For assessment of transient viremia, blood samples were also collected at euthanasia from each animal in the time-course study and RNA extracted using a Ribopure blood extraction kit. Previous work showed this extraction method to be the most sensitive for qPCR detection of HeV RNA in experimentally infected horses (A. Foord, personal communication). In mice, a low level (C T of 39.2) of viral RNA was detected in blood from only one animal (#111); this level of genome was very close to our cut off of C T of 39.6 (equivalent to 1 copy RNA from the standard curve).