Fig 1.
The effect of ZIKV-DAK on pregnancy was evaluated in three groups of pregnant rhesus macaques.
Three groups of pregnant macaques were inoculated early in gestation with 44 PFU of ZIKV-DAK at either gd 30 (Group 30/7 and Group 30/14) or gd 45 (Group 45/14). Viremia in the dams was monitored via blood collection. Pregnancies were surgically terminated at either 7 dpi (Group 30/7) or 14 dpi (Group 30/14 and Group 45/14) and MFI and fetal tissues were collected for evaluation. After pregnancy termination, the dams were monitored and blood was collected and evaluated weekly until ZIKV was no longer detected in the blood. When ZIKV was no longer detected in the blood for two consecutive blood draws, the dam was switched to monthly blood collection through 87 dpi.
Fig 2.
Plasma viremia following inoculation with 44 PFU of ZIKV-DAK.
Plasma ZIKV loads were determined by RT-qPCR and are represented as vRNA copies/mL from 0 up to 16 dpi. Only values above the limit of detection of 150 copies/mL are shown. Experimental groups are represented by different colors, symbols represent individual animals within experimental groups.
Fig 3.
Neutralizing antibody titers following inoculation with 44 PFU of ZIKV-DAK.
Plaque reduction neutralization tests (PRNT) were performed on serum samples collected at 14 dpi (45/14-4 and 30/14-1) or approximately 28 dpi (all other animals). Data are expressed relative to infectivity in the absence of serum. A) Neutralization curves. B) PRNT90 and PRNT50 values were estimated using nonlinear regression analysis and are indicated with dotted lines in Panel A.
Fig 4.
ZIKV-DAK detection in MFI and fetal tissues and fluids.
A) Illustration of the rhesus macaque conceptus depicting evaluated tissues and fluids. The pregnant uterus is lined by the maternal endometrium of pregnancy called the decidua. The layers of the placenta are shown as the trophoblastic shell where the trophoblasts and the decidua meet, the placental villi, and the chorionic plate made up of the chorion and large fetal vessels. The chorionic membrane is the outer fetal membrane and the inner fetal membrane is the amniotic membrane. As the fetus grows the amniotic membrane is pushed up against the chorionic membrane and the membranes are fused. Fused fetal membranes were only observed in one case, 45/14-4. B) ZIKV RNA burden as detected by RT-qPCR on RNA isolated from indicated tissues and fluids. The level of ZIKV burden is summarized as high (>1000 ZIKV RNA copies/mg of tissue or mL of fluid), medium (>100 ZIKV RNA copies/mg or mL), low (< 100 ZIKV RNA copies/mg), or below the limit of detection. The limit of detection for all the fluids listed (CSF, amniotic fluid, umbilical cord blood) is 150 copies/mL. The theoretical limit of detection for all of the tissues is 3 copies/mg. Placental viral loads are presented as the mean of three tissue biopsies per disc. Placental biopsies represent a general survey of the placenta as these samples contain maternal blood, chorionic plate, and portions of the trophoblastic shell in addition to placental villi. C) ZIKV RNA presence as detected by ISH. ZIKV RNA was noted as present (+) or absent (-) in the indicated tissues. In some instances, a histological sample was not obtained, thus ZIKV RNA presence or absence could not be properly evaluated (/).
Fig 5.
ZIKV burden in fetal tissues and fluids.
A) Amniotic fluid ZIKV RNA levels determined by RT-qPCR (left panel) and infectious ZIKV determined by plaque assay (right panel). Limit of detection for the RT-qPCR fluid assay shown on the left is 150 copies/mL, this is represented by the dashed line. On the right, the dashed line represents the limit of detection of 0.7 PFU/mL for the plaque assay. Four amniotic fluid samples were analyzed by plaque assay based on positive detection of vRNA by RT-qPCR. B) ZIKV RNA isolated from fetal tissues and fluids; viral loads are measured in vRNA copies/mL of fluid (CSF) or vRNA copies/mg of tissue. Limit of detection for the RT-qPCR fluid assay (CSF) is 150 copies/mL and the theoretical limit of detection for the RT-qPCR assay for the tissues is 3 copies/mg. Only samples above the limit of detection are shown.
Fig 6.
ZIKV RNA detected in MFI by in situ hybridization (ISH).
Photomicrographs of paraffin embedded sections of the placenta and fetal membranes from 45/14-4. Panels A, C, E, G I, and K Positive ISH indicated by the red/pink chromogenic stain. Panels B, D, F, H, J, and L are corresponding H&E-stained sections to provide histological detail. Panels E, F K and J are higher magnification images of the corresponding regions in the adjacent photomicrographs. Scale bar represents 100 μm. Abbreviations: MBS maternal blood space, STB syncytiotrophoblasts, CP chorionic plate, PV placental villi, AM amniotic membrane, CH chorionic membrane. Black arrows indicate STB layer of placental villi in Panels E and F, red arrows indicate epithelial cells in the AM in Panels K and L, and * indicate the chorionic membrane layer of the fetal membranes in Panels K and L.
Table 1.
Histopathological evaluation of MFI tissue sections.
The presence or absence of specific histological lesions in the MFI are indicated for mock-infected control pregnancies, and ZIKV-inoculated pregnancies. The criteria for acute villitis excluded interface necrosis in the villi anchored to maternal decidua.
Fig 7.
ZIKV RNA in decidual spiral arteries.
Photomicrographs of paraffin embedded sections of the decidua basalis from 30/7-2. Spiral arteries are outlined by dashed lines. A) Pink staining shows ZIKV RNA detected using ISH in multiple profiles of a decidual spiral artery with invading luminal extravillous trophoblasts (EVTS). B) Immunohistochemical staining for CD56 reveals large EVT cells in outlined area, as well as smaller immune cells outside the outlined area. C) H&E stained serial section. Panels D, E and F represent higher magnification of images of indicated regions of Panels A, B, and C respectively. Scale bar represents 100 μm.