Viral Heterogeneity in an Avian Reovirus Isolate Despite Plaque Purification and Differences in Segment-Wise Abundance Between Different Amplification and Purification Steps.

Abstract

Avian reovirus (ARV) remains a significant concern in the poultry industry. 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 usually been taken for granted, especially in older publications, when there was no means of checking the purity. 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). WGS analysis of multiple samples across different amplification and purification steps revealed persistent mixed viral populations despite three rounds of plaque purification on chicken embryo liver cells. Both S1133-like and divergent contigs were found for each segment in all assembled genomes, with differential strain representation patterns observed before and after purification. As expected, inoculation of mixed population into embryos greatly favored growth of embryo-adapted strain S1133, with highest selection for S1 segment of this strain. Although conventional PCR targeting the sigma C (σC) gene failed to amplify the AL isolate using standard primers,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 the genetic cluster GC4, which was phylogenetically farthest from the vaccine group GC1. These findings demonstrate that plaque purification in chicken embryo liver cells may not ensure clonal isolation of ARV and highlight the dynamic nature of mixed viral populations during different propagation conditions.