Molecular characterisation of IBDV isolates in Turkey revealed reassortant strains.

1. Infectious bursal disease (IBD) is an acute, highly contagious viral disease of chickens caused by a virus (IBDV) which has a bi-segmented, double-stranded RNA genome. It has five viral proteins in its structure; the VP1 gene is encoded in segment B and the other four are in segment A.2. In this study, bursae of Fabricius and spleen samples taken from chickens suspected of having clinical or subclinical IBD from a total of 50 chicken flocks located in different geographical regions of Turkey were examined.3. The RT-PCR analysis of the VP2 gene showed that 30 of the 50 samples (60%) tested positive. Eight positive isolates were chosen and RT-PCR was performed to amplify the VP1 gene.4. The study showed that reassortant field strains that cause clinical or subclinical disease are currently circulating in broiler flocks across Turkey.

Chicken PRMT3 facilitates IBDV replication.

Protein arginine methyltransferases (PRMTs) in mammals play a role in various signaling pathways, such as virus infection, inflammasome responses, and cancer growth. While some PRMTs have been found to regulate interferon production in mammals, the mechanism in chickens remains to be fully understood. This study focused on investigating the function of chicken PRMTs. Our findings indicate that chicken PRMTs act as inhibitors of interferon production in response to dsRNA or MDA5 stimulation. Each PRMT is involved in different stages of interferon induction through the MDA5-MAVS-TBK1 pathway. Furthermore, we observed the colocalization of multiple PRMTs with the viral protein VP3 of infectious bursal disease virus (IBDV). Among the chicken PRMTs studied, PRMT3 was found to be widely expressed in various organs and its expression was upregulated during IBDV infection. Notably, PRMT3 supported IBDV replication, as demonstrated by ectopic expression and inhibition studies using SGC-707. Silencing of PRMT3 led to enhanced interferon production and inhibition of IBDV replication. This study provides novel insights into the role of chicken PRMTs, particularly PRMT3, in promoting IBDV replication by suppressing interferon signaling.

Genetic Sequence and Pathogenicity of Infectious Bursal Disease Virus in Chickens in Egypt During 2017-2021.

The continued circulation of infectious bursal disease virus (IBDV) in Egypt, despite the use of various vaccines, is a serious problem that requires continuous detection of IBDV. In the current study, real-time reverse transcriptase polymerase chain reaction testing of 100 diseased chicken flocks during 2017-2021 revealed the presence of very virulent IBDV (vvIBDV) in 67% of the flocks, non-vvIBDV in 11%, and a mixture of both vvIBDV and non-vvIBDV in 4%. Twenty-nine IBDV isolates were submitted for partial sequencing of the viral protein 2 hypervariable region (VP2-HVR), and 27 isolates were confirmed to be genogroup A3 (vvIBDV) with 96.3%-98.5% similarity to the global A3 (vvIBDV) and 88.9%-97% similarity to genogroup A1 vaccine strains. The remaining two isolates were non-vvIBDV and showed 91.1% and 100% identity with classical genogroup A1 strains, respectively. Furthermore, the sequence and phylogenetic analysis of VP1 (amino acids 33-254) of two selected isolates of A3, 5/2017 and 98/2021, clustered them as B2, vvIBDV-like, strains with high similarity (99.5%) to four Egyptian, 99% to Chinese and European, and 97.7% to Chinese and Polish vvIBDV isolates. Experimental infection of commercial broiler chickens with two vvIBDV-A3B2 isolates (5/2017 and 98/2021) showed no mortality despite typical tissue lesions, clear histopathological changes, and strong ELISA antibody response. Isolate 98/2021 was more pathogenic, as confirmed by histopathology, whereas isolate 5/2017 induced a stronger serological response. In conclusion, vvIBDV (A3B2) strains with two amino acid (aa) substitutions in VP1 as V141I and V234I as well as VP2 as Y220F and G254S are still circulating in Egypt.

