Defining the Impact of Cell-Type and Species on the Molecular RNA Replication Kinetics of Seoul Virus.

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the virus's ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR (ssqRT-PCR) assay and a SYBR-based qRT-PCR (Sb-qRT-PCR) assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release, but instead followed a trend of M>L>S. Overall, this study validates two qRT-PCR assays to measure SEOV RNA, details the accumulation and release of each viral segment, and demonstrates the impact of host cell type on hantavirus replication. Impact Statement: Hantavirus infections in humans lead to significant immune-mediated morbidity and mortality around the world and pose a threat to public health. Zoonotic transmission occurs from persistently infected rodents which show no overt signs of disease. Understanding the molecular interactions that drive divergent infection outcomes is incomplete in the field of hantavirus research due to limited tools and reagents. This novel strand- and segment-specific quantitative RT-PCR assay uncovers the unique kinetics of hantavirus genome replication in reservoir and non-reservoir cells. Further, this assay will serve as a valuable tool to investigate antiviral therapies and virus-host interactions critical for polymerase activity.