Harrell Telvin L, Alvarez-Narvaez Sonsiray, Read Quentin D, Conrad Steven J
US Department of Agriculture, Agricultural Research Service, US National Poultry Research Center, Athens, Georgia, USA.
US Department of Agriculture, Agricultural Research Service, Raleigh, North Carolina, USA.
Microbiol Spectr. 2025 Jun 3;13(6):e0314024. doi: 10.1128/spectrum.03140-24. Epub 2025 Apr 30.
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. TCID is 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 × 10 ng 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.
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.
禽呼肠孤病毒（ARV）是一种普遍存在的双链RNA（dsRNA）病毒，可感染野生和家养禽类。虽然许多禽呼肠孤病毒毒株不会引起临床疾病，但致病性变异株与多种临床症状有关。尽管采取了生物安全措施和疫苗接种措施，但在过去二十年中，致病性田间分离株有所增加，给家禽养殖户带来了沉重负担。ARV病例和疫情数量的增加促使对这种病原体及其在家禽中驱动ARV发病机制的分子机制进行更深入的分子特征分析。半数组织培养感染剂量（TCID）是定量ARV的首选方法，但它不能提供有关样品中存在的基因组数量的具体信息。因此，需要一种分子方法来准确量化ARV基因组。在此，我们提出了一种链特异性定量聚合酶链反应（SS-qPCR）检测方法，以定量病毒基因组拷贝数，而不受病毒mRNA的干扰，并更准确地表征病毒复制。即使样品中存在低至5×10 ng的cDNA，SS-qPCR也能检测到ARV，每个PCR反应的检测限估计为200个基因组拷贝。在鸡肝癌（LMH）和鹌鹑纤维肉瘤（QM5）的病毒生长曲线期间，将SS-qPCR与传统qPCR检测方法在ARV基因组定量方面进行了比较，揭示了传统qPCR观察到的基因组计数高估现象。令人惊讶的是，在这个验证过程中，我们注意到ARV毒株在细胞结合程度上存在明显差异，这可能会促使在未来的实验中进行进一步研究。
禽呼肠孤病毒（ARV）是家禽和野生鸟类的病原体，并已成为近年来家禽业重大的干扰源之一。在此，我们详细介绍了一种新的qPCR检测方法的验证，该方法仅定量ARV基因组的负义部分，即使在存在病毒基因表达的情况下也能准确量化病毒基因组。该检测方法将使研究人员能够精确量化ARV基因组，而不会像传统qPCR方法那样高估。