Análisis de las secuencias genéticas y de la patogenicidad del virus de la enfermedad infecciosa de la bolsa de pollos en Egipto durante los años 2017­2021. La circulación continua del virus de la enfermedad infecciosa de la bolsa (IBDV) en Egipto, a pesar del uso de varias vacunas, continua siendo un problema serio que requiere la detección continua de este virus. En el presente estudio, se realizó una prueba de transcripción reversa y reacción en cadena de la polimerasa en tiempo real de 100 parvadas enfermas de pollos durante los años 2017­2021 y reveló la presencia de virus muy virulentos (vvIBDV) en el 67% de las parvadas, otros tipos diferentes a los muy virulentos en el 11%, y una mezcla de virus muy virulentos y otros tiposen un 4% de las parvadas. Se enviaron veintinueve aislados del virus de la enfermedad infecciosa de la bolsa para la secuenciación parcial de la región hipervariable de la proteína viral 2 (VP2-HVR), y se confirmó que 27 aislados pertenecían al genogrupo A3 (vvIBDV) con una similitud del 96.3% al 98.5% con el genogrupo A3 global (vvIBDV) y de 88.9% a 97% de similitud con las cepas vacunales del genogrupo A1. Los dos aislamientos restantes no resultaron ser muy virulentos y mostraron un 91.1% y un 100% de identidad con las cepas clásicas del genogrupo A1, respectivamente. Además, la secuencia y el análisis filogenético de la proteina VP1 (aminoácidos 33-254) de dos aislados seleccionados de genogrupo A3, 5/2017 y 98/2021, los agruparon como cepas B2, similares a virus muy virulentos, con alta similitud (99.5%) con cuatro aislamientos de Egipto, con similitud de 99% con aislados chinos y europeos, y de 97.7% con aislados muy virulentos chinos y polacos. La infección experimental de pollos de engorde comerciales con dos aislados muy virulentos tipo A3B2 (5/2017 y 98/2021) no mostró mortalidad a pesar de las lesiones tisulares típicas, los cambios histopatológicos claros y la fuerte respuesta de anticuerpos por ELISA. El aislado 98/2021 fue más patógeno, según lo confirmado por histopatología, mientras que el aislado 5/2017 indujo una respuesta serológica más fuerte. En conclusión, las cepas muy virulentas (A3B2) con dos sustituciones de aminoácidos (aa) en la proteina VP1 como V141I y V234I, así como en VP2 tales como Y220F y G254S, todavía circulan en Egipto.

Generation of a novel attenuated IBDV vaccine strain by mutation of critical amino acids in IBDV VP5.

Infectious bursal disease virus (IBDV) is an acute and highly infectious RNA virus known for its immunosuppressive capabilities, chiefly inflicting rapid damage to the bursa of Fabricius (BF) of chickens. Current clinical control of IBDV infection relies on vaccination. However, the emergence of novel variant IBDV (nVarIBDV) has posed a threat to the poultry industry across the globe, underscoring the great demand for innovative and effective vaccines. Our previous studies have highlighted the critical role of IBDV VP5 as an apoptosis-inducer in host cells. In this study, we engineered IBDV mutants via a reverse genetic system to introduce amino acid mutations in VP5. We found that the mutant IBDV-VP5/3m strain caused reduced host cell mortality, and that strategic mutations in VP5 reduced IBDV replication early after infection, thereby delaying cell death. Furthermore, inoculation of chickens with IBDV-VP5/3m effectively reduced damage to BF and induced neutralizing antibody production comparable to that of parental IBDV WT strain. Importantly, vaccination with IBDV-VP5/3m protected chickens against challenges with nVarIBDV, an emerging IBDV variant strain in China, reducing nVarIBDV loads in BF while alleviating bursal atrophy and splenomegaly, suggesting that IBDV-VP5/3m might serve as a novel vaccine candidate that could be further developed as an effective vaccine for clinical control of IBD. This study provides a new clue to the development of novel and effective vaccines.

Exploring Variability: Inflammation Mediator Levels across Tissues and Time in Poultry Experimentally Infected by the G1a and G6 Genogroups of Infectious Bursal Disease Virus (IBDV).

