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

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

The Cellular Targets of LCMV within the Cerebral Cortex Depend on Host Age

<div><p>Shown are 50-μm-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.</p><p>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.</p><p>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.</p><p>Inoculation on PD 21 leads to infection of neither neurons nor astrocytes.</p></div

LCMV Infection Disrupts Neuronal Migration in the Developing Brain of Humans and Rats

<div><p>(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.</p><p>(B and C) are 2-μm-thick sections through the cerebellar cortex of uninfected control (B) and LCMV-infected (C) rats.</p><p>(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.</p><p>(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.</p><p>Magnification bars represent 100 um in (B and C).</p></div

Critical Role for Glial Cells in the Propagation and Spread of Lymphocytic Choriomeningitis Virus in the Developing Rat Brain

Inoculation of the neonatal rat with lymphocytic choriomeningitis virus (LCMV) results in the selective infection of several neuronal populations and in focal pathological changes. However, the pathway by which LCMV reaches the susceptible neurons has not been described, and the nature and time course of the pathological changes induced by the infection are largely unknown. This study examined the sequential migration of LCMV in the developing rat brain and compared the pathological changes among infected brain regions. The results demonstrate that astrocytes and Bergmann glia cells are the first cells of the brain parenchyma infected with LCMV and that the virus spreads across the brain principally via contiguous glial cells. The virus then spreads from glial cells into neurons. However, not all neurons are susceptible to infection. LCMV infects neurons in only four specific brain regions: the cerebellum, olfactory bulb, dentate gyrus, and periventricular region. The virus is then cleared from glial cells but persists in neurons. LCMV induces markedly different pathological changes in each of the four infected regions. The cerebellum undergoes an acute and permanent destruction, while the olfactory bulb is acutely hypoplastic but recovers fully with age. Neurons of the dentate gyrus are unaffected in the acute phase but undergo a delayed-onset mortality. In contrast, the periventricular region has neither acute nor late-onset cell loss. Thus, LCMV infects four specific brain regions in the developing brain by spreading from glial cells to neurons and then induces substantially different pathological changes with diverse time courses in each of the four infected regions

Congenital LCMV Infection of Humans Induces Periventricular Calcifications

<div><p>The neonatal rat model demonstrates that periventricular neurons are selectively vulnerable to infection with LCMV.</p><p>(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.</p><p>(B) Horizontal section (50-μm-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 = lateral ventricle, S = septum, BG = basal ganglia.</p><p>(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).</p><p>Magnification bars represent 500 um in (B) and 100 um in (C).</p></div

LCMV Infection Induces Focal Destructive Lesions within the Developing Brains of Humans and Rats

<div><p>(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.</p><p>(B) Section (50-μm-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).</p><p>(C) Nissl-stained section (2-μm-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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030149#ppat-0030149-b084" target="_blank">84</a>].</p><p>(D) Section (2-μm-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.</p><p>(E) Section (50-μm-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 CD8+ antigen, which labels a subset of lymphocytes. Note the dense infiltration of CD8+ lymphocytes (arrows).</p><p>Magnification bars represent 1 cm in (A), 100 um in (B), 500 um in (C), 500 um in (D), and 100 um in (E).</p></div