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. 2023 Mar 27;97(4):e01812-22. doi: 10.1128/jvi.01812-22

FIG 1.

FIG 1

Intrahepatic inoculation of NrHV genomic RNA in immunocompromised mice identifies mouse-adaptive mutations. (A) Variant frequency table for NrHV whole ORF sequencing of the rat inoculum used for long-term or serial passage in NRG mice, of the resulting serum pools, and of WT mice inoculated with the serial pool. The corresponding fractions of the NrHV subpopulations “A” (blue) and “B” (red) are shown at the top. The table includes mutations present at <10% in the rat inoculum and either >40% in the long-term pool (equivalent to >50% of subpopulation A, constituting ~85%) or >30% in the serial pool (equivalent to >50% of subpopulation B, constituting ~65%). Mutations selected for further study are highlighted in gray. (B) Amino acid differences between the RHV-rn1 (GenBank accession no. KX905133), NrHV-A (MF113386), and NrHV-B (ON758386) clones. Numbering is according to the NrHV polyprotein. Dots indicate identity with the RHV-rn1 reference. The NrHV-A and -B UTRs are identical to those of RHV-rn1 except for the single nucleotide substitution G271A. (C) Intrahepatic inoculation of IVT genomic RNA of NrHV-A and NrHV-B consensus clones into 4- to 8-week-old NRG mice. (D) Intrahepatic inoculation of IVT genomic RNA of NrHV-BS, NrHV-BL, NrHV-BSL, and NrHV-BSLIS into 4- to 8-week-old NRG mice. (E) NrHV whole ORF sequencing analysis of serum from NRG mice at week 2. Shown are substitutions with a frequency of >20% in at least one sample against the respective reference genome, NrHV-A or NrHV-B. Engineered mutations are highlighted in boldface. Asterisks indicate residues of predicted glycosylation sites. The blanked position 2214 represents sequence differences between NrHV-A and NrHV-B. The full data set is available at GEO (accession no. GSE225619). (F) Immunoblot of whole-cell lysate derived from HEK-293T cells expressing the E2 protein of RHV-rn1, NrHV-B, NrHV-BSL, or NrHV-BSLIS with or without deglycosylation by PNGase F amidase treatment. (G) Infection of 4-week-old C57BL/6J WT mice with week 2 serum from RNA-inoculated NRG mice. LLOQ, lower limit of quantification; MW, molecular weight; NRG, NOD-Rag1−/−IL2Rγ−/−.