Quantitative synthetic Powassan virus lineage II RNA can be used for assay development, verification, and validation as well as monitoring of day-to-day test variation and lot-to-lot performance of molecular-based assays. The quantitative format allows for the generation of a standard curve for quantitative PCR (qPCR) to determine viral load. This preparation includes fragments from the E, NS1, NS5, and 3’ UTR regions.