Ocular and uteroplacental pathology in a macaque pregnancy with congenital Zika virus infection

<div><p>Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.</p></div

Uteroplacental histopathology from the primary placental disc.

<p>(A) Maternal neutrophils invading chorionic plate (arrow) is diagnostic of acute chorioamnionitis. (B) Villi show increased perivillous fibrin deposition (arrow) and there are multiple remote infarctions (arrow, C). (D) Radial arteries in the myometrium show a pronounced leukocytoclastic vasculitis (arrow) defined as an infiltrative mixture of lymphocytes, eosinophils, and plasma cells into the smooth muscle wall of these vessels.</p

ZIKV RNA levels in maternal body fluids.

<p>vRNA was measured by quantitative RT-PCR in plasma, urine, amniotic fluid and CSF. The limit of detection is 33 copies/mL and the assay limit of quantification is 100 copies/mL, which are indicated by dashed lines.</p

Amniotic fluid (AF) markers confirm rupture of membranes and contamination of pan-collected urine.

<p>(A) An AmniSure^® test, which measures PAMG-1 protein, was performed on pan urine collection (28 dpi, 45 dpi, 49 dpi) and AF (28 dpi) from the pregnant animal. Nonpregnant control animal urine and pregnant animal AF are included as controls. (B) Relative pixel density of the Amnisure^® test strip test band and control band. (C) Amniotic fluid protein IGFBP-1 ELISA. Body fluids from the pregnant animal (pan urine collection 28, 42, 45, 49 dpi and AF 28 dpi), nonpregnant negative control male and female urine samples, amniotic fluid from a control pregnancy were evaluated for the presence of IGFBP-1. Dashed lines indicate the upper and lower limits of quantitative accuracy of the assay. In Panels B and C, white bars denote body fluids from the experimental animal and grey bars denote control fluids from other animals in the colony.</p

Maternal and fetal necropsy images.

<p>(A) The uterus was removed in entirety from the abdominal cavity of the dam using sterile instruments and a syringe was used to aspirate the purulent fluid from inside the uterine cavity. (B) The fetus was removed from the uterus and was covered in thick fibrinous material. Placental discs 1 (C) and 2 (D) were covered in the same thick fibrinous maternal that covered the fetus.</p

Fetal growth measured by ultrasonography.

<p>(A) Head circumference (HC), biparietal diameter (BPD), and femur length (FL) were measured in weekly ultrasounds. All measurements are depicted as millimeters (mm). The solid lines were derived from reference ranges from Tarantal et al. 2005 to show the mean (black lines) and one, two, and three standard deviations from the mean (grey lines). The HC, BPD, and FL were then plotted along these reference ranges to observe any deviations from the mean. (B) The pGA is plotted against the aGA (based on gestational age estimated from breeding and menstrual history). The pGA is shown separately for each measurement: BPD (triangle), HC (square), and FL (circle).</p

Neutralizing antibody titers following ZIKV infection.

<p>PRNT titers were measured pre and post infection. The x-axis represents the reciprocal serum dilution (log₁₀) and the y-axis represents the percent reduction. The dashed lines indicate 90% and 50% reduction.</p

Tissue histology and viral localization of maternal spleen, maternal uterus, amniotic/chorionic membrane, and fetal lung.

<p>Each tissue was stained with H&E, ISH, and mFISH. ISH shows localization of ZIKV vRNA. mFISH shows replicative intermediates by staining the negative sense RNA strands green and positive sense RNA strands red. Co-localization (yellow) demonstrates dsRNA intermediates. Black arrows denote a germinal center. Asterisks indicate neutrophils. Blue arrows highlight green, red, or yellow fluorescence.</p

Timeline depicting body fluid sampling and procedures throughout pregnancy.

<p>Blood, urine, saliva, amniotic fluid, and CSF were collected as indicated in the schedule above, and ultrasounds were performed weekly. The axes are not drawn to scale.</p

Tissues with detectable ZIKV RNA from mother and fetus.

<p>Tissues with detectable ZIKV RNA from mother and fetus.</p