Infectious bursal disease virus polyprotein expression arrests growth and mitogenic stimulation of B lymphocytes

Summary. Infectious bursal disease virus (IBDV) causes lymphocytolysis and immunosuppression in infected poultry. The IBDV genome encodes a polyprotein VP243 that is post-translationally cleaved by the VP4 protease into the two structural proteins pVP2 and VP3. The objective of the present study was to determine if IBDV polyprotein induced suppression of bursal B lymphocyte growth and their capacity for proliferation. Bursal B cells were examined both for chickens infected with IBDV and for chickens orally inoculated with a DNA construct expressing IBDV VP243 polyprotein. Bursae were collected at 0, 12, 24 and 48 hours after inoculation. Proliferation of bursal B cells (purified AvBu1⁺ cells) in response to concanavalin A mitogenic stimulation was significantly suppressed by infection at 1 day old with either the classical STC or variant E strains of IBDV. Oral administration of DNA constructs expressing the IBDV VP243 polyprotein from either the classical STC or variant E strains in the pCR3.1 vector resulted in persistent, moderate levels of construct in the bursa until at least 48 hours after inoculation. The VP243 DNA construct similarly induced suppression of proliferation for bursal lymphocytes independently of the virus infection. Expression of VP243 polyprotein in transiently transfected DT40 B lymphocyte culture also suppressed cell growth and proliferative responses to mitogen stimulation. Polyprotein expression did not affect cell viability and suppression of proliferation probably occurred by means of cell cycle arrest. The expression of the mature viral proteins VP2, VP4 or VP3 did not change the rate of cell proliferation or response of B cell cultures to mitogen. The results suggested that IBDV polyprotein is a mediator of immunosuppression.     

An overview of infectious bursal disease

Infectious bursal disease (IBD) is a viral immunosuppressive disease of chickens attacking mainly an important lymphoid organ in birds [the bursa of Fabricius (BF)]. The emergence of new variant strains of the causative agent [infectious bursal disease virus (IBDV)] has made it more urgent to develop new vaccination strategies against IBD. One of these strategies is the use of recombinant vaccines (DNA and viral-vectored vaccines). Several studies have investigated the host immune response towards IBDV. This review will present a detailed background on the disease and its causative agent, accompanied by a summary of the most recent findings regarding the host immune response to IBDV infection and the use of recombinant vaccines against IBD.     

Identification of a European interserotypic reassortant strain of infectious bursal disease virus

Infectious bursal disease virus (IBDV, family Birnaviridae) is a bi-segmented double-stranded RNA virus for which two serotypes are described. Serotype 1 replicates in the bursa of Fabricius and causes an immunosuppressive and potentially fatal disease in young chickens. Serotype 2 is apathogenic in poultry species. Up to now, only one natural event of interserotypic reassortment has been described after the introduction of very virulent IBDV (vvIBDV) in the USA in 2009, resulting in an IBDV strain with its segment A related to vvIBDV and its segment B related to US serotype 2 strain OH. Here, we present the first European isolate illustrative of interserotypic reassortment. The reassorting isolate, named 100056, exhibits a genomic segment A typical of current European vvIBDV but a segment B close to European serotype 2 viruses, supporting an origin distinct from US strains. When inoculated into SPF chickens, isolate 100056 induced mild clinical signs in the absence of mortality but caused a severe bursal atrophy, which strongly suggests an immunosuppressive potential. These results illustrate that interserotypic reassortment is another mechanism that can create IBDV strains with a modified acute pathogenicity. As a consequence, and for a more precise inference of the possible phenotype, care should be taken that the molecular identification of IBDV strains is targeted to both genome segments.     

Pathogenesis of infectious bursal disease in cyclophosphamide-treated chickens

Broiler chickens inoculated with cyctophosphamide showed atrophic bursae and severe immuno-suppression. Infectious bursal disease virus (IBDV) inoculation of the birds at 5 weeks of age caused neither clinical signs nor gross lesions. IBDV was re-isolated from some bursae samples. Cyclophosphamide non-treated (CYNT) chickens inoculated with IBDV showed bursal enlargement followed by atrophy. Examination of sections of the organ showed severe lymphocytic necrosis and depletion. IBDV was re-isolated from the bursa and spleen. The geometric mean titres of the IBDV serum neutralizing antibodies for infected cyclophosphamide treated (CYT) and CYNT chickens were 4.3 and 955.4, respectively. The above observations show that CYT chickens did not develop clinical IBD in the presence of infection and severe immunosuppression. They confirm the hypothesis that a healthy bursa is essential for the development of clinical IBD in chickens within the susceptible age range.     

Propagation and titration of infectious bursal disease virus,including non-cell-culture-adapted strains,using ex vivo-stimulated chicken bursal cells

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.     

