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Cl inic a l I m pl ic a t ions of B a sic R e se a rch


ElizabethG. Phimister, Ph.D., Editor

Modeling Zika Virus Infection in Pregnancy


IndiraU. Mysorekar, Ph.D., and MichaelS. Diamond, M.D., Ph.D.
There were few studies of Zika virus (ZIKV), a
flavivirus, until this past year, when large epidemics in the Americas were accompanied by
unexpectedly severe clinical manifestations. Infection in pregnant women has emerged as a major
global concern because of its linkage to congenital abnormalities including microcephaly,
spontaneous abortion, and intrauterine growth
restriction.1 In addition, ZIKV infection in other
age groups has been associated with severe neurologic disease and the GuillainBarr syndrome.2
Transmission cycles between humans and
Aedes aegypti mosquitoes in urban settings can
cause large-scale epidemics of ZIKV infection.
Although mosquitoes clearly are the primary
cause of ZIKV outbreaks, other modes, such as
sexual transmission,3 have been reported. So far,
all cases of sexual transmission of ZIKV have
been from infected men to their partners. Viral
persistence in the testes and semen has been described, and the window of sexual transmission
remains uncertain, which has increased the apprehension about ZIKV infection during pregnancy.
The question of in utero transmission attained
prominence as the emergence of ZIKV in Brazil
and other parts of Latin America became associated with an increase in the number of cases of
microcephaly. Data that support vertical infection
and a causal role for ZIKV in the development of
congenital malformations include the detection
of ZIKV RNA or antigen in the amniotic fluid,
the placenta, or the brain tissues of fetuses or
infants in whom microcephaly is diagnosed after
death in utero or soon after birth.4,5 Moreover, a
prospective study involving ZIKV-infected pregnant women in Brazil showed that 29% of fetuses had gestational abnormalities including
microcephaly and intrauterine growth restriction,
which in a subgroup of cases resulted in fetal

death.1 Apart from this evidence, the development of an in utero animal model of ZIKV transmission and infection was considered to be important in order to establish causality, satisfy the
criteria for proof of teratogenicity, and provide
a means for screening candidate vaccines and
therapeutic agents.
Four recently published mouse-model studies
have addressed the causal relationship between
ZIKV infection in pregnancy and pathologic
changes in fetuses.6-9 Three of the studies introduced different ZIKV strains into pregnant mice
through peripheral inoculation routes (intravenous, intraperitoneal, or subcutaneous),6,7,9 and
two studies inoculated ZIKV directly into the
fetal brain.8,9 All the studies established ZIKV
infection in fetuses with resultant injury to cells
in the central nervous system; one study showed
the cellular basis of transmission of ZIKV from
maternal blood to the fetus.
Flaviviruses must overcome type I interferon
receptor signaling in order to replicate in cells;
in part, ZIKV achieves this by inducing the degradation of the STAT2 signaling molecule (a messenger protein that is downstream of the interferon receptor) in human cells. Because ZIKV
inefficiently promotes the degradation of mouse
Stat2,10 Miner et al.6 used mice that were deficient
in type I interferonreceptor signaling so that a
sufficient level of viremia could be achieved to
seed the placenta. This group inoculated a contemporary Asian ZIKV strain into the skin of
pregnant mice early in gestation (at E6.5, equivalent to the first trimester of pregnancy in humans) and observed placental infection, injury,
and insufficiency, a high rate of fetal resorption,
and fetal brain injury and neuronal-cell death.
These observations recapitulate findings in humans that occur in some ZIKV-infected women
during the first and second trimesters of preg-

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of

Maternal
spiral arteries

ZIKV

Junctional Zone
MATERNAL
SIDE

Glycogen
trophoblast

Spongiotrophoblast
Placenta
Labyrinth Zone

Mononuclear
trophoblast

Decidua
FETAL
SIDE

Trophoblast
giant cell

Maternal
red cell
MATERNAL
BLOOD-FILLED
SINUS

Fetal
red cell
FETAL
CAPILLARY

FETUS

Fetal
endothelium

Syncytiotrophoblast
bilayer

B
CORTICAL PL ATE
Neocortex

SU BPL ATE

Cortex
INTERMED IATE
ZONE
Neuron
Ventricle
Ventricular
zone

Radial glial
fiber

BRAIN

VENTRICU L AR
ZONE
NEU RAL PROGENITOR CEL L S

Figure 1. Cellular Sites of Zika Virus (ZIKV) Infection in Pregnancy.


The mouse placenta comprises both maternal- and fetal-derived components, including the junctional and labyrinth zones (Panel A).
ZIKV can infect placental trophoblasts including the trophoblast giant cells, glycogen trophoblasts, and spongiotrophoblasts in the junctional zone and the mononuclear trophoblasts, maternal sinusoids, and fetal endothelial cells that line fetal capillaries in the labyrinth
zone (insets). ZIKV infects the mouse fetal brain with tropism for cells of the cerebral cortex and ventricular zone, including cortical
neural progenitor cells and radial glial cells (Panel B). ZIKV infection leads to reduced cortical layers and ventricular cavity owing to increased cell death in cortical neural progenitors and their processes, especially in the intermediate and ventricular zones (inset).

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Clinical Implications of Basic Research

nancy.1 Moreover, this group identified ZIKV


RNA and virus within trophoblasts and fetal
endothelial cells in the placenta, findings that
are consistent with a tropism for cells lining the
maternalfetal interface and a transplacental
route of infection (Fig.1).
Cugola et al.7 inoculated pregnant immunocompetent SJL mice through an intravenous
route with an extraordinarily high dose (1010 to
1012 plaque-forming units) of a Brazilian strain
of ZIKV. Possibly, this dose allowed some of the
virus inoculum to seed the placenta directly
without requiring much replication in peripheral
organs, where it would be expected to be inhibited by type I interferon. As in the findings of
Miner et al., they observed intrauterine growth
restriction that resulted in smaller pups at birth.
Examination of the fetal and neonatal brains revealed a reduced number of cortical neurons and
reduced cortical-layer thickness (Fig.1). Cellular
analysis showed that neurons in the cerebral
cortex, thalamus, and hypothalamus had evidence of deterioration. In addition, this group
noted ocular abnormalities that were similar to
those that have been described in humans.11
Li et al.8 and Wu et al.9 injected contemporary
Asian ZIKV strains directly into the lateral left
ventricle of fetal brains at E13.5 (equivalent to
the late second trimester of pregnancy in humans) and analyzed tissues before8 or after9
birth. Both groups observed a reduction in the
number of cortical neural progenitors in the dorsal ventricular and subventricular zones, and
this loss was associated with cell death. Wu et al.
also injected ZIKV through an intraperitoneal
route into pregnant immunocompetent mice at
E13.5. They found that ZIKV could cause a transient viremia with seeding of the placenta in a
subgroup of pregnant mice. Somewhat unexpectedly, they found ZIKV RNA in the dorsal
ventricular zone of the fetal brain but in no
other brain regions. They concluded that ZIKV
can cross the fetalplacental barrier and specifically target the cortical neural progenitors of
fetal mice.
Three of the four studies suggest that ZIKV
gains access to the fetus during pregnancy after
crossing the placental barrier, which is composed
of different types of trophoblasts and ancillary
cells (Fig.1). On the basis of the mouse models
as well as recent studies of infection in primary

human cells and tissue samples,12,13 investigators


have found that ZIKV probably replicates in subgroups of trophoblasts, fetal endothelial cells,
and Hofbauer placental macrophages. Access to
these cell types may be enhanced by the binding
of ZIKV to a cell-surface tyrosine kinase receptor
called AXL.8,14 Early in pregnancy, ZIKV infection may lead to severe placental vascular damage and a reduction in fetal blood vessels and
blood flow. Alternatively, ZIKV could cross the
placental barrier without excessive damage and
spread to the fetal brain, where it preferentially
infects and injures neuronal progenitor cells.
This outcome may be more typical of infection
later in pregnancy because of enhanced inter
feron-induced innate immunity in trophoblasts.12 Infection and death of neuroprogenitor
cells could inhibit neuronal-cell differentiation,
which would explain the cortical thinning, malformation of brain structures, and microcephaly
that are observed during pregnancy in humans.
Investigators must consider the differences in
morphologic, spatial, and temporal placentation
and in utero brain development in rodents and
humans. However, many elements of placental
development and trophoblast function are similar in the two species. Although the time scale
differs substantially, neuronal developmental processes in rodents and humans are remarkably
parallel. Collectively, the findings from the studies in mice support the hypothesis that infection
during pregnancy with contemporary ZIKV
strains causes placental infection and injury and
that ZIKV spreads to the fetal brain and kills
neuronal progenitor cells, which probably contributes to the microcephaly and other congenital malformations that have been observed in
human neonates. The new mouse models provide a foundation for defining mechanisms of
pathogenesis and represent a resource for determining the effects of ZIKV strain variation on
clinical manifestations and for testing candidate
therapeutic agents and vaccines.
Disclosure forms provided by the authors are available at
NEJM.org.
From the Departments of Pathology and Immunology (I.U.M.,
M.S.D.), Obstetrics and Gynecology (I.U.M.), and Medicine
and Molecular Microbiology (M.S.D.), and the Center for Human Immunology and Immunotherapy Programs (M.S.D.),
Washington University School of Medicine, St. Louis. Address
reprint requests to Dr. Mysorekar at
mysorekari@
wudosis
.wustl.edu or to Dr. Diamond at diamond@wusm.wustl.edu.

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This article was published on July 13, 2016, at NEJM.org.


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DOI: 10.1056/NEJMcibr1605445
Copyright 2016 Massachusetts Medical Society.

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