Figure 3.

MHV-68 is transmitted during latency despite an intact type I IFN system. (A) Schematic of adoptive transfer model. Splenocytes were isolated and pooled from latently infected CD45.2⁺ mice (≥21 days after infection) and adoptively transferred (1 × 10⁷ cells/mouse) into CD45.1⁺ recipient mice (n = 8). Recipient mice were sacrificed after 10 days, and CD45.1⁺ splenocytes were purified by FACS and adoptively transferred (7.5 × 10⁶ cells/mouse) into Ifnar1^−/− mice (n = 8). Total RNA was isolated from splenocytes 11 days after the transfer into Ifnar1^−/− recipient mice. (B) Quantification of MHV-68 ORF52 expression by qPCR in splenocytes of Ifnar1^−/− mice 11 days after adoptive transfer. The dotted line represents the level of relative ORF52 expression in pooled CD45.1⁺ donor splenocytes before adoptive transfer into Ifnar1^−/− mice. (C) Conventional RT-PCR on MHV-68 transcripts M3, M9, and ORF52 in spleen tissue of Ifnar1^−/− mice 11 days after the second adoptive transfer. (D) Schematic of adoptive transfer model. Splenocytes were isolated from latently infected (MHV-68 H2bYFP), tamoxifen-inducible Ifnar1^−/− (R26CreERT2 × Ifnar1^fl/fl) mice and adoptively transferred (1 × 10⁷ cells/mouse) into Ifnar1^−/− recipient mice. Recipient mice were subsequently treated orally with (+Tam, n = 17) or without (-Tam, n = 18) a single dose of 2 mg tamoxifen. (E–G) Relative quantification of MHV-68 genomic DNA (gB) by qPCR, frequency of YFP⁺ splenocytes, and quantification of recoverable infectious by TCID₅₀ assay in spleen tissue of recipient mice seven days after adoptive transfer (geometric mean is indicated). WT recipient mice received latently infected splenocytes in the absence of tamoxifen (n = 12). Red dots indicate measurements for the same three mice. p-values were calculated by Mann-Whitney’s U test. ****, p ≤ 0.0001.