Novel strand-specific qPCR assay elucidates different cell associations between different strains of avian orthoreovirus

Authors: Harrell, Telvin; Alvarez Narvaez, Sonsiray; Read, Quentin; Conrad, Steven

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/8/2025
Publication Date: 4/30/2025
Citation: Harrell, T.L., Alvarez Narvaez, S., Read, Q.D., Conrad, S.J. 2025. Novel strand-specific qPCR assay elucidates different cell associations between different strains of avian orthoreovirus. Microbiology Spectrum. 13:e03140-24. https://doi.org/10.1128/spectrum.03140-24.
DOI: https://doi.org/10.1128/spectrum.03140-24

Interpretive Summary: Avian orthoreoviruses (ARVs) are widespread RNA viruses that infect both wild and domestic birds, such as chickens and turkeys. Although many strains don’t cause illness, some pathogenic versions lead to symptoms that impact poultry health and production. Despite vaccination and containment efforts, pathogenic strains have increased over the past 20 years, creating challenges for poultry farmers and the industry. Consequently, there is significant interest in understanding the molecular processes that cause ARV infections to develop in various bird species used in poultry farming. By studying these mechanisms, scientists aim to find ways to better control and prevent disease outbreaks in these important production animals. Traditional methods to quantify how much ARV is on a sample, estimate the viral infectious dose but don’t give exact molecular information such as the exact number of viral genomes in a sample. To address this, we have developed a more accurate detection method called strand-specific quantitative PCR (SS-qPCR). This technique is a modification of the traditional qPCR that counts ARV genomes, avoiding interference from viral RNA produced during infection, and can detect as few as 200 viral copies per test. When tested on chicken and quail cells, SS-qPCR provided more precise genome counts than traditional qPCR, which tends to overestimate. Interestingly, our study also found that different ARV strains vary in how closely they associate with cells, a detail that warrants further research.

Technical Abstract: Avian orthoreoviruses (ARVs) are ubiquitous double-stranded RNA (dsRNA) viruses that can infect both wild and domestic birds. Although many avian orthoreovirus 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. TCID50 is the prefer 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 when as little as 5 × 10-7 ng of cDNA was 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), evidencing 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 will need to be further assessed in future experiments.