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.

TRIM25 inhibits infectious bursal disease virus replication by targeting VP3 for ubiquitination and degradation.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3-6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3.

Characterizations of glucose-rich polysaccharides from Amomum longiligulare T.L. Wu fruits and their effects on immunogenicities of infectious bursal disease virus VP2 protein.

The aim of this study is to explore the characterization of Amomum longiligulare T.L. Wu fruits polysaccharide (ALP) and their immune enhancement effects. Two homogeneous polysaccharides (ALP1 and ALP2) were isolated from the fruits. The structural characterization results showed that ALP1 (26.10 kDa) and ALP2 (64.10 kDa) were both mainly composed of glucose. Furthermore, ALP1 was consisted of (1,2)-α-D-Glcp, (1,2,3)-α-D-Glcp and T-α-D-Glcp, while ALP2 was consisted of T-α-D-Glcp, (1,3)-α-D-Glcp and (1,3,6)-α-D-Glcp. Afterwards, the immune enhancement effects of two polysaccharides were evaluated by determining their effects on immunogenicities of infectious bursal disease virus (IBDV) VP2 protein. Chickens were immunized with IBDV VP2 protein accompanied with ALP1/ALP2. And the results indicated both ALP1 and ALP2 promoted the weights and bursa of fabricius indexes of chickens. In addition, both two polysaccharides increased specific IBDV antibody levels, while ALP1 possessed higher immune enhancement ability and was expected to be an adjuvant for IBDV VP2 protein.

Characterization of antigenic variant infectious bursal disease virus strains identified in South Korea.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases of young chickens, causing considerable economic losses to the poultry industry. More than 30 years ago, an antigenic variant (av) pathotype of the IBD virus (IBDV) was reported to originate in, and subsequently spread among, poultry farms in the USA. Recently, a novel avIBDV lineage was identified in China and was shown to exhibit clear differences in its pathogenicity as well as molecular characteristics compared with the previously isolated variant strains. In this study, we conducted a passive surveillance of chicken carcasses submitted to our research division from June-December 2019, and detected the IBDV strains by reverse transcription PCR. Five avIBDV strains were isolated, and their pathogenicity was determined by necropsy and molecular analysis. Additionally, a coinfection field case involving an avIBDV strain and a very virulent IBDV (vvIBDV) strain was identified. Multiple sequence alignment and phylogenetic analysis of partial viral protein 1 (VP1) and hypervariable region (hv) VP2 genes revealed that those strains originated from two different avIBDV lineages. The co-occurrence of two sub-groups of avIBDVs in South Korea confirms for the first time the evolution of antigenic variant IBDV strains, and highlights the urgency for the development of new strategies for IBDV intervention in South Korea.RESEARCH HIGHLIGHTS Five avIBDV strains were identified in South Korea by passive surveillance test in 2019.A coinfection between two IBDV strains from different genogroups was reported in a field case.By phylogenetic analysis, Korean avIBDVs belonged to two distinct lineages of antigenic variant genogroup.

Recombinant Lactococcus Expressing a Novel Variant of Infectious Bursal Disease Virus VP2 Protein Can Induce Unique Specific Neutralizing Antibodies in Chickens and Provide Complete Protection.

Recent reports of infectious bursal disease virus (IBDV) infections in China, Japan, and North America have indicated the presence of variant, and the current conventional IBDV vaccine cannot completely protect against variant IBDV. In this study, we constructed recombinant Lactococcus lactis (r-L. lactis) expressing a novel variant of IBDV VP2 (avVP2) protein along with the Salmonella resistance to complement killing (RCK) protein, and Western blotting analysis confirmed that r-L. lactis successfully expressed avVP2-RCK fusion protein. We immunized chickens with this vaccine and subsequently challenged them with the very virulent IBDV (vvIBDV) and a novel variant wild IBDV (avIBDV) to evaluate the immune effect of the vaccine. The results show that the r-L. lactis-avVP2-RCK-immunized group exhibited a 100% protection rate when challenged with avIBDV and 100% survival rate to vvIBDV. Furthermore, this immunization resulted in the production of unique neutralizing antibodies that cannot be detected by conventional ELISA. These results indicate that r-L. lactis-avVP2-RCK is a promising candidate vaccine against IBDV infections, which can produce unique neutralizing antibodies that cannot be produced by other vaccines and protect against IBDV infection, especially against the variant strain.

