Modulation of macrophages by infectious bursal disease virus

Infectious bursal disease is one of the most important naturally occurring viral diseases of chickens worldwide. The causative agent, infectious bursal disease virus (IBDV), belongs to the family Birnaviridae. This virus causes an acute, highly contagious and immunosuppressive disease in chickens. The virus infects and destroys actively dividing IgM-bearing B cells. Although B cells are the principal targets for IBDV, recent data show that the virus also infects macrophages. IBDV-infected macrophages produce various cytokines and chemokines which may play an important role in the protection and/or pathogenesis of IBDV. In this review, the modulatory effects of IBDV on macrophages will be discussed.     

Effects of chicken intestinal antimicrobial peptides on humoral immunity of chickens and antibody titres after vaccination with infectious bursal disease virus vaccine in chicken

Sixty chickens were randomly divided into two groups (30 chickens in each group) to determine the effect of oral administration of chicken intestinal antimicrobial peptides (CIAMP) on the humoral immune response. Chickens of both groups were fed the same diet. In the treatment group chickens received drinking water supplemented with CIAMP (1 microg/ml) right after hatching. Samples of blood, bursa of Fabricus, spleen and intestine were taken at day 1, 4, 7, 10 and 17 of experiment. CIAMP supplementation enhanced the content of IgG and IgM in serum from day 4-10 and day 10-17, respectively, (p < 0.05), IgM-forming cells in bursa of Fabricus and spleen at the age of 7 days (p < 0.05) and IgG-forming cells in bursa of Fabricus at the age of 4 days (p < 0.05). In addition, CIAMP enhanced the IgA-forming cells in caecal tonsils diffuse area at day 4 (p < 0.05). Furthermore, CIAMP enhanced the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens 21 days following IBDV vaccine administration (p < 0.05). These results suggested that CIAMP could modulate the humoral immune response of chickens and increased the antibody titres of infectious bursal disease virus vaccine.     

Infiltration by CD4+ and CD8+ lymphocytes in bursa of chickens infected with Infectious Bursal Disease Virus (IBDV): strain-specific differences

In order to investigate if there is any definite correlation between the degree of T-cell response in the bursa of Fabricius (BF) and the virulence of Infectious Bursal Disease (IBD) virus strains, chickens were infected with strains of different virulence i.e. mild (Lukert strain), intermediate (Georgia strain) or invasive intermediate (IV-95 strain). At various times post-inoculation, bursal samples were collected to study virus specific histopathological lesions, the distribution of viral antigen and the extent of T-cell infiltration in the bursa. Most severe bursal lesions were induced by IV-95 strain (the invasive intermediate strain), whereas Lukert, the mild strain caused the least severe lesions. The number of virus positive cells in the bursa was highest in chickens infected with IV-95 strain. Substantial infiltration of CD4+ and CD8+ T-cells in the bursal follicles of virus-infected groups was observed from 4 d.p.i. onwards. The magnitude of T-cell response was more in the birds infected with intermediate (Georgia) or invasive intermediate strains of virus than chickens inoculated with mild (Lukert) strain, even when 10-fold higher doses of the inoculums were used. PHA responses to peripheral lymphocytes were found suppressed in all the groups of chickens only transiently. The results indicate that the magnitude of T-cell responses in BF during IBDV infection is influenced more by the virulence of virus strain rather than the quantum of viral load in BF. Over all these studies may have implications in understanding the role of T-cells in pathogenesis and immunity in IBD.     

Effects of feeding a Fusarium toxin-contaminated diet to infectious bursal disease virus-infected broilers on the protein turnover of the bursa of Fabricius and spleen

Two experiments were carried out to examine the effects of feeding an uncontaminated control diet (CON) or a Fusarium toxin-contaminated diet (FUS; 10.7 mg deoxynivalenol [DON]/kg diet) to growing broilers, which were either uninfected or infected with infectious bursal disease virus (IBDV) beginning at 1 day post hatch. Broilers had been infected at three weeks post hatch with either a classical virulent infectious bursal disease virus (IBDV-IM, Exp. 1) or a very virulent IBDV (vvIBDV, Exp. 2) strain. The effects of the DON-contaminated diet in combination with the virus-infection on the bursa of Fabricius and spleen were determined at 3 and 6-7 days post infection. The transient development of the bursa oedema and the bursa atrophy was not significantly affected by the diet after infection with the different IBDV-strains. The histopathological lesions were more severe in IBDV-IM-infected birds at 6 days post infection when additionally exposed to the FUS diet as compared to the FUS-free feed. Most parameters of the bursa of Fabricius and spleen protein turnover (e.g. fractional protein synthesis rate, protein, DNA and RNA content and derived indices) were significantly and interactively influenced by infection and stage of infection. The vvIBDV-infected birds responded with a more pronounced depressing effect on the fractional protein synthesis rate after feeding the DON-containing FUS diet when compared to their IBDV-IM-infected counterparts, where the opposite effect was observed. It can be concluded that feeding a FUS diet to IBDV-infected broilers might modulate the virulence-dependent pathogenesis of an IBDV infection.     

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.     

