Susceptibility of chicken mesenchymal stem cells to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the causative agent of one of the most important viral diseases affecting the poultry industry worldwide. The virus causes an acute, highly contagious and immunosuppressive disease in chickens. Previous studies have demonstrated that in addition to B cells, macrophages can support the replication of IBDV. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of hematopoietic precursors, the interaction between IBDV and mesenchymal stem cells was investigated. Mesenchymal stem cells were isolated from chicken bone marrow. The classical IM strain and the variant strain-E of IBDV, both adapted to grow in a chicken macrophage cell line, were used to infect mesenchymal stem cells. Primary chicken mesenchymal stem cells were highly susceptible to replication of IBDV. Both viruses induced cytopathic effects and replicated to high titers in mesenchymal stem cells. The finding that IBDV can replicate in mesenchymal stem cells provides new information on the susceptible target cell population within the host and contributes to the understanding of the pathogenic potential of the virus.     

Immunosuppressive effect of infectious bursal disease virus on vaccination against infectious bronchitis

Groups of broiler chicks hatched with parental antibodies to infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) were vaccinated against IBV at 1 day of age via the oculonasal routes and inoculated with virulent IBDV at 1, 5, 10, 15 or 20 days of age. While the non-IBDV inoculated birds were solidly immune against IBV challenge at an age of 29 days, immunity in the IBDV infected birds was depressed. This depression, which was most serious in the birds IBDV inoculated at 1 or 5 days of age, coincided with a delayed infiltration of the Harderian gland by lymphocytes and immunoglobulin-bearing cells. In the groups inoculated at older ages infiltration did not seem to be delayed but moderate destruction of plasma cells was observed 7 to 14 days later. The neutralisation index to IBV, which was significantly higher in the non-IBDV infected than in the infected birds at the day of challenge, increased sharply after challenge in the IBDV infected but not in the non-infected birds. All IBDV-inoculated birds developed typical lesions when 17 to 26 days old whereas precipitins reappeared when birds were 29 to 33 days old.     

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.     

Alteration of chicken heterophil and macrophage functions by the infectious bursal disease virus

The effects of the infectious bursal disease virus (IBDV) on chicken heterophils and macrophages are reported. Short-term incubation of IBDV with heterophils causes an activation of cellular oxidative burst, a decrease in phagocytosis and cellular migration, and an increase in cellular adherence to the IBDV-infected chicken embryo fibroblasts. Under electron microscopic examination, heterophils show condensed chromatin and packets of intact granules, either free or being phagocytized by macrophages, indicating that the heterophils are undergoing apoptosis. The HD-11 cells, a virus-transformed macrophage cell, after 1–2 h of infection by IBDVin vitro, become less responsive of chemoattractant and less capable of performng phagocytosis. After a longer period of infection, IBDV is able to multiply in the HD-11 cells, and starts to cause cells to undergo apoptosis. This IBDV-induced alteration in heterophil and macrophage functions and the death of these phagocytic cells may play a role in the subsequent immunosuppression in the affected chickens.     

Replication of infectious bursal disease virus in lymphoid cells

Summary Replication of infectious bursal disease virus in lymphoid cells from the bursa of Fabricius occurs preferentially in a population of proliferating cells characterized by increased thymidine incorporation and by blast cell formation. This was demonstrated by enrichment of virus producing cells in gradients of bovine serum albumin and by mitogenic stimulation of cells with fetal calf serum or an extract prepared from bursae of susceptible chickens. Susceptibility of lymphoid bursa cells is not correlated with the expression of immunoglobulins on their surface. This conclusion was reached after the enrichment of Ig-bearing cells by rosetting techniques and by separation through a cell sorter.With 3 Figures     

Competitive replication of different genotypes of infectious bursal disease virus on chicken embryo fibroblasts

Infectious bursal disease virus (IBDV) causes immunosuppression in chickens. We investigated the molecular changes in chicken embryo fibroblasts (CEF) adapted IBDV by genomic sequencing. IBDV were serially passaged in CEF and chickens were infected with the IBDV obtained after different numbers of passages in CEF. Chicken infections showed that 16th, 20th, and 21st passage viruses were pathogenic, while 26th and 36th passage viruses were non-pathogenic. Sequencing demonstrated that the initial changes during the serial passage comprised of a single-nucleotide deletion in the 3′ non-coding region of segment B of the virus after 19th passage, followed by changes in the VP1 gene after the 20th passage of the virus and changes in VP2, VP5 after the 21st passage of the virus. These data suggested that the attenuation of very virulent IBDV was due to multigenic mutations and there are in vitro and in vivo competitive replications in IBDV quasispecies.     

The immunoglobulin M response in chicken serum to infectious bursal disease virus

Despite the severe bursal damage observed in 5-week-old chicks as a result of the replication of a virulent strain of infectious bursal disease virus, the virus-specific antibody response in serum followed a typical pattern. Three methods were used to monitor the IgM response, an indirect ELISA using serum and ELISAs, carried out on gel chromatography fractions and eluants from affinity chromatography of serum. The profile of IgM production was found to be similar by each method. Possible future uses for the indirect ELISA are discussed.     

Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift

We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV + IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV + IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.     

In vitro characterization of chicken bone marrow-derived dendritic cells following infection with very virulent infectious bursal disease virus

Infectious bursal disease is caused by infectious bursal disease virus (IBDV), an immunosuppressive virus that targets immune cells such as B cells and macrophages. However, the involvement of dendritic cells (DCs) during IBDV infection is not well understood. In this study the in vitro effects of live and inactivated very virulent IBDV (vvIBDV) UPM0081 on bone marrow-derived DCs (BM-DC) were characterized and compared with BM-DC treated with lipopolysaccharide (LPS). Morphologically, BM-DC treated with LPS and vvIBDV showed stellate shape when compared to immature BM-DC. In addition, LPS-treated and both live and inactivated vvIBDV-infected BM-DC expressed high levels of double positive CD86 and major histocompatibility complex class II antigens (>20%). vvIBDV-infected BM-DC showed significantly higher numbers of apoptotic cells compared to LPS. Replication of vvIBDV was detected in the infected BM-DC as evidenced by the increased expression of VP3 and VP4 IBDV antigens based on flow cytometry, real-time polymerase chain reaction and immunofluorescence tests. Levels of different immune-related genes such as interleukin-1β (IL-1β), CXCLi2 (IL-8), IL-18, interferon gamma (IFN-γ, IL-12α, CCR7 and Toll-like receptor-3 (TLR3) were measured after LPS and vvIBDV treatments. However, marked differences were noticed in the onset and intensity of the gene expression between these two treatment groups. LPS was far more potent than live and inactivated vvIBDV in inducing the expression of IL-1β, IL-18 and CCR7 while expression of Th1-like cytokines, IFN-γ and IL-12α were significantly increased in the live vvIBDV treatment group. Meanwhile, the expression of TLR3 was increased in live vvIBDV-infected BM-DC as compared to control. Inactivated vvIBDV-treated BM-DC failed to stimulate IFN-γ, IL-12α and TLR3 expressions. This study suggested that BM-DC may serve as another target cells during IBDV infection which require further confirmation via in vivo studies.     

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     

Isolation of infectious bursal disease virus from turkeys

A virus, which is morphologically identical and antigenically related to two previously known isolates of infectious bursal disease (IBD) virus, was isolated from pooled faeces of 6-week-old turkeys with diarrhoea. It is concluded that this virus, designated TY89, is an isolate of IBD virus. The isolation of TY89 was heavily dependent upon the use of electron microscopy and the immunofluorescence technique. Antibody to TY89 virus was detected by both indirect immunofluorescence and serum neutralisation tests in 28 of 95 (29%) sera collected from 20-week-old turkeys. The available evidence suggests that this virus has only recently been introduced into turkeys in Northern Ireland. It is believed that this is the first report of natural infection of turkeys with IBD virus.     

Reassortant infectious bursal disease virus isolated in China

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.     

Small interfering RNAs inhibit infectious bursal disease virus replication in Vero cells

Small interfering RNA (siRNA) molecules are considered to be a promising antiviral therapeutics. This study was performed to analyze the application of siRNA against infectious bursal disease virus (IBDV) replication. Two siRNAs were designed to target common coding sequences of four IBDV proteins. Corresponding vectors were constructed to express anti-IBDV short hairpin RNAs (shRNA) that were tested for their antiviral effect in Vero cells. The results showed that expressed shRNA inhibited the virus replication to a significant extent (92%) as determined by the virus titration in cell culture. This outcome demonstrated the effectiveness of RNA interference (RNAi) based mechanism against the IBDV in vitro.     

Effect of reticuloendotheliosis virus and infectious bursal disease virus on Marek's disease herpesvirus latency

Birds infected with reticuloendotheliosis virus (REV) were exposed to Marek's disease virus (MDV) to determine if the establishment of MDV latency was affected by REV-induced immunosuppression, while other chickens, already latently infected with MDV, were challenged with REV or infectious bursal disease virus (IBDV) to determine if the consequent immunosuppression caused a return to cytolytic infection. Immunosuppression was assessed by in vitro mitogen stimulation assays with spleen cells. Latently MDV-infected cells were free of viral internal antigen(s) (VIA) but could be identified by their ability to produce VIA after in vitro cultivation. The results were unexpected: chickens infected with either of these viruses had very low, and often undetectable, levels of MDV infection when compared with appropriate controls. REV infection interfered with early cytolytic MDV infection, and IBDV and REV both failed to activate latent MDV infection in the face of inferred (IBDV) or demonstrated (REV) immunosuppression by these viruses. Apparently, both viruses reduced the number of MDV infected cells since neither cytolytic nor latent infection could be demonstrated. This was based on an absence of cells with VIA either before or after cultivation and, in the case of REV infection, on failure to detect MDV-DNA using a dot-blot hybridisation technique.     

Infectivity and propagation of attenuated infectious bursal disease virus in the chicken B-lymphocyte cell line DT40

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN₃, directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.