High Sensitivity Detection of Neurotropic Viruses in Postmortem Brain Tissue by In Situ PCR and In Situ RT-PCR



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.


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.