An Infectious Bursal Disease Virus (IBDV) Reverse Genetics Rescue System and Neutralization Assay in Chicken B Cells

Abstract

Infectious bursal disease virus (IBDV) is a major threat to the productivity of the poultry industry due to morbidity, mortality, and immunosuppression that exacerbates secondary infections and reduces the efficacy of vaccination programs. Field strains of IBDV have a preferred tropism for chicken B cells, the majority of which reside in the bursa of Fabricius (BF). IBDV adaptation to adherent cell culture is associated with mutations altering amino acids in the hypervariable region (HVR) of the capsid protein, which affects immunogenicity and virulence. Until recently, this has limited both the application of reverse genetics systems for engineering molecular clones, and the use of in vitro neutralization assays, to cell-culture-adapted strains of IBDV. Here, we describe the rescue of molecular clones of IBDV containing the HVR from diverse field strains, along with a neutralization assay to quantify antibody responses against the rescued viruses, both using chicken B cells. These methods are readily adaptable to any laboratory with molecular biology expertise and negate the need to obtain wild-type strains. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: A chicken B-cell rescue system for IBDV Basic Protocol 2: A chicken B-cell neutralization assay for IBDV.

Keywords: chicken B-cells; infectious bursal disease virus; neutralization assay; reverse genetics.

Figure 1

Overview of the chicken B‐cell rescue system for IBDV. To rescue IBDV strains, electroporate reverse genetics plasmids for segments A and B into DT40 cells. Reverse genetics plasmids, in this example for the IBDV strain PBG98 (pRG‐PBG98‐A and pRG‐PBG98‐B), are constructed to contain an insert consisting of the self‐cleaving hammerhead ribozyme (purple box) followed by the 5′ noncoding region (black box), the coding region of either segment A or B, the 3′ noncoding region (black box), and a self‐cleaving hepatitis delta ribozyme (purple box), in an expression vector under the control of the CMV promoter. Then, plasmids pRG‐PBG98‐A and pRG‐PBG98‐B electroporated into DT40 cells and incubated for 48 hr at 37°C. Next, cells are passaged by the addition of fresh DT40 cells to the cultures in a 3:1 ratio and the cells are incubated for 72 hr at 37°C. After two such passages, an aliquot of the cell suspension is harvested, and virus rescue is confirmed through immunofluorescence staining of DT40 cells and RT‐qPCR quantification of the cell pellet. Finally, the cells are passaged further and assessed to determine whether viral titers have increased with each passage by titrating the supernatant for viral titers and quantifying the cell pellet by RT‐qPCR. The figure was created using templates from the Motifolio toolkit (Motifolio Inc., Ellicott City, MD, USA).

Figure 2

Overview of the chicken B‐cell neutralization assay for IBDV. Antibodies in polyclonal serum samples can be assessed for their neutralization capacity against diverse IBDV strains. After serial dilution of the heat‐inactivated serum samples, the virus to be tested is added at 10³ TCID₅₀/ml and incubated for 1 hr at 37°C. DT40 cells are added to the serum/virus mix and the combination is incubated for 72 hr at 37°C. Cells are then fixed and stained with a monoclonal antibody against an IBDV antigen to identify wells that are positive or negative for IBDV, and finally the viral neutralization titers (VNT) are determined. Sample VNT titers are shown in the table. The figure was created using templates from the Motifolio toolkit (Motifolio Inc., Ellicott City, MD, USA).