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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #423288
Research Project: Systems Biology Approaches to Develop Medical Countermeasures to Detect, Prevent, and Control Poultry Production Viral Diseases

Location: Endemic Poultry Viral Diseases Research

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

Author
item ZUBAIR, KHALID - Auburn University
item Alvarez Narvaez, Sonsiray
item Harrell, Telvin
item CHOWDHURY, ERFAN - Auburn University
item Conrad, Steven
item HAUCK, RUEDIGER - Auburn University

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2025
Publication Date: 12/3/2025
Citation: Zubair, K., Alvarez Narvaez, S., Harrell, T.L., Chowdhury, E.U., Conrad, S.J., Hauck, R. 2025. Viral Heterogeneity in an Avian Reovirus Isolate Despite Plaque Purification and Differences in Segment-Wise Abundance Between Different Amplification and Purification Steps. Microorganisms. Volume 70: Issue 1. https://doi.org/10.1637/aviandiseases-D-25-00068.
DOI: https://doi.org/10.1637/aviandiseases-D-25-00068

Interpretive Summary: This study is focused upon the avian reoviruses (ARVs), which are important pathogens of chickens and turkeys. Scientists routinely use field isolates, or viruses obtained from sick animals, in their research. But, before a particular field isolate can be used for experimental infections, researchers must first verify that the virus is pure (which means that the isolate is composed of one and only one strain of ARV). The usual procedure to ensure that you are working with a single ("clonal") strain of a virus is to do plaque purification assays through three iterations. But, as this paper shows, three rounds of plaque purification are not sufficient to ensure a researcher is working with a clonal sample of virus. This is an important point for anyone working with ARVs because it indicates that the current method of producing a "pure" virus is not adequate to separate two co-infecting ARV strains in a field isolate.

Technical Abstract: 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-isolate) using conventional PCR, Sanger sequencing, and whole genome sequencing (WGS). While conventional PCR targeting the sigma C (sC) gene failed to amplify the AL isolate using standard primers, de novo primers designed based on WGS successfully detected its presence. Sanger sequencing of the sC 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.