Infectious bursal disease (IBD), a globally important immunosuppressive disease of poultry caused by infectious bursal disease virus (IBDV), continues to pose a substantial threat to the poultry industry. For the past three decades, very virulent IBDV (vvIBDV), associated with high mortality, have been the predominant epidemic threat. While intensive vaccination is effectively controlling the acute mortality caused by vvIBDV, the global poultry industry now confronts the widespread prevalence of atypical IBD, which induces severe immunosuppression without lethality. In China, two primary IBDV strains are responsible for atypical IBD: the novel variant IBDV (nVarIBDV) and the mutated vvIBDV (mvvIBDV). However, the potential for co-infection and subsequent viral evolution between these prevalent strains remained unexplored. This study provides the first evidence of a natural co-infection by mvvIBDV and nVarIBDV in a single chicken and demonstrates that this co-infection state can be stably maintained. Initial RT-PCR analysis of a field bursa sample, designated IBDV-SD23-1903, for the VP2 hypervariable region (HVR) revealed a sequencing chromatogram with overlapping peaks, indicating a mixed infection. Based on T-vector cloning and Sanger sequencing of the VP2-HVR and VP1-B-marker amplicons, we identified two distinct viruses in this sample: an mvvIBDV (A3B3 genotype) with the characteristic VP2 mutations A222T/G254D/I256L/D279N and an nVarIBDV (A2dB1 genotype). Remarkably, the stability of this co-infection through four serial passages in SPF chickens was consistently observed, with overlapping peaks in sequencing chromatograms from F2 and F4 bursa samples. Moreover, next-generation sequencing (NGS) analysis of the F4 bursa sample confirmed the simultaneous presence of both mvvIBDV and nVarIBDV genomes, supported by the finding that the abundance of their segment A strongly correlated with that of segment B. Co-infection by mvvIBDV and nVarIBDV within a host poses a considerable risk for the generation of novel IBDV variants through genome reassortment or homologous recombination, thereby creating a new concern for the poultry industry. Our findings underscore the need for enhanced epidemiological surveillance and novel vaccines that simultaneously protect against both mvvIBDV and nVarIBDV to mitigate the evolving threat of atypical IBD.

Keywords: Atypical IBD; Co-infection; Mutated very virulent IBDV (mvvIBDV); Novel variant IBDV (nVarIBDV); Reassortment.