Chicken B Lymphoma DT40 Cells as a Useful Tool for in vitro Analysis of Pathogenic Infectious Bursal Disease Virus

Abstract

ABSTRACT. Susceptibility of DT40 cells to pathogenic field strains of infectious bursal disease virus (IBDV) including very virulent and classical virulent strains were studied. After the first and second passage of the virus in DT40 cells, IBDV-specific antigen was readily detected in DT40 cells inoculated with the pathogenic field strain infected bursal homogenates. Nucleotide sequence analysis in the VP2 hypervariable domain, which is critical for the virulence of IBDV, revealed no common amino acid substitutions among the pathogenic IBDVs in accordance with the propagation in DT40 cells. These results indicate that DT40 cells are a useful tool for rapid isolation of pathogenic field strains and successive in vitro analysis of IBDV. KEY WORDS: DT40, IBDV, virus isolation.J. Vet. Med. Sci. 70(4): 407-410, 2008 Infectious bursal disease virus (IBDV) is a member of the Birnaviridae, whose genome consists of two segments (A and B) of double-stranded RNA [6]. Genome segment B (2.9 kb) encodes a 90-kDa protein VP1, which is an RNAdependent RNA polymerase [1]. Segment A (3.4 kb) encodes a nonstructural protein VP5 [16] and a 106-kDa precursor polyprotein which is processed to capsid proteins VP2 and VP3, and the viral protease VP4 [10,11].IBDV is the etiologic agent of infectious bursal disease (IBD), a highly contagious immunosuppressive disease of young chickens [13]. IBDV infection destroys lymphoid organs, especially the bursa of Fabricius [9,19]. IBD exists worldwide and its immunosuppressive effect increases the incidence and severity of many other infectious diseases [7,15,22]. IBDV isolates have been classified into classical virulent (cv) and very virulent (vv) types. Classical virulent type IBDV mainly causes immunosuppression and moderate mortality, while vv type IBDV, which emerged in 1986, causes an acute course of disease with high mortality.In general, virus isolation and subsequent propagation in cultured cells are essential for definite diagnosis of the viral disease and further characterization of the isolated virus. In the case of IBDV, chicken embryo fibroblast (CEF) cells have been used for such in vitro analyses. However, IBDV pathogenic field strains are reported to be attenuated in the process of the adaptation in CEF cells. Alternate cultured cell in which pathogenic field strains maintain its pathogenicity during replication was not reported. In addition, amino acid changes D279N and A284T in the VP2 hypervariable domain, which is known as the minimum region required for reaction with virus neutralizing monoclonal antibody, are essential for propagation in CEF cells [26]. In the case of adaptation of IBDV to Vero cells, identical amino acid changes were observed [14]. Therefore, for adaptation of pathogenic field strains to propagate in CEF cells, long-term passage on the chorioallantoic membrane (CAM) or in the yolk sac of embryonated eggs was required until these amino acid changes occur [8,26]. DT40, an avian leukosis virus-induced chicken B cell line [2], is widely used for gene knoc...

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV)

“…Taken together, these data demonstrate that the chicken primary B cells could support the replication of cell culture-adapted and vv IBDV strains. This is in contrast to primary chicken embryo fibroblasts (CEFs) or the immortalized chicken fibroblast cell line, DF-1, which do not support the replication of vv IBDV without prior adaptation that can lead to viral attenuation [20].…”

Infectious bursal disease virus persistently infects bursal B-lymphoid DT40 cells

“…A simple interpretation of our data suggests that a large proportion of the cell population is naturally resistant to IBDV infection. Low infected-cell ratios were also reported by Terasaki et al (2008) after infecting fresh DT40 cultures with several pathogenic IBDV field isolates. Data described in that report show that the number of infected DT40 cells increases significantly after three consecutive virus passages in this cell line, indicating that IBDV undergoes an adaptation process that results in the selection of variants with an enhanced ability to infect fresh DT40 cell cultures.…”

“…In a recent report, Terasaki et al (2008) showed that very virulent IBDV isolates can be grown directly in chicken lymphoid DT40 tumour cells infected persistently with avian leukosis virus (ALV) (Baba et al, 1985), without the requirement for a preliminary adaptation process. Most importantly, serial passage in this cell line does not result in the incorporation of mutations at the specific VP2 residues.…”

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

“…The surface expression of the VP3 antigen and viral RNA (based on the VP4 gene) were detected as early as 3 hpi and the peak of infection was at 12 hpi. Previous studies have shown the presence of IBDV antigen in KUL01 + bursal-derived macrophages (Khatri et al, 2005), while IBDV antigen was readily detected by IFAT in the chicken B cell line, DT40, following infection (Terasaki et al, 2008). These findings might suggest that vvIBDV is able to replicate in BM-DCs and induce their maturation indicating that recognition of vvIBDV by BM-DCs do occur and that DCs are susceptible to IBDV infection.…”