of 98.80% (positive concordance 87.27%, negative concordance 100%).DiscussionThe TaqMan qPCR assay detected seven additional positive samples missed by conventional PCR, all confirmed as true positives by Sanger sequencing, demonstrating superior sensitivity. These results show that the established TaqMan qPCR assay is a rapid, sensitive, specific, and reproducible tool for GoAdV-4 detection, providing a valuable diagnostic instrument for the surveillance and control of this emerging waterfowl pathogen.

Introduction:Duck hepatitis A virus type 3 (DHAV-3) causes acute fatal hepatitis in ducklings. Methods:This study compared host transcriptomic responses in duck livers following infection with virulent (HB) or attenuated (HB80) strains. Results:RNA sequencing (RNA-Seq) revealed that the virulent HB strain induced 2,355 differentially expressed genes (DEGs) at 2 days post-infection (dpi), compared to only 322 DEGs triggered by the attenuated HB80 strain. Functional analysis showed that the HB strain robustly activated immune pathways, particularly Toll-like receptor (TLR) and RIG-I-like receptor (RLR) signaling, leading to a potent type I interferon (IFN-I) response and marked chemokine upregulation. In contrast, the HB80 strain elicited a markedly milder immune reaction. Among the DEGs, 77 immune-related genes were identified, with significant enrichment in the IFN-I signaling pathway, suggesting their critical role in initiating an interferon storm and subsequent chemokine upregulation. Selected key genes (IFN-α2, RSAD2, RIG-I, MDA5, TBK1, TLR7) were validated by RT-qPCR and ELISA for targeted protein confirmation. Discussion:These findings delineate divergent host transcriptomic responses to virulent vs. attenuated DHAV-3 and highlight IFN-I signaling as a central axis in antiviral immunity.

Duck adenovirus 3 (DAdV-3) causes liver damage and bleeding, with morbidity rates ranging from 40 to 55% and mortality rates between 35 and 43%. Co-infection with other pathogens complicates disease control, significantly impacting the duck breeding industry. Currently, there have been no effective vaccines or treatments for DAdV-3. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling this virus. Our study developed a lateral flow strip (LFS) detection method using recombinase polymerase amplification (RPA) and CRISPR/Cas12a. The RPA-CRISPR/Cas12a-LFS method, performed at 37°C, allowed for result visualization without sophisticated equipment. It targeted the DAdV-3 Fiber-2 gene and achieved a detection limit of 3.0 gene copies. Additionally, this method demonstrated high specificity, with no cross-reactivity to eight other avian viruses. The reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. Analysis of 95 waterfowl samples showed 98.95% consistency and agreement with quantitative polymerase chain reaction using the Fiber-2 RPA-CRISPR/Cas12a-LFS method. These findings highlighted the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DAdV-3 detection.

Pasteurella multocida is a zoonotic pathogen responsible for severe diseases in domestic and wild animals, posing threats to public health and causing substantial economic losses. Here, we describe a naturally attenuated P. multocida strain, FCF147, isolated from a mortality event involving black-necked swans (Cygnus melancoryphus) in a wildlife habitat in Fujian, China. Genomic and phylogenetic analyses revealed that FCF147 is evolutionarily distant from other P. multocida lineages and lacks the entire capsule gene cluster. Morphological observations revealed that the loss of the capsule exposed proteins on the bacterial surface. Phenotypic characterization demonstrated reduced capsule production, enhanced biofilm formation, and increased tolerance to heat stress. In vivo infection models confirmed that FCF147 exhibits markedly attenuated virulence in both mice and poultry. However, immunization with FCF147 did not provide effective protection against the challenge of a virulent capsular type A strain. These findings suggest that while FCF147 is poorly virulent, its ability to form robust biofilms and survive thermal stress may facilitate persistence in wild bird reservoirs and potential transmission routes. These findings offer novel insights into the ecological adaptation and pathogenic potential of naturally capsule-deficient P. multocida in wildlife, highlighting their relevance to wildlife surveillance and disease ecology.

Interferon regulatory factor 7 (IRF7)-mediated type I interferon antiviral response is crucial for regulating the host following viral infection in chickens. Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that induces immune suppression and high mortality rates in chickens aged 3-6 weeks. Previous studies have shown that IBDV infection antagonizes the type I interferon production to facilitate viral replication in the cell, and IRF7 signaling might play an important role. However, the underlying mechanisms that enable IBDV to block the IRF7 pathway remain unclear. In this study, we found that IRF7 and IFN-β expression were suppressed in DF-1 cells during infection with very virulent IBDV (vvIBDV), but not with attenuated IBDV, while the virus continued to replicate. Overexpression of IRF7 inhibits IBDV replication while knocking down IRF7 promotes IBDV replication. Overexpression of IRF7 couldn’t compensate the IRF7 protein level in vvIBDV-infected cells, which suggested that IRF7 protein was degraded by IBDV infection. By using inhibitors, the degradation of IRF7 was found to be related to the proteasome pathway. Further study revealed that IRF7 was observed to interact and colocalize with the IBDV VP3 protein. Consistent with IBDV infection results, IBDV VP3 protein was observed to inhibit the IRF7-IFN-β expression, affect the degradation of IRF7 protein via proteasome pathway. All these results suggest that the IBDV exploits IRF7 by affecting its expression and proteasome degradation via the viral VP3 protein to facilitate viral replication in the cells. These findings revealed a novel mechanism that IBDV uses to evade host antiviral defense.

Since 2021, an epidemic disease characterized with hydrosalpinx fluid syndrome (HFS) has been circulating in the laying Sheldrake ducks in China, which seriously endangers the healthy development of the duck industry. The causative agent of this disease has been traced to avian metapneumovirus subtype C (aMPV/C), known to cause acute upper respiratory tract infections and egg-drop in poultry. To date, no reports have been made to isolate and characterize aMPV/C infection in Sheldrake ducks in China. Here, a strain of virus, designated aMPV-FJ21, was successfully isolated from the diseased ducks exhibiting HFS. Transmission electron microscopy revealed that the virus is an enveloped particle exhibiting a spherical or pleomorphic morphology. Indirect immunofluorescence assays demonstrated that the aMPV-FJ21 strain had an obvious reactive activity with the ploy-antibody against aMPV/C F protein. The complete genome of aMPV-FJ21 was determined to be 14,149 nucleotides in length. Notably, the amino acid sequence of the G protein was only 55.6%-78.7% identical to those of other aMPV/C reference strains. Phylogenetic analysis indicated that aMPV-FJ21 forms a distinct lineage within the aMPV/C group and is genetically distant from the North American and Eurasian lineages, suggesting that it may represent a novel genetic lineage. In challenge experiments, laying Sheldrake ducks with aMPV-FJ21 reproduced the typical clinical symptoms and pathological lesions observed in the field cases. Altogether, we had isolated a novel aMPV/C variant from Sheldrake ducks exhibiting HFS, distinct from previously reported strains, and provided the first evidence confirming its role as the causative agent of HFS in ducks.

Infectious Bursal Disease (IBD) is an immunosuppressive viral disease caused by the Infectious Bursal Disease Virus (IBDV). It primarily affects young chickens, targeting the bursa of Fabricius, and poses significant economic threats to the poultry industry. To date, in addition to strict biosecurity measures, large-scale immunization is the optimal strategy and effective method to prevent and control IBDV infection. The emergence of new variant strains has made it more urgent to develop new vaccination strategies against IBD. Over the past few decades, many high-quality vaccines have been available on the market for the control of IBD, which can provide solid protection against the infections and diseases caused by classic IBDV to very virulent IBDV that had been continuously evolving and were endemic worldwide. However, viruses are not static. As they continue to circulate and evolve in the fields, novel antigenic variant viruses have been emerged in the last few years, and vaccines need to keep up with their pace. Collectively, this review summarizes the strategic evolution of IBDV vaccines from traditional methods to cutting-edge molecular platforms, providing promising strategies for developing the next-generation vaccines with higher safety, efficacy, and the ability to keep pace with the antigenic drift in IBDV.

Infectious bronchitis virus (IBV) is one of the major diseases affecting the poultry industry worldwide. The high mutation rates of viral genome pose serious challenges to the prevention and control of IB. Based on our previous studies on IBV isolates in southern China during 2009-2017, we continued to carry out the genetic evolution analyses of S1 gene from IBV isolates during 2018-2023. Furthermore, we investigated the temporal and geographical origin of 245 IBV strains from southern China during 1985-2023, and reconstructed their transmission dynamics. Our findings revealed that GI-19 (LX4-type) was still the most dominant genotype, with the coexistence of multiple genotypes. Notably, the GI-19 strains were further classified into three subgroups, one of which was a new GI-19 subtype with nucleotide sequence similarities less than 90 % compared to the vaccine strain QXL87, along with five and eight consecutive amino acid substitutions, respectively. GI-28 (LDT3-A-type) strains re-emerged in 2019 and remained predominant thereafter. In addition, there was an increasing isolation rate of GI-7 (Taiwan-I-type). The purification pressure was detected in the S1 protein IBV isolates during 2018-2023, but eight positive selection sites with high entropy values were identified. Five isolates were confirmed to be recombinants, and GI-22 strains involved in all five recombinants as major or minor parents. The estimated times for the most recent common ancestors based on the S1 gene was the years of 1905. Bayesian skyline analysis indicated a rapid increase in genetic diversity of the S1 gene in IBV strains since late 2015. Bayesian geographical analysis demonstrated that the southern China IBVs originated from Nanning and identified four principal transmission pathways. In conclusion, our findings revealed the emergence of a new GI-19 subtype of IBV, the re-emergence of GI-28, and multiple transmission routes in southern China during 2018-2023, underscoring the importance of real-time and continuous surveillance of circulating strains and the urgency of developing safe, effective, and broad-spectrum new vaccines against emerging and re-emerging IBVs.

New genotype Muscovy Duck Parvovirus (N-MDPV), a member of the Parvoviridae family, exhibits broad host tropism affecting Muscovy ducks, semi-Muscovy ducks, and white Kaiva duck. This pathogen causes severe morbidity and mortality in ducklings under 3 weeks of age, characterized by classic parvoviral lesions, beak atrophy, and growth retardation, posing substantial economic threats to China’s duck industry. To address diagnostic challenges, we developed an equipment-free detection platform targeting the conserved VP3 gene of N-MDPV. By integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated lateral flow strip (LFS) visualization, this method achieved isothermal amplification at 37°C within 35 min, eliminating dependency on thermocyclers. Validation experiments demonstrated exceptional sensitivity with a detection limit of 1.3 gene copies. Specificity testing revealed no cross-reactivity with eight common avian pathogens, confirming target exclusivity. Clinical validation using 98 field-collected duck tissue samples showed 98.98% concordance between our RPA-CRISPR/Cas12a-LFS and quantitative PCR. This study establishes the first CRISPR/Cas12a-based on-site diagnostic tool for N-MDPV, combining rapidity, sensitivity, accuracy and field-deployability.

