Retention of Viral Heterogeneity in an Avian Reovirus Isolate despite Plaque Purification

Avian reovirus (ARV) remains a significant concern in the poultry industry due to its economic impact and genetic diversity. Three rounds of plaque purification are routinely employed to obtain clonal populations of a viral isolate for experimental purposes. However, the effectiveness of this approach in achieving viral homogeneity has not been evaluated. This study aimed to determine the purity of a plaque-purified ARV isolate (strain AL) using conventional PCR, Sanger sequencing, and whole genome sequencing (WGS). While conventional PCR targeting the sigma C (σC) gene failed to amplify the AL isolate using standard primers, de novo primers based on WGS successfully detected its presence. Sanger sequencing of the σC gene confirmed sequence divergence from the reference S1133 strain with only ~44% amino acid identity. This placed the AL isolate in genetic cluster GC4, which was phylogenetically farthest from the vaccine group GC1. WGS analysis revealed the presence of mixed viral populations despite three rounds of plaque purification on chicken embryo liver cells (CELi) and both S1133-like and divergent contigs were found in the assembled genome. These findings indicate that plaque purification in chicken embryo liver cells may not ensure clonal isolation of ARV.