FIG 3.

NrHV mouse adaptation causes attenuated but persistent infection in rats. (A) Intrahepatic inoculation of IVT genomic RNA of NrHV variants into 10-week-old inbred Lewis rats. (B) NrHV whole ORF sequencing analysis of serum from Lewis rats. Polyprotein positions with a combined frequency of one or several mutations of >50% in at least two rats are included. Individual substitutions with minimal contribution (<20%) at such positions were not included. Engineered mutations are highlighted in boldface. An asterisk indicates the predicted glycosylation site. Blanked positions represent sequence differences between RHV-rn1 (reference for r05, r06, and r09) and NrHV-B (reference for others). The full data set is available at GEO (accession no. GSE225619). (C) Percentage neutralization of RHVcc-1 infection relative to naive preinfection serum at indicated time points post-RNA inoculation of Lewis rats with RHV-rn1, NrHV-B, or NrHV-B_SLIS. Each data point represents triplicate experiments. (D) Representative microscopy images of HEK-293T cells expressing an NrHV-B C₅₆-E2 construct after staining with sera collected from rat r03, r04, or r10 (inoculated with NrHV-B) or expressing NrHV-B_SLIS C₅₆-E2 and stained with r07 or r08 serum (inoculated with NrHV-B_SLIS) taken 20 weeks post-RNA inoculation. Images were taken 2 days posttransfection at ×200 magnification. LLOQ, lower limit of quantification. Data for r05, r06, and r09 were previously published (15) but are included here for direct comparison.