Postbiotic-based recombinant receptor activator of NF-κB ligand enhanced oral vaccine efficiency in chicken.

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Examination of Pyroptosis by Flow Cytometry.

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

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.

Growth and replication of infectious bursal disease virus in the fish cell line as an experimental vaccine.

Recently, several attempts have been made to replace egg-based with cell-based vaccines to prevent and control Infectious Bursal Disease Virus (IBDV). This study aimed to evaluate a new fish cell line (M99) for culturing and replicating IBDV. After observing complete cytopathic effects (CPE) on the M99 cell line, virus titers were determined using the TCID50 test, and the presence of the virus was confirmed using an RT-PCR test. Subsequently, 135 broiler chickens (14 days old) were randomly divided into three equal groups for immune response measurements: G1: immunized with a commercial vaccine, G2: immunized with an experimental vaccine, and G3: control. Antibody responses, bursal index, and histopathological evaluations were examined on different days after immunization. Based on the results, CPE of the virus was noticeable from the first passage, becoming complete by the third passage. The infectious titer of the virus was log106.9. Antibody titer measured 21 days after immunization in both vaccinated groups were significantly differed from the control group (p < 0.05). The results obtained from examining the bursal index and histopathological evaluations showed no significant difference between the studied groups at different times. Overall, this research is the first report on the successful cultivation of infectious bursal virus on a permanent cell line of fish origin, with the advantages of tolerance to a wide temperature range (26-40 degrees Celsius). Therefore, this cell line has potential for use to attenuate, cultivate, and adapt other pathogens to cold temperatures in future studies.

Protective effects of rabbit sacculus-derived antimicrobial peptides on SPF chicken against infection with very virulent infectious bursal disease virus.

Previous studies here have demonstrated that the rabbit sacculus rotundus-derived antimicrobial peptides (RSRP) could alter the intestinal mucosal immune responses in specific-pathogen-free (SPF) chickens, however, the protective effects of RSRP on chickens against infection remain questionable. In the present study, eighty SPF chickens were randomly divided into five groups and challenged with very virulent infectious bursal disease virus (vvIBDV) to determine the protective effects and its underlying mechanism of RSRP. Histopathology examination found that vvIBDV-infection caused severe damage in the bursa of Fabricius, especially the bursal lymphoid follicles underwent severe necrosis, depletion, hemorrhage, and edema. Unexpectedly, RSRP intervention significantly reduced the necrosis and depletion of lymphoid follicles in the vvIBDV-infected chickens. Moreover, RSRP treatment significantly decreased the expression of Bax (P < 0.01) as well as remarkably promoted the expression of Bcl-2 (P < 0.01), concomitantly alleviated the excessive apoptosis in the immune organs such as the bursa of Fabricius during vvIBDV infection. Notably, consistent with our previous reports that increased mast cell activation and degranulation in the bursa after vvIBDV infection, RSRP administration considerably reduced the mast cell density and the expression of tryptase, a marker for activated mast cells. Collectively, the present study indicates that rabbit sacculus rotundus-derived antimicrobial peptides could effectively protect the major immune organs including the bursa of Fabricius from the damage caused by vvIBDV infection, which provides the possibility and a promising perspective for the future application of antimicrobial peptides for poultry production.

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.

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.

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.

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.

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.

Research Note: Characterization and phylodynamic analysis of new infectious bursal disease virus variants circulating in Argentina.

Infectious Bursal Disease is a highly contagious disease that affects young chickens and leads to significant economic losses. Its causal agent is a double-stranded RNA virus that, due to its high error rate during the replication process, gives rise to a constant generation of new virus variants. Until 2014, strains of Infectious Bursal Diseases Virus (IBDV) belonging to genogroup 4 predominated in Argentina, but there have been no reports since then regarding the circulating genogroups in poultry. In this study, 11 recent sequences of Argentine from the hypervariable region of VP2 protein (hvVP2) were analyzed to determine their genogroup, origin, evolution, and amino acid sequence. Samples from chickens showing signs of IBDV infection were collected, and the hvVP2 region was amplified using RT-PCR, followed by sequencing. The results indicated that the analyzed strains belong to genogroup 2, with an estimated evolutionary rate of 1.74 × 10-3 substitutions/site/year. It is speculated that the predominant group of sequences began to spread in Argentina around 2014 and had its origins in China. Another sample is related to strains from South Korea and is not closely linked to the main group. Furthermore, the predicted amino acid sequences show similarity to strains that can evade vaccine-induced immunity. These findings underscore the importance of active surveillance in poultry to mitigate losses caused by IBDV.

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.

Continuous clinicopathological and molecular recognition of very virulent infectious bursal disease virus in commercial broiler chickens.

