HIGH
SENSITIVITY DETECTION OF NEUROTROPIC VIRUSES IN POSTMORTEM BRAIN
Regina W. von
Einsiedel1*, Ingrid W. Samorei1,
Michael Pawlita2, Michael Schmid3,
Christoph Mundt1, Thorsten Schäfer1,
Thomas W. Vahlenkamp4, Hermann Mueller4,
Anja Kipar5, Harry V. Vinters6.
1Psychiatrische
Universitätsklinik Heidelberg, Molekularbiologisches Labor,
Heidelberg, Germany; 2Deutsches
Krebsforschungszentrum (DKFZ) Heidelberg, Germany; 3Institut
fò r Virologie, Infektiologie und Epidemiology e.V.,
Medizinish-diagnostisches Gemeinschaftslabor, Stuttgart, Germany; 4Institut fò r Virologie,
Veterinärmedizinische Falkuhät, Universität Leipzig, Germany; 5Institut
fò r Pathologie, Veterinärmedizinische Falkuhät, Universität
Gricb en, Germany; 6Department of
Pathology and Lab Medicine (Neuropathology) Brain Research
Institute and Neuropsychiatric Institute, UCLA Medical Center,
Los Angeles, USA
For the investigation of
neurotropic DNA and RNA-viruses the highly sensitive in situ
polymerase chain reaction (ISPCR) method and reverse
transcriptase ISPCR (RT-ISPCR) were employed. With the indirect
(RT-)ISPCR the target-DNA or RNA was amplified
intracellularly and detected by a specific labeled probe for
JC-virus-DNA or Bornavirus-RNA.
Progressive multifocal
leukoencephalopathy (PML) is a fatal opportunistic JC virus
infection of the central nervous system leading to
neuropsychiatric deficits. JC virus has been studied manifold
using conventional methods and was therefore ideal for the
validation of ISPCR. In detail, formalin-fixed and
paraffin-embedded postmortem brain tissue of 10 AIDS-patients
with PML was studied. 12 controls were included to disclose false
results and a semi-hot start method was employed. The study
revealed that ISPCR has a significantly higher sensitivity than
conventional in situ hybridization (ISH), the amount of
JCV-positive oligodendrocytes always exceeded the ISH results two
to three-fold, and some additional hitherto unreported
neuropathological observations could be made.
The ISPCR technique is now being
transferred to the detection of Bornavirus-RNA. A human infection
with Bornavirus might cause psychiatric diseases such as
depression and psychosis. Whether the virus is not only
neurotropic but also haematotropic remains to be elucidated. The
aim of the study is the assessment of the clinical relevance of
Bornavirus for humans. In situ RT-PCR should prove to be a
reliable method to diagnose the presence of Bornavirus RNA in
tissue and cell suspensions. To establish RT-ISPCR for Bornavirus
RNA, Bornavirus-positive postmortem formalin-fixed and
paraffin-embedded equine brain tissue as well as persistently
Bornavirus-infected MDCK-cells are used. Liquid RT-PCR in these
materials have been established. An examination of postmortem
brain tissue of patients with a psychiatry history, as well as
the detection of Bornavirus-RNA in peripheral blood mononuclear
cells and in cerebrospinal fluid of acute and chronically ill
psychiatric patients and healthy controls will be undertaken.