Infectious bursal disease virus (IBDV) is a significant burden for poultry production and market due to both direct disease and induced immunosuppression. In the present study, the expression of different cytokines in the bursa of Fabricius and thymus was evaluated during a 28-day-long experimental infection with two strains classified in the G1a (Classical) and G6 (ITA) genogroups. Although both strains significantly affected and modulated the expression of different molecules, the G6 strain seemed to induce a delayed immune response or suppress it more promptly. A recovery in the expression of several mediators was observed in the G1a-infected group at the end of the study, but not in the G6 one, further supporting a more persistent immunosuppression. This evidence fits with the higher replication level previously reported for the G6 and with the clinical outcome, as this genotype, although subclinical, has often been considered more immunosuppressive. However, unlike other studies focused on shorter time periods after infection, the patterns observed in this paper were highly variable and complex, depending on the strain, tissue, and time point, and characterized by a non-negligible within-group variability. Besides confirming the strain/genogroup effect on immune system modulation, the present study suggests the usefulness of longer monitoring activities after experimental infection to better understand the complex patterns and interactions with the host response.

Genomic characterization of infectious bursal disease virus in Argentina provides evidence of the recent transcontinental spread of Chinese genotype A2dB1b.

The infectious bursal disease virus (IBDV) is a significant pathogen affecting the poultry industry worldwide. Its epidemiological history has been marked by the emergence of strains with different antigenic, pathogenic, and genetic features, some of which have shown notable spread potential. The A2dB1b genotype, also known as novel variant, has become widespread and gained increased relevance in IBDV epidemiology. This genotype was described in China in the 2010s and rapidly spread in Asia and Africa. The present study describes the circulation of the A2dB1b genotype in Argentina. Applying a next-generation sequencing approach, we obtained the complete coding sequence of 18 Argentine viruses. The high level of genomic homogeneity observed amongst these viruses, their monophyletic clustering in both partial and complete segments A and B derived phylogenies, and their close relatedness to some Chinese strains suggest that a unique transcontinental spread event from China to Argentina occurred recently. The apparent success of the A2dB1b genotype spreading throughout Asia, Africa, and South America may partially be due to specific amino acid characteristics. Novel residues in the hypervariable region of VP2 may help A2dB1b IBDVs evade the protection elicited by the applied commercial vaccines. Our findings underscore the importance of continuous characterization of field samples and evaluation of the control measures currently applied to fight against this specific IBDV genotype.

Emergence of the novel infectious bursal disease virus variant in vaccinated poultry flocks in Egypt.

Since the detection of antigenically atypical very virulent Infectious bursal disease viruses (vvIBDV) in Egypt in 1999, the country has been experiencing recurrent outbreaks with high mortality rates and typical gross lesions associated with typical vvIBDV. However, a significant change occurred in 2023, marked by a notable increase in reported subclinical IBDV cases. To evaluate the field situation, samples from 21 farms in 2023 and 18 farms from 2021 and 2022, all of which had experienced IBD outbreaks based on clinical diagnosis, were collected, and subjected to VP2-HVR sequencing. Phylogenetic analysis revealed that all samples collected in 2021 and 2022 clustered with classical virulent strains and vvIBDV. In 2023, one sample clustered with the Egyptian vvIBDV, another with classical virulent IBDV, and the rest with the novel variant IBDV (nVarIBDV) circulating in China. The alignment of deduced amino acid sequences for VP2 showed that all Egyptian classic virulent strains were identical to the Winterfield or Lukert strains, while vvIBDV strains exhibited two out of the three typical residues found in Egyptian vvIBDV, namely Y220F and G254S, but not A321T. Meanwhile, all Egyptian variant strains exhibited typical residues found in nVarIBDV. However, all Egyptian variants showed a mutation at position 321 (321V), which represents the most exposed part of the capsid and is known to have a massive impact on IBDV antigenicity, except for one sample that had 318G instead. This report highlights the emergence of a new variant IBDV in Egypt, clustered with the Chinese new variants, spreading subclinically in broiler farms across a wide geographic area.RESEARCH HIGHLIGHTS New variant IBDV which emerged in Egypt clustered with Chinese nVarIBDV.nVarIBDV spread subclinically across a wide geographic area.Mutation at 321 represents capsid's most exposed part, a defining feature.Antigenically modified vvIBDV still circulating in Egypt with typical lesions.

Characterization and functional analysis of chicken CDK protein.