Susceptibility of chicken blood lymphoblasts and monocytes to infectious bursal disease virus (IBDV)

Summary PHA-M stimulated lymphoblasts obtained from peripheral blood and separated from small lymphocytes by ×1g velocity sedimentation, unstimulated blood lymphocytes, monocytes and cells isolated from the bursa of Fabricius of chickens, were infected in vitro by the pathogenic strain CU-1 of infectious bursal disease virus (IBDV). Six hours after infection 32.5 per cent of the bursal cells reacted immunocytologically with IBDV antiserum and had high infectivity titers in plaque assays. Separated lymphoblasts showed a marked lower degree of virus replication and only 2.5 per cent reacted positively when studied by immunocytology, while monocytes ranged between these two cell types with regard to both the degree of virus replication and the positive reaction with IBDV antiserum. Small lymphocytes, however, were found to be totally resistant to IBDV infection. When studied by electron microscopy, virus particles arranged in a crystalloid pattern could only be detected in bursal cells. The results of this study indicate that proliferating lymphoid cells at a certain stage of cellular differentiation are the target cells for IBDV, and that in infected chickens monocytes may play a role in the spreading of the virus.With 2 Figures     

Efficacy of inactivated infectious bursal disease (IBD) vaccines: Comparison of serology with protection of progeny chickens against IBD virus strains of varying virulence

The efficacy of inactivated infectious bursal disease vaccines was determined by measuring both the antibody response of vaccinated chickens and clinical protection of progeny chicks from vaccinated dams. Similar virus neutralizing (VN) antibody titres were obtained in 4-week-old chickens and mature hens after vaccination with one vaccine dose. VN titres below 10 log 2 increased considerably between the fourth and seventh week after vaccination in 4-week-old chickens as well as in mature chickens. All 2-week-old progeny chicks with serum VN antibody titres of at least 9 log 2 were clinically protected against the classical virulent 52/70 infectious bursal disease virus (IBDV) strain, as well as against the very virulent IBDV (vvIBDV) strain D6948. However, vaccination often did not prevent subclinical infection in these 2-week-old progeny chicks, which often resulted in severe lymphocyte depletion in the bursa of Fabricius. Even a serum VN titre of 11 log 2 was not always sufficient to prevent severe bursal damage. Although 52/70 IBDV and vvIBDV were equally pathogenic in 2-weekold specific pathogen free chickens, significant higher maternal antibody titres were required to prevent the adverse effects of vvIBDV in comparison with 52/70 IBDV. The relation between the serological response of chickens after application of inactivated IBD vaccines and the protection of progeny chicks of vaccinated dams depended on both the virulence of the IBDV challenge strain and the IBDV strain in the vaccine.     

Characterization of the antigenic,immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa,embryo, and cell culture). III. Pathogenicity

Differences in the relative pathogenicity of variant (1084 E and GLS) and standard (Edgar and STC) infectious bursal disease virus (IBDV) strains were observed after propagation in the bursa of Fabricius, embryos, or cell cultures. Bursa-derived IBDV induced the most severe lesions in the bursa of Fabricius when compared with strains propagated in embryos or cell cultures. Embryo-derived IBDV induced moderate gross bursal lesions, whereas cell culture-derived IBDV did not damage the bursa grossly. A high frequency of virus re-isolations was obtained from bursal, spleen, and thymic samples collected from birds inoculated with bursa-derived or embryo-derived IBDV. Virus re-isolation occurred much less frequently from birds inoculated with cell culture-adapted IBDV. Serological evaluations demonstrated that bursa-derived IBDV strains induced a higher neutralizing antibody response than did embryo-derived or cell culture-derived strains. These results document that the relative pathogenicity and immunogenicity of IBDV is reduced following propagation in embryos or cell cultures.     

Enhanced immunopathology induced by very virulent infectious bursal disease virus.

Immunohistochemical and flow cytometric analyses of the bursa, spleen and thymus following infection with the very virulent infectious bursal disease virus (vvIBDV) strain UK661 revealed discrete differences from classical virulent infectious bursal disease virus strains. Bu-1+, immunoglobulin (Ig)M+ and IgG+ cells were all depleted from the bursa, spleen and thymus, suggesting loss of both immature and mature B lymphocytes. Small numbers of Bu-1+ cells repopulated the bursa 14 days post-infection but few of these expressed IgM or IgG. A transient increase in macrophages at 3 to 5 days post-infection was followed by a later influx of CD4+ and CD8+ T cells into the bursa. Loss of cortical thymocytes during the acute phase of infection suggested disruption of the T-cell system. The results showed that vvIBDV strain UK661 caused earlier and more severe pathology than classical virulent strains of infectious bursal disease virus. The marked influx of T cells into the infected bursa indicates that cell-mediated immunity is likely to be important in the clearance of vvIBDV.     

Antigenic and sequence heterogeneity of infectious bursal disease virus strains isolated in Australia

Summary.  Six recently isolated field strains of infectious bursal disease virus (IBDV) were compared to vaccine strains at the antigenic and genetic level to ascertain the level of heterogeneity among Australian IBDV strains. Five strains, 01/94, 02/95, 03/95, 04/95 and 08/95, isolated at four locations in the state of Victoria, were antigenic variants. They failed to react with monoclonal antibodies directed against two different epitopes on the VP2 protein which were present in vaccine strains and one field isolate (06/95) from the state of New South Wales. Serum neutralization tests confirmed that these strains were antigenic variants as they were of a different subtype to that of vaccine strains. Sequence comparison of the hypervariable region of the VP2 proteins showed that the five Victorian strains had between 13 and 16 amino acid substitutions in comparison with vaccine strains. Four to six of these substitutions were in the two hydrophilic domains previously identified as being of importance in the formation of protective virus neutralizing antibodies. Comparison of these five variants to those isolated previously in the USA revealed little similarity at both the antigenic and genetic level. Phylogenetic analysis showed that Australian IBDV strains belong to a separate and distinct genetic group which is considerably heterogeneous. Overall the results indicate that the current Australian IBDV situation resembles that seen in the USA, with the existence of classical and variant IBDV strains, but neither the classical nor the variant strains found in Australia are closely related to those prevalent in the USA. Received May 25, 1999/Accepted September 24, 1999     

Antigenicity,pathogenicity and immunosuppressive effect caused by a South American isolate of infectious bursal disease virus belonging to the “distinct” genetic lineage

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described “distinct IBDV” (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage.

Research Highlights

• A South American strain of the dIBDV lineage was phenotypically characterized.

• The strain produced subclinical infections with a marked bursal atrophy.

• Infected chickens were severely immunosuppressed.

• The dIBDV strains are antigenically divergent from other IBDV lineages.

     

T-Cell suppression by cyclosporin-A enhances infectious bursal disease virus infection in experimentally infected chickens

In this study, the role of T lymphocytes was investigated in chickens experimentally infected with infectious bursal disease virus (IBDV). Chickens were treated with cyclosporin-A (CS-A), a selective T-cell suppressant drug, by the intramuscular route, starting 3 days before virus infection and every third day thereafter,and infectious bursal disease pathogenesis was compared in such T-cell suppressed and intact chickens using a vaccine strain; namely, Georgia and a field isolate of IBDV. Treatment of chickens with CS-A caused a significant suppression of phytohaemagglutinin-A specific proliferative responses of peripheral blood mononuclear cells. The virus neutralizing antibody titres in such CS-A treated chickens were not suppressed. T-Cell suppression resulted in an increase in the severity of gross lesions caused by IBDV and extensive muscular haemorrhages were observed in such chickens between 15 and 21 days post-inoculation. Similarly, there was a marked increase in the severity of infectious bursal disease-specific microscopic lesions in the bursa of T-cell suppressed chickens. Consistently higher titres of virus were observed in bursa of CS-A treated chickens. Virus titres were 1 to 2 log10 higher in the T-cell suppressed chickens as compared with the intact ones. These studies suggest that T cells play a role in limiting the IBDV infection.     

Effects of infectious bursal disease virus and reticuloendotheliosis virus infection of chickens on the incidence of Marek's disease and on local tumour development of the non-producer JMV transplant

Dual infection of chickens with the HPRS-16 or HPRS-B14 strains of Marek's disease (MD) virus and non-transforming reticuloendotheliosis virus (REV-C) was shown not to affect significantly the incidence of MD or the incidence of MD lymphomas. Simultaneous infection of chickens with a pathogenic strain of infectious bursal disease virus (IBDV) and the HPRS-B 14 strain of MD virus resulted in a consistently reduced incidence of MD and lengthening of the latent period, but the incidence of MD lymphomas was unaffected. Unmixed IBDV infection did not have any effect on the oncogenicity of the JMV non-producer MD tumour transplant or its attenuated variant JMV-A. In dually pre-infected chickens the JMV tumour development at the site of transplantation as stimulated by MD virus-induced immunosuppression was enhanced by IBDV infection. This phenomenon was attributed to an additive effect of MDV- and IBDV-induced immunosuppression.     

