METHOD FOR DETECTING ACUTE BORNA DISEASE VIRUS (BDV) INFECTIONS AND A DIAGNOSTIC KIT FOR THERETO, IN PARTICULAR IN COMBINATION WITH METHOD FOR DIFFERENTITING ACUTE FROM CHRONIC AND CALM BDV INFECTIONS AND DIAGNOSTIC KITS FOR THERETO

Description

[0001] In a first aspect, the present invention is directed to a method for detecting acute Borna Disease Virus (BDV) infections. According to the invention, the presence of heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein in a sample is determined using a sandwich ELISA as set forth in the claims. This determination involves the use of specific, monoclonal primary antibodies against both the p24 BDV phosphoprotein and the p40 BDV nucleoprotein.

[0002] In a second aspect, the use of a diagnostic kit for a sandwich ELISA for detecting acute BDV infections is provided. This diagnostic kit comprises, as set forth in the claims, a first primary antibody and a second primary antibody, one of these primary antibodies being directed against the p24 BDV phosphoprotein, while the other primary antibody is directed against the p40 BDV nucleoprotein, with at least one secondary antibody labeled with a reporter molecule, means for immobilizing a primary antibody to a surface, and instructions for carrying out the method according to the invention.

[0003] In a third aspect, the present invention is directed to distinguishing acute BDV infections from chronic and dormant infections by combining the method according to the invention and the use of the diagnostic kit provided therewith, on the one hand, with a method for detecting BDV infections in which the freely circulating immune complexes (CIC) composed of BDV antigens and specific antibodies bound thereto are detected by immunological tests, as described, for example, in DE 19758017 C2, and, on the other hand, by further utilizing a method for detecting BDV infections in which freely circulating BDV antibodies in the infected host are detected by immunological tests. Accordingly, the use of diagnostic kits according to the invention comprises means for detecting BDV CIC and a corresponding diagnostic kit for detecting BDV antibodies.

[0004] Finally, the use of the method according to the invention for the diagnosis of acute BDV infections and / or their monitoring is provided. Particularly in combination with the methods for detecting BDV-CIC and BDV antibodies, the method according to the invention is provided for differentiating acute BDV infections from chronic and dormant infections and / or monitoring their progression and ruling out BDV infection. It is equally suitable for humans and animals. The state of the infection and its progression have a direct impact on the feasibility of antiviral therapy in clinically ill individuals. State of the art

[0005] The Borna virus (Borna Disease Virus; BDV or BoDV-1 according to the latest taxonomic designation) is taxonomically the leading virus of the species "Mammalian 1 bomavirus" in the virus family "Bornaviridae", which are for the order of the "Mononegavirales". Its virus strains, which are more than 95% genetically identical, infect the brain and blood cells of humans and many mammals and are widespread worldwide. In horses, it is the causative agent of Borna disease. Borna viruses are enveloped viruses (90 nm in diameter) with a negative-stranded non-segmented single-stranded (NSS) RNA of 8,910 kilobases, which encodes six genes. Borna viruses replicate in the nucleus of the host cell and are among the oldest viruses in evolutionary history, having coexisted with primate ancestors for at least 40 million years and even left lasting traces in their genetic makeup down to humans. Today, BDV infections occur in many farm animals and domestic animals (including horses, sheep, cattle, cats) and in humans.They remain in the infected organism for life (persistent infection without cell destruction), preferentially attack the developmentally older part of the brain (limbic system) and are involved in behavioral and mood changes.

[0006] It is estimated that one in three adults in Germany is infected with Borna viruses without being noticed, i.e., without clinical symptoms (Bode and Ludwig, 2003). One in six infected individuals (16-17%) is at increased risk of developing a mental disorder during their lifetime. In the population as a whole, one in twenty (5 out of 100 people; 5%) is at increased risk of disease. Chronic stress is thought to be the most significant risk factor, which weakens the immune system over the long term and promotes the activation of dormant Borna viruses.

[0007] The Borna virus preferentially infects the nerve cells of the limbic system of the brain. These central brain areas regulate behavior and emotions and are involved in learning processes and memory formation. The virus replicates primarily in nerve cells and can spread throughout the nervous system via their processes. The viral proteins N (p40 nucleoprotein) and P (p24 phosphoprotein) (antigens) are produced in excess. In the brains of symptomatic patients (e.g., in depression), they contribute to dysfunction of the balance of brain neurotransmitters (evidence from animal experiments).

[0008] BDV infections in animals are associated with intermittent behavioral disorders, including apathy, loss of attention, and impaired motor coordination. In humans, acute or chronic Borna virus infections have been found much more frequently, for example, in acute depressive episodes, obsessive-compulsive disorders, and chronic fatigue syndrome than in healthy individuals. Therefore, a contribution of Borna viruses to this highly complex disease process has been suspected for years, although this is still considered controversial (Bode and Ludwig, 2003). The proven benefit of antiviral therapy with amantadine supports this contribution. Endogenous recurrent depression, whether unipolar or bipolar, is one of the most significant psychiatric disorders in Germany and worldwide, with a lifetime prevalence of at least 5%.

[0009] There is therefore a need for reliable diagnostic systems to detect BDV infections, both for monitoring the infection phases in infected individuals and for monitoring the course and therapy, as well as for detecting undetected infected asymptomatic carriers, on a population-based basis for epidemiological reasons or on a person-by-person basis to determine the individual risk of disease.

[0010] For assessing the phases of infection (acute, chronic, dormant), the elevated expression of BDV antigens (N and P) in the blood has proven to be the decisive activity parameter. It correlates well with clinical disease (in humans and animals), is quantitatively measurable, and important for assessing the clinical course. Antigen determination cannot be replaced by the detection of viral nucleic acid via amplification with nested RT-PCR or real-time RT-PCR in PBMCs, as this only detects the virus itself, not its activity. Today, however, the diagnosis of Borna virus infections in many places is based on detection of the viral genome or is limited to the detection of antibodies using various serological methods.However, a negative detection of specific antibodies alone is not a suitable tool to exclude a BDV infection, since free antibodies are bound in the form of circulating immune complexes (CIC) in the serum of the infected person during an antigen-releasing replication phase and can temporarily fall below the detection limit.

[0011] In recent years, diagnostic procedures based on ELISA techniques have been performed, measuring BDV-specific immune complexes (CICs) as well as viral proteins and, where appropriate, freely circulating antibodies (Bode et al., 2001). Specific monoclonal antibodies are used. The amino acid sequences of the BDV nucleoprotein p40 and the phosphoprotein p24 have been known since the first two complete genomes of two BDV virus strains were sequenced in 1994 (Briese et al., 1994; Cubitt et al., 1994). A ribbon model of the three-dimensional structure of the nucleoprotein has been constructed (Rudolph et al., 2003).

[0012] The key reagents for the BDV ELISA tests, the monoclonal antibodies W1 (anti-N) and Kfu2 (anti-P), have been characterized in their basic properties (Ludwig et al., 1993). The BDV CICs are the most frequently detectable infection markers and have proven to be optimal for corresponding diagnostic tests.

[0013] Based on this, DE 19758017 C2 concerns corresponding methods for the detection of BDV infections, in which the freely circulating immune complexes of BDV antigens and the specific antibodies bound to them are detected by immunological tests. This patent also concerns a corresponding diagnostic kit, as mentioned above. An extension of this can be found in DE 19860255 C2, directed at a method for the detection of BDV infections, in which the antigen detection is carried out in a plasma sample, cerebrospinal fluid, or urine.

[0014] Corresponding blood tests for BDV CIC are used. The use of the immune complex (CIC), which is only detectable when the viruses have replicated in phases, is well-suited to indicate chronic infections in positive cases. The additional prior antigen test indicated a flare-up in positive cases. A negative CIC test required the additional antibody ELISA to detect a dormant infection or to rule out infection.

[0015] Current diagnostic systems and methods are based on the detection of CIC using a sandwich ELISA with monoclonal antibodies from a first species to bind the antigen portion in the CIC and then to detect the antibody portion of the bound CIC using an enzyme-labelled secondary anti-antibody from another species and a substrate reaction (colour change) corresponding to the relative amount of CIC in the sample.

[0016] The CIC-ELISA has proven itself as a basic test for the detection of chronic BDV infection. This test alone is not suitable for the detection of acute BDV infection, which is particularly important for clinically ill patients. The current antigen test is limited by high sample volumes and limited resources, making it unsuitable as a diagnostic kit in the long term.

[0017] Hsu TA, et. al., 1994, Virology, 204, 854 - 859 describes Borna Disease Virus p24 and p38 / 40 synthesis in a baculovirus expression system.

[0018] The present invention closes this gap by providing a completely new test method for acute BDV infections. Combined with the CIC test and antibody test, the new method is capable of distinguishing between acute, chronic, and dormant infections, thus raising BDV diagnostics to a previously unattained level of quality.

[0019] The present invention provides a completely new test method for acute BDV infections. The specific binding profiles of the monoclonal antibodies "W1" and "Kfu2" used in the methods according to DE 19758017 C2, which recognize potent conformational epitopes on the BDV N and P proteins, respectively, have now been identified (Bode, 2008). The present invention aims at detecting the active, phosphorylated form of the P protein and N / P heterodimers. The monoclonal antibodies described here are therefore particularly well suited for the new test method and the test methods combined with it. Description of the invention

[0020] The object of the present invention is to provide a method for detecting acute BDV infections and the corresponding use of diagnostic kits based on ELISA, particularly sandwich ELISA techniques. A further object is to provide improved methods for differential diagnosis.

[0021] In a first aspect, the invention is directed to a method for detecting acute Borna Disease Virus (BDV) infections according to claim 1, comprising the step of determining the presence of heterodimers of p24 BDV phosphoprotein (Seq. ID No. 2) and p40 BDV nucleoprotein (Seq. ID No. 1) in a sample by means of a sandwich ELISA, wherein a first primary antibody is immobilized and a second primary antibody is added in non-immobilized form after incubation of the sample with the immobilized first antibody, wherein the first primary antibody is directed against the p24 BDV phosphoprotein and the second primary antibody is directed against the p40 BDV nucleoprotein.

[0022] It was shown that the detection of an acute, currently active infection is possible if the first primary antibody only recognizes phosphorylated P protein (Bode, 2008). The phosphorylated form is essentially only detectable in the active state of the infection.

[0023] In a second aspect, the present invention is directed to the use of a diagnostic kit for a sandwich ELISA for the detection of acute BDV infections as set out in the claims. This diagnostic kit comprises a first primary antibody and a second primary antibody, wherein this first primary antibody is directed against the p24 BDV phosphoprotein and the other primary antibody against the p40 BDV nucleoprotein, wherein the primary antibody against the p24 BDV phosphoprotein is immobilized. The kit further comprises at least one secondary antibody labeled with a reporter molecule, optionally means for immobilizing the primary antibody to a surface, in particular means for immobilizing the primary antibody against the p24 BDV phosphoprotein, and instructions for carrying out the method according to the invention.

[0024] In a third aspect, the present invention is directed to distinguishing acute BDV infections from chronic and dormant infections, as set forth in the method in the claims. To distinguish from chronic infections, the present invention is further combined with methods such as those described, for example, in DE 19758017 C2 for detecting BDV infections, in which the freely circulating immune complexes (CIC) composed of BDV antigens and specific antibodies bound to them are detected by immunological tests.

[0025] To distinguish dormant infections, the method according to the invention is combined with methods for detecting BDV infections, in which freely circulating BDV antibodies against the p24 protein and / or the p40 protein in the infected host are detected by immunological tests. Detailed description of the invention

[0026] The present invention relates to a method for detecting acute Borna Disease Virus (BDV) infections according to the claims, comprising the step of determining the presence of heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein in a sample by means of a sandwich ELISA, wherein a first primary antibody is immobilized and a second primary antibody, non-immobilized, is added after incubation of the sample with the immobilized first antibody, wherein the first primary antibody is directed against the p24 BDV phosphoprotein and the second primary antibody is directed against the p40 BDV nucleoprotein. It has been shown that by using the two said primary antibodies, namely the antibody against the p24 BDV phosphoprotein on the one hand and the primary antibody against the p40 BDV nucleoprotein on the other hand, heterodimers formed from these two proteins can be detected.Such heterodimers, in which p24 is present in a phosphorylated form, are particularly present in the acute phase. This makes it possible to definitively detect an acute BDV infection.

[0027] The CIC-ELISA has proven itself as a basic test for the detection of chronic BDV infection. This test alone is not suitable for the detection of acute BDV infection, which is particularly important for clinically ill patients. The current antigen test is limited by high sample volumes and limited resources, making it unsuitable as a diagnostic kit in the long term.

[0028] The present invention closes this gap by providing a completely new test method for acute BDV infections. Combined with the CIC test and antibody test, the new method is capable of distinguishing between acute, chronic, and dormant infections, thus raising BDV diagnostics to a previously unattained level of quality.

[0029] The present invention provides a completely new test method for acute BDV infections because, among other things, the binding profiles of the monoclonal antibodies "W1" and "Kfu2" used in the method DE 19758017 C2 could be deciphered, which in particular determine the high specificity of the new method. The amino acid sequences of the binding sites that form the respective conformational epitopes are named here for the first time (Seq. ID. Nos. 3 to 7 for p40 and Seq. ID. Nos. 8 to 10 for p24). They represent essential molecular components of the properties of the aforementioned monoclonal antibodies.

[0030] The specificity requirements of the primary antibodies are such that they are monoclonal and recognize conformational epitopes that do not overlap with the binding domain of the BDV phosphoprotein on the BDV nucleoprotein, so that heterodimers can be recognized. Furthermore, the requirements of antibodies directed against the phosphoprotein are preferably such that the epitope includes the major phosphorylation site (Schwemmle et al., 1997) and recognizes only the active phosphorylated form of the protein.

[0031] According to the invention, the first primary antibody is therefore directed against the phosphorylated p24 BDV phosphoprotein. According to the invention, only p24 phosphorylated heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein are detected.

[0032] The BDV antigens p24 (P protein) and p40 (N protein) are produced in increased amounts during the viral activity surge and released into the blood plasma. These p24 and p40 proteins then preferentially form heterodimers of p24 and p40 in the blood, with the p24 protein in the heterodimer being phosphorylated after active viral replication and secretion into the plasma. Phosphorylation is an active process in the host cell following formation of the viral P protein.

[0033] The detection of an acute, currently active BDV infection is particularly possible by detecting heterodimers with phosphorylated BDV p24 phosphoprotein using the method according to the invention. According to the invention, this antibody directed against the phosphorylated form is a monoclonal antibody, for example, a mouse-derived monoclonal antibody. This antibody is the first primary antibody immobilized on a surface.

[0034] Detection of an acute, currently active BDV infection is assured by the fact that the monoclonal antibody binding the phosphorylated form, such as the primarily used mAb "Kfu2" (see Ludwig et al., 1993), recognizes a conformational epitope formed by three binding sites on the active phosphorylated BDV P protein (SEQ ID NOs. 8, 9, and 10). The second primary monoclonal antibody, such as the primarily used mAb "W1" (see Ludwig et al., 1993), recognizes a conformational epitope formed by five binding sites on the BDV N protein (SEQ ID NOs. 3, 4, 5, 6, and 7). There is no overlap between these binding sites on either protein and the binding domain of the P protein (Berg et al., 1998) on the N protein. This allows N / P heterodimers to be detected (Bode, 2008).

[0035] In one embodiment, the first primary antibody is immobilized to the surface directly; in an alternative embodiment, the immobilization is carried out via a first secondary antibody which, for example, binds the primary antibody in a species-specific, in particular subclass-specific manner, without impairing the activity of this primary antibody for binding to the heterodimer of p24 BDV phosphoprotein and p40 BDV nucleoprotein, in particular the p24 phosphorylated BDV phosphoprotein.

[0036] The immobilization of the primary antibody or the first secondary antibody to a surface is carried out according to conventional methods. Such methods are known to those skilled in the art. Immobilization is usually carried out via the Fc region of the secondary antibody or the primary antibody.

[0037] The method described here for detecting infection in serum / plasma is primarily based on the infection of peripheral blood mononuclear cells (PBMCs) in such a way that the virus's proliferation in phases leads to the secretion of N and P proteins (antigens) from PBMCs (and as yet unknown sources in the body) into the blood plasma (acute infection). Subsequently, antibodies are formed in the host, and these bind to the antigens to form circulating immune complexes (CICs), which indicate a chronic infection. The term "detection" is used here to indicate both initial qualitative detection and quantitative determination of the concentration of heterodimers.

[0038] In a further embodiment of the method according to the invention, the non-immobilized primary second antibody is determined via a second secondary antibody comprising a reporter molecule.

[0039] The term "reporter molecule" is understood here to mean a molecule that, on the one hand, allows for direct measurement, for example, also known as a label or tag. On the other hand, this reporter molecule can have a first binding parameter, which is detected with another labeled molecule (second binding partner) as a binding pair. Finally, the reporter molecule can comprise a catalyst, such as an enzyme.

[0040] Examples of suitable reporter molecules include known reporter molecules, such as dyes, including fluorescent dyes, or radioactive molecules. Alternatively, this reporter molecule can be a known binding pair, such as a biotin-streptavidin or biotin-avidin pair. Finally, the reporter molecule can be a catalyst, particularly an enzyme. Known enzymes include alkaline phosphatase, peroxidase, particularly horseradish peroxidase, and other common enzymes.

[0041] The person skilled in the art is familiar with corresponding reporter molecules, such as those used in particular in immune-based detection methods, in particular in ELISA methods.

[0042] In one embodiment, the second secondary antibody is one that contains biotin as a reporter molecule. This antibody is then detected using an appropriate binding partner, such as avidin or streptavidin, coupled with an enzyme, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

[0043] Alternatively, this second secondary antibody can be directly labeled with an enzyme such as AP or HRP.

[0044] In one embodiment, the two secondary antibodies are from the same species; in another embodiment, the two secondary antibodies originate from different species. In one embodiment, the two secondary antibodies are those that, originating from the same species or different species, recognize different immunoglobulin subclasses of the species of the primary antibodies.

[0045] In one embodiment of the present invention, the primary antibodies can be those in which the first primary antibody and the second primary antibody originate from different species, and in which these primary antibodies are monoclonal antibodies. In one embodiment, the primary antibodies originate from the same species but from different immunoglobulin classes or different immunoglobulin subclasses. In one embodiment, the primary antibodies originate from the mouse and are monoclonal antibodies, for example, in which one of the primary antibodies is one of the IgG1 subclass and the second primary antibody is a mouse antibody of the IgG2 subclass.

[0046] If the primary antibodies originate from different subclasses, such as IgG1 and IgG2, then the secondary antibodies are preferably those that specifically detect these subclasses, i.e., bind to these subclasses. This can minimize cross-reactivity.

[0047] The primary antibodies can be modified to contain other components or to be single-chain antibodies. The primary antibodies are characterized by the fact that they specifically recognize the corresponding BDV protein and, if required, contain the necessary binding site for the secondary antibody. If the primary antibody is directly coupled to the surface, this binding site does not need to be present for the secondary antibody. With direct coupling, the primary antibody can be modified to promote binding to the surface.

[0048] Those skilled in the art are familiar with suitable methods for modifying primary antibodies using appropriate molecular biological techniques. This preserves the specificity of the primary and, if applicable, secondary antibodies, while removing components that may cause cross-reactivity. These modifications support the improvement of specificity.

[0049] The term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, and chimeric antibodies, unless otherwise stated. The antibodies can be produced from recombinant sources and / or by hybridoma cells. Alternatively, transgenic animals, etc., can be used. The antibodies can also be present as antibody fragments, provided they retain specificity for the p24 BDV phosphoprotein and p40 BDV nucleoprotein. The term "antibody fragments" as used herein includes suitable scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, as well as fab, fab, or f(ab')2 fragments.

[0050] Such fragments can be produced by appropriate recombinant techniques.

[0051] In the present invention, preference is given to monoclonal antibodies produced using suitable conventional techniques, i.e., hybridoma techniques. This technique, in combination with infection and immