Latent Cytomegalovirus Infection of the Central Nervous System

Latent Cytomegalovirus Infection of the Central Nervous System

Lorraine Jones-Brando

It has been proposed that some cases of schizophrenia result

from a latent viral infection; one that may have been acquired in vitro. A

primary goal of this laboratory is to study a human neurotropic virus known to

establish latent infections and examine its effects on the cells of the central

nervous system (CNS).  Learning more about the pathogenesis of a latent

virus infection in the CNS should lead to a better understanding of how, at a

cellular level, a virus infection could lead to schizophrenia. In the current

study, human cytomegalovirus (HCMV) has been used as a model of a human virus

known to establish latent infections in utero.  Congenital (in

utero) HCMV infection has been associated with late manifestations of

disorders of neuronal migration and cortical organization.  A latent

infection of human neuronal precursor cells has been established in vitro.

Immunofluorescence assay results indicate that only the major

immediate early region of the viral genome is being transcribed in this latent

state.  However, infectious virion production can be induced if latently

infected cells are treated with retinoic acid, which causes the cells to

differentiate.  Characterization of the latent infection from both the

virus and the cellular aspect will be discussed.  Future plans include

investigation of up or down regulation of some cellular proteins reportedly

important in the pathogenesis of schizophrenia, e.g., neural cell adhesion


Determination of Reverse Transcriptase (RT) Activity in

Clinical and Post-Mortem Samples Obtained from Individuals with Schizophrenia

and Bipolar Disorder

Frances Yee

Our laboratory is interested in studying the role of

retroviruses in the etiology of neuropsychiatric illnesses such as schizophrenia

and bipolar disorder.  Retroviruses may represent an important link between

genetic and environmental factors, as this would account for horizontal and

vertical transmission, and these viruses may be involved with the disease

process in subpopulations affected with these serious mental illnesses.  An

important component of infectious retroviruses is that they contain the reverse

transcriptase (RT) enzyme, and consequently they can be detected by assays for

RT activity.

In this study, we set out to examine the levels of RT activity

in various samples, e.g. cerebrospinal fluid (CSF) from first episode patients

with schizophrenia, post-mortem cisternal fluids and various brain regions from

individuals with schizophrenia, bipolar disorder, depression without psychosis,

as well as unaffected individuals.  Before proceeding with these

experiments, it is important to determine the baseline RT activity present in

these samples, which may be due to the presence of endogenous

retroviruses.  We have modified an ultrasensitive RT assay called

product-enhanced RT (PERT; Pyra et al., PNAS 91:1544-1548, 1994), by adding a

second round of polymerase chain reaction (PCR) amplification.  When the

resulting PCR products are visualized with the nucleic acid dye SYBR-green

(Molecular Probes) and analyzed with the Fluorimager SI (Molecular Dynamics) we

are able to detect as little as 10-7 units of Moloney murine leukemia

virus-reverse transcriptase (MMLV-RT) activity in the PERT assay.  With

this PERT assay the background RT activity in the cerebellum of unaffected

individuals (n=10) was found to be less than 10-4 units.  Data

on the RT activity in the cerebellum from the affected groups, as well as the

CSFs from the first episode patients with schizophrenia, will be presented.

Simultaneous Analysis of Expression Levels of Many Genes in

Post-Mortem Brain Tissue From Individuals with Schizophrenia and Bipolar


Kia Faridi

The analysis of expression levels of genes in the post-mortem

brains of individuals with schizophrenia and bipolar disease is a potentially

important method in understanding the molecular basis of these diseases. 

The simultaneous analysis of levels of a large number of genes is particularly

valuable, allowing the evaluation of functionally related gene groups.

We used the Clontech Atlas Human I cDNA Expression Array to

simultaneously analyze levels of 588 genes.  Total RNA was extracted from

cerebellar brain tissue.  This RNA was used to generate 32P-labelled

cDNA probes, which were hybridized to the membrane array.  Autoradiograph

images of each array were then analyzed using ImageQuaNT software (Molecular

Dynamics) generating a value for each gene.  A total of 22 samples were

analyzed, including four brains from schizophrenics, six from bipolar

individuals, five from clinically depressed individuals, and seven normal

controls.  ANOVA analysis was performed to compare the levels of each gene

between the four disease groups.  Several genes were found to be

significantly differentially expressed in patients and controls. In addition for

each disease, total RNA from four samples was pooled, and this mixture was

hybridized to the array.  The largest differences were seen in levels of glutathione

S-transferase microsomal (raised in schizophrenic and bipolar) and glutathione

S-transferase theta-1 (depressed in schizophrenic).

Protein Alterations in the Postmortem Frontal Lobes of

Individuals with Severe Psychiatric Disorder

Nancy L. Johnston

Two-dimensional gel electrophoresis is an objective means to

compare the levels of individual proteins in different samples.  We

analyzed 89 postmortem frontal cortex brain regions from individuals with

schizophrenia, bipolar disorder, major depression and unaffected normal

controls.  We used a multivariate analysis to identify changes specific to

psychiatric disease.  Eight spots were altered, with 6 decreasing and 2

increasing in level in one or more illnesses.  Four of those which were

decreased were variants of glial fibrillary acidic protein (GFAP). 

Ubiquinone cytochrome c reductase complex protein 1 was reduced in depression,

and dihydropyrimidinase related protein 2 was reduced in all three

illnesses.  Carbonic anhydrase 1 was elevated in depressed individuals and

a second as yet unidentified protein was elevated in all three diseases.

The GFAP protein formed a family of spots that likely include

modified and unmodified forms of this protein.  It is noted that two forms

were significantly decreased in all disorders while the other two were only

altered in non-psychotic depression.  The pattern of change was distinct

between the three diseases and could not be attributed to any known confounding

variables.  Two-dimensional gel electrophoresis offers a sensitive and

viable means to measure changes in levels and modifications that would not be

detected by one-dimensional electrophoresis or analysis of the corresponding

nucleic acids.

Detection of Retroviral RNA in the CSF of Individuals with


Håkan Karlsson

We have explored the hypothesis of retroviral involvement in the

etiology of schizophrenia by testing 18 CSF’s obtained from first-episode

individuals with schizophrenia-spectrum disorder and 18 CSF’s from individuals

without psychiatric disease.

RNA’s were isolated from pelleted CSF’s reverse transcribed, and

subjected to PCR using primers directed towards a conserved region of the

retroviral pol-gene (Tuke et al, Acta Neurol Scand 1997;169:16-21).

A PCR-product was detected in seven of the 18 schizophrenia

samples and in three of the 18 control samples.  Subsequent cloning and

sequencing of these products showed that at lest six samples from the

individuals with schizophrenia contained a single retroviral species, showing

90-95% homology to the multiple sclerosis associated retrovirus (Perron et al,

PNAS 1997;94:7583-7588).  Individual control samples were found to contain

multiple species, homologous to human endogenous retroviral sequences (including

multiple sclerosis associated retrovirus), probably originating from

contaminating human nucleic acids.

These results document the presence of specific retroviral

particles in the CSF of acutely ill individuals with schizophrenia.  The

retroviral particles may have arisen from the differential activation of

endogenous sequences or may be the result of exogenous retroviral infection.