High antigenic diversity of serotype 1 infectious bursal disease virus revealed by antigenic cartography

Abstract

The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

Keywords: Antigenic cartography; Primary chicken B cells; Serotype 1 IBDV; Virus neutralization test.

Fig. 1

Phylogenetic trees of segments A and B. (A, B) Phylogenetic trees of analyzed viruses based on nucleotide sequences for segment A (A) and B (B). Genogroups are indicated in dashed squares. See Fig. S1 for complete trees used to determine studied viruses genotype. (C, D) Phylogenetic trees based on VP2 hypervariable region (HVR) amino acid sequences (positions 177–360) for segment A (C) and based on a 474 bp sequence (amino acid positions 100–257) for segment B, corresponding to the VP1 N-terminal domain and the finger subdomain of the central polymerase (D). The trees based on amino acid sequences were generated using Neighbour Joining method based on Jones-Taylor-Thornton (JTT) model with 1000 bootstrap replicates implemented in MEGA software version 7. Bootstrap values>75% are indicated.

Fig. 2

Amino acid alignment of VP2 HVR for the sixteen IBDV used in the present work. Boxes indicate exposed loop in VP2 P domain. *Residues associated to antibody neutralization-escape mutants. †Residues associated to virulence and adaptation to cell culture. Variants found for a specific position are shown in blue, with major or minor variants indicated by bigger or smaller letter, respectively.

Fig. 3

Antigenic relatedness by Antigenic Cartography. (A) Two-dimensional (2D) antigenic map of the sixteen viruses used in the present work against eleven sera from IBDV infected chicken. The positions of these viruses were based on the neutralizing titre at a virus dose of 100 TCID₅₀. Colors represent the antigenic clusters identified by a K-means clustering algorithm. The vertical and horizontal axes both represent antigenic distances. The space between grid lines is 1 unit of antigenic distance, corresponding to a two-fold dilution of serum in the VN test. B. Correlation between antigenic distances and R values (Archetti and Horsfall). The antigenic distances between viruses were derived from the 2D antigenic map and the R values from the neutralizing antibody titres. Spearman's correlation coefficient was - 0.73 (P<6.2 × 10⁻¹¹). C. Analysis of variance for serum-virus distances. A Bartlett's test was used for variance comparison of serum-virus distances for each serum. Different letters indicate significant difference (p<0.05) between the variances.