During the initial dose titration experiment, 60% of mice infected with RVFVmiR-122 at higher doses (2,000 TCID50 and 200,000 TCID50) survived until the end of the experiment (Fig. 3A). As this was not expected, we conducted a serial euthanasia study to compare the disease progression of mice infected with RVFVmiR-122 at 20 TCID50 and 2,000 TCID50. Groups of mice underwent scheduled euthanasia at 3, 6, and 9 dpi. Euthanasia was required for a portion of mice due to clinical disease between 10 and 12 dpi, so these animals were included with mice euthanized at 12 dpi as scheduled; all but one of these mice demonstrated clinical symptoms. Mice infected with either dose had a decrease in viral RNA load in the liver and an increase in viral RNA load in the brain over time, which was consistent with their disease manifestation (Fig. 6A and B). However, whereas the level of viral RNA in the liver of mice infected with 20 TCID50 RVFVmiR-122 remained the same at 6 dpi as that at 3 dpi (P = 0.5015), mice infected with 2,000 TCID50 had viral RNA significantly lower at 6 dpi compared to that at 3 dpi (PFig. 6A and B). This was confirmed by RVFV-specific IHC data, in which no antigen staining was detected in the liver of mice infected with 2,000 TCID50 RVFVmiR-122 as early as 6 dpi, whereas mice infected with 20 TCID50 RVFVmiR-122 still showed antigen staining until 9 dpi in the liver (Fig. 6C). Viral RNA
Limiting viral replication in hepatocytes alters Rift Valley fever virus disease manifestations
