Fig 1: ZIKV Can Be Transmitted via Corneal Transplantation in AG129 Mice but Infects Human Corneal Explants Inefficiently(A) Survival of AG129 mice inoculated with Ifnar1−/− corneal homogenates 7 days after subcutaneous inoculation with ZIKV (n = 11) or uninfected Ifnar1−/− cornea control homogenates (n = 4).(B and C) ZIKV mRNA expression in tissues was quantitated using qRT-PCR. Tissues were harvested from cornea donors (n = 2) and recipient (n = 6) mice on days 6 and 7 after infection.(D) ZIKV (6 × 105 FFU) was stored in cornea storage medium (Optisol) at 4°C for 28 days. Infectious virus was measured by focus forming assay at the indicated time points.(E) Human donor corneas (n = 4 technical replicates per donor from three independent donor corneas) were inoculated with 3 × 106 FFU/mL of ZIKV (Brazil strain), and infectious virus was quantitated at the indicated time points by focus forming assay. Data represent the mean ± SEM.(F) RNA ISH and histological analysis was performed on uninfected (left panel) and ZIKV-infected (right panel) human corneal explants in order to determine virus tropism. The black arrow indicates infected corneal epithelial cells.(G) Baseline expression of IFNL1, IFNL2, and IFIT1 was measured using qRT-PCR in three human donor corneas in independent experiments. Data represent the mean ΔCT ± SEM relative to expression of GAPDH. Each cornea was sectioned to produce eight technical replicates per experiment (n = 24) from three biological replicates in independent experiments.
Fig 2: The IFN-λ Receptor (IFNλR1) Regulates Antiviral ISG Expression and Restricts HSV-1 Growth in Human Corneal Explants(A) RNA ISH and histological analysis were performed on human donor corneas to determine IFNLR1 mRNA expression in the corneal epithelium. Panels shown are positive control housekeeping gene (left), negative control probe (middle), and IFNLR1 (right). Scale bar, 10 μm. Data are representative of three independent experiments with unique donor corneas. Black arrows indicate positive staining of cells for IFNLR1.(B–F) Human donor corneas were treated with IFN-λ or PBS vehicle control, and ISG expression from human corneal explants was quantitated using qRT-PCR. Data represent the mean ± SEM of 12 samples, including 4 cornea segments per donor from 3 donor corneas. Results were pooled from two independent experiments.(G) ZIKV replication in a representative human donor cornea sample treated with anti-IFNλR1 or isotype control antibody. Infectious units of ZIKV were quantitated by focus forming assay. ZIKV replicated in only 2 of 5 human cornea samples under these conditions. Results were analyzed using two-way ANOVA.(H) HSV-1 replication in human donor corneas treated with anti-IFNλR1 or isotype control antibody. Infectious units of HSV-1 were quantitated at the 3 h eclipse phase and every 24 h thereafter by plaque assay. Data represent the mean ± SEM of n = 12 replicates, including 4 technical replicates per group from 3 corneas. Results in (H) were pooled from three independent experiments and were analyzed using two-way ANOVA.(I and J) IFNL1 and IFNL2 mRNA expression levels were quantitated using qRT-PCR 24 h after infection with HSV-1. Data represent the mean ± SEM of mock-infected (n = 3) and infected (n = 3) human donor corneas with 4 cornea segments per donor in two independent experiments.(K) RNA ISH and histological analysis were performed on human donor corneas to determine IFNL1 mRNA expression in the cornea. Scale bar, 10 μm. Data are representative of 10 samples from seven independent donors. Samples from two donors repeatedly did not stain for positive control (or target) and were excluded. Black arrows indicate positive staining of cells for IFNL1.(L) SARS-CoV-2 replication in human donor corneas samples treated with anti-IFNλR1 or isotype control antibody. SARS-CoV-2 mRNA expression was measured using qRT-PCR. Data represent the mean ± SEM of n = 7–11 replicates, with two to four technical replicates per group from 3 donor corneas. Results in (L) were pooled from two independent experiments and were analyzed by two-way ANOVA.Results in (B)–(F), (I), and (J) were pooled from two independent experiments and were analyzed using the Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S1.
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