Fig 1.
ZIKV in vitro infection and genomics.
(A) Vero cells were infected with ZIKV for 72 h and submitted to indirect immunofluorescence with the anti-flaviviral 4G2 monoclonal antibody. Inset: higher magnification of 4G2 staining surrounding the nuclei of infected Vero cells. Red: Zika virus labeling; Blue: Cell nuclei stained with DAPI; scale bars, 0.1 mm. (B) The linear ssRNA (+) (10,640 bp) complete cds (+ partial UTRs) sequence of ZIKV (HS-2015-BA-01), highlighting the 339 bp amplicon of the E protein gene (see DNA bands from five independent samples in the inset), which was cloned and sequenced.
Fig 2.
Window of susceptibility in a wild-type (FVB/NJ and C57BL/6J), immunocompetent model of ZIKV infection in mice.
(A) Scheme of intravascular injection via the external jugular vein of 105 plaque-forming units (pfu). ZIKV produces a host of morphological disruptions at days 5.5, 7.5 and 9.5 days, but not at 12.5 days post coitum (dpc). This outlines a window of sensitivity to ZIKV teratogeny in mice spanning late gastrulation to late neurulation. Schematic syringes and embryos indicate injection and harvest days, respectively. (B-B') Scheme representing maternal, fetal and placental outcomes after intravascular injection of ZIKV into pregnant females at 5.5 dpc (see Table 1), as determined by quantitative real time PCR (qRT-PCR). ZIKV mRNA detection is graphically represented by pink filling and the developmental stage at time of harvest is indicated for every litter (in dpc). Affected litters in B were all dead at the time of harvest and those affected in B' were alive. MF indicates malformed embryos. (C-D) Schematic representation of the ZIKV burden in pregnant females, embryos/fetuses and placentas as indicated by the number of viral copies detected by qRT-PCR. (E) qRT-PCR results from embryos (n = 32, 14 ZIKV positive), placentas (n = 32, 12 ZIKV positive), maternal spleen (n = 13, 6 ZIKV positive), kidney (n = 13, 4 ZIKV positive), liver (n = 13, 2 ZIKV positive), and brain (n = 13, no ZIKV positive).
Table 1.
ZIKV (105 plaque-forming units)-injected pregnant mouse dams and litters.
Table 2.
PBS-injected mouse dams and respective litters.
Fig 3.
Detection of ZIKV E protein in an immunocompetent mouse model of ZIKV infection.
Western Blot detection of flaviviral immunoreactivity in total protein extracted from maternal organs (A) and individual placentas (B) from pregnant females injected with ZIKV at 5.5 dpc and harvested at 12.5 dpc. Samples from ZIKV-infected tissues display a specific 50 kDa band, which corresponds to the mature flaviviral protein E (arrow). Higher immunoreactive bands represent immature polyprotein precursors such as prM/E (arrowheads). Note that no flaviviral protein was detected in the maternal brain. Nonspecific bands at 55 kDa observed in control samples (except the brain) correspond to tissue-derived immunoglobulin heavy chain (IgH).
Fig 4.
ZIKV infects female organs, placentas and embryos from wild-type immunocompetent mice (FVB/NJ and C57BL/6J), despite the absence of detectable viremia.
Maternal blood samples were collected at 1h (n = 5), 12h (n = 3) and 24h (n = 3) after intravascular ZIKV injection at 5.5 days post-coitum (dpc), or at 12.5 dpc. Further blood samples, maternal organs, placentas and embryos/fetuses were collected at the day of harvest 10.5 dpc (n = 4) and 11.5 dpc (n = 2), or collected at 16.5 dpc (n = 4) in females injected at 12.5 dpc. Samples were assessed for viral load using a tissue culture infectious dose (TCID50) assay in Vero cells. Embryos and respective placentas (n = 8) and maternal organ samples from two 16.5 dpc ZIKV-injected pregnant females (injected in 12.5 dpc), as well as two 12.5 PBS-injected control females were assessed for viral content using a TCID50 assay. ZIKV was detected in the spleen of both females and in the liver of one female. Notably, all placentas had significant amounts of ZIKV, whereas just half of the embryos were ZIKV positive. No malformations were observed.
