Nonsynonymous variants detected in two or more samples during in vivo passage.

Mutation frequencies (shown as percent) with a frequency of >1% were identified using CLC Workbench. Sample sequences were compared to the Genbank sequence for each strain to identify variant sites. Low frequency mutations were verified using overlapping read pairs (ORP) and error modeling [40]. Sites with 2 different variants present have the second variant amino acid noted next to the frequency (i.e. Thr605Ser detected for sample M4.Te at 40%). NC = no coverage at this site; LC = Low coverage (less than 300 reads). Frequencies were derived by alignment of reads to the appropriate reference sequence from GenBank. Mice are numbered 1–4. AF amniotic fluid, LN lymph node, Va vagina, Spl spleen, Te testes. Br Brain, d day post infection.