An in vivo experimental model to determine antigenic variations among infectious bursal disease viruses

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus causing infectious bursal disease in chickens. IBDV undergoes antigenic drift, so characterizing the antigenicity of IBDV plays an important role for identification and selection of vaccine candidates. In this study, an in vivo experimental model was developed to differentiate a new antigenic variant of IBDV. To this end, a hyper-immune serum to IBDV E/Del-type virus was generated in specific pathogen-free chickens and a standard volume of the hyper-immune serum was serially diluted and injected in specific pathogen-free birds via intravenous, subcutaneous, or intramuscular routes. The chickens were bled at different time points in order to evaluate the dynamics of virus neutralization titres. Based on the results, chickens were injected with different serum dilutions by the subcutaneous route. Twenty-four hours later, chickens were bled and then challenged with 100 median chicken infectious doses of the E/Del virus and a new IBDV variant. Chickens were euthanized at 7 days post infection and the bursa of Fabricius was removed for microscopic evaluation to determine the bursal lesion score. The determined virus neutralization titre along with the bursal lesion score was used to determine the breakthrough titre in the in vivo chicken model. Based on the data obtained, an antigenic subtype of IBDV was identified and determined to be different from E/Del. This model is a sensitive model for determination of IBDV antigenicity of non-tissue culture adapted IBDV.     

Infectious bursal disease virus of chickens: pathogenesis and immunosuppression

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. The virus is ubiquitous and, under natural conditions, chickens acquire infection by the oral route. IgM+ cells serve as targets for the virus. The most extensive virus replication takes place in the bursa of Fabricius. The acute phase of the disease lasts for about 7-10 days. Within this phase, bursal follicles are depleted of B cells and the bursa becomes atrophic. Abundant viral antigen can be detected in the bursal follicles and other peripheral lymphoid organs such as the cecal tonsils and spleen. CD4(+) and CD8(+) T cells accumulate at and near the site of virus replication. The virus-induced bursal T cells are activated, exhibit upregulation of cytokine genes, proliferate in response to in vitro stimulation with IBDV and have suppressive properties. Chickens may die during the acute phase of the disease although IBDV induced mortality is highly variable and depends, among other factors, upon the virulence of the virus strain. Chickens that survive the acute disease clear the virus and recover from its pathologic effects. Bursal follicles are repopulated with IgM(+) B cells. Clinical and subclinical infection with IBDV may cause immunosuppression. Both humoral and cellular immune responses are compromised. Inhibition of the humoral immunity is attributed to the destruction of immunoglobulin-producing cells by the virus. Other mechanisms such as altered antigen-presenting and helper T cell functions may also be involved. Infection with IBDV causes a transient inhibition of the in vitro proliferative response of T cells to mitogens. This inhibition is mediated by macrophages which are activated in virus-exposed chickens and exhibit a marked enhancement of expression of a number of cytokine genes. We speculate that T cell cytokines such as interferon (IFN)-gamma may stimulate macrophages to produce nitric oxide (NO) and other cytokines with anti-proliferative activity. Additional studies are needed to identify the possible direct immunosuppressive effect of IBDV on T cells and their functions. Studies are also needed to examine effects of the virus on innate immunity. Earlier data indicate that the virus did not affect normal natural killer (NK) cell levels in chickens.     

Inflammatory response of different chicken lines and B haplotypes to infection with infectious bursal disease virus.

Chickens representing two different inbred lines (layer and meat-type) and three different B haplotypes (BW1, B19 and B131) were infected with infectious bursal disease virus (IBDV) at 21 days of age. Mortality was recorded, and surviving chickens were killed and examined either 3 or 17 days post-infection. Non-infected control chickens were run in parallel. The variables recorded were all significantly changed by the virus infection, including the serum concentration of mannan-binding lectin that was increased 2-fold 3 days post-infection. A significantly increased mortality, liver to body weight ratio and erythrocyte sedimentation rate (ESR) was seen among chickens from the layer type line compared with the meat-type line. In addition, the haplotype of the chickens influenced the atrophy and hypertrophy of the thymus. It was concluded that the meat-type chicken line was more resistant to IBDV infection than the layer-type line, and that mortality rate, liver to body weight ratio and ESR were valuable variables for evaluation of the level of IBDV infection-induced inflammation and disease.     

Induction of apoptosis in the bursa of Fabricius by vaccination against Gumboro disease

Infectious bursal disease is a severe acute viral disease of young chickens, affecting mainly the B-lymphocytes in the bursa of Fabricius, leading to severe immunosuppression as a result of the death of lymphoid cells. In the bursa infected with infectious bursal disease virus, viral replication is associated with apoptosis of lymphoid cells, inflammatory change and atrophy. Vaccination has appeared to be a crucial factor for control, with live attenuated vaccines being the most used. However, the apoptotic effect of these vaccines on the bursa has not been tested. We determined the apoptotic effect caused by the most used vaccines in local production on the bursa of Fabricius cells and the correlation with histological changes. In this study, it was demonstrated that apoptosis levels in the vaccinated groups were higher than those observed in the non-vaccinated birds leading to the conclusion that the action of the live virus vaccine strains modifies the boundary of the bursa and shapes processes of cell death by apoptosis. In contrast to other studies, the vaccine strains used did not show the phenomenon of bursal atrophy during the experimental period.