Term Paper: Detection of the Borna Disease

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[. . .] [Author not available 2003]

BDV and Neuropsychiatric Disease reason for renewed interest and research into the pathogenesis and characteristics of the BDV is the possibility recently discovered of the BDV being an etiological factor in human behavioral disorders as well. [Jurgen 1997] Numerous scientific studies are being done to accumulate evidence to clarify this issue.

One study showed that 10-15% of psychiatric patients had anti-BDV antibodies as compared to only 2% in the general population. [Schizophrenia and Borna Disease Virus as a Zoonosis? 2000] Another on patients with severe depression detected anti-BDV antibodies in 30% of the hospitalized cases. [Schizophrenia and Borna Disease Virus as a Zoonosis? 2000] BDV was also detected and isolated in patients with mood disorders while BDV nucleic acids and antigens have been detected in both sera and brain tissue of psychiatric patients. [Schizophrenia and Borna Disease Virus as a Zoonosis? 2000]

It is the very ambiguity of classification of psychiatric illnesses that makes it difficult to find a conclusive etiology or at the very least some association. The same syndrome may have numerous and variable causes. Thus, proving that BDV indeed was linked to psychiatric disease would probably imply that that the virus would only be the cause of disease in a small fraction of cases. [Author not available 2003] There are also serious doubts as to the specificity of both the serologic and RT-PCR assays. [Schwemmle, 2001]

The association between the BDV and human psychiatric diseases has yet to be proven but the evidence supporting this theory is enough to warrant further study. [Author not available 2003]

Analytic Techniques Used for Viral Detection

There are multiple diagnostic tests available for detecting viruses at the present. Each has its own value depending on the virus involved and often results from separate test can brew controversy. In general they can be divided into three main categories i.e.: [Rott, 1995]

1. direct detection, 2. indirect examination (virus isolation), and 3. serology.

I. Direct

Direct examination involves the detection of virus particles, antigen or nucleic acids by examination of the clinical specimen directly. Indirect examination on the other hand attempts to grow the virus in a cell culture, eggs or animals, the method also being known as virus isolation. [Rott, 1995] Serological diagnosis is made by detection of antibodies and is the most reliable method to date. [Rott, 1995]

The site from which the specimen under examination is obtained is also important, positive results having more value if obtained from the site of the pathology. [Rott, 1995] Examples of techniques using this concept include:

Electron Microscopy morphology / immune Electron microscopy, Light microscopy for histological appearance, Antigen detection immunofluorescence, ELISA etc. And Molecular techniques for the direct detection of viral genomes.

Antigen Detection

Immunofluorescence testing of nasopharyngeal aspirates for respiratory viruses, detection of rotavirus antigen in feces, the detection of HSV and VZV in skin scrapings, and the detection of HBsAg in serum (also considered serological) are several examples of these types of studies. These assays are easy and quick in terms of the result being available in a few hours. The method is however tiresome and time consuming, the result difficult to read and interpret, and the sensitivity and specificity poor. The quality of the specimen obtained is of utmost importance in order for the test to work properly. [Rott, 1995]

II. Indirect

Options available in this technique are, Cell Culture to detect cytopathic effect, haemadsorption, confirmation by neutralization, interference, immunofluorescence etc. Also egg pocks on CAM can show haemagglutination, and inclusion bodies while animal studies reveal disease or death confirmation by neutralization. [Rott, 1995]

III. Serology

This method looks at the rising titers of antibodies during the illness especially between acute and convalescent stages, or the verification of primary or active infection through the isolation of IgM.

Diagnostics in BDV Detection

There are still no accepted standards to detect the BDV in animal and human tissue though there are some techniques that have greater application as compared to others. These include anti-BDV antibody detection, IFA - indirect immunofluorescence assay, Western immunoblot method, radiommunoprecipitation, enzyme linked immunosorbent assay (ELISA) and to a lesser extent in human studies, actual virus isolation. [Rott, 1995]

The obstacles to the development of a standard detection technique include the wide variety of antigen preparations. Also the BDV can persist at very low levels making it extremely difficult to detect. The methods also show differences in specificity and sensitivity, none of them being optimal. [Rott, 1995] The validity of using animal antigens for making assumptions about human studies is also under question.

The nested RT-PCR is a commonly used technique in BDV diagnosis but has a high possibility for contamination and the results of the presence of virus-specific antibodies is shown to conflict with presence of the disease as we know it. Also there is a lack of correlation between detection of BDV RNA in blood and the serum antibodies to BDV.[5] Furthermore the inadequacy of current methods is also emphasized by the recent discovery of a new BDV strain with 15% genomic divergence that was not detected by current BDV methods employed. It also underscores the possibility of a more widespread epidemiology than previously thought. [Schizophrenia and Borna Disease Virus as a Zoonosis?, 2000]

Indirect Immunofluorescence Assay (IFA)

The cerebrospinal fluid (CSF) changes in Borna disease may be similar to those caused by other forms of viral meningoencephalitis (Bilzer et al., 1996; Grabner & Fischer, 1991;). These include such general indicators as raised protein content and mononuclear pleocytosis. It is the detection of BDV specific antibodies in the sera or CSF that is more significant towards the diagnosis of BDV. Currently the most reliable method of detecting these antibodies is the indirect immunofluorescence assay (IFA). (Durrwald, 1993)

Even then the results vary in sera and CSF giving rise to doubt. One study showed BDV specific antibodies in all its sera and CSF samples (Bilzer et al., 1996), while another had a ratio of 41% sera positive as compared 61% of the CSF (Grabner & Fischer, 1991). Herzog et al. (1994) detected BDV-specific antibodies in 100% of serum samples but only in 73% of CSF samples from horses with BD. Also in only two out of the three ponies induced with Borna disease experimentally could BDV-specific antibodies be detected (Katz et al., 1998).

Reasons for non-uniformity in the obtained results include low titers of the BDV-specific antibody in diseased animals, (Metzler et al., 1979; Durrwald, 1993; Herzog et al., 1994; Bilzer et al., 1996; Katz et al., 1998; Caplazi & Ehrensperger, 1998). Discrepancies also arise from the different cell systems used by the different for IFA. Thus post-mortem histological examination of brain tissue is also required for confirmation. [Staeheli 2000]

Immunohistological analyses

Encephalitis produces a similar picture in the brain no matter what the etiology, thus a BDV infection of the CNS must be proven. Isolation of the virus from brain cells is not usually successful. (Herzog et al., 1994; Durrwald, 1993) Joest-Degen inclusion bodies in nuclei of infected neurons have long been considered BDV-specific markers, but again are not consistently seen in diseased samples. Using monoclonal antibodies on tissue samples increases sensitivity of virus detection. This could also be very complicated because viral markers (RNA and antigen), both vary significantly in individual animals with BD. (Bilzer et al., 1995,)

Immunohistological analyses shows that virus infected cells are non-uniformly distributed in brains of diseased animals and the antigen-positive neurons being more prominent in the hippocampus (Bilzer et al., 1995; Caplazi & Ehrensperger, 1998). In some animals with overt neurological disease only very few virus-infected cells can be visualized (Caplazi & Ehrensperger, 1998), indicating that sometimes BD cases might escape detection by this method. [Staeheli 2000]

Molecular Methods: (Reverse transcription polymerase chain reaction RT-PCR)

Methods based on the detection of viral genome are also commonly known as molecular methods. [Metzler, Ehrensperger & Wyler, 1978] Molecular biology methods are used to compare relationships between resembling organisms based on genomic similarities. Genetic markers of distinct strains can be recognized by detecting and sequencing portions of a viral genome and then replicating them. [Metzler, Ehrensperger & Wyler, 1978,Schizophrenia and Borna Disease Virus as a Zoonosis?, 2000] Thus has evolved the science of genetic epidemiology (i.e. disease tracing). On a global scale, the progression of different genetic strains (genotypes) of different viruses can be followed in terms of changing geography and morphology. [Metzler, Ehrensperger & Wyler, 1978]

Analysis of protein and antigen biochemistry using modern molecular methods has improved the specificity of virus detection. [Metzler, Ehrensperger & Wyler, 1978] There are numerous modifications and adaptations to this method ad well and though most researchers use the RT-PCR others apply a method sensitive to 100 to 300 copies of RNA template (nested RT-PCR, 80 to 100 cycles), for e.g. In the detection of the BDV. [Metzler, Ehrensperger & Wyler, 1978] Molecular methods also include polyacrylamide gel electrophoresis (PAGE) of protein fragments,

Western blotting, and identification of specific proteins with labeled probes

Polymerase chain reaction (PCR),… [END OF PREVIEW]

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