Detection methods and epidemiology of Borna disease virus

¶ … detection of the Borna disease virus relating them to the epidemiology.

The first cases of Borna disease were described in the 17-19th century in Southern Germany. It was discovered to be a fatal disease affecting the neurological systems of horses and sheep, (Ludwig et al., 1985; Durrwald, 1993) causing behavioral and neurological symptoms. It was proven to be caused by a 2003]

Today it is being realized that the scope of the disease is not limited to just a few countries as was previously believed but encompassed the world. Also it was realized that far from affecting just horses and sheep as was originally thought virus, the Borna Disease Virus (BDV) in the early 1900's by Zwick and his team in Giessen Germany. [Author not available, it in fact affected other animals and even human beings.[Staeheli, Sauder; Schwemmle, et al., 2000]

Research into the epidemiology and pathogenesis of the BDV is impeded however by a lack of universally accepted standard for its detection, especially in humans. Many different techniques are available and used, including indirect immunofluorescence assay (IFA), Western immunoblot (WB), anti-BDV antibody detection, radioimmunoprecipitation, enzyme-linked immnosorbent assay (ELISA), RT-PCR and very rarely direct isolation of the virus. None however are considered to be completely reliable and valid.[Diagnostic Methods In Virology, 2003,Nakamura et al. 2000] The sensitivity and selectivity of these assays are under suspicion due to the various results obtained in the same population when different methods are used. [Author not available 2003] This also raises difficulties during epidemiological studies.

This paper is an attempt to cover in detail the various methods used to detect the BDV virus and analyze their efficiency in doing so. This will be done with reference to the epidemiology of the virus that is changing significantly and speedily with the use of the analytical techniques. An attempt is also made to compare these processes and give suggestions for future research in the light of modern day studies.

About the Disease

The Borna Disease and its symptomology has been recognized for almost two centuries now but it was only after the isolation of its etiologic factor, the Borna Disease Virus, that any scientific research into the actual pathogenesis, transmission and management of the disease could be done.

It was found that the Borna disease virus (BDV) was a nonsegmented, enveloped, negative strand RNA virus that had a tropism for limbic and dopamine circuitry of the brain, in particular the hypothalamus. [Author not available 2003] The virus also showed special replication properties being highly genetically stable in its natural host, the horse. [Diagnostic Methods In Virology, 2003] It is thought that the disease process is not induced directly by the replication of the virus; rather it is the virus-induced T-cell mediated immune reaction that produces the pathology. This has been concluded through studies in the Lewis rat, and the discovery of BDV reactive antibodies in the sera of patients with neuropsychiatric disorders. [Diagnostic Methods In Virology, 2003]

Borna Disease is a disseminated non-purulent meningo-encephalomyelitis that infiltrates mononuclear cells (Jurgen 1997, Schizophrenia and Borna Disease Virus as a Zoonosis?, 2000) and polio encephalomyelitis especially affecting the gray matter of the cerebrum and the brain stem (Schizophrenia and Borna Disease Virus as a Zoonosis? 2000). [Diagnostic Methods In Virology, 2003] Clinically it manifests in horses as behavioral changes [Durrwald & Ludwig, 1997] resembling human psychiatric diseases including bipolar disorder, schizophrenia, and autism. [Author not available 2003]

Transmission of the BDV is thought to be through various routes including, salival, nasal, or conjunctival secretions as BDV-specific RNA has been found in all of these. (Schwemmle, 2001, Hagiwara, 1998, Hagiwara 1997) Small quantities have also been found in the feces and urine of infected animals. [Nakamura et al. 2000] It is either through direct contact with these secretions or by contaminated food and water that the virus can be transmitted. Experimentally, the BDV virus has been administered intracerebrally, intraocularly, intranasally and intramuscularly. The virus however has been found to be not very infectious, [Nakamura et al. 2000] though it can recur in those animals that survive the acute attack, especially after stress. (Jurgen 1997)[Diagnostic Methods In Virology, 2003]

The incubation period of the BDV in horses is thought to be about four weeks; [Diagnostic Methods In Virology, 2003] the clinical illness lasting for about 1-3 weeks with neurological signs such as ataxia, depression, circular movement, standing in awkward positions, collapsing, running into obstacles, and paralysis, during the acute phase. [Diagnostic Methods In Virology, 2003] The mortality rates for diseased horses are high, 80% to 100% (Jurgen 1997).

As yet, a definite reservoir for the virus has not been discovered but suspicion through scientific study has fallen on various rodents, seropositive horses with subclinical infections, and even insects due to the seasonal characteristic of Borna Disease epidemics. (Hagiwara 1997)[Diagnostic Methods In Virology, 2003] BDV is more common in some years than in others and the number of documented infections increases in the spring and early summer. [Diagnostic Methods In Virology, 2003]

Epidemiology

That the BDV may have a larger geographical and host range than previously believed is being proven by epizootiology studies in animals and epidemiology in human beings. [Diagnostic Methods In Virology, 2003] This research has shown Borna Disease to be a widespread zoonosis and that BDV or a similar virus can also infect human beings. [Diagnostic Methods In Virology, 2003] These conclusions have been arrived at through results of seroepidemiological studies, preliminary data from studies on the isolation of BDV from the CSF and the detection of BDV-specific antigen and BDV-RNA in the human brain. [Diagnostic Methods In Virology, 2003]

Most of the cases of the Borna Disease in animals have been discovered and described in Central Europe, especially in Germany and Switzerland. Liechtenstein (Weissenbck et al., 1998 b; Caplazi et al., 1999). Conclusive evidence to support its presence in other countries has not yet been obtained. [Diagnostic Methods In Virology, 2003] Natural infections however have been documented in North America, East Africa and parts of Asia (including Japan, Israel, Thailand and Taiwan). BDV-specific antibodies were found in horses in several European countries, Israel (Hagiwara, 1998, Hagiwara 1997), Japan (Hausmann, 2001), Iran (Durrwald, 1993), and the United States.[Diagnostic Methods In Virology, 2003] It is seen that BDV antibodies are found in otherwise clinically healthy animals, the natural infections remaining asymptomatic, thus requiring a high index of suspicion for detection. [Diagnostic Methods In Virology, 2003] Using the various techniques for virological studies available and being evolved the scope of this disease could be found to be much larger than believed.

Natural infections have been detected in other Equidae such as, donkeys (Zimmermann et al., 1994; Durrwald, 1993; Caplazi et al., 1999), goats (Caplazi et al., 1999) and cattle (Caplazi et al., 1994; Bode et al., 1994 b) to a certain extent. Some of these are cases detected on farms in areas of Germany where BD is not endemic in horses and sheep (Bode et al., 1994 b). BDV antigen and virus have been detected in the CNS of two rabbits with neurological symptoms. Ruminants, rabbits, (Metzler et al., 1978) cats, and ostriches have also been found to be hosts while it has been seen that non-human primates can be exogenously infected. Recently BDV antigen and RNA has been shown in the brain of a dog with severe CNS inflammation and neurological disease in Austria (Weissenbck et al., 1998 a).

That BDV may be involved in causing 'staggering disease' in cats is supported by initial data showing that a high percentage (44%) of Swedish cats suffering from this encephalitic disease had serum antibodies to BDV (Lundgren et al., 1993; Lundgren & Ludwig, 1993). According to analytical studies viral markers shown to be low in the brains of only three out of 24 diseased cats (Lundgren et al., 1995 a, b). Cats with Staggering Disease in Austria had no detectable levels of BDV markers in the CNS (Nowotny & Weissenbck, 1995).