Fig 5.
Fetuses from two FVB/NJ pregnant females injected with ZIKV at 5.5 days post coitum (dpc) and harvested at 10.5 dpc.
(A-D) All fetuses were alive at time of harvest. Lateral view of severely affected embryos. (C’-D’) Ventral views of embryos depicted in C-D, respectively. (E) Ventral view of an affected embryo, which was dead at harvest. (E’) Dorsal view of embryo depicted in E. Note the increased volume of the fourth ventricle and that the neural tube remains opened far below the otic capsule, at the level of the fifth somite, which is abnormally low for 9.5 dpc embryos. In all images: arrow, optic vesicle; asterisks, dorsal edemas; dashed circle, otic vesicle; dotted circle, cardiac situs inversus; square bracket, posterior hypotrophy; triangles in C, small, compacted somites; cross, dysraphia; 4V, fourth ventricle; LV, lateral ventricle. Scale bars, 0.5 mm.
Fig 6.
Phenotypes of malformed embryos and fetuses harvested from wild-type pregnant females injected with ZIKV.
(A-B) Embryos from a 12.5 days post coitum (dpc) litter harvested from a C57BL/6J female injected with ZIKV at 5.5 dpc. A, Malformed embryo displaying pericardial edema, unfused mandibular arch, missing hyoid arch, abnormal forelimbs and hindlimb rudiments, consistent with developmental delay and growth restriction (staged at 9.5 dpc, or Theiler stage 15). (B) Outwardly normal littermate of the embryo depicted in A (staged at 11.5 dpc, or Theiler stage 19). (C) Control FVB/NJ embryo from a PBS-injected 12.5 dpc pregnant female (staged at 12.5 dpc, or Theiler stage 20–21). (D) Malformed fetus from an 18.5 dpc litter harvested from a C57BL/6J female injected with ZIKV at 7.5 dpc. Note the abnormal postures of the right fore and left hind limbs, which are reminiscent of arthrogryposis in humans (white arrowheads), with splayed out forelimb and hindlimb digits, typical of 14.5 dpc (Theiler stage 22). In contrast, eye and ear landmarks are compatible with 11.5 dpc (Theiler stage 19), suggesting that the conceptus was already developmentally delayed at the time of its death (see S2, S5 Tables). The malformed conceptus is displayed along with the maternal side of the placenta. (E) Outwardly normal littermate of the fetus in D. The conceptuses were staged at 18.5 dpc (Theiler stages 26–27). (F) Control C57BL/6J fetuses from a pregnant female injected with PBS at 7.5 dpc and harvested at 18.5 dpc. The fetuses were staged at 18.5 dpc (Theiler stages 26–27). Scale bars, 1.0 mm.
Fig 7.
Fetuses from FVB/NJ pregnant female injected with ZIKV at 9.5 days post coitum (dpc).
Fetuses were harvested at 16.5 dpc. (A-H) Fetuses are displayed in an order of increased severity of developmental malformations. (I-M) Outwardly normal littermates. (N-P) Fetal phenotypes recorded immediately after harvesting and initial dissection. Prominent features of malformed embryos ranged from vascular rarefaction and pallor in the cephalic region (A, P) (square bracket), generalized edema (B, C) (arrows) to complete disruption of normal development (E-H). Note the intra amniotic hemorrhage in N, clouding of the amniotic fluid in O and the preferential pallor and/or vascular rarefaction in the cephalic region in P (square bracket). White arrowhead indicates abnormal forelimb postures suggestive of arthrogryposis. Scale bar, 2.0 mm.
Fig 8.
Morphometric analyses in three litters of ZIKV-injected pregnant mouse females.
(A) Crown rump length (CRL). (B) Occipital-frontal diameter (OFD). (C) Cephalic proportion relative to body size as indicated by the ratio between OFD/CRL. All data are displayed in relation to an interval defined as the average ± three standard deviations from respective PBS-injected controls. Arrows in C indicate two conceptuses displaying marginal evidence for microcephaly. m, malformed conceptuses, ***p<0.001.