The family of cell cycle-dependent kinases (CDKs) serves as catalytic subunits within protein kinase complexes, playing a crucial role in cell cycle progression. While the function of CDK proteins in regulating mammalian innate immune responses and virus replication is well-documented, their role in chickens remains unclear. To address this, we cloned several chicken CDKs, specifically CDK6 through CDK10. We observed that CDK6 is widely expressed across various chicken tissues, with localization in the cytoplasm, nucleus, or both in DF-1 cells. In addition, we also found that multiple chicken CDKs negatively regulate IFN-ß signaling induced by chicken MAVS or chicken STING by targeting different steps. Moreover, during infection with infectious bursal disease virus (IBDV), various chicken CDKs, except CDK10, were recruited and co-localized with viral protein VP1. Interestingly, overexpression of CDK6 in chickens significantly enhanced IBDV replication. Conversely, knocking down CDK6 led to a marked increase in IFN-ß production, triggered by chMDA5. Furthermore, targeting endogenous CDK6 with RNA interference substantially reduced IBDV replication. These findings collectively suggest that chicken CDKs, particularly CDK6, act as suppressors of IFN-ß production and play a facilitative role in IBDV replication.

Life cycle of chicken bursal secretory dendritic cell (BSDC).

The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.

Loop PDE of viral capsid protein is involved in immune escape of the emerging novel variant infectious bursal disease virus.

Infectious bursa disease (IBD) is an acute, highly contactable, lethal, immunosuppressive infectious disease caused by the Infectious bursa disease virus (IBDV). Currently, the emerged novel variant IBDV (nVarIBDV) and the sustainedly prevalent very virulent IBDV (vvIBDV) are the two most prevalent strains of IBDV in China. The antigenic properties of the two prevalent strains differed significantly, which led to the escape of nVarIBDV from the immune protection provided by the existing vvIBDV vaccine. However, the molecular basis of the nVarIBDV immune escape remains unclear. In this study, we demonstrated, for the first time, that residues 252, 254, and 256 in the PDE of VP2 are involved in the immune escape of the emerging nVarIBDV. Firstly, the IFA-mediated antigen-antibody affinity assay showed that PBC and PDE of VP2 could affect the affinity of vvIBDV antiserum to VP2, of which PDE was more significant. The key amino acids of PDE influencing the antigen-antibody affinity were also identified, with G254N being the most significant, followed by V252I and I256V. Then the mutated virus with point or combined mutations was rescued by reverse genetics. it was further demonstrated that mutations of V252I, G254N, and I256V in PDE could individually or collaboratively reduce antigen-antibody affinity and interfere with antiserum neutralization, with G254N being the most significant. This study revealed the reasons for the widespread prevalence of nVarIBDV in immunized chicken flocks and provided innovative ideas for designing novel vaccines that match the antigen of the epidemic strain.

OASL suppresses infectious bursal disease virus replication by targeting VP2 for degrading through the autophagy pathway.

Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE: OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.

Novel variant infectious bursal disease virus diminishes FAdV-4 vaccination and enhances pathogenicity of FAdV-4.

Infectious bursal disease virus (IBDV) caused an acute and highly contagious infectious disease characterized by severe immunosuppression, causing considerable economic losses to the poultry industry globally. Although this disease was well-controlled under the widely use of commercial vaccines in the past decades, the novel variant IBDV strains emerged recently because of the highly immunized-selection pressure in the field, posting new threats to poultry industry. Here, we reported novel variant IBDV is responsible for a disease outbreak, and assessed the epidemic and pathogenicity of IBDV in this study. Moreover, we constructed a challenge model using Fowl adenovirus serotype 4 (FAdV-4) to study on the immunosuppressive effect. Our findings underscore the importance of IBDV surveillance, and provide evidence for understanding the pathogenicity of IBDV.

The effect of ghrelin on bursa and cecal tonsils of chickens infected with an attenuated virus strain of infectious bursal disease virus.