A study done in Sweden (Berg et al., 2000) described a free-ranging lynx with non-suppurative meningoencephalitis showing similarities to Borna disease. Immunohistochemistry showed that the astrocytes from the lynx's brain stained positive for BDV. [Staeheli 2000] In contrast a RT-nested PCR performed on a sequence of a BDV p24 fragment from the same brain tissue differed by more than 2% from known BDV strains. This underlines the observation emphasizes the idea that the host range of BDV is wider than previously thought. [Staeheli. et al. 2000]

BDV is also considered to be genetically invariable forming a single type in the genus Bornavirus of the family Bornaviridae. This makes detection of the virus difficult as does the low levels of viral load found in diseased cases. [Author not available 2003] The virus exists as BDV-specific circulating immune complexes during the chronic phase of the disease, which impedes the detection of viral antibodies. [Author not available 2003] Scientists also doubt the reliability of using antigens from strains of BDV found in only in animals and not in humans. They have an increased chance of contamination. [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]

Neutralization

In the process of neutralization of a virus it is reacted with a specific antibody thus rendering it non-infective. Virus and serum are mixed under appropriate condition and then inoculated into cell culture, eggs or animals. The presence of unneutralized virus may be detected by reactions such as CPE, haemadsorption/haemagglutination, plaque formation, and disease in animals. There are two types of neutralization; - [Diagnostic Methods In Virology, 2003]

Reversible neutralization - This occurs due to the interference in the ability of the virions to attach to the cell receptors. If antibodies saturate the cell surface then viruses can be neutralized, the process being reversed by diluting the Ab-Ag mixture within a short time of the formation of the Ag-Ab complexes. [Diagnostic Methods In Virology, 2003]

Stable neutralization - Once the Ag-Ab complexes become stable with time the process of neutralization become s irreversible. Neither the virions nor the Antibodies are permanently changed in stable neutralization, for the unchanged components can be recovered. The number of antibody molecules needed for stable neutralization is much smaller than that of reversible neutralization, [Diagnostic Methods In Virology, 2003]

Viral evolution occurs through mutations selected for antigens involved in neutralization without the need to affect other antigenic sites. [Diagnostic Methods In Virology, 2003] Different varieties thus evolve but some of the original A virus mosaic of antigenic determinants recognizable by CFTs remains. Neutralization has a high immunological specificity and is often used as a standard against which the specificity of the other serological techniques is evaluated. [Diagnostic Methods In Virology, 2003]

Titration

To identify a virus isolate, a known pretitred antiserum is used, and to measure the antibody response of an individual to a virus, a known pretitred virus is used. To titrate a known virus, serial tenfold dilutions of the isolate is prepared and inoculated into a host system such as cell culture or animal. [Diagnostic Methods In Virology, 2003]

The virus endpoint titer is the reciprocal of the highest dilution of virus that infects 50% of the host system. This endpoint dilution contains one 50% tissue culture infecting dose (TCID50) or one 50% lethal dose (LD50) of virus per unit volume. The concentration of virus generally used in the neutralization test is 100 TCID50 or 100 LD50 per unit volume. [Diagnostic Methods In Virology, 2003]

The antiserum is titrated in the neutralization test against its homologous virus. Serial twofold dilutions of serum is prepared and mixed with an equal volume containing 100TCID50 of virus. The virus and serum mixtures are incubated for 1 hour at 37oC.[Diagnostic Methods In Virology, 2003] The time and temperature for incubation varies with different viruses. The mixtures are then inoculated into a susceptible host system. The endpoint titration contains one antibody unit and is the reciprocal of the highest dilution of the antiserum protecting against the virus. Generally 20 antibody units of antiserum are used in the neutralization tests. [Diagnostic Methods In Virology, 2003]

The intra-vitam detection of BDV is made difficult in humans and animal studies, as the virus is associated with the CNS. Using virological methods becomes almost impossible.[Diagnostic Methods In Virology, 2003]

Serological Methods Elisa, immunofluorescence western blotting wide variety of assays (IFA, Western immunoblot, radioimmunoprecipitation, and enzyme-linked immunosorbent assay) and antigen preparations (infected cells, infected cell extracts, and recombinant proteins produced in prokaryotic or baculovirus systems) have been used for BDV serology. Due to the limited data about inter-assay comparability for serology within individual and separate laboratories discrepancies between investigators may simply be due to differences in clinical populations, assay sensitivity, or other factors.

Serology is based on the primary humoral immune response mechanism to antigen. The first antibody to appear is the IgM and then followed by IgG after initial exposure to an antigen. In cases of reinfection, the level of specific IgM may remain the same or rise slightly but IgG levels increase rapidly than in a primary infection. [Diagnostic Methods In Virology, 2003] Many different types of serological tests are available. The sensitivity and specificity of the assays depend greatly on the antigen used. Assays that use recombinant protein or synthetic peptide antigens tend to be more specific than those using whole or disrupted virus particles are. [Diagnostic Methods In Virology, 2003]

The disadvantages to serological methods include the increased duration for making a diagnosis for paired acute and convalescent sera. Also mild local infections may not produce a humoral response significant enough to be detected. Conflicting results are produced when cross reactivity on a broad level takes place between antigens of related viruses. There is suddenly an increase of false positive cases. [Rott, 1995] The humoral response is reduced or lacking in the immunocompromised again producing false data. Patients who have been transfused blood or blood products may give a false positive result due to the transfer of antibody. [Diagnostic Methods In Virology, 2003]

Western blotting

Western blot analysis can detect and specify the size of a specific protein in a complex and multiple protein mixture. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein thus a small portion of the protein must be supplied from a cloned DNA fragment. That antibody then probes the particular protein. [Diagnostic Methods In Virology, 2003]

Western blotting can also specify how much protein has accumulated in cells. To detect the rate of synthesis of a protein, and to detect a protein that degrades rapidly, Radio-Immune Precipitation (RIP) assay would be a better choice instead of Western blotting.[Diagnostic Methods In Virology, 2003]

Immunofluorescense (IF)

Immunofluorescence (IF) detects viral antigen in clinical specimens, as well as virus-specific IgG or IgA or IgM antibody. The technique uses a fluorescein- labeled antibody to stain specimens containing specific virus antigens, so that the stained cells fluoresce under UV illumination. In the case of direct IF, the specimen is probed directly with a specific labeled antibody against a particular virus antigen. In the case of indirect IF, the specimen is first probed with a non-labeler specific antibody, followed by a labeled antibody against the first antibody. [Diagnostic Methods In Virology, 2003]

Direct or indirect IF can be used for the detection of virus antigen, whereas indirect IF is virtually always used for the detection of antibody. [Diagnostic Methods In Virology, 2003]

IF is highly dependent on the quality of the specimen. A conclusive diagnosis can be made on the identification of a few cells only that contain fluorescence of the right color and with the correct antigen distribution. IF however is labor intensive needing highly skilled staff for reading the specimen. [Diagnostic Methods In Virology, 2003] It is however simple to set up and thus usually used to for newly discovered viruses, in particular arboviruses [Diagnostic Methods In Virology, 2003]

ELISA

ELISA was developed in 1970 and became rapidly accepted. Many techniques were utilized in this process but the main ones remain:[Diagnostic Methods In Virology, 2003]

1. Competitive methods

2. Sandwich methods

3. Antibody captures methods

4. Competitive methods

Antibody capture methods

They are widely used for the diagnosis of acute infections by IgM detection but may also be used for detecting IgG and IgA. The samples to be tested are first reacted with for e.g. insolubilized anti-IgM, and subsequently with either enzyme labeled antigen followed by enzyme linked antibody. [Diagnostic Methods In Virology, 2003,Durrwald, 1993]

Researchers have discovered that during persistent infection, BDV-specific circulating immune complexes are present and can interfere with the serological tests. A new enzyme immunoassay was developed to test for these complexes. Using this new assay, infection rates of both humans and horses were determined to be dramatically higher (x10) than the levels determined in the same populations using the earlier serological tests. The results of this study confirmed earlier studies that find increased prevalence of BDV in psychiatric patients. [Hausmann, 2001].