Fig 9.
Morphometric analyses in three litters of ZIKV-injected pregnant mouse females.
(A-C) To better normalize the morphometric results we plotted Crown rump length (CRL) and Occipital-frontal diameter (OFD) as a function of individual embryonic/fetal stages, rather than as nominal litter stages. All conceptuses were classified according to Kauffman [20] and Theiler [22]. (A) CRL data. (B) OFD data. (C) OFD/CRL data. All data are displayed in relation to intervals defined as the average ± three standard deviations of PBS-injected (light grey), or reference controls (dark grey). Stage normalization eliminated all evidence for specific changes in cephalic proportions, but for two conceptuses. One fetus (shown in Fig 7H) displayed an abnormally increased OFD/CRL, which resulted from cephalic collapse in the cranio-caudal axis (arrowhead), while the other fetus (shown in Fig 6D) constitutes the only specific evidence for microcephaly in our study (arrow).
Fig 10.
Placental damage associated with exposure to ZIKV.
(A) Immunoperoxidase reactivity for the flaviviral E protein utilizing the 4G2 monoclonal antibody counterstained with Mayer's Hematoxylin in the labyrinth of a placenta from an outwardly normal fetus from a pregnant females exposed to ZIKV at 9.5 and harvested at 16.5 dpc. Scale bar, 20 μm. Placentas from embryos without structural abnormalities (A) show immunoperoxidase reactivity for ZIKV (brown) in the labyrinthine area mainly in the coating of trophoblast cells in the channels filled by maternal blood (tc). (B) Negative control of the immunohistochemical reaction. Placentas were harvested from conceptuses of a 16.5 dpc ZIKV-exposed litter and from a non-injected counterpart. (C-D') Macroscopic views of placentas from: an outwardly normal, ZIKV exposed, fetus (C-C'); a control fetus from a PBS-injected dam (D-D'). C-D, fetal side; C'-D', maternal side. Scale bars C-D', 1 mm. (E-F) Panoramic views of the histological structure in placentas from an outwardly normal, ZIKV exposed, fetus (E) and a control fetus from a PBS-injected dam (F). Scale bars, 400 μm. (G, H) A higher magnification view of the placentas respectively displayed in E, F. Scale bars, 50 μm. Panels (G) to (H) show the major placental layers: decidua (d), junctional zona with spongiotrophoblast (st), and labyrinth (la). (I-J) A labyrinthine view. Placentas from an outwardly normal, ZIKV exposed, fetus (I) and a control fetus from a PBS-injected dam (J). Scale bars, 20 μm. The inset in (J) depicts the interhemal membrane (↔) between fetal capillaries (fc) and trophoblast channels (tc) filled with maternal blood. Nucleated red blood cells (arrow in I) are seen in all ZIKV infected placentas. Scale bar, 20 μm. d, decidua; Cp, chorionic plate; H & E, Hematoxylin and Eosin stain, st, spongiotrophoblast; fc, fetal capillary.
Fig 11.
Early placental changes associated with exposure to ZIKV.
Immunoperoxidase reactivity (brown) for CD31, EpCAM and SCL in labyrinthine placental area. Sections were counterstained with Mayer's hematoxylin. Pregnant females were PBS-injected (A, C, E) or ZIKV-exposed (B, D, F) on 5.5 dpc and the placentas were harvested on 12.5 dpc. At the maternal-fetal barrier, there was a less intense staining for CD31 and for SCL in ZIKV-infected mothers (B, F) in comparison with PBS-injected controls (A and E, respectively). A higher-magnification view of the placentas respectively show staining on endothelial cells (arrows, inserts in A and B) and syncytial layers (arrowheads, inserts in E and F). Immunoreactivity to EpCAM is restricted to the chorionic plate (cp) in control placenta (C and insert) and in cell agglomerates (*) throughout the labyrinth area of placentas exposed to ZIKV (D and insert). Scale bars, 100 μm and 50 μm in inserts.