Whole Genome Sequencing of Infectious Bursal Disease Viruses Isolated from a Californian Outbreak Unravels the Underlying Virulence Markers and Highlights Positive Selection Incidence.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84-100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains.

Molecular epidemiology of endemic and very virulent infectious bursal disease virus genogroups in backyard chickens in California, 2009-2017.

Pathogenic strains of infectious bursal disease virus (IBDV) are associated with increased morbidity, mortality, and immunosuppression in susceptible chickens. Backyard poultry is increasing in popularity in the United States, but very little is known about the prevalence and molecular epidemiology of IBDV within these flocks. We performed a retrospective study and phylogenetic analyses of IBDV detected in backyard chickens (BYCs) submitted to the California Animal Health and Food Safety (CAHFS) diagnostic laboratory system in 2009-2017. There were 17 CAHFS autopsy cases of very virulent IBDV (vvIBDV) segment A detected by RT-rtPCR in BYC flocks from 7 counties in California from 2009-2017. During this same time period, non-vvIBDV genotypes were detected by RT-rtPCR in 16 autopsy cases originating from BYC premises in 10 counties in California. Subsequent RT-PCR and phylogenetic analysis of a segment of the hvVP2 and VP1 gene identified vvIBDV, interserotypic reassortant IBDV (vvIBDV segment A and serotype 2 segment B), and non-vvIBDV (variant/subclinical IBDV and classic IBDV) strains in BYC flocks in California.