Differential gene expression in chicken primary B cells infectedex vivowith attenuated and very virulent strains of infectious bursal disease virus (IBDV)

Abstract

28Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically 29 important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius 30 (BF), causing immunosuppression and morbidity in young chickens. In addition to strains 31 that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in 32 circulation, causes more severe disease and increased mortality. IBDV has traditionally been 33 controlled through the use of live attenuated vaccines, with attenuation resulting from serial 34 passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated 35 phenotypes are poorly understood. In order to address this, we aimed to investigate host 36 cell-IBDV interactions using a recently described chicken primary B cell model, where 37 chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken 38 CD40L. We demonstrated that these cells could support the replication of IBDV when 39infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of 40 B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by 41 microarray. We found that key genes involved in B cell activation and signaling (TNFSF13B, 42 CD72 and GRAP) were down-regulated following infection relative to mock, which we 43 speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells 44 responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the 45 induction was far less pronounced upon infection with UK661, which we speculate could 46 contribute to its virulence.

Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammationin vivoand more rapid induction of pro-inflammatory responses in primary bursal cells stimulatedex vivo

“…However, it should be noted that RNA-seq was only performed using samples from infected birds, and we did not compare the gene expression between mock and infected birds. It is likely that ISGs were up-regulated in infected birds relative to mock as has previously been shown (21). We also observed genes that were up-regulated in line 15 birds compared to line W were involved in a variety of pathways, but no pathway was more represented than another, suggesting that a more balanced transcriptional response existed in the BF of birds that are more resistant to severe disease.…”

“…Primary bursal cells were obtained from the BF of line 15 and line W birds as previously described (20, 21). Briefly, the BF was harvested aseptically post-mortem and washed in sterile PBS.…”

“…Cells were re-suspended in RPMI before undergoing centrifugation at 2,000 rpm for 20 min at 4°C over Histopaque 1077 (Sigma-Aldrich). Bursal cells that banded at the interface of the medium and histopaque were collected, washed in PBS, counted, and re-suspended in Iscove’ s modified Dulbecco’ s medium (IMDM) supplemented with FBS, chicken serum (Sigma-Aldrich), insulin transferrin selenium (Gibco), beta-mercaptoethanol (Gibco), penicillin, streptomycin, nystatin and chCD40L (20, 21) (complete B cell media).…”