Infectious bursal disease (IBD) significantly affects the poultry industry, causing substantial economic losses. This study aimed to investigate the effects of ghrelin on chicks infected with an attenuated virus strain of IBDV (aIBDV). Chicks were divided into 3 groups: a control group (group I), an aIBDV infection group (group II), and a ghrelin + aIBDV infection group (group III). Mice in groups II and III were fed until they reached 19 d of age and then inoculated with aIBDV to establish a subclinical infection model. Group III received an intraperitoneal injection of 0.5 nmol/100 g ghrelin from d 17 to 23. The present study utilized paraffin sectioning, H&E staining, and immunohistochemical staining to examine the effects of ghrelin on the bursa of fabricius and cecum tonsils in aIBDV-infected chicks. The results indicated that at 3 d postinfection (dpi), the average body weight of group III was significantly greater than that of group II (P < 0.05). At 3 and 7 dpi, the proportion of large lymphoid follicles in the bursa of fabricius in group III was notably greater than that in group II (P < 0.05). aIBDV infection resulted in bleeding, edema, and fibrosis in the cecal mucosal layer of chicks, but ghrelin administration mitigated these pathological changes. At 3 and 7 dpi, the thickness of the lamina propria in the cecal tonsils of group III was significantly lower than that in the cecal tonsils of group II (P < 0.05). Additionally, the percentage of large lymphoid follicles in the cecal tonsils of group III was significantly greater than that in group II at 3 and 5 dpi (P < 0.05). There were significantly fewer macrophages in the cecal tonsils of group III than in those of group II at 1, 3, and 5 dpi (P < 0.05). In conclusion, ghrelin supplementation improved performance and mitigated bursal atrophy in aIBDV-infected chicks. It also reduced histological lesions and immune responses in the cecum tonsil. Notably, the reduction in macrophages in the cecum tonsil following ghrelin administration may decrease the risk of aIBDV spread.

The booster immunization using commercial vaccines effectively protect chickens against novel variants of infectious bursal disease virus (genotype A2dB1).

The novel variant IBDV (nVarIBDV, genotype A2dB1), characterized by bursal atrophy of fabricius and decreased lymphocytes, has been emerging on a large scale in Asia (including China) since late 2018. nVarIBDV is a new threat to the poultry industry, yet the currently licensed commercial vaccines, including the live viral vector vaccine, IBDV immune complex vaccine or VP2 subunit vaccine, are ineffective against nVarIBDV infection. In this study, specific-pathogen-free (SPF) chickens and broilers divided into 3 groups were vaccinated with the live viral vector vaccine, the VP2 subunit vaccine or the IBDV immune complex vaccine at 1 day-old, respectively. The SPF chickens received a secondary vaccination with the live B87 strain vaccine at 11-day-old. The bursa/body weight ratio, histopathology lesion of the bursa, and the differentiation between infected and vaccinated animals (DIVA) by qRT-PCR confirmed that the live viral vector vaccine or immune complex vaccine plus live B87 strain booster could provide at least 80% protection against the FJ2019-01 strain of nVarIBDV in SPF chickens. The broilers also received a secondary vaccination using a live W2512 G-61 strain vaccine at 14-day-old, and analyses showed that the VP2 subunit vaccine or immune complex vaccine plus the live W2512 G-61 strain booster also provided more than 80% protection against the FJ2019-01 strain of nVarIBDV. Unfortunately, the live viral vector vaccine plus live W2512 G-61 strain booster provided poor to moderate protection against FJ2019-01 in broilers. These findings suggest that combining commercial vaccines with rational booster immunization can effectively protect chickens against an nVarIBDV challenge.

ADP-ribosylation factor 6 promotes infectious bursal disease virus replication by affecting the internalization process via clathrin.

ADP-ribosylation factor 6 (ARF6) is a small G protein with extensive functions, including regulation of cellular membrane transport and viral infection. Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV), which mainly invades the bursa of Fabricius and causes low immunity in poultry. Our study demonstrated that IBDV infection could promote the expression of ARF6; however, the underlying mechanism remains unclear. Herein, the function of ARF6 in IBDV infection was explored, and it was revealed that viral replication was significantly promoted by ARF6 overexpression and hampered by siRNA-mediated inhibition of ARF6. Using two site mutants of ARF6 (ARF6-T27N and ARF6-Q67L), we found that IBDV replication was repressed by ARF6-T27N, indicating that ARF6 promotes IBDV replication. Further exploration of its mechanism revealed that ARF6 affects the copy number of IBDVs entering cells. A clathrin inhibitor (pitstop 2) impeded the early replication of IBDV, even when ARF6 was overexpressed. These results indicated that ARF6 promotes viral replication by affecting the internalization of IBDV, which may involve clathrin-dependent endocytosis. Our findings improve the understanding of the processes governing IBDV infection and provide insights into its prevention and control.

Genetic and pathogenic characterizations of a naturally occurring reassortant and homologous recombinant strain of the classical infectious bursal disease virus re-emerging in chickens in southern China.