[Co-expression, purification and bioassay of three avian viral antigens].

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

T cell subset profile and inflammatory cytokine properties in the gut-associated lymphoid tissues of chickens during infectious bursal disease virus (IBDV) infection.

While infectious bursal disease virus (IBDV) mainly targets immature B cells and causes T cell infiltration in the bursa of Fabricius (BF) of chickens, the effect of IBDV infection on the properties of T cells and relevant cytokine production in avian gut-associated lymphoid tissues (GALTs) remains unknown. Here, we show that while the CD8+ T cell subset is not affected, IBDV infection decreases the percentage of CD4+ T cells in the cecal tonsil (CT), but not in esophagus tonsil, pylorus tonsil, and Meckel's diverticulum of GALTs, in contrast to BF and spleen, in which the proportion of CD4+ cells increases upon IBDV infection. Further, IBDV infection upregulates IFN-γ, IL-10, and the T cell checkpoint receptor LAG-3 mRNA expression in BF. In contrast, in CTs, IBDV infection significantly increases the production of IFN-ß and CTLA-4 mRNA, while no significant effect is seen in the case of IFN-γ, IL-10 and LAG-3. Together, our data reveal differential modulation of T cell subsets and proinflammatory cytokine production in different lymphoid tissues during the course of IBDV infection.

Development of a recombinant VP2 vaccine for the prevention of novel variant strains of infectious bursal disease virus.

Since 2017, novel variant strains of infectious bursal disease virus (nvIBDV) have been detected in China, while the current vaccines on the market against very virulent IBDV have limited protection against this subtype virus. In this context, a strain of the virus has been isolated, and sequencing alignment and bird regression experiments showed that the virus was IBDV, belonging to the nvIBDV subtype (and named IBDV FJ-1812). Furthermore, the Escherichia coli expression system was used to successfully express soluble nvIBDV rVP2, which is specifically recognized by an anti-IBDV standard serum and anti-nvIBDV positive serum, and could be assembled into 14 - 17 nm virus-like particles. Based on the purified nvIBDV rVP2, we developed an IBDV FJ-1812 VP2 VLP vaccine at a laboratory scale to evaluate protection by this vaccine; in addition, we also prepared an IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV and evaluated its cross-protection against nvIBDV. Results of bird experiments showed that the nvIBDV rVP2 vaccine could induce high titres of specific antibodies, completely protect the bursa of Fabricius from viral infection, and provide 100% immune protection to SPF and Ross 308 broiler chickens. Furthermore, the IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV could provide 60% protection for SPF chickens and 80% protection for Ross 308 broiler chickens. This report provides important technical supports for the prevention and control of nvIBDV in the future.

Generation of recombinant VP3 protein of infectious bursal disease virus in three different expression systems, antigenic analysis of the obtained polypeptides and development of an ELISA test.

Infectious bursal disease virus (IBDV), which infects young chickens, is one of the most important pathogens that harm the poultry industry. Evaluation of the immune status of birds before and after vaccination is of great importance for controlling the disease caused by this virus. Therefore, the development of low-cost and easy-to-manufacture test systems for IBDV antibody detection remains an urgent issue. In this study, three expression systems (bacteria, yeast, and human cells) were used to produce recombinant VP3 protein of IBDV. VP3 is a group-specific antigen and hence may be a good candidate for use in diagnostic tests. Comparison of the antigenic properties of the obtained polypeptides showed that the titres of antibodies raised in chickens against bacteria- or human-cell-derived recombinant VP3 were high, whereas the antibody level against yeast-derived recombinant VP3 was low. The results of an enzyme-linked immunosorbent assay (ELISA) of sera from IBDV-infected chickens demonstrated that the recombinant VP3 produced in E. coli would be the best choice for use in test systems.

Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt.

Infectious bursal disease virus (IBDV), the agent of an immunosuppressive and sometimes lethal disease in chickens, is causing recurrent outbreaks in broiler chickens in Egypt. In particular, an antigenically modified isolate of very virulent IBDV (vvIBDV) called 99323 was detected in Egypt nearly twenty years ago; this isolate was shown to be experimentally controlled by an antigenically classical live vaccine. However, acute IBD is still reported, even in vaccinated flocks, and little is known about the genetic and antigenic properties of viruses currently circulating in Egypt. In the present study, ten samples collected in Egyptian broiler farms in 2015 as well as five samples collected in 2001 were analyzed. Genetic analyses of partial VP2 sequences revealed that 8 isolates clustered with vvIBDV strains, and 5 with tissue culture adapted and vaccine strains. Similar results were observed for partial VP1 sequences with the exception of isolate 160019, for which VP2 clustered with the vaccine strain Bursine while VP1 clustered with vvIBDV, suggesting reassortment. For isolates genetically related to vvIBDV, antigenic profiling revealed two patterns: while some isolates exhibited typical European vvIBDV reactivity with lack of binding of mAbs 5, other revealed extensive antigenic modifications, with lack of binding of mAbs 3, 5, 6, 8 and 9, similar to isolate 99323. These different patterns were associated with a single amino acid mutation at position 321 of VP2 that is located within peak PHI. Full genome sequencing was performed for three isolates, among which two were representative of the two antigenic patterns observed for vvIBDV as well as the reassortant isolate 160019. This study highlights the co-circulation of both antigenically typical and modified vvIBDV during the last fifteen years in Egypt.

Coupling metabolomics analysis and DOE optimization strategy towards enhanced IBDV production by chicken embryo fibroblast DF-1 cells.

Infectious bursal disease (IBD) caused by IBD virus (IBDV) is highly contagious viral and vaccination in chicken embryo has been an effective mean to prevent acute infection. However, the current production of IBDV vaccine faces serious batch instability and external contamination. The chicken embryonic fibroblast cell line DF-1 is widely used for the proliferation of avian viruses and vaccine production. Thus, optimizing the production of IBDV by DF-1 cells has an important application value. Combining metabolomics analysis and a Design of Experiments (DOE) statistical strategy, this study successfully optimized the process of IBDV production by DF-1 cells. Differential analysis and time series analysis of metabolite data in both IBDV-infected and uninfected DF-1 cells were performed by multivariate statistical analysis. The results showed that the intracellular metabolite intensities of glycolysis, the pentose phosphate pathway, the nucleoside synthesis pathway, lipid metabolism, and glutathione metabolism were upregulated, and the TCA cycle underwent a slight downregulation after IBDV infection of DF-1 cells. Based on the metabolome results and DOE statistical optimization method, the additive components suitable for IBDV proliferation were determined. The IBDV titer increased by 20.7 times upon exogenous addition of cysteine, methionine, lysine and nucleosides in the control medium, which is consistent with the predicted result (20.0 times) by a multivariate quadratic equation. This study provides a strategy for the efficient production of IBDV vaccines and could potentially be utilized to improve the production of other viral vaccines and biologics.

Expressing the immunodominant projection domain of infectious bursal disease virus fused to the fragment crystallizable of chicken IgY in yellow maize for a prospective edible vaccine.

Control of Infectious bursal disease virus (IBDV) in endemic countries has been based on early immunization of chicks using conventional live or inactivated vaccines that became not fully effectual and have biosafety concerns. This endeavor seeks generating a recombinant chimeric protein merging the projection domain (PD) of IBDV VP2 capsid with the fragment crystallizable (Fc) of avian IgY (FcIgY), in maize as a prospective poultry edible vaccine. The PD sequence was built on the basis of very virulent IBDV isolates circulating in Egypt. After optimization of codon-usage in maize, sequences of PD and FcIgY were effectively expressed in two elites of yellow maize via bombardment transformation in immature embryos. Chimeric protein amount in stable transgenic samples ranged from1.36% to 3.03% of the total soluble protein based on tissue age and maize cultivar. IBDV VP2 coding sequence was amplified from viral RNA, cloned, and expressed in E. coli. A group of Balb/C mice were hyper-immunized with purified recombinant VP2 protein for raising anti- recombinant VP2 antibodies (anti-rVP2 Ab). Proper expression in maize and immunoreactivity of the chimeric protein (PD-FcIgY) to chicken anti- IBDV and anti-rVP2 Ab were confirmed by both direct and indirect double antibody sandwich (DAS)-ELISAs as well as western blotting. Seeds of regenerated transgenic maize will be validated for chickens as edible vaccination in further studies.

Infectious Bursal Disease Virus: Molecular Epidemiologic Perspectives and Impact on Vaccine Efficacy Against Avian Influenza and Newcastle Disease Viruses.

Infectious bursal disease (IBD) virus (IBDV) is the causative agent of a highly contagious and immunosuppressive disease of chickens with huge economic losses to the poultry industry despite extensive vaccination. Analysis of isolated IBDV field strains from vaccinated birds would greatly improve the current immunization regimens and support the development of vaccines that offer better immunity. The study investigated the genetic characteristics and pathologic features of IBDVs in commercial broiler chicken farms, as well as the effect of IBDV infection on the efficacy of vaccination against avian influenza virus (AIV) and Newcastle disease virus (NDV) under field conditions. A preliminary diagnosis of IBD was made on the basis of the flock history and the characteristic gross pathologic findings. Microscopically, lymphoid depletion in bursal follicles with infiltration of lymphomononuclear cells along with cystic cavitations reflected the IBDV infection. The molecular analysis confirmed the IBDV infection in (57.1%) of tested flocks. Upon phylogenetic analysis of the VP2 hypervariable region of 14 Egyptian IBDVs, most viruses (n = 12) were clustered within the genogroup 3, while two viruses were closely related to attenuated vaccine isolates in genogroup 1. The analysis of the amino acid (aa) sequences revealed that most of the strains possessed five consistent aas at the VP2 protein (222A, 242I, 256I, 294I, and 299S), which are characteristic for the very virulent IBDV (vvIBDV). Serology indicated the immunosuppressive effect of IBDV, which is represented by a decrease (1.6-2.6 and 1.4-2.6 mean log 2) in the hemagglutination inhibition titer of the low pathogenic AIV subtype H9N2 and NDV, respectively. The examined IBDVs showed a high mutation rate within the hypervariable domain of the VP2 peptide. The results highlighted the need for carrying out an inclusive surveillance of IBDV infections in chicken flocks in Egypt.

Virus de la enfermedad de la bolsa infecciosa: perspectivas epidemiológicas moleculares e impacto sobre la eficacia de la vacunación contra los virus de influenza aviar y de la enfermedad de Newcastle. El virus de la enfermedad de la bolsa infecciosa (IBD) es el agente causante de una enfermedad altamente contagiosa e inmunosupresora de los pollos con grandes pérdidas económicas para la industria avícola a pesar de la vacunación extensiva. El análisis de cepas de campo del virus de Gumboro aisladas de aves vacunadas mejoraría en gran medida los regímenes de inmunización actuales y respaldaría el desarrollo de vacunas que ofrezcan una mejor inmunidad. Este estudio investigó las características genéticas y patológicas de los virus de la enfermedad infecciosa de la bolsa en granjas comerciales de pollos de engorde, así como el efecto de la infección por el virus de Gumboro en la eficacia de la vacunación contra el virus de la influenza aviar (AIV) y el virus de la enfermedad de Newcastle (NDV) bajo condiciones de campo. Se realizó un diagnóstico preliminar de la enfermedad infecciosa de la bolsa con base en la historia de la parvada y de los hallazgos patológicos macroscópicos característicos. Microscópicamente, la despoblación linfoide en los folículos bursales con infiltración de células linfomononucleares junto con formaciones quísticas reflejó la infección por el virus de Gumboro. El análisis molecular confirmó la infección por este virus en 57.1% de las parvadas analizadas. Después del análisis filogenético de la región hipervariable del gene VP2 de 14 virus egipcios, la mayoría de los virus (n = 12) se agruparon dentro del genogrupo 3, mientras que dos virus estaban estrechamente relacionados con los aislamientos vacunales atenuados del genogrupo 1. El análisis de las secuencias de aminoácidos reveló que la mayoría de las cepas poseían consistentemente cuatro aminoácidos en la proteína VP2 (222A, 242I, 256I, 294I y 299S), que son características de cepas muy virulentas del virus de Gumboro (vvIBDV). La serología indicó el efecto inmunosupresor del virus de Gumboro, que está representado por una disminución (1.6­2.6 y 1.4­2.6 log2) en los títulos de anticuerpos por inhibición de la hemaglutinación contra el virus de influenza aviar de baja patogenicidad subtipo H9N2 y del virus de Newcastle, respectivamente. Los virus de Gumboro examinados mostraron una alta tasa de mutación dentro del dominio hipervariable del péptido VP2. Los resultados resaltaron la necesidad de llevar a cabo una vigilancia intensiva de las infecciones por el virus de Gumboro en parvadas de pollos en Egipto.

The Roles of MicroRNAs (miRNAs) in Avian Response to Viral Infection and Pathogenesis of Avian Immunosuppressive Diseases.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of various biological processes including cell development and differentiation, apoptosis, tumorigenesis, immunoregulation and viral infections. Avian immunosuppressive diseases refer to those avian diseases caused by pathogens that target and damage the immune organs or cells of the host, increasing susceptibility to other microbial infections and the risk of failure in subsequent vaccination against other diseases. As such, once a disease with an immunosuppressive feature occurs in flocks, it would be difficult for the stakeholders to have an optimal economic income. Infectious bursal disease (IBD), avian leukemia (AL), Marek's disease (MD), chicken infectious anemia (CIA), reticuloendotheliosis (RE) and avian reovirus infection are on the top list of commonly-seen avian diseases with a feature of immunosuppression, posing an unmeasurable threat to the poultry industry across the globe. Understanding the pathogenesis of avian immunosuppressive disease is the basis for disease prevention and control. miRNAs have been shown to be involved in host response to pathogenic infections in chickens, including regulation of immunity, tumorigenesis, cell proliferation and viral replication. Here we summarize current knowledge on the roles of miRNAs in avian response to viral infection and pathogenesis of avian immunosuppressive diseases, in particular, MD, AL, IBD and RE.

Screening and Identification of a Chicken Dendritic Cell Binding Peptide by Using a Phage Display Library.

Dendritic cells (DCs), as antigen-presenting cells, can initiate adaptive immune responses efficiently. Although the DC-targeting strategy has attracted more attention, relevant studies on chicken are rare. Here, specific chicken bone marrow DC-binding peptides were selected using a phage display peptide library and confirmed through ELISA, flow cytometry, fluorescence microscopy, and laser confocal microscopy. The peptide candidate SPHLHTSSPWER, named SP, was fused to the infectious bursal disease virus (IBDV) structural protein and protective antigen VP2. In vitro, the expression of DC markers (CD80, CD83, CD86, DEC205, and MHCII) and some cytokines (IFN-γ, IL-12, TNF-α, IL-1ß, IL-6, and CXCLi1) by VP2-SP-stimulated DCs was significantly higher than that by DCs treated with the VP2-control peptide at 4 h (p < 0.001). In addition, an oral vaccine targeting DCs was generated using chicken-borne Lactobacillus saerimneri M11 (L. sae M11) to deliver VP2 fused with SP. Anti-IBDV mucosal and humoral immune responses were induced efficiently via oral administration, resulting in higher protective efficacy in the VP2-SP group than the VP2 group. Therefore, chicken DC targeting of IBDV protective antigen VP2 delivered by L. sae provides effective immune protection in chicken. Our study may promote research on the DC-targeting strategy to enhance the effectiveness of chicken vaccines.

Immune responses upon in ovo HVT-IBD vaccination vary between different chicken lines.

The genotype of chickens is assumed to be associated with variable immune responses. In this study a modern, moderate performing dual-purpose chicken line (DT) was compared with a high-performing layer-type (LT) as well as a broiler-type (BT) chicken line. One group of each genotype was vaccinated in ovo with a recombinant herpesvirus of turkeys expressing the virus protein VP2 of the infectious bursal disease virus (HVT-IBD) while one group of each genotype was left HVT-IBD unvaccinated (control group). Genotype associated differences in innate and adapted immune responses between the groups were determined over five weeks post hatch. HVT-IBD vaccination significantly enhanced humoral immune responses against subsequently applied live vaccines compared to non-HVT-IBD vaccinated groups at some of the investigated time points (P < 0.05). In addition HVT-IBD vaccination had depending on the genotype a significant impact on splenic macrophage as well as bursal CD4+ T-cell numbers (P < 0.05). On the other hand, the detectable genotype influence on Interferon (IFN) γ and nitric oxide (NO) release of ex vivo stimulated spleen cells was independent of HVT-IBD vaccination. The results of our study suggest considering a genotype specific vaccination regime in the field.

Co-Expression of Chicken IL-2 and IL-7 Enhances the Immunogenicity and Protective Efficacy of a VP2-Expressing DNA Vaccine against IBDV in Chickens.

Chicken infectious bursal disease (IBD) is still incompletely controlled worldwide. Although IBD virus (IBDV) VP2 DNA vaccine was considered a safe vaccine for IBD prevention, the immunogenicity by itself remains poor, resulting in the failure of effectively protecting chickens from infection. We and others demonstrated that chicken IL-2 (chIL-2) and chIL-7 have the capacity to enhance the immunogenicity of the VP2 DNA vaccine. However, whether chIL-2 and chIL-7 can mutually enhance the immunogenicity of VP2 DNA vaccine and thereby augment the latter's protection efficacy remains unknown. By using chIL-2/chIL-7 bicistronic gene vector to co-immunize the chickens together with the VP2 DNA vaccine, we now show that chIL-2 and chIL-7 significantly increased IBDV VP2-specific antibody titers, T cell proliferation, and IFN-γ production, resulting in the ultimate enhancement of vaccine-induced protection efficacy relative to that of chIL-2 or chIL-7 gene vectors alone. These results suggest that chIL-2 and chIL-7 can mutually enhance VP2 DNA vaccine's efficacy, thereby establishing a concrete foundation for future optimization of IBDV VP2 DNA vaccine to prevent/treat chicken IBD.

Identification of four serotypes of fowl adenovirus in clinically affected commercial poultry co-infected with chicken infectious anaemia virus in Trinidad and Tobago.

Fowl adenovirus (FAdV), which causes the high-impact diseases such as inclusion body hepatitis and hepatitis-hydropericardium syndrome, is of major concern to the poultry industry internationally. This study was carried out in direct response to mortality rates of up to 75% in commercial broiler flocks in Trinidad, West Indies. Symptoms in 3- to 8-week-old broilers and 13- to 18-week-old pullets pointed to infection with an immunosuppressive viral pathogen. The objectives of the study were to determine whether the infectious agent FAdV, along with other viral pathogens, was responsible for the clinical disease, and to obtain information on the serotypes of FAdV that were infecting the birds. Tissue samples from clinically affected birds from eight different farms were tested for chicken infectious anaemia virus (CIAV) and infectious bursal disease virus (IBDV) by real-time reverse transcription polymerase chain reaction (PCR) and for FAdV by conventional PCR. The birds tested positive for FAdV and CIAV, but negative for IBDV. The gene corresponding to the L1 loop of the hexon protein for FAdV was amplified and sequenced. Phylogenetic analysis of seven FAdV strains inferred that four serotypes were likely to be circulating in the chickens. Well supported genetic relatedness was observed for serotype 8a (97.8%), 8b (97.8%), 9 (95.8%) and 11 (98.8%-99.5%). This is the first published report from Trinidad and Tobago on the presence and circulation of pathogenic FAdV strains, in combination with CIAV, in poultry. The data demonstrate a possible need for the introduction of serotype-specific vaccines against FAdV, as well as vaccines against CIAV, in broilers in the region and emphasize the importance of maintaining high levels of biosecurity on farms to prevent the spread of these potentially devastating viruses between farms.

Detection of infectious bursal disease virus (IBDV) antibodies using chimeric plant virus-like particles.

The aim of the present study is to use Physalis mottle virus (PhMV) coat protein (CP) as a scaffold to display the neutralizing epitopes of Infectious bursal disease virus (IBDV) VP2. For this, three different chimeric constructs were synthesized by replacing the N-terminus of PhMV CP with tandem repeats of neutralizing epitopes of IBDV VP2 and expressed in Escherichia coli. Expression analysis revealed that all the three recombinant chimeric coat protein subunits are soluble in nature and self-assembled into virus-like particles (VLPs) as evidenced through sucrose density gradient ultracentrifugation. The chimeric VLPs were characterized by various biochemical and biophysical techniques and found that they are stable and structurally sound. When the chimeric VLPs were used as coating antigen, they were able to detect IBDV antibodies. These results indicated that the chimeric VLPs can be used as potential vaccine candidates for the control of IBDV, which needs to be further evaluated in animal models.

Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha.

Infectious bursal disease virus (IBDV) belongs to the Birnaviridae family and is the etiological agent of a highly contagious and immunosuppressive disease (IBD) that affects domestic chickens (Gallus gallus). IBD or Gumboro disease leads to high rates of morbidity and mortality of infected animals and is responsible for major economic losses to the poultry industry worldwide. IBD is characterized by a massive loss of IgM-bearing B lymphocytes and the destruction of the bursa of Fabricius. The molecular bases of IBDV pathogenicity are still poorly understood; nonetheless, an exacerbated cytokine immune response and B cell depletion due to apoptosis are considered main factors that contribute to the severity of the disease. Here we have studied the role of type I interferon (IFN) in IBDV infection. While IFN pretreatment confers protection against subsequent IBDV infection, the addition of IFN to infected cell cultures early after infection drives massive apoptotic cell death. Downregulation of double-stranded RNA (dsRNA)-dependent protein kinase (PKR), tumor necrosis factor alpha (TNF-α), or nuclear factor κB (NF-κB) expression drastically reduces the extent of apoptosis, indicating that they are critical proteins in the apoptotic response induced by IBDV upon treatment with IFN-α. Our results indicate that IBDV genomic dsRNA is a major viral factor that contributes to the triggering of apoptosis. These findings provide novel insights into the potential mechanisms of IBDV-induced immunosuppression and pathogenesis in chickens.IMPORTANCE IBDV infection represents an important threat to the poultry industry worldwide. IBDV-infected chickens develop severe immunosuppression, which renders them highly susceptible to secondary infections and unresponsive to vaccination against other pathogens. The early dysregulation of the innate immune response led by IBDV infection and the exacerbated apoptosis of B cells have been proposed as the main factors that contribute to virus-induced immunopathogenesis. Our work contributes for the first time to elucidating a potential mechanism driving the apoptotic death of IBDV-infected cells upon exposure to type I IFN. We provide solid evidence about the critical importance of PKR, TNF-α, and NF-κB in this phenomenon. The described mechanism could facilitate the early clearance of infected cells, thereby aiding in the amelioration of IBDV-induced pathogenesis, but it could also contribute to B cell depletion and immunosuppression. The balance between these two opposing effects might be dramatically affected by the genetic backgrounds of both the host and the infecting virus strain.