Fig 1.
Infection of Stat1-/- mice with ZIKV.
(A) 12–16 week-old Stat1-/- mice (n = 4–7) were infected intraperitoneally (i.p.) with various doses of ZIKV (4×103, 4×104, or 4×106 pfu/mouse). Weights and survival rates were monitored daily. Nine week-old Ifnar-/- mice were infected i.p. with 4×104 pfu/mouse in parallel. (B) Serum samples were collected from ZIKV infected Stat1-/- mice (4×104 pfu/mouse) and virus titers were measured by plaque assay. (C) Photograph of liver and spleen collected from ZIKV infected (1×103 pfu/mouse) (D7#) and uninfected Stat1-/- mice (Mock) on D7 post-infection. (D) ZIKV and cyclophilin A (CPH, a house keeping gene) RNA expression in various tissues was measured by quantitative RT-PCR (n = 3–4) to obtain quantification cycle (Cq) values. Relative ZIKV gene expression level was calculated as ZIKV(Cq)/CPH(Cq). D3 and D5 samples were collected from mice infected with 4×104 pfu/mouse and D7# samples were from mice infected with 1×103 pfu/mouse. (E) ZIKV viral protein expression in spleens harvested at D3 and D5 post-infection (4×104 pfu/mouse) and D7 post-infection (1×103 pfu/mouse) were examined by immunoblotting with specific antibodies for NS1 and capsid. (F) NS1 protein expression in spleens and brains 7 days post-infection (1×103 pfu/mouse) was also evaluated. (G) Determination of serum NS1 concentrations by ELISA. Culture medium collected from Vero cells infected with DENV or ZIKV were used as a positive control for NS1. *, p ≤ 0.05; **, p ≤ 0.01.
Fig 2.
ZIKV replication and assessment of apoptosis in various organs in infected Stat1-/- mice.
(A) Liver, spleen, and brain tissues harvested from ZIKV-infected and uninfected Stat1-/- mice were subjected to immunohistochemistry with an anti-NS1 antibody. (B) Cell death in infected tissues was determined using the TUNEL method. Tissues were collected 3 and 7 days post-infection with 4×104 pfu/mouse. TUNEL+ signals were examined by fluorescence microscopy and images from 5–10 randomly selected 200X magnification fields were analyzed by ImageJ software (NIH).
Fig 3.
Proinflammatory cytokines were not involved in ZIKV-dependent mortality.
(A) Stat1-/-, Stat1-/-× Il6-/- and Stat1-/-× Tnfa-/- (n = 3) were challenged with ZIKV (1×103 pfu/mouse, ip) and monitored daily. (B) Stat1-/-, Stat1-/-× Il6-/-, and Stat1-/-× Tnfa-/- mice were challenged with Dengue virus by intravenous injection (D2Y98P strain, 1×103 pfu/mouse) and mouse survival rates were determined daily.
Fig 4.
ZIKV infection caused the expansion of PDCA+ dendritic cells but not the activation of F4/80+Ly6G- macrophages in Stat1-/- mice.
Splenocytes isolated from wild type (n = 1), Stat1-/- mice (n = 3) and Ifnar-/- mice (n = 3) without or with ZIKV infection (60 h post-infection, 4x104 pfu/mouse) were subject to FACS analysis using side scattered light (SSC) for granulocyte (A) and specific markers for dendritic cells (C,E,G) and macrophage cells (I,K). 7-Aminoactinomycin D (7-AAD) and CD45 were used to exclude dead and non-hematopoietic cells, respectively. For quantitation analysis, the percentage of specific subpopulations to the gated population was calculated for each splenocyte preparation. Quantifications of the subpopulations of immune cells were shown in (B,D,F,H,J,L). *, p ≤ 0.05; **, p ≤ 0.01 Stat1-/- mice were less competent to activate splenic cDCs and macrophages than Ifnar-/- mice after ZIKV infection.
Fig 5.
Establishing a complete mosquito-mediated ZIKV transmission cycle.
(A) Experimental design for establishing a complete ZIKV transmission cycle. (B) A. aegypti mosquitoes were injected in the thorax with ZIKV (400 pfu/mosquito) and viral titers were determined by plaque assay by homogenization whole mosquitoes 7 days later (n = 15). (C) To confirm the infectivity, viral titers were determined by focus-forming assay by homoginizing mosquito salivary gland 4 or 7 days later (n = 8 or 2). The ZIKV-infection rate (I.R.) was calculated. (D) Stat1-/- mice (n = 3) were bitten by 1–3 or 6–12 ZIKV-carrying mosquitoes (day 4 or 7 post-thoracic injection). Weights and survival were monitored daily. Virus titers in serum collected from mosquito-bitten mice day 2 post-mosquito exposure were determined by focus-forming assay. (E) Mosquitoes were starved overnight and allowed to take blood meals from the ZIKV-infected mice (Group b and d mice in D; day 2 post-ZIKV infection). The Group e and f mosquitoes took blood from Group b and d mice, respectively. Virus titers in mosquito midguts collected right after the blood meal (Day 0), 4 and 7 days later were measured by focus forming assay (n = 5–20). Infection rate was calculated. (F) Legs and wings collected from the Group e and f mosquitoes were also subject to virus detection (n = 10–12). (G) Stat1-/- mice (Group g or h; n = 4) were bitten by the ZIKV-infected mosquitoes from Group e or f, respectively (day 7 post-infection in E, 6–9 mosquitoes/mouse). Weights and survival rates were monitored daily.
Fig 6.
Threshold of ZIKV infection in mosquitoes.
(A) Experimental design. (B) Six Stat1-/- mice were infected with 5, 50 or 500 pfu of ZIKV intraperitoneally. Mouse body weights and survival rates were monitored daily. Virus titers in serum from day 2, 3 and 4 post-infection were determined (n = 2). (C) Starved mosquitoes were allowed to take blood meals from the ZIKV-infected mice (#1 to #6) in B on day 2 post-infection. Mosquitoes obtained blood meal from the same mouse were housed and grouped together. Virus titer and infection rate in each mosquito group were measured on day 7 post-blood meal (n = 7–14). (D) Based on the mouse serum data and their corresponding infection rate in mosquitoes, the mosquito infectious dose of 50% (MID50) was estimated.