Volume 21, Issue 6, Pages 1588-1599 (November 2017) Outcomes of Congenital Zika Disease Depend on Timing of Infection and Maternal-Fetal Interferon Action  Jinling Chen, Yuejin Liang, Panpan Yi, Lanman Xu, Hal K. Hawkins, Shannan L. Rossi, Lynn Soong, Jiyang Cai, Ramkumar Menon, Jiaren Sun  Cell Reports  Volume 21, Issue 6, Pages 1588-1599 (November 2017) DOI: 10.1016/j.celrep.2017.10.059 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 ZIKV Infection in IFNAR−/− Dams Results in Fetal Demise and Intrauterine Growth Retardation at Mid-pregnancy (A) The changes of maternal body weight in IFNAR−/− females, which were crossed to IFNAR−/− males. Pregnant dams at E5.5, E10.5, and E15.5 were inoculated with 1 × 105 PFU/mice i.p., followed by harvest on E18.5. Mice were monitored for weight loss until E18.5. (B) Representative images showed pathological features of a fetus at E18.5. Nearly 30% fetuses had undergone fetal demise (white arrow). (C) Fetus size was assessed by the crown-rump length and occipito-fronal diameter of the fetal head. N.D., no detected fetus. Symbols represent individual fetuses. Bars indicate the mean of 20 to 21 fetuses per group. Red dots indicate the fetal demise. p values were determined using a one-way ANOVA. ∗p < 0.05; N.S., p > 0.05. (D) Representative H&E staining showed pathological features of placentas at E18.5. The normal placenta in a mouse is composed of the maternal decidua (De), JZ, and LZ. ZIKV infection of the mice placenta at E10.5, with trophoblast necrosis (white arrow), which is present in the decidua but not in the junctional and labyrinth zones. The decidua of placenta in animals infected with ZIKV at E15.5 contains a focus of inflammatory cells. Infiltrated neutrophils (black arrow) are evident. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 ZIKV Infection in IFNAR−/+ Dams Results in Intrauterine Growth Retardation at Mid-pregnancy (A) The changes of maternal body weight in IFNAR−/− females, which were crossed to wild-type males (KO × wild-type). Pregnant dams at E5.5, E10.5, and E15.5 were inoculated with 1 × 105 PFU/mice i.p., followed by harvest on E18.5. Mice were monitored for weight loss until E18.5. (B) Representative images showed the pathological features of the fetus at E18.5. All of the fetuses at E5.5 died in utero and had undergone resorption, leaving only the residual placenta (white arrow). (C) Fetus size was assessed by crown-rump length × occipito-frontal. Symbols represent individual fetuses. Bars indicate the mean of 22–24 fetuses per group. Statistical analyses were performed using a one-way ANOVA. N.D., not detected; ∗p < 0.05; N.S., p > 0.05. (D) Representative H&E staining showed pathological features of placentas at E18.5. The normal placenta in a mouse is composed of the maternal De, JZ, and LZ. Destructive decidua contains a focus of necrosis (white arrow) and hemorrhage (black arrow) in animals infected with ZIKV at E5.5. ZIKV infection of the mice placenta at E10.5, with less trophoblast necrosis (white arrow). No destruction was present in mice placenta infected with ZIKV at E15.5. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 The Fetomaternal Interface of IFNAR−/+ Dams Was Partially Resistant to ZIKV-Related Fetal Injury (A) The viral burden in muscle (MCS), spleen (SPL), brain (BRN), heart (HRT), kidney (KDY), lung (LNG), lymph node (LYN), placenta (PLA), residual placenta (RPL), and liver (LIV) from IFNAR−/− dams (KO × KO) and IFNAR−/+ dams (KO × wild-type) was measured by quantitative real-time PCR. Data are expressed as FFU equivalents per gram after normalization to a standard curve generated in parallel. N.D., no virus was detected; N.A., not applicable. Data are shown as mean ± SD. (B) Viral burden of fetal organs in IFNAR−/− and heterozygote at E10.5 and E15.5 was assessed by quantitative real-time PCR. Data are expressed as FFU equivalents per gram after normalization to a standard curve generated in parallel. (C) The viral loads in the fetal brain were measured by titration at E10.5 and E15.5. Statistical analyses in (B and C) were performed using an unpaired two-tailed Student’s t test. ∗p < 0.05. N.D., no virus was detected. (D) The relative expression of IFN-γ was assessed by quantitative real-time PCR. p values were determined using a one-way ANOVA. N.S., p > 0.05. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 IFN-λ Treatment Improves Fetal Growth Restriction at Mid-pregnancy Mice were inoculated with 2 μg IFN-λ2 i.p. prior to and after ZIKV at E5.5, E10.5, and E15.5. (A) The analysis of maternal body weight after the administration of IFN-λ2. Mice were monitored for weight loss until E18.5. p values were determined using two-way ANOVA. (B) Representative images showed pathological features of a fetus at E18.5. Nearly 30% fetuses had undergone fetal demise (white arrow). (C) The fetus sizes were measured by crown-rump length × occipito-frontal after the administration of IFN-λ2. Symbols represent individual fetuses. Bars indicate the mean of 23–27 fetuses per group. Statistical analyses were performed using a one-way ANOVA. (D) Pathological scores in the placentas of mice were assessed in a blinded manner on a scale of 0–4. Data are shown as mean ± SD. p values were determined using two-way ANOVA. N.S., p > 0.05; ∗p < 0.05; ∗∗p < 0.01. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 IFN-λ Treatment Reduced ZIKV Replication in the Maternal and Fetal Organs at Mid-pregnancy (A) The viral burden in maternal muscle (MCS), spleen (SPL), brain (BRN), heart (HRT), kidney (KDY), lung (LNG), lymph node (LYN), placenta (PLA), and liver (LIV) was measured by quantitative real-time PCR. (B) The viral burden of fetal organs was assessed by quantitative real-time PCR. Data are expressed as FFU equivalents per gram after normalization to a standard curve generated in parallel. Data are shown as mean ± SD. Statistical analyses in the figure were performed using a one-way ANOVA; ∗p < 0.05 compared with the PBS group. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 MX1 Is Critical for ZIKV Reduction Induced by IFN-λ and IFN-α in Human Trophoblast Cells JEG-3 cells were pretreated with IFN-λ1 (p-IFN-λ1) or IFN-α2 (p-IFN-α2) for 24 hr, followed by ZIKV infection, or treated with IFN-λ1 (100 ng/mL) or IFN-α2 (100 ng/mL) and ZIKV. (A) Viral load in JEG-3 cells was detected by quantitative real-time PCR. (B) Viral burden in cellular supernatant was assayed by titration. (C) The relative expression of MX1 was measured by quantitative real-time PCR. (D) The expression of MX1 was measured by western blot. (E) JEG-3 cells transfected with control siRNA (Control) or MX1 siRNA (Si-MX1) for 24 hr prior to ZIKV infection. Viral load was measured by quantitative real-time PCR. (F) Viral burden in cellular supernatant was assayed by titration. Data are shown as mean ± SD. Statistical analyses in the figure were performed using a one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. N.D., no detected virus. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 IFN-λ and IFN-α Induced MX1 Upregulation and Conferred Protection against ZIKV in Primary Human Amnion Epithelial Cells Human amnion epithelial cells were pretreated with IFN-λ1 (100 ng/mL) or IFN-α2 (100 ng/mL) for 24 hr, followed by ZIKV infection, or treated with IFN-λ1 or IFN-α2 and ZIKV. (A) Viral load in human amnion epithelial cells was detected by quantitative real-time PCR. (B) The relative expression of MX1 was measured by quantitative real-time PCR. (C) Western blot analysis of MX1 from primary human amnion epithelial cells was performed. (D) The expression of MX1 in primary human amnion epithelial cells was measured by immunofluorescence staining. Bar, 50 μm. Data are shown as mean ± SD. Statistical analyses in (A–C) were performed using a one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Error bars represent mean ± SEM. Cell Reports 2017 21, 1588-1599DOI: (10.1016/j.celrep.2017.10.059) Copyright © 2017 The Author(s) Terms and Conditions