For RNA extraction, homogenised tissue samples or swab samples from studies 1-3 were centrifuged at 16 000 g for 2 minutes to pellet debris and 100 ml supernatant was mixed with 265 ml MagMAX Lysis/Binding solution (Ambion, Victoria, Australia) and removed from BSL4. RNA was extracted using the MagMax-96 viral RNA isolation kit (Ambion). For the susceptibility studies, 100 ml of EDTA blood was mixed with 265 ml MagMAX Lysis/Binding solution (Ambion, Victoria, Australia) and RNA extracted as described above. For the time course study, RNA was extracted from whole blood mixed in RNAlater using the Mouse RiboPure-Blood RNA Isolation Kit (Ambion). TaqMan qPCR was performed using the AgPath-ID one-step reverse transcription-PCR kit (Applied Biosystems, Victoria, Australia), targeting the N gene of HeV as previously described [46] . Positive results were defined by a cycle threshold (C T ) value of ,39.6 based on a standard curve where this C T represented one copy of target RNA. All samples were normalised against the housekeeping gene 18S and expressed as copy numbers HeV/10 12 copy numbers of 18S.