Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters

Abstract

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

Development of the Neuro-Immune-Vascular Plexus in the Ventricular Zone of the Prenatal Rat Neocortex

“…Endothelial cells may thus serve as a route for spreading infection widely throughout the body and also an entry point for infectious diseases into the brain. ZIKV has been shown to infect NPCs (Dang et al 2016;Garcez et al 2016;Tang et al 2016), and has also been associated with enlarged cerebral vessels exhibiting abnormal morphology in fetal rhesus monkey and ferret (Hutchinson et al 2019;Tarantal et al 2020), and with impaired development of cerebral vasculature in mouse models (Shao et al 2016;Garcez et al 2018). Given the complex interconnectivity we show here between vessels, NPCs, and microglia in the normally developing cerebrum (Fig.…”

“…9), endothelial cells that become functionally impaired through infection may further compromise nervous system development. Indeed, we have found that Zika administration to fetal rhesus monkeys produced abnormalities in microglial cell and Tbr2 + cell distribution, blood vessel morphology, and is correlated with thinner cortical gray matter (Tarantal et al 2020). Other conditions including schizophrenia, hydrocephaly, and children born of pre-eclamptic pregnancies have also been correlated with vascular abnormalities (Webster et al 2001;Lara et al 2018;Fehnel et al 2019).…”

Early embryonic loss following intravaginal Zika virus challenge in rhesus macaques

“…Because vRNA was not detected in any fetal tissues, our results may suggest that vertical transmission did not occur between dam 049-101 and the developing fetus. Alternatively, the results may suggest immunologic elimination of virus at later gestational ages as previously suggested by a study using direct fetal ZIKV inoculation (43). Our decision to challenge the animals in this study early in pregnancy (~30 days gestation) was based on findings in humans suggesting that during the first trimester, ZIKV infection is associated with a higher risk of adverse fetal and pregnancy outcomes (27,50-53)( 43) (27,(50)(51)(52)(53).…”

“…Hysterotomy and tissue collection Dams 049-102 and 049-103 were scheduled for hysterotomies in the late first trimester (nonviable embryos detected days prior to hysterotomy). The hysterotomy for dam 049-101 was performed at approximately 155 days gestational age according to the original study design (Figure 1) and following established protocols (43). The gestational sac was removed for fetal tissue assessments, with a modified collection protocol for nonviable specimens (see below).…”

“…Following isolation, cDNA synthesis was performed using the Qiagen Sensiscript RT kit (cat# 205213, Qiagen, Germantown MD) according to the manufacturer's protocol. Quantification of vRNA load was performed by real-time PCR using the Taqman amplification system and the QuantStudio 12 K Flex Real-Time PCR System (ThermoFisher Scientific, Grand Island, NY) as described previously (43). The estimated limit of quantification of the assay is 50-ZIKV RNA copies/mg tissue (average = 75 copies/mg).…”