Fig. 1: Design of RHV mutants evaluated as LAV vaccine candidates and their infection outcomes in rats.

A The minimum folding energy differences (MFED) in the RHV-rn1 genome were plotted as the blue line in the upper panel and for different HCV genotypes in the lower panel. MFED values were calculated for consecutive 240 base fragments, incrementing by 15 bases across the genome. MFED values were calculated by subtraction of the MFE of the native sequence from the mean value of the same sequence scrambled in base order while maintaining native dinucleotide frequencies. R2-4 depicts the genomic regions selected for mutagenesis, and their MFED values are shown in red fonts. B Table shows mononucleotide and dinucleotide compositions of wild-type (RHV-rn1), CDLR, and UpA- and CpG-maximized sequences in R-2 and R-3 regions. The yellow highlighted numbers indicate the final number of dinucleotides in the R-2 and R-3 regions of the UpA and CpG mutants. *Ratio of observed frequencies of CpG or UpA frequencies to expected values based on mononucleotide composition. C Schematics of in vivo rescue of infectious virus from mutated genomes using intrahepatic injection of transcribed RNA in rats. Each rat was injected with 10 μg viral RNA in PBS. D Course of viremia of CDLR mutants and wild-type RHV-rn1. Note that the CDLR-4 mutant failed to produce consistent and high-titer viremia observed in R2 and R-3 CDLR mutants. E Course of viremia in rats injected with R-2 and R-3 variants with elevated UpA or CpG dinucleotide frequencies. Source data are provided as a Source Data file.