Background: Research has demonstrated that apolipoprotein L1 (APOL1) has a role in the emergence and progression of a number of malignant cancers. It is unclear, however, how APOL1 functions in colorectal cancer (CRC). In this study, we examined the possible molecular processes underlying APOL1's biological role in CRC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to identify APOL1 expression in patients with CRC and the cell line of cancer tissue. Following transfection of human colon carcinoma cells (HCT116) and human colon adenocarcinoma cells (SW1116) with sh-APOL1, the effects of APOL1 on the biological behavior of CRC cell lines were examined. In nude mice, the effect of APOL1 on tumor growth was noted. The protein interaction between APOL1 and RUNX1 was detected via coimmunoprecipitation. The expression of relevant proteins and cell biological behaviors were examined to confirm the APOL1-RUNX1 pathway in CRC cell lines. Results: The CRC tissues and cells exhibited elevated expression of APOL1. HCT116 and SW1116 cells' proliferation, migration, and invasion were suppressed by sh-APOL1, and sh-APOL1 also increased the expression of E-cadherin and decreased the expression of RUNX1, cyclin D1, (3-catenin, N-cadherin, and vimentin. APOL1 bound to the RUNX1 protein and regulated its protein levels. The counteractive effect of sh-APOL1 epithelial-mesenchymal transition (EMT), proliferation, migration, and invasion of CRC cells was counteracted by the overexpression of RUNX1. By silencing APOL1, the Wnt-(3-catenin pathway was able to restrain EMT and regulate the biological behavior processes in CRC cells. Conclusions: APOL1 has potential as a diagnostic biomarker for CRC. By preventing the Wnt-(3-catenin pathway from being activated, the sh-APOL1-binding protein RUNX1 inhibited the EMT and biological behavior of CRC cells.

With the virus continuing to evolve, very virulent IBDV (vvIBDV) and novel variant IBDV (nvIBDV) have become the predominant epidemic strains in China, exacerbated by the widespread use of attenuated vaccine strains (attIBDV), making a complex infection situation of IBDV in the field. Therefore, developing a rapid and accurate high-resolution melting curve quantitative reverse transcription PCR (HRM-qRT-PCR) for the identification and pathotyping of IBDV is crucial for clinical monitoring and disease control. Extensive data analysis and genome-screening of the three dominant IBDV pathotypes identified a specific region (nucleotides 2450–2603 in segment A) with distinct GC content as the detection target. Experimental testing of HRM-qRT-PCR revealed distinct melting curves and high sensitivity, with the detection limits of 61.2 copies/μL, 61.1 copies/μL and 67.5 copies/μL for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no inter-genotypes cross-reactivity among the three pathotypes and no reactivity to other common avian pathogens. Applied to samples with double and triple co-infections of different IBDV pathotypes, the method displayed specific melting peaks corresponding to the viruses present in the samples, with an accuracy rate of 100 %. This method precisely identifies and differentiates all the single or co-infected samples, generating distinct peaks corresponding to the Tm values of each virus pathotype in traditional melting curve plots. Furthermore, the method overcomes the limitations of traditional pathotyping methods, requiring only one reaction to achieve rapid viral pathotyping and facilitating quantitative analysis of viruses within the samples. This study introduces an innovative HRM-qRT-PCR method, offering new technology to rapid and accurate identification, pathotyping and quantification of vvIBDV, nvIBDV, and attIBDV. With strong discriminatory power, user-friendliness and a short processing time, this method is highly attractive for the rapid IBDV pathotyping in real-time large-scale epidemiological surveillance during outbreaks.

Infectious bursal disease virus (IBDV) infection causes highly contagious and immunosuppressive disease in poultry. The thymus, serving as the primary organ for T cell maturation and differentiation, plays an important role in the pathogenicity of IBDV in the infected chickens. However, there are no reports on the molecular pathogenesis of IBDV in the thymus currently. The aim of the study was to elucidate the molecular mechanisms underlying the pathogenicity of a field very virulent (vv) IBDV strain NN1172 in the thymus of SPF chickens using integrative transcriptomic and proteomic analyses. Our results showed that a total of 4,972 Differentially expressed genes (DEGs) in the thymus of NN1172-infected chickens by transcriptomic analysis, with 2,796 up-regulated and 2,176 down-regulated. Meanwhile, the proteomic analysis identified 726 differentially expressed proteins (DEPs) in the infected thymus, with 289 up-regulated and 437 down-regulated. Overall, a total of 359 genes exhibited differentially expression at both mRNA and protein levels, with 134 consistently up-regulated and 198 genes consistently down-regulated, as confirmed through a comparison of the RNA-seq and the proteomic datasets. The gene ontology (GO) analysis unveiled the involvement of both DEGs and DEPs in diverse categories encompassing cellular components, biological processes, and molecular functions in the pathological changes in IBDV-infected thymus. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the host mainly displayed severely disruption of cell survival/repair, proliferation and metabolism pathway, meanwhile, the infection triggers antiviral immune activation with a potential emphasis on the MDA5 pathway. Network inference analysis identified seven core hub genes, which include CDK1, TYMS, MCM5, KIF11, CCNB2, MAD2L1, and MCM4. These genes are all associated with cell-cycle regulating pathway and are likely key mediators in the pathogenesis induced by NN1172 infection in the thymus. This study discovered dominant pathways and genes which enhanced our understanding of the molecular mechanisms underlying IBDV pathogenesis in the thymus.

Novel variant infectious bursal disease virus (nvIBDV) is an emerging genotype (A2dB1b) that can cause severe and prolonged immunosuppression in young chickens. Despite current commercial vaccines being proven to lack complete protection against nvIBDV, it remains unclear whether the oil emulsion inactivated vaccines (OEVs) of the homologous and heterologous virus or booster immunization can provide effective protection. In this study, OEVs with two types of nvIBDV isolates QZ191002 (A-nv/B-nv) and YL160304 (A-nv/B-HLJ0504-like) were prepared and evaluated the protective effects of OEVs plus the booster immunizations with different current commercial vaccines against the challenge of nvIBDVs. The results from vaccination-challenge experiments showed that nvIBDV could break through the protection provided by only one immunization dose of the commercial vaccines, with the protection rates ranging from 40% to 60%. Interestingly, even with booster immunization with different commercial vaccines, the protection rates could only be increased to 60%–80%. As expected, only the OEVs of the homologous virus could provide 100% protection against the homologous nvIBDV, which could induce high-level specific antibodies, ameliorate target organ damage, and significantly reduce the viral load of the bursal in the challenged chickens. Notably, YL160304-OEV performed better than QZ191002-OEV, providing 100% protection not only against the challenge of homologous strain but also against that of heterologous QZ191002 strain. Antibody levels of the immunized chickens gradually increased after a short decline and reached the highest level on the age of 28 days. Similarly, the percentages of lymphocytes CD4+, CD8+ T, and B in peripheral blood lymphocytes (PBLs) were significantly increased on 21 d and 28 d. Notably, despite the nvIBDV, OEVs initially induced a delayed responses in the early stages but ultimately reach higher levels of CD4+ and CD8+ T lymphocytes. The results of study suggest that even booster immunization with different commercial vaccines cannot provide complete protection against nvIBDV, while the OEVs made by the nvIBDVs can provide full protection. Moreover, YL160304-OEV exhibits a broader protective spectrum against different nvIBDV strains, making it a potential candidate for the development of new vaccine.

为了对某鸡群疑似传染性法氏囊病(IBD)的病例进行分子诊断、病毒分离和分子特征分析,本试验通过核酸检测调查该鸡群法氏囊组织和病毒分离物中传染性法氏囊病病毒(IBDV)的感染情况,将分离得到的毒株命名为GL1906,继而对该病毒基因组双节段的VP2高变区(vVP2)序列和VPl-b序列进行分析.结果显示,GL1906 vVP2基因的特征性氨基酸位点在222A、256I、284A、294I和299S上均符合超强毒株(vvIBDV)的特征,但279N符合致弱毒株的特征;VP1-b基因在242D、390L、393E与弱毒株一致,287A与vvIBDV 一致,此外第777～782位核苷酸序列为GGTGCC,与弱毒株一致;GL1906 vVP2的核苷酸、氨基酸序列与vvIBDV的同源性最高,在系统进化树中与vvIBDV同为A3分支;GL1906 VP1-b的核苷酸、氨基酸序列与B节段属于独特来源的NN1172同源性最高,在系统进化树中同属于B3独特分支.结果表明,本试验分离株GL1906的基因组双节段vVP2和VP1-b具有不同的来源,是基因型为A3 B3的基因自然重排毒株.

