Generation and characterization of transgenic mice carrying the IRF3 R278Q allele and susceptibility to infections with HSV-2 and IAV. (A) Mice carrying the patient-specific mIRF3 R278Q amino acid substitution were made using CRISPR microinjection in C57Bl6/J zygotes. The CRISPR guide used was 5′-GTG​GGA​GTG​GCC​TAG​GCG​CTG​GG-3′. (B) Litter size of Irf3^R278Q/R278Q and Irf3^−/− pubs bred at the Aarhus University animal core facility in 2024 compared with average litter size of C57Bl6/JRj mice bred by Janvier. (C) Weight of C57Bl6/JRj, Irf3^WT/R278Q, Irf3^R278Q/R278Q, and Irf3^−/− mice at experiment start (square, female; triangle, male). (D) WB analysis of IRF3 protein and phosphorylation of IRF3 Ser379 in lysates of BMDMs from WT, Irf3^WT/R278Q, Irf3^R278Q/R278Q, and Irf3^−/− mice stimulated with 100 μg/ml cGAMP for 2 h. (E and F)Ifnb and Isg15 gene expression response of murine astrocyte cultures to HSV-1 infection at MOI 1.0 for 24 h. (G–L)Ifnb and Isg15 gene expression response to stimulation with PRR agonists poly-IC (25 μg/ml) or cGAMP (100 μg/ml) for 4 h. Murine astrocyte (G and H), murine microglia (I and J), and murine neurons (K and L). CNS cell culture gene expression was measured by RT-qPCR, and data were normalized to β-actin (Actb) and are represented as fold change normalized to expression in UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Statistical analyses of gene expression in CNS cell cultures (E–L) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. (M–X) WT, Irf3^WT/R278Q, Irf3^R278Q/R278Q, and Irf3^−/− mice were infected with (M–T) HSV-2 by the vaginal route or (U–X) IAV via the nasal route and were followed for disease development over time until reaching humane endpoint or recovering 100% of starting weight. (M, Q, and U) % weight change. (N and R) Symptom score. (O, S, and V) Survival curve. Dead animals were censored in the graphs and thus represented in the graphs with weight and symptom score at time of death. HSV-2 longitudinal (M–O) (WT, n = 8; Irf3^R278Q/R278Q, n = 8; Irf3^−/−n = 8), (Q–S) (WT, n = 8; Irf3^WT/R278Q, n = 8; Irf3^R278Q/R278Q, n = 8), and IAV longitudinal (U and V) (WT, n = 11; Irf3^R278Q/R278Q, n = 11; Irf3^−/−, n = 7; UI, n = 6). Viral load was assessed by (P and T) HSV-2 TCID50% assay of vaginal washes on day 2 after infection; (P) WT, n = 8; Irf3^R278Q/R278Q, n = 7; Irf3^−/−n = 7; and (T) WT, n = 8; Irf3^WT/R278Q, n = 8; or (X) IAV M-Protein gene transcripts in lung homogenates on day 4 postnasal inhalation infection measured by RT-PCR (WT, n = 5; Irf3^R278Q/R278Q, n = 5; Irf3^−/−n = 5). Disease development (M, N, Q, R, and U) was compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Error bars; SEM. Survival (O, S, and V) was analyzed using log-rank Mantel–Cox test. Viral load (P, T, and X) was analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: SourceData FS2.