Novel strand-specific qPCR assay elucidates differences in viral replication kinetics between different strains of avian reovirus.

Abstract

UNLABELLED: Avian reovirus (ARVs) is an ubiquitous double-stranded RNA (dsRNA) virus that can infect both wild and domestic birds. Although many avian reovirus strains cause no clinical disease, pathogenic variants have been associated with a variety of clinical symptoms. Despite biocontainment measures and vaccination efforts, there has been an increase in pathogenic field isolates in the past two decades, placing a major burden on poultry producers. The increase in the number of ARV cases and outbreaks has prompted a deeper molecular characterization of this pathogen and the molecular mechanisms that drive ARV pathogenesis in poultry. TCIDis the preferred method to quantify ARV, but it does not provide specific information about the number of genomes present in a sample. Therefore, there is a need for a molecular method to accurately quantify ARV genomes. Herein, we present a strand-specific quantitative polymerase chain reaction (SS-qPCR) assay to quantify viral genome copy numbers without interference from viral mRNAs and to more accurately characterize viral replication. The SS-qPCR can detect ARV even with as little as 5 × 10ng of cDNA present in the sample, and the limit of detection was estimated to be 200 genome copies per PCR reaction. SS-qPCR was compared to a traditional qPCR assay in the quantification of ARV genomes during viral growth curves in chicken hepatocellular carcinoma (LMH) and quail fibrosarcoma (QM5), revealing the overestimation of genome counts observed with traditional qPCR. Surprisingly, during this process of validation, we noted distinct differences between ARV strains in their degree of cell association that can prompt further investigation in future experiments. IMPORTANCE: Avian reovirus (ARVs) is a pathogen of poultry and wild birds that have become a significant source of disruption in the poultry industry in recent years. Herein, we detail the validation of a new qPCR assay that quantitates only the negative-sense portion of the ARV genome, which allows for the accurate quantification of viral genomes even in the presence of viral gene expression. This assay will enable researchers to precisely quantitate the ARV genome without the overestimates provided by traditional qPCR methods.