Development of real-time reverse transcriptase PCR assays for the detection of Punta Toro

29 30 Background: Research with high biocontainment pathogens such as Rift Valley fever virus 31 (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select 32 institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and 33 Pichinde virus (PICV) for LASV infection allow research to be performed under more 34 permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no 35 standard real-time RT-PCR assays to detect and quantify pathogenesis. PTV is also a human 36 pathogen, making a standardized detection assay essential for biosurveillance. Here, we 37 developed and characterized two real-time RT-PCR assays for PICV and PTV by optimizing 38 assay conditions and measuring the limit of detection (LOD) and performance in multiple 39 clinical matrices. 40 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.