To improve the sensitivity of BDV antigen detection, an antigen-capture ELISA was introduced which uses a cocktail of monoclonal antibodies and a monospecific polyclonal antiserum for antigen capture and detection, respectively (Durrwald, 1993). It was found that a large fraction of brain samples which tested positive with this ELISA did not contain infectious virus (Durrwald, 1993). Surprisingly, the antigen-capture ELISA also detected large amounts of viral antigen in PBMCs and peripheral organs like liver, kidney and spleen of some diseased horses (Durrwald, 1993; Bode et al., 1994 a). Unfortunately, the specificity of this ELISA has never been compared systematically with more traditional BDV antigen detection systems. It thus remains unclear whether a positive result with this antigen-capture ELISA is firm proof of infection. [Staeheli 2000]

To determine the frequency of clinically unapparent BDV infections of horses in Germany large-scale serological studies using sensitive IFA were done. (Herzog et al., 1994). These studies showed that approximately 12% of the horses from both endemic and non-endemic regions had serum antibodies against the BDV antigen. As in diseased horses, the titers of BDV-specific serum antibodies in healthy animals were also low (1:5 to 1:320). Using IFA, Western blot analysis or ELISA, variable proportions (ranging from 3% to 58%) of horses from the United States of America, Japan, Iran and Sweden were found to be seropositive (Herzog et al., 1994; Bahmani et al., 1996). By contrast, a survey by IFA of healthy horses from an endemic region in Switzerland suggested that only about 1% were seropositive.[Staeheli 2000]

Suggestions and Future Directions

The broad potential host range of BDV suggests that humans are targets for infection. The sources and routes of potential human infection are not clear, no detailed epidemiology has been done in animal populations, and transmission from domestic animals to humans has not been demonstrated. Viral gene products are readily detected in CNS of natural and experimental hosts without such sensitive methods as nested RT-PCR.[22]

Due to low viral loads in infected humans sensitive diagnostic methods need to be employed. To address the issues of BDV epidemiology and pathogenesis, multicenter groups in Europe and the United States are collaborating to collect and perform blinded analysis of human clinical materials by standardized methods and reagents.[Richt 1992] These projects are to determine the prevalence of serum antibodies to BDV in patients and controls and the extent to which various assays for antibodies are in accord. To detect the prevalence of BDV nucleic acids in brains and PBMC of patients and controls, and the correlation between antibodies to BDV or viral nucleic acids and a particular neuropsychiatric disease [Metzler, Ehrensperger & Wyler, 1978,Bechter et al. 1992]

Numerous alternative in-vitro amplification techniques have been developed, after PCR of which some are now available commercially. Examples of these alternative techniques include ligase chain reaction (LCR), nucleic acid sequence based amplification/isothermal amplification (NASBA), strand displacement amplification, Qb Replicase method, and branched DNA probes. [Diagnostic Methods In Virology, 2003]

In spite of discrepant views between laboratories on the interpretation of RT-PCR results, a wealth of serological data continues to indicate that BDV or an antigenically related agent might be associated with human psychiatric disorders. Challenging tasks for future research include confirming the role of BDV in human disease or else identifying the related immunogen. [Staeheli 2000]

Due to intrinsic difficulties of BDV diagnostics, epidemiological studies are not straightforward. Since the humoral immune response to natural infection with BDV is usually weak, it is necessary to push the limits of presently available serological assays with the drawback of reduced specificity.[Staeheli 2000,Durrwald, 1993] Therefore, diagnostic laboratories should initiate a concerted action to carefully evaluate the various serological tests for specificity and sensitivity. Before such standardization has been achieved, the interpretation of serological studies will remain difficult.[Staeheli 2000]