Proteome dynamics in primary target organ of infectious bursal disease virus

Viruses induce dramatic changes in target tissue during pathogenesis, including host cellular responses that either limit or support the pathogen. The infectious bursal disease virus (IBDV) targets primarily the bursa of Fabricius (BF) of chickens, causing severe immunodeficiency. Here, we characterized the cellular proteome changes of the BF caused by IBDV replication in vivo using 2DE followed MALDI-TOF MS identification. Comparative analysis of multiple 2DE gels revealed that the majority of protein expression changes appeared between 24 and 96 h after IBDV infection. MS identified 54 altered cell proteins, 12 of which were notably upregulated by IBDV infection. Meanwhile, the other 42 cellular proteins were considerably suppressed by IBDV infection and are involved in protein degradation, energy metabolism, stress response, host macromolecular biosynthesis, and transport process. The upregulation of β-actin and downregulation of dynamin during IBDV infection were also confirmed by Western blot and immunofluorescence analysis. These altered protein expressions provide a response profile of chicken BF to virulent IBDV infection. Further functional study on these altered proteins may lead to better understanding of pathogenic mechanisms of virulent IBDV infection and to new potential therapeutic targets.     

Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens.     

Effects of chicken intestinal antimicrobial peptides on humoral immunity of chickens and antibody titres after vaccination with infectious bursal disease virus vaccine in chicken

Abstract

Sixty chickens were randomly divided into two groups (30 chickens in each group) to determine the effect of oral administration of chicken intestinal antimicrobial peptides (CIAMP) on the humoral immune response. Chickens of both groups were fed the same diet. In the treatment group chickens received drinking water supplemented with CIAMP (1 µg/ml) right after hatching. Samples of blood, bursa of Fabricus, spleen and intestine were taken at day 1, 4, 7, 10 and 17 of experiment. CIAMP supplementation enhanced the content of IgG and IgM in serum from day 4 – 10 and day 10 – 17, respectively, (p < 0.05), IgM-forming cells in bursa of Fabricus and spleen at the age of 7 days (p < 0.05) and IgG-forming cells in bursa of Fabricus at the age of 4 days (p < 0.05). In addition, CIAMP enhanced the IgA-forming cells in caecal tonsils diffuse area at day 4 (p < 0.05). Furthermore, CIAMP enhanced the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens 21 days following IBDV vaccine administration (p < 0.05). These results suggested that CIAMP could modulate the humoral immune response of chickens and increased the antibody titres of infectious bursal disease virus vaccine.     

Exchange of the C-terminal part of VP3 from very virulent infectious bursal disease virus results in an attenuated virus with a unique antigenic structure

Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).     

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains,field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeⅠ site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into nonimmunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.     

In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus

VP1, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV), has been suggested to play an essential role in the replication and translation of viral RNAs. In this study, we first expressed the complete VP1 protein gene in Escherichia coli (E. coli), and then the produced polyclonal antibody and four monoclonal antibodies (mAbs) to recombinant VP1 protein (rVP1) were shown to bind the IBDV particles in chicken embryo fibroblast and Vero cells. The epitopic analysis showed that mAbs 1D4 and 3C7 recognized respectively two distinct antigenic epitopes on the rVP1 protein, but two pair of mAbs 1A2/2A12 and 1E1/1H3 potentially recognized another two topologically related epitopes. Immunocytochemical stainings showed that VP1 protein formed irregularly shaped particles in the cytoplasm of the IBDV-infected cells. These results demonstrated that the mAbs to rVP1 protein could bind the epitopes of IBDV particles, indicating that the rVP1 protein expressed in E. coli was suitable for producing the mAb to VP1 protein of IBDV, and that the cytoplasm could be the crucial site for viral genome replication of IBDV.     

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.     

表达鸡传染性法氏囊病毒VP2蛋白的干酪乳杆菌免疫保护效力

利用干酪乳杆菌作为传染性法氏囊病毒(IBDV)VP2抗原传递系统,探讨口服雏鸡的免疫次数、免疫剂量、免疫途径和攻毒保护效果。用pLA-VP2重组干酪乳杆菌对5日龄雏鸡进行二次和三次免疫,并设108、109、1010 CFU/mL的重组干酪乳杆菌组,间接ELISA检测血清IgG和小肠洗液sIgA,末免后7 d攻毒,计算保护效果。根据确定的2次免疫和109 CFU/mL免疫剂量免疫5日龄雏鸡,分别口服、滴鼻/点眼pLA-VP2/L.casei,口服、肌注商品活苗及口服pLA/L.casei和PBS为对照,监测IgG和sIgA抗体水平;末免后7 d检测脾淋巴细胞增殖情况并攻毒,7 d后剖检,观察法氏囊损伤程度并记录病变得分和保护率。结果表明各组的特异性sIgA、IgG抗体水平显著高于对照组(P0.01);口服pLA-VP2/L.casei组的淋巴细胞刺激指数显著高于其他组(P0.01),保护率高达83.3%,免疫保护效果优于滴鼻/点眼组。因此,构建的重组干酪乳杆菌的安全性优于商品活苗,可以作为IBDV候选疫苗。     

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.     

Infectious bursal disease virus uptake involves macropinocytosis and trafficking to early endosomes in a Rab5‐dependent manner

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant‐negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post‐infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post‐infection. We compared IBDV infection to the internalization of well‐established ligands with defined endocytic pathways: transferrin, cholera‐toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics‐blocked cells. Our results indicate that IBDV endocytic internalization was clathrin‐ and dynamin‐independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.