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.

Oral Immunization with Recombinant Saccharomyces cerevisiae Expressing Viral Capsid Protein 2 of Infectious Bursal Disease Virus Induces Unique Specific Antibodies and Protective Immunity.

Infectious bursal disease (IBD), as a highly infectious immunosuppressive disease, causes severe economic losses in the poultry industry worldwide. Saccharomyces cerevisiae is an appealing vehicle used in oral vaccine formulations to safely and effectively deliver heterologous antigens. It can elicit systemic and mucosal responses. This study aims to explore the potential as oral an vaccine for S. cerevisiae expressing the capsid protein VP2 of IBDV. We constructed the recombinant S. cerevisiae, demonstrated that VP2 was displayed on the cell surface and had high immunoreactivity. By using the live ST1814G/Aga2-VP2 strain to immunize the mice, the results showed that recombinant S. cerevisiae significantly increased specific IgG and sIgA antibody titers, indicating the potential efficacy of vaccine-induced protection. These results suggested that the VP2 protein-expressing recombinant S. cerevisiae strain was a promising candidate oral subunit vaccine to prevent IBDV infection.

Isolation and genomic characteristics of the novel variant infectious bursal disease virus in China.

The infectious bursal disease virus (IBDV) is a member of the viruses that can induce immunosuppression in chickens. In recent years, more and more IBDV-infected cases by the novel variant IBDV were reported in China, and it has been demonstrated that currently used vaccines could not provide complete protection against these new IBDV variants. However, a lack of comprehensive analysis of the genomic characteristics of the novel variant strain IBDV has hampered its vaccine development. In this study, a strain of IBDV, designated HB202201, was phylogenetically analyzed, and it was found that the hypervariable region (HVR) of VP2 belonged to the novel variant strain. Furthermore, the 5'- and 3'-ends of segments A and B were analyzed using the rapid amplification of cDNA end (RACE) method. After the full-length of segment A and segment B were determined, the phylogenetic analysis of the segment A and segment B showed that the isolated HB202201 belonged to A2dB1 genotype, which demonstrated the HB202201 belonged to the novel variant strain. In addition, the specific mutations in VP1-VP5 amino acids were analyzed, which showed that there were multiple typical mutations in novel variant IBDV proteins, including VP1 (G24, I141, V163, and E240), VP2 (K221, and I252), VP3 (Q167 and L196), and VP5 (R7, P44, R92, G104, and E147), whereas there was no typical mutation in VP4. This study provides insights into the genomic and antigenic characteristics of the novel variant IBDV, which will promote the development of novel vaccine against the novel variant IBDV.

Virus-like Particle Vaccines of Infectious Bursal Disease Virus Expressed in Escherichia coli Are Highly Immunogenic and Protect against Virulent Strain.

OBJECTIVES: Infectious bursal disease virus (IBDV) is a highly contagious, acutely infectious agent that causes immunosuppression in chickens. We expressed IBDV VP2 proteins in Escherichia coli (E. coli) to develop an effective virus-like-particles (VLPs) vaccine and evaluated its immunogenicity. METHODS: The VLPs produced in E. coli were used as an immunogen mixed with a water-in-mineral-oil adjuvant (MontanideTM ISA 71 VG, ISA 71 RVG) or a white oil (7#) adjuvant. VLPs without an adjuvant, commercial subunit vaccine, inactivated vaccine, and attenuated vaccine were used as controls. These test vaccines were intramuscularly injected into 19-day-old SPF chickens, which were challenged with the IBDV virulent strain at 30 days after vaccination. RESULTS: The adjuvants boosted antibody production, and the adjuvant groups (except white oil) produced higher antibody levels than the non-adjuvanted controls and the commercial vaccine groups. In terms of cellular immunity, the VLPs plus adjuvant combinations produced higher levels of cytokines, IL-2, IL-4, and IFN-γ than the controls. CONCLUSION: IBDV VLPs plus the ISA 71 RVG adjuvant can be used as an optimal vaccine combination for improving the immune efficacy of IBD subunit vaccines, which can protect against the virulent strain.

Whole Genome Sequencing of Infectious Bursal Disease Viruses Isolated from a Californian Outbreak Unravels the Underlying Virulence Markers and Highlights Positive Selection Incidence.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84-100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains.