Congenital Viral Infections of the Brain: Lessons Learned from Lymphocytic Choriomeningitis Virus in the Neonatal Rat

Abstract

The fetal brain is highly vulnerable to teratogens, including many infectious agents. As a consequence of prenatal infection, many children suffer severe and permanent brain injury and dysfunction. Because most animal models of congenital brain infection do not strongly mirror human disease, the models are highly limited in their abilities to shed light on the pathogenesis of these diseases. The animal model for congenital lymphocytic choriomeningitis virus (LCMV) infection, however, does not suffer from this limitation. LCMV is a well-known human pathogen. When the infection occurs during pregnancy, the virus can infect the fetus, and the developing brain is particularly vulnerable. Children with congenital LCMV infection often have substantial neurological deficits. The neonatal rat inoculated with LCMV is a superb model system of human congenital LCMV infection. Virtually all of the neuropathologic changes observed in humans congenitally infected with LCMV, including microencephaly, encephalomalacia, chorioretinitis, porencephalic cysts, neuronal migration disturbances, periventricular infection, and cerebellar hypoplasia, are reproduced in the rat model. Within the developing rat brain, LCMV selectively targets mitotically active neuronal precursors. Thus, the targets of infection and sites of pathology depend on host age at the time of infection. The rat model has further shown that the pathogenic changes induced by LCMV infection are both virus-mediated and immune-mediated. Furthermore, different brain regions simultaneously infected with LCMV can undergo widely different pathologic changes, reflecting different brain region-virus-immune system interactions. Because the neonatal rat inoculated with LCMV so faithfully reproduces the diverse neuropathology observed in the human counterpart, the rat model system is a highly valuable tool for the study of congenital LCMV infection and of all prenatal brain infections In addition, because LCMV induces delayed-onset neuronal loss after the virus has been cleared, the neonatal rat infected with LCMV may be an excellent model system to study neurodegenerative or psychiatric diseases whose etiologies are hypothesized to be virus-induced, such as autism, schizophrenia, and temporal lobe epilepsy.

Full text

Review
Congenital Viral Infections of the Brain:
Lessons Learned from Lymphocytic
Choriomeningitis Virus in the Neonatal Rat
Daniel J. Bonthius
*
, Stanley Perlman
ABSTRACT
T
he fetal brain is highly vulnerable to teratogens,
including many infectious agents. As a consequence
of prenatal infection, many children suffer severe
and permanent brain injury and dysfunction. Because most
animal models of congenital brain infection do not strongly
mirror human disease, the models are highly limited in their
abilities to shed light on the pathogenesis of these diseases.
The animal model for congenital lymphocytic
choriomeningitis virus (LCMV) infection, however, does not
suffer from this limitation. LCMV is a well-known human
pathogen. When the infection occurs during pregnancy, the
virus can infect the fetus, and the developing brain is
particularly vulnerable. Children with congenital LCMV
infection often have substantial neurological deficits. The
neonatal rat inoculated with LCMV is a superb model system
of human congenital LCMV infection. Virtually all of the
neuropathologic changes observed in humans congenitally
infected with LCMV, including microencephaly,
encephalomalacia, chorioretinitis, porencephalic cysts,
neuronal migration disturbances, periventricular infection,
and cerebellar hypoplasia, are reproduced in the rat model.
Within the developing rat brain, LCMV selectively targets
mitotically active neuronal precursors. Thus, the targets of
infection and sites of pathology depend on host age at the
time of infection. The rat model has further shown that the
pathogenic changes induced by LCMV infection are both
virus-mediated and immune-mediated. Furthermore,
different brain regions simultaneously infected with LCMV
can undergo widely different pathologic changes, reflecting
different brain region–virus–immune system interactions.
Because the neonatal rat inoculated with LCMV so faithfully
reproduces the diverse neuropathology observed in the
human counterpart, the rat model system is a highly valuable
tool for the study of congenital LCMV infection and of all
prenatal brain infections In addition, because LCMV induces
delayed-onset neuronal loss after the virus has been cleared,
the neonatal rat infected with LCMV may be an excellent
model system to study neurodegenerative or psychiatric
diseases whose etiologies are hypothesized to be virus-
induced, such as autism, schizophrenia, and temporal lobe
epilepsy.
Introduction
Multiple viruses, bacteria, and parasites can infect the
developing human fetus, resulting in a range of outcomes that
include fetal demise, developmental anomalies, and disease of
the newborn [1]. Disease may also be clinically silent at birth
and not become evident until after the first few months or
years of life. Factors important in determining outcome
include pathogen identity, gestational timing of infection,
pathogen load, tissue tropism, inflammatory response, and
immune status of the mother and fetus.
For example, human infection with the rubella virus during
the first 11 weeks of gestation results in teratogenic changes
in most fetuses that survive the acute infection, with
abnormalities commonly detected in the heart, eye, and
central nervous system [2]. At later times in gestation (11–16
weeks), infection is less likely to result in congenital
anomalies, but may still result in hearing loss, mental
retardation, and growth deficits [3]. Sequelae from congenital
rubella may become manifest at even later times of postnatal
life, with insulin-dependent diabetes apparent in up to 20%
of infected humans by adulthood [4]. Although congenital
infections caused by rubella virus have been greatly decreased
as a consequence of vaccination, effective vaccines are not
available for infections caused by other pathogens, such as
cytomegalovirus, which are also important causes of
congenital infection.
Perinatal infection by less common or emerging pathogens
may become increasingly prevalent. One such emerging viral
pathogen is lymphocytic choriomeningitis virus (LCMV), an
arenavirus that has been increasingly recognized as a
teratogen in recent years [5–11].
LCMV was initially isolated by Armstrong and Lillie in 1933
[12] from the cerebrospinal fluid of a woman who was
thought to have St. Louis encephalitis. This patient had
presented with general malaise, but her condition worsened,
and she died. The virus isolated from her cerebrospinal fluid
was passaged five times through monkeys and, with each
passage, produced a disease resembling St. Louis encephalitis.
Editor: B. Brett Finlay, University of British Columbia, Canada
Citation: Bonthius DJ, Perlman S (2007) Congenital viral infections of the brain:
Lessons learned from lymphocytic choriomeningitis virus in the neonatal rat. PLoS
Pathog 3(11): e149. doi:10.1371/journal.ppat.0030149
Copyright: Ó 2007 Bonthius and Perlman. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abbreviations: LCMV, lymphocytic choriomeningitis virus; PD, postnatal day
Daniel J. Bonthius is with the Departments of Pediatrics, Neurology, and Anatomy
and Cell Biology at the Carver College of Medicine, University of Iowa, Iowa Cit y,
Iowa, United States of America. Stanley Perlman is with the Departments of
Pediatrics and Microbiology at the Carver College of Medicine, University of Iowa,
Iowa City, Iowa, United States of America.
* To whom correspondence should be addressed. E-mail: daniel-bonthius@uiowa.
edu
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On the sixth passage, the virus was inoculated into a monkey
that was immune to St. Louis encephalitis. However, the virus
still produced the disease, indicating that the virus was not St.
Louis encephalitis virus. This new infectious agent was named
‘‘ lymphocytic choriomeningitis virus’’ for the pathologic
changes that it induced in the choroid plexus and meninges
of infected mice and monkeys [12].
The virus was subsequently isolated from cerebrospinal
fluid of multiple patients with aseptic meningitis. Thus, it was
established that LCMV was an important etiologic agent of
aseptic meningitis in humans. Subsequent clinical and
etiologic studies identified LCMV as one of the most frequent
infectious causes of aseptic meningitis in humans [13].
The first recognized case of congenital infection with
LCMV was reported in England in 1955 [14]. In the decades
that followed, multiple cases of congenital LCMV infection
were reported throughout Europe [15,16]. Although LCMV
has been recognized as an important cause of aseptic
meningitis in the United States for decades, the first cases of
congenital LCMV infection were not reported in the United
States until 1993 [17,18].
Like all arenaviruses, LCMV utilizes rodents as its principal
reservoir [19–21]. Mus musculus, the common house mouse, is
both the natural host and reservoir for the virus, which is
transferred vertically from one generation to the next within
the mouse population by intrauterine infection. Although
heavily infected with LCMV, mice that acquire the virus
prenatally often remain asymptomatic because the virus is
not cytolytic and because congenital infection provides the
mice with immunological tolerance for the virus [22,23].
Throughout their lives, mice prenatally infected with LCMV
shed the virus in large quantities in nasal secretions, saliva,
milk, semen, urine, and feces.
Postnatal humans acquire LCMV by inhalation of
aerosolized virus or by direct contact with fomites
contaminated with infectious virus. LCMV infection during
postnatal life (childhood or adulthood) typically consists of a
brief febrile illness from which the patient fully recovers.
Symptoms include headache, fever, myalgia, photophobia,
and vomiting. In as many as one-third of postnatal infections,
the disease is asymptomatic.
Human-to-human horizontal infection has not been
documented, except for the unusual circumstances in which
the virus was acquired through transplantation of infected
tissues [24]. In contrast, human-to-human vertical
transmission does occur and is the basis for congenital LCMV
infection.
Because LCMV is prevalent in the environment and has a
great geographic range, the virus infects large numbers of
humans. An epidemiologic study has demonstrated that 9%
of house mice in urban Baltimore are infected with LCMV,
and that substantial clustering occurs where the prevalence is
higher [25]. Serological studies have demonstrated that 5.1%
of healthy black women in Birmingham and 4.7% of adults in
Baltimore possess antibodies to LCMV, indicating prior
exposure and infection [25,26].
The prevalence and incidence of congenital LCMV
infection are unknown. While case reports of infection
during pregnancy demonstrate that LCMV can induce severe
defects in brain structure and function [6–8,11,14,15], it is not
known whether the profoundly affected infants described in
the case reports represent the typical outcome of gestational
LCMV infection, or whether they represent only the most
severely affected cases. Prospective clinical or
epidemiological studies of congenital LCMV infection have
not been conducted. The fact that LCMV is not one of the
infectious agents for which infants with a suspected
congenital infection are routinely evaluated further limits
information regarding the incidence and spectrum of LCMV-
induced teratogenicity. Therefore, congenital LCMV
infection might produce a spectrum of pathologic effects
ranging from minimal to severe [11]. The high prevalence of
infected mice and of sero-positive postnatal humans suggest
that congenital LCMV infection is an underdiagnosed disease
and that the virus is responsible for more cases of congenital
neurologic and vision dysfunction than has previously been
recognized [6,7,27,28].
Transplacental infection of the fetus is the basis for most
cases of congenital LCMV infection, presumably during
maternal viremia [5]. In some cases, the fetus may acquire
LCMV during the intrapartum period [14]. Within the human
fetus, the brain is the principal target of LCMV infection and
the most important site of pathology [8,11]. Mitotically active
neuronal precursors are particularly vulnerable to LCMV
infection and an important site of LCMV replication
[5,29,30]. Microencephaly, periventricular calcifications, gyral
dysplasia, cerebellar hypoplasia, and focal cerebral
destruction are common pathologic effects of congenital
LCMV infection [11]. These pathologic changes reflect both
the viral tropism for replicating neuroblasts and disrupted
brain development induced by loss or dysfunction of
immature or replicating neurons [5,8,30,31]. The mechanism
by which LCMV damages the fetal human brain is unknown.
LCMV is not a cytolytic virus in most cell types, including
neurons. Thus, unlike herpes and several other pathogens
that directly damage the brain by killing host brain cells [1],
LCMV neuroteratogenicity must have some other underlying
pathogenesis [30].
Progress toward understanding the pathogenesis of most
perinatal infections is limited by the absence of animal
models that mirror human disease. However, this is not the
case for congenital LCMV infection. Neonatal rats infected
with LCMV develop virtually all of the neuropathological
abnormalities observed in infected humans [29,30], including,
but not limited to encephalomalacia (Figure 1), disrupted
neuronal migration (Figure 2), and periventricular infection
(Figure 3). Most strikingly, the rat model of LCMV illustrates
the complex interactions among timing of infection relative
to animal age, cellular tropism, and the host immune
response in determining acute disease and ultimate outcome
[30].
In the rat model, rat pups receive intracerebral injections
of LCMV during the neonatal period [30–32]. Postnatal rat
pups model human congenital LCMV infection because the
rat brain is immature at the time of birth, relative to the
human brain [33]. Thus, in terms of brain development, the
first two postnatal weeks in the rat mimic the second half of
human gestation [34].
The neonatal rat model of congenital LCMV infection was
pioneered by Monjan and coworkers during the 1970s and
early 1980s. They discovered that LCMV induces a distinct
pattern of infection in which certain neuronal populations
are infected, while others are spared [31,32]. They further
found that the virus can induce retinopathy [35], cerebellar
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destruction [32], disrupted neurotransmission [36], and
altered behavior [37]. In addition, they found that much of
the pathology induced by LCMV is immune-mediated [38].
This finding of an important role for the immune system in
LCMV infection of the neonatal rat complemented the many
landmark studies by others over the past 70 years, in which
immunologists and virologists have utilized LCMV to gain a
deeper understanding of immunology and immunopathology
[39–41]. Recently, interest in the neonatal rat model of LCMV
infection has been re-ignited, as the importance of human
congenital LCMV infection and the value of the rat model
system for studying that infection have become clear.
Critical Role of Host Age in LCMV-Induced Disease
In the rat model, host age profoundly affects the outcome
of LCMV infection [30]. The cellular targets of infection,
maximal viral titers, and the nature and the severity of
neuropathology all depend strongly on host age at the time of
infection. Figure 4 illustrates the importance of host age in
determining the cellular targets of infection. Inoculation of
the rat pup on postnatal day (PD) 1 leads to a widespread
infection of the cerebral cortex, in which both astrocytes and
neurons are infected. However, if the infection occurs just 3 d
later, on PD4, only astrocytes are infected, and neurons are
completely spared. By PD21, no cells of the cerebral cortex—
neither astrocytes nor neurons—are infectable with LCMV.
Thus, over the course of a short period of developmental
time, the cellular targets of infection within the cerebral
cortex change from a combination of astrocytes and neurons,
to astrocytes alone, to no infectable cells [30].
This progressive restriction in the cellular targets of
infection is reflected by a decline in the maximal viral titers.
When the infection occurs on PD1, maximal viral titer
exceeds 10
8
plaque-forming units per gram of tissue. By PD4,
maximal viral titer falls 100-fold to 10
6
, and by PD21, no live
virus is detectable in the cerebral cortex (though the virus
remains detectable in some other brain regions, including the
olfactory bulb and ventricular ependyma).
The neuropathology induced by LCMV likewise depends
strongly on host age [30]. Specific neuropathologic changes
are reliably produced by infection at certain ages and reliably
absent at others. For example, LCMV induces cerebellar
hypoplasia, in which the cerebellum is small but has a normal
cytoarchitecture, only if the infection occurs on PD1. In
contrast, if the infection occurs on PD4 or PD6, then LCMV
induces a neuronal migration disturbance, in which the
cerebellar granule cells fail to migrate properly in the ventral
lobules, and encephalomalacia, in which the dorsal lobules
undergo a destructive process and are obliterated. In further
contrast, if the infection occurs on PD21, then LCMV induces
no cerebellar pathology at all (though it continues to induce
olfactory bulb destruction and hydrocephalus, because of the
continued infectability of the olfactory bulb and ventricular
ependyma).
Children congenitally infected with LCMV can have a
diverse set of neuropathologic changes that vary from case to
case [11]. Virtually all of these neuropathologic changes
observed among children with congenital LCMV infection
can be recapitulated in the rat model by infecting the host at
different ages (Table 1). This finding strongly suggests that the
diversity in pathology and outcome among children with
doi:10.1371/journal.ppat.0030149.g001
Figure 1. LCMV Infection Induces Focal Destructive Lesions within the
Developing Brains of Humans and Rats
(A) Head CT scan from a 4-mo-old child with congenital LCMV infection.
The scan reveals bilateral asymmetric regions of encephalomalacia
(asterisks), strongly suggestive of a focal destructive process. Note also
the periventricular calcifications (arrow), characterisitic of a prenatal viral
infection.
(B) Section (50-lm-thick) through the cerebellar cortex of a neonatal rat
infected with LCMV. The section has been immunohistochemically
stained for LCMV antigens. The virus infects both Purkinje cells (arrows)
and granule cells (arrowheads).
(C) Nissl-stained section (2-lm-thick) through the cerebellar vermis of an
uninfected (control) 30-d-old rat. The ten lobules of the cerebellar vermis
(I–X) are labelled according to the system of Larsell [84].
(D) Section (2-lm-thick) through the cerebellar vermis of a 30-d-old rat
infected 3 wk earlier with LCMV. The dorsal cerebellum has undergone a
destructive process (arrows). Most of lobules V, VI, VII, and VIII have been
obliterated, while lobules I, II, III, and X have been relatively spared.
(E) Section (50-lm-thick) through the cerebellar cortex of a 14-d-old rat
infected 10 d earlier with LCMV. The animal was sacrificed at the time
that acute destruction of the cerebellum was occurring. The section has
been immunohistochemically stained for CD8þ antigen, which labels a
subset of lymphocytes. Note the dense infiltration of CD8þ lymphocytes
(arrows).
Magnification bars represent 1 cm in (A), 100 um in (B), 500 um in (C), 500
um in (D), and 100 um in (E).
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congenital LCMV infection is due, at least in part, to
differences in gestational age at the time of infection.
Glial Cells Play a Critical Role in LCMV Infection of
the Developing Brain
Glial cells play central roles in the entry, replication, and
dispersion of LCMV in the developing rat brain [29].
Astrocytes and Bergmann glia are the initial parenchymal
brain cells infected by LCMV and are the principal cell types
in which LCMV replicates. Furthermore, it is via the
sequential movement of virus from one astrocyte to its
neighbor that LCMV spreads throughout the developing rat
brain. Thus, glial cells are the portals of entry, the principal
sites of replication, and the conduits through which LCMV
disseminates through the brain parenchyma.
doi:10.1371/journal.ppat.0030149.g002
Figure 2. LCMV Infection Disrupts Neuronal Migration in the Developing Brain of Humans and Rats
(A) MRI scan of a 3-y-old child with congenital LCMV infection. The MRI scan demonstrates microencephaly and a deficit of white matter (arrowheads)
with a compensatory enlargement of the lateral ventricles (asterisks). There is also a diminished number of cortical sulci and an abnormally smooth
cortical surface (white arrow). This is strongly suggestive of pachygyria, a developmental defect due to abnormal neuronal migration.
(B and C) are 2-lm-thick sections through the cerebellar cortex of uninfected control (B) and LCMV-infected (C) rats.
(B) Normal cerebellar cortex from a control (uninfected) adult rat demonstrating the trilaminar cytoarchitecture of the cortex, which consists of the
molecular layer (M), Purkinje cell layer (P), and granule cell layer (G). Within the molecular layer, a few stellate cells and basket cells (arrowheads) are
normally present. In contrast, granule cells (arrows) have migrated through the molecular layer to the granule cell layer. Granule cells no longer reside in
the molecular layer in the normal cerebellum.
(C) Cerebellar cortex from an adult rat infected during early postnatal life with LCMV. Many granule cells (arrows) remain abnormally placed within the
molecular layer. As a result of LCMV infection, these neurons have failed to migrate properly to their normal location within the granule cell layer and
remain permanently ectopic within the molecular layer.
Magnification bars represent 100 um in (B and C).
doi:10.1371/journal.ppat.0030149.g003
Figure 3. Congenital LCMV Infection of Humans Induces Periventricular Calcifications
The neonatal rat model demonstrates that periventricular neurons are selectively vulnerable to infection with LCMV.
(A) Head CT scan from an infant with congenital LCMV infection. The scan reveals microencephaly and prominent periventricular calcifications (arrows).
In addition, this scan reveals an abnormal cortical gyral pattern (arrowheads), suggestive of disturbed cortical neuronal migration.
(B) Horizontal section (50-lm-thick) through a 49-d-old rat brain immunohistochemically stained for LCMV. The rat was inoculated as a neonate with
LCMV. Infection is localized to the periventricular region (arrows). L ¼ lateral ventricle, S ¼ septum, BG ¼ basal ganglia.
(C) Higher magnification of the boxed area in (B) shows that the infected cells are neuronal in morphology. Viral antigen is present in neuronal cell
bodies (arrows) and neurites (arrowheads).
Magnification bars represent 500 um in (B) and 100 um in (C).
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While glial cells are the major target of LCMV in the
developing rat, this does not appear to be the case in
developing mice, where neurons are the principal target [42].
Thus, the cellular targets of infection vary among host
species. Whether glial cells are an important target of LCMV
in the developing human brain is unknown.
LCMV Preferentially Targets Neuroblasts
Following infection of glial cells, LCMV infects neurons.
However, not all brain regions in the rat are vulnerable to
neuronal infection. On the contrary, LCMV infects neurons
only in four specific brain regions. These four regions are the
cerebellum, olfactory bulb, dentate gyrus, and periventricular
region [29]. Neurons elsewhere are spared.
Why LCMV infection of neurons is restricted to these four
regions is unknown. One possibility is that neurons in these
vulnerable regions selectively express a host cell receptor for
the virus. Alpha-dystroglycan is a receptor for at least some
strains of LCMV [43–45]. Whether alpha-dystroglycan is a
receptor for LCMV within the brain is unknown. Alpha-
dystroglycan is developmentally regulated in the rat brain
[46], which, if it is the relevant receptor, may explain why the
cellular targets of infection change with developmental age
[30].
A second possibility is that the restricted pattern of
neuronal infection reflects not the restricted ability of LCMV
to enter cells, but to replicate within them. An important trait
common to all four of the vulnerable brain regions in the
neonatal rat is possession of at least one population of
mitotically active neuronal precursors (Figure 5). Unlike all
other brain regions in the neonatal rat, in which neuronal
populations are uniformly post-mitotic, these four regions all
possess neuroblasts that are generating new neurons [47–53].
Thus, LCMV infects neurons only in brain regions in which
neurons are mitotically active. The converse is also true: all
brain regions with mitotically active neuroblasts are
infectable with LCMV [30]. This suggests that the metabolic
machinery that accompanies neurogenesis promotes LCMV
propagation.
The cerebral cortex is the exception that proves this rule.
As described above, neurons of the cerebral cortex are
doi:10.1371/journal.ppat.0030149.g004
Figure 4. The Cellular Targets of LCMV within the Cerebral Cortex Depend on Host Age
Shown are 50-lm-thick sections through the cerebral cortex of rats infected on PD 1 (A), PD 4 (C), or PD 21 (E). In each case, the animals were sacrificed
25 d post-inoculation, and the brain sections were immunohistochemically stained for LCMV antigens.
Inoculation on PD 1 leads to infection of cerebral cortical neurons (arrows) and astrocytes (arrowheads). Note that a greater proportion of neurons are
infected in superficial cortical layers than in deep cortical layers.
Inoculation on PD 4 leads to infection of astrocytes alone (arrowheads). Whereas countless neurons were infectable 3 d earlier, virtually no neurons are
infectable by PD4.
Inoculation on PD 21 leads to infection of neither neurons nor astrocytes.
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infectable on PD1 and not thereafter. However, on PD1,
cerebral cortical neurons are all post-mitotic [54]. Why, then,
would they be infectable? Close inspection of the infection
pattern within the cerebral cortex reveals that the cortical
neurons are not uniformly infectable. Rather, a much greater
proportion of neurons are infectable in the superficial
cortical layers than in the deep cortical layers (Figure 4).
During histogenesis, the cerebral cortex develops in an
‘‘ inside out’’ sequence, so that neurons of the superficial
cortical layers were generated after those of the deep cortical
layers [55,56]. Thus, the neurons of the superficial cortical
layers underwent mitosis more recently than those of the
deeper layers and likely contain more of the metabolic
machinery driving mitosis than do the deeper layers.
Therefore, the ‘‘ outside in’’ gradient of cortical LCMV
infection reflects the ‘‘ inside out’’ pattern of cortical
neurogenesis and is exactly the pattern expected if LCMV
neuronal infection depends upon neuronal mitotic
machinery.
Another possibility is that LCMV replication depends not
on neuronal mitotic activity per se but on an immature stage
of neuronal differentiation. The brain regions that contain
mitotically active neuroblasts also contain neurons at early
stages of differentiation. Thus, multiplication of LCMV
within neurons may depend upon cellular genes whose
expression is restricted to the early stages of neuronal
differentiation.
LCMV Simultaneously Infects Multiple Brain
Regions and Induces Different Forms of Pathology
in Each Region
Following infection of neurons, LCMV induces
neuropathological changes. However, the nature and time
course of the neuropathology differ substantially among
brain regions [29,30]. For example, when rat pups are
inoculated with LCMV on PD4, the virus infects neurons of
the cerebellum, olfactory bulb, dentate gyrus, and
periventricular region and induces neuropathology unique to
each region. In the cerebellum, LCMV induces an acute
immune-mediated destruction of the dorsal lobules (Figure
2). This destructive process is driven by CD8þ lymphocytes,
which infiltrate the cerebellum in large numbers and destroy
the dorsal lobules. In the cerebellar ventral lobules, LCMV
induces a neuronal migration disturbance, which causes
cerebellar granule cells to be permanently ectopically located
within the molecular layer (Figure 3).
In the olfactory bulb, LCMV infection at this same age leads
to an acute hypoplasia of the olfactory bulb, due to a reduced
production of granule cells. This hypoplasia is temporary,
and the olfactory bulb can rebound to a normal size by
adulthood [29].
In the hippocampal formation, a completely different
pattern of neuropathology is observed. Here, the dentate
gyrus initially appears histologically normal, despite a heavy
viral burden within the dentate granule cells. However,
several months post-inoculation, the previously infected
granule cells begin to die. By 4 mo post-inoculation, there has
been a profound loss of granule cells selectively from the
dentate gyrus [29,57]. Thus, within the hippocampal
formation, LCMV induces a delayed- onset selective mortality
of dentate granule cells.
In the periventricular region, still a different pattern is
observed. The periventricular region has neither an acute
destructive process (like the dorsal cerebellum), nor an acute
migration defect (like the ventral cerebellum), nor an acute
hypoplasia (like the olfactory bulb), nor a delayed-onset drop-
out of neurons (like the hippocampus). The periventricular
region never shows any pathologic changes, despite infection
of its neurons.
Why four brain regions simultaneously infected with a
single viral species have such different pathologic responses is
unknown. Part of the difference must be due to regionally
different interactions with the immune system. Perhaps, in
response to LCMV, the different brain regions produce
different patterns of chemokines and cytokines, leading to
regional differences in lymphocytic infiltration and different
degrees of immune-mediated tissue injury. The regional
differences in pathology may be due to differences in the host
innate response to LCMV among brain regions. LCMV
infection can trigger a robust innate immune response,
including interferon expression and natural killer cell
activation [58,59]. The innate immune response can differ in
different parts of the brain. For example, microglia, which
respond to initial insults to the central nervous system (CNS),
are present in different densities throughout the CNS, which
may result in region-specific pathological changes [60]. Thus,
regional differences in the innate immune response may lead
to different patterns and intensity of neuroinflammation,
ultimately leading to different patterns of neuronal loss and
other forms of tissue injury.
LCMV-Induced Pathology of the Developing Brain Is
Both Immune- and Virus-Mediated
The pathologic changes induced by LCMV infection of the
developing brain are due to both the immune response and
to the virus itself. Following inoculation of rats on PD4,
LCMV induces destruction of the dorsal cerebellar lobules
and hypoplasia of the olfactory bulbs [29]. The destructive
process in the cerebellum is immune-mediated. Evidence for
Table 1. Pathology in Human Congenital LCMV Infection and in
the Rat Model of the Disease
Pathology
in Humans
Pathology in
the Rat Model
Infection
Day
Microencephaly Microencephaly PD1–PD6
Cerebellar hypoplasia Cerebellar hypoplasia PD1
Encephalomalacia
Encephalomalacia
(cerebellum) PD4–PD6
Encephalomalacia
(dentate gyrus) PD6
Porencephalic cyst Porencephalic cyst PD10–PD60
Periventricular cyst Periventricular cyst PD10–PD60
Periventricular
calcification
Periventricular
infection PD1–PD10
Neuronal migration
disturbance
Neuronal migration
disturbance PD4-PD6
Hydrocephalus Hydrocephalus PD10–PD60
Chorioretintis Chrorioretintitis PD1
Table adapted from Bonthius et al. [30].
doi:10.1371/journal.ppat.0030149.t001
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this lies in the facts that 1) the spatio-temporal pattern of
tissue destruction corresponds perfectly with the spatio-
temporal infiltration of lymphocytes [29], 2) suppression of
the immune response with anti-lymphoid serum blocks the
destructive process [38], and 3) the destructive process does
not occur in congenitally athymic (nude) rats, which lack T
lymphocytes [61].
In contrast, the olfactory bulb hypoplasia appears to be
virus-mediated, as evidenced by the facts that the hypoplasia
occurs in the absence of a lymphocytic infiltration following
infection on PD4 [29] and that an identical hypoplasia occurs
following infection on PD1 [30], a time when the animal is
tolerant to the virus and does not mount an immune
response to it.
Whether the pathology in any particular brain region is
immune-mediated or virus-mediated, however, depends on
the age of the host at the time of infection [30]. In the
cerebellum, LCMV induces immune-mediated destruction if
the infection occurs on PD4, but virus-mediated hypoplasia if
the infection occurs on PD1. In the olfactory bulb, LCMV
induces virus-mediated hypoplasia if the infection occurs on
PD4, but immune-mediated destruction if the infection
occurs on PD6 or later. Thus, different brain regions
simultaneously infected with LCMV can undergo different
forms of pathology with different underlying mechanisms,
and the type of pathology in any particular brain region can
shift from virus-mediated to immune-mediated, depending
on host age at the time of infection [30].
Speculations and Future Directions for Research
Despite recent advances, much remains unknown
regarding the biology of LCMV infection of the developing
brain. Why there is so much variability in the pathology and
outcome among cases of human congenital LCMV infections
is one key question [11]. As discussed above, differences in
gestational timing of infection probably account for much of
the variability. But other factors may contribute, as well.
Children with congenital LCMV infection may have variable
outcomes because they were infected with different viral
strains. Different strains of LCMV have different avidities for
the receptor alpha-dystroglycan [45]. In the adult mouse
spleen, these different viral strains have distinct infection
kinetics and tissue tropisms, and they cause distinct diseases
[62]. The same may be true in the developing brain.
Another possible explanation for variability in outcome is
differences in host genetics. LCMV can alter gene expression
within neurons and thereby induce neuronal dysfunction
[63,64]. Genetic differences among individuals may underlie
different host gene–LCMV interactions and lead to different
patterns of neurological dysfunction [65,66].
How LCMV induces a neuronal migration defect is another
important question with clear clinical implications. Within
the rat’s developing cerebellum, LCMV disrupts the
doi:10.1371/journal.ppat.0030149.g005
Figure 5. Within the Developing Brain, LCMV Induces a Specific Pattern
of Infection, in which Brain Regions Containing Mitotically Active
Neuronal Precursors Are Selectively Infected
(A) During the early postnatal period in the rat, neurogenesis continues
in four brain regions. These regions include the olfactory bulb (OB),
periventricular region (PV), dentate gyrus (DG), and cerebellum (C). These
four regions in which neurogenesis occurs are precisely the same four
regions in which neurons are vulnerable to LCMV infection.
(B) Micrograph illustrating the selective infection of the olfactory bulb by
LCMV. This is a 50-lm-thick section through the anterior forebrain of a
30-d-old rat inoculated with LCMV on PD4. The section has been
immunohistochemically stained for LCMV antigens. Note that the
olfactory bulb (OB) is selectively infected. The immediately adjacent
olfactory stalk (OS) is free of infection. The arrowheads demarcate the
line between the olfactory bulb (right of arrowheads) and olfactory stalk
(left of arrowheads) and demonstrate the highly selectivity pattern of
LCMV infection.
(C) Micrograph illustrating the selective infection of the dentate gyrus.
This section was obtained from the same LCMV-infected animal shown in
(B) and is immunohistochemically stained for LCMV antigen. Note that
the neurons of the dentate gyrus (DG) are selectively infected, while the
neurons of the immediately adjacent hilus (H) are entirely spared.
Magnification bars represent 1 mm in (B) and 100 um in (C).
PLoS Pathogens | www.plospathogens.org November 2007 | Volume 3 | Issue 11 | e1491547

migration of granule cells from the external granule cell layer
to the internal granule cell layer and leaves many of the cells
permanently ectopic within the molecular layer [29].
Cerebellar granule cells are a major target of LCMV [29–32].
Thus, the migration defect may be due to a direct effect of the
virus on the migrating cells. However, LCMV also heavily
targets Bergmann glia [29], which constitute the scaffolding
along which granule cells migrate [67,68]. Thus, the migration
defect may be due to a virus-induced corruption of
Bergmann glia structure or function. A third possibility is
that the migration defect is due to an alteration in the
chemical environment of the cerebellum. During migration,
cerebellar granule cells respond to gradients of chemicals,
including chemokines, to direct their migratory movements
[69,70]. LCMV infection and its accompanying inflammatory
response likely alter the chemical environment, including
chemokine concentrations, which may misdirect the forward
progress of migrating neurons. Recently, in vitro systems have
been developed that allow systematic study of the role of
Bergmann glia, cell surface molecules, and diffusible
substances in cerebellar granule cell migration [71].
Application of these systems may elucidate the relative roles
of cytokines, chemokines, altered Bergman glia, and altered
granule cell function in the neuronal migration disturbance
induced by LCMV.
LCMV infection of the developing rat leads to substantial
regional differences in pathology across the brain. Similarly,
humans with congenital LCMV infection can have different
forms and severity of pathology in different brain regions
[11]. The differences in pathology are not due to regional
differences in viral titer, since titers are often similar among
regions with substantially different pathologies [29,30]. One
possibility, as discussed above, is regional differences in
cytokine and chemokine production leading to regional
differences in lymphocytic infiltration and tissue destruction.
Another possibility is regional differences in glial cell biology.
Astrocytes perform multiple functions vital to the health of
their neuronal neighbors and are a principal target of LCMV
[29]. Furthermore, astrocytes are not homogeneous across the
brain. Substantial regional differences exist in glial cell
physiology [72–75]. Thus, regional differences in LCMV-
induced pathology may be due to regional differences in
astrocytic responses to the virus.
One of the most fascinating aspects of LCMV infection
occurs in the developing hippocampal formation. Following
inoculation on PD4, the dentate gyrus is selectively and
heavily infected [29,57]. Despite a heavy viral load, the
dentate gyrus suffers no acute injury, and a full complement
of histologically healthy-appearing dentate granule cells is
generated. However, several months post-inoculation, a
selective drop-out of dentate granule cells begins [31]. This
delayed-onset cellular mortality progresses over months and
eventually results in a severe depletion of dentate granule
cells [57] and a corresponding decrease in the seizure
threshold [76]. Importantly, the virus has been cleared from
the animal before much of the neuronal loss occurs [29,57].
Thus, LCMV infection in the neonatal rat brain initiates a
pernicious and progressive pathological phenomenon that
continues even after the virus has been cleared. The
mechanism underlying this delayed-onset cell loss is
unknown, but may be due to excitotoxicity. Dentate granule
cells previously infected with LCMV are
electrophysiologically abnormal and hyperexcitable [57].
Furthermore, GABAergic (inhibitory) interneurons of the
dentate gyrus are infected with LCMV before the loss of
dentate granule cells begins [77]. Thus, the delayed-onset loss
of dentate granule cells may be due to viral-induced
disruption of inhibitory circuits that persists even after the
virus is gone.
Many prominent childhood- and adult-onset neurological
and psychiatric diseases are hypothesized to be due to viral
infections that occurred at a much younger age. Such diseases
include Alzheimer disease, Parkinson disease, multiple
sclerosis, temporal lobe epilepsy, schizophrenia, bipolar
disorder, and autism [78–83]. The fact that LCMV infection of
the neonatal rat brain leads to delayed-onset neuronal loss
after the virus has been cleared establishes the neonatal rat
model of LCMV as a potentially valuable model system for the
study of many important neurological diseases whose etiology
has remained elusive.
The pathogenesis of congenital LCMV infection in humans
is a mystery. However, because the neonatal rat model so
reliably reproduces the pathology observed in humans, use of
the animal model holds great promise for understanding the
biology and pathogenesis of this emerging infectious disease.
Lessons learned from the LCMV-infected rat may also shed
light on the pathogenesis of other congenital infections.
&
Acknowledgments
Author contributions. DJB and SP wrote the article.
Funding. DJB was supported by Public Health Service grant K08
NS02007 from the National Institute of Neurological Disorders and
Stroke, research grant 1-01-217 from the March of Dimes Birth
Defects Foundation, The John Martin Fund for Neuroanatomical
Research, the Children’s Miracle Network, a Carver Medical Research
Initiative grant, an Iowa Research Experience for Undergraduates
grant, and a grant from the University of Iowa Biological Sciences
Funding Program. SP was supported by Public Health Service grant
R01 NS36092 from the National Institute of Neurological Disorders
and Stroke.
Competing interests. The authors have declared that no competing
interests exist.
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