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses worldwide. The current study aims to conduct a molecular characterization of chicken farms for the infectious bursal disease virus (IBDV). Based on postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV) by sequence analysis. Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the 5 chosen isolates with strains representing different governorates in Egypt during 2022. All strains were clustered with vvIBDV with no evidence of reassortment in the VP1 gene. The VP1 and VP2 genes are divided into groups (I, II). The strains in our study were related to group II, and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs compared with HK46 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the loop PBC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the loop PDE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E, 390M, and 393D which were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in IBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of molecular monitoring to keep up to date on the circulating IBDV for regular evaluation of commercial vaccination programs against circulating field viruses.

Evasion of maternal antibody protection by an IBDV Argentine variant.

Infectious bursal disease (IBD) is a viral disease that affects the ability of chickens to produce humoral immune responses. One way to prevent the disease is the passage of maternally derived antibodies (MDA) from dams to offsprings via the yolk. Despite sanitary measures, which include immunization with genogroup 1 (G1) vaccines, infections with IBDV genogroup 4 (G4) in young animals have been detected. The aim of this study was to determine whether a local IBDV isolate belonging to G4 could evade the immunity generated by MDAs. Twelve-day-old animals positive for MDA, were inoculated with G1 or G4 isolates or phosphate buffered saline (PBS) as a control. After 1 wk, the animals were sacrificed and the following parameters were evaluated: bursa-body (BB) ratio, viral load, and histologic damage in the bursa of Fabricius. Results showed that G4-infected animals had significant differences in the BB ratio compared to the PBS group. In addition, viral load was significantly higher in the G4 group than in the G1 group. Histologic damage in the bursa of Fabricius was detected only in G4-infected MDA chickens. Our results suggest that infection with G4 local isolate can circumvent the immunity generated by MDA and, furthermore, that G4 isolate does not differ in its pathogenicity from G1 isolate, which underlines the need to include variant strains in vaccine formulations to reduce potential losses caused by these viruses.

Infectious bursal disease virus VP5 triggers host shutoff in a transcription-dependent manner.

Viruses have evolved intricate mechanisms to evade host antiviral responses and exploit cellular resources by manipulating the expression profile of host genes. During infection, viruses encode proteins with shutoff activity to globally inhibit host protein synthesis, which is an effective strategy for immune evasion. In this study, compelling evidence shows that infectious bursal disease virus (IBDV) infection triggers the suppression of host protein synthesis. Furthermore, using both in vitro and in vivo viral infection models, we have identified that IBDV specifically impedes the transcription of host genes via the shutoff activity of viral VP5, simultaneously conferring advantages to IBDV infection in these circumstances. The proposed mechanism suggests that VP5 competitively binds to RanBP1, disrupting the RanGDP/GTP gradient. This disruption interferes with cellular nucleocytoplasmic transport, impairing the nuclear import of proteins bearing nuclear localization signals. The nuclear transport of pivotal transcriptional regulatory factors, such as p65 and IFN regulatory factor 7, is also compromised, leading to the inhibition of pro-inflammatory cytokines and interferon expression. This newly discovered strategy employed by IBDV enables them to manipulate host gene expression, providing novel insights into how viruses evade host immune responses and establish infections.IMPORTANCEViruses manipulate host processes at various levels to regulate or evade both innate and adaptive immune responses, promoting self-survival and efficient transmission. The "host shutoff," a global suppression of host gene expression mediated by various viruses, is considered a critical mechanism for evading immunity. In this study, we have validated the presence of host shutoff during infectious bursal disease virus (IBDV) infection and additionally uncovered that the viral protein VP5 plays a pivotal role in inhibiting the overall synthesis of host proteins, including cytokines, through a transcription-dependent pathway. VP5 competitively binds with RanBP1, leading to disruption of the Ran protein cycle and consequently interfering with nucleocytoplasmic transport, which ultimately results in the suppression of host gene transcription. These findings unveil a novel strategy employed by IBDV to evade host innate immunity and rapidly establish infection. This study also suggests a novel supplement to understanding the pathway through which viruses inhibit host protein synthesis.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

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.

Protective effects of a novel chimeric virus-like particle vaccine against virulent NDV and IBDV challenge.

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

Molecular epidemiology of infectious bursal disease virus in the Near East and Persian Gulf regions.

RESEARCH HIGHLIGHTS: Different field IBDVs were found to circulate in the Near and Middle East.Multiple atypical genotypes (A3B1, A4B1, A6B1) were found to circulate extensively.Traditional very virulent IBDVs (A3B2) were a minority of the detected strains.Viral exchanges can be hypothesized between the region and different continents.