In order to clarify the pathogen types and molecular characteristics of chickens with suspected infectious bursal disease virus（IBDV） infection in a farm in Guangxi, virus isolation, gene amplification, sequencing, nucleotide homology analysis, construction of phylogenetic tree, amino acid site variation analysis, recombination and selection pressure analysis were conducted in the experiment. The results showed that a novel reassortant strain of IBDV（named GX-NN200111） was successfully isolated, which belonged to the newly discovered genotype A3B1b. The isolated strain GX-NN200111 could proliferate well in chicken embryos, bleeding of which mainly occurred in the skins of head, neck and back; Sequence analysis showed that the isolated strain GX-NN200111 vVP2 belonged to the branch A3 of the very virulent strain, with a nucleotide homology of 93.7% to97.9%, and had the characteristic amino acid sites 212N, 222A, 256I and 294I of the very virulent strains. Its VP1-b belonged to the branch B1b of Chinese novel variant strain, and its nucleotide homology was 95.8% to 99.7%, and it had the characteristic amino acid site 240E of the novel variant strain in China. The recombination analysis showed that there was no evidence to confirm that there was an obvious recombination event in the isolate GX-NN200111, and the selection pressure analysis found that the isolate GX-NN200111 had 3（position 205, 222, 249） and 2（position 331, 426） positive selection sites in vVP2 and VP1-b, respectively. Amino acid exchange entropy analysis found that the isolate GX-NN200111 had 10（position 213, 222, 242, 249, 253,254, 256, 279, 294, 299） and 4（position 242, 287, 390, 393） mutant sites in vVP2 and VP1-b, respectively. The study confirmed the isolate GX-NN200111 was the first discovery of a novel reassortant strain（A3B1b）, the A segment was derived from the very virulent strain, and the B segment was derived from the Chinese novel variant strain.

Infectious bursal disease (IBD) classical virus strain (cIBDV) can cause morbidity and mortality in young chickens with severe long-term immunosuppression. However, since the emergence and widespread prevalence of very virulent strain (vvIBDV) in China from 1991, reports of cIBDV have become rare. A novel reassortant and recombinant strain GXYL211225 (genotype A1aB1a) with segment A originating from the classical strain (A1a) and segment B from the attenuated vaccine strain (B1a) was characterized in the study. Notably, segment A resulted from recombination between the cIBDV strains 150127-0.2 and Faragher52-70, expressing as a backbone from 150127-0.2, where a fragment located at the position of nucleotide (nt) 519-1 410 was replaced by the corresponding region of Faragher52-70. The infection of GXYL211225 caused mortality in SPF chicken embryos, despite lacking the critical amino acid (aa) residues 253H, 279 N and 284A associated with the cellular tropism, and induced significant cytopathic effect (CPE) on a wide range of cells, confirming its natural cell-adapted character. Furthermore, the challenge experiment of GXYL211225 was performed on the commercial Three-yellow chickens of 4-week-old, and with the vvIBDV HLJ-0504-like strain NN1172 and the novel variant (nv) IBDV strain QZ191002 as the comparison. All the challenged birds experienced reduced body-weight gain. QZ191002 infected birds showed no obvious clinical symptoms or mortality, while those of NN1172 and GXYL211225 showed typical IBD symptoms and resulted in 20% (2/10) and 10% (1/10) of mortality rates, respectively. At 7 days post-challenge (dpc), the damages of bursal of Fabricius (BF) varied among groups, with NN1172 causing the most severe lesions, followed by GXYL211225, and then QZ191002. It was also found that the pathogenicity was correlated positively with the viral load, aligning with the histopathological severity in BF. The study confirms the rapid and diverse evolution of the re-emerged classical strains in the field and emphasizes the need to monitor the changes of IBDV on both the genetic and pathogenic aspects for the effective control of the disease.