Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus

Abstract

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4⁺ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4⁺ and CD8⁺ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Keywords: B cells; Ig knockout chickens; Marek’s disease virus; lymphomagenesis; transgenic chickens.

Fig. 1.

Confirmation of the absence of B cells in infected KO chickens. Quantification of total IgM (A) and IgY (B) in serum from wt, JH^+/−, and JH^−/− chickens by ELISA at 28 dpi. The mean values with the SD (error bars) of seven chickens per group are shown. (C) Representative images of chicken B cell marker (AV20) immunofluorescence staining of JH^+/− (Left) and JH^−/− (Right) spleen tissues (4 dpi). (Scale bars: 20 μm.)

Fig. 2.

MDV pathogenesis in the absence of B cells. (A) qPCR analysis of MDV genome copies in the blood of wt, JH^+/−, and JH^−/− chickens at 4, 7, 10, 14, and 28 dpi. Mean MDV genome copies per 1 million cells with the SD (error bars) are shown for eight chickens per group (JH^−/− vs. wt and JH^+/−: P > 0.05, Kruskal–Wallis test). Disease (B) and tumor incidence (C) of infected animals at termination of the experiment. Results are shown as the percentage of affected animals per group [wt (n = 17), JH^+/− (n = 22), and JH^−/− (n = 21)]. No significant difference was observed (Kruskal–Wallis test). Necropsies were performed on chickens upon onset of clinical symptoms or after termination of the experiment. MD, Marek’s disease. (D) Mean number of affected organs harboring gross tumors per animal with the SD (error bars). No significant difference was observed (Kruskal–Wallis test).

Fig. 3.

B cells are also dispensable in natural MDV infection. Naive wt (n = 11), JH^+/− (n = 8), and JH^−/− (n = 11) chickens were housed together with infected animals. The percentages of animals with disease (A) and tumors (B) are shown upon infection via the natural route (P > 0.05, Kruskal–Wallis test). MD, Marek’s disease.

Fig. 4.

Virus spread to lymphoid organs in the absence of B cells. qPCR analysis of MDV genome copies in the bursa (A), thymus (B), and spleen (C) in JH^+/− and JH^−/− chickens at 4, 7, 10, and 14 dpi. Mean MDV genome copies per 1 million cells with the SD (error bars) are shown for three chickens per group and time point. Significant differences between JH^+/− and JH^−/− chickens (*P = 0.05 compared with JH^+/−, Wilcoxon–Mann–Whitney test) are indicated with an asterisk.

Fig. 5.

Infection of CD4⁺ and CD8⁺ T cells compensates for the lack of B cells. (A) Representative immunohistochemistry images of infected B cells (AV20) and CD4⁺ and CD8⁺ T cells in the spleens of JH^+/− and JH^−/− chickens at 7 dpi. Images show cell nuclei (DAPI, blue), the indicated cellular marker (red), and virus antigens (green). Infected AV20⁺ cells were not detected (n.d.) in JH^−/− chickens. (Scale bars: 10 μm.) (B) Quantification of infected B cells and CD4⁺ and CD8⁺ T cells in the spleen (n = 304).