Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples

Abstract

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

Keywords: Astrovirus; Coronaviridae; Gallus gallus; Igacovirus; Metagenomics; Next-Generation Sequencing; Sicinivirus.

Fig. 1

Maximum likelihood phylogenetic analysis of the complete S1 coding sequence of 204 infectious bronchitis virus strains. A GTR + G + I evolutionary model was used with partial deletion of missing data and gaps and 500 bootstrap replicates to evaluate the significance of nodes. For visual clarification, subtrees representing the identified lineages GI-1 and GI-13 were extracted from the full-dataset phylogenetic tree. ♦: sequence determined in the present study. IBV/chicken/Belgium/4439_001_PTLB/2020 and IBV/chicken/Belgium/4439_001_iPTLB/2002 correspond to the genome sequences determined from the tissue sample pool and the derived embryonated egg-passaged virus, respectively, while Infectious bronchitis virus strain IBV/chicken/Belgium/4134_001/2019 corresponds to the sequence determined from the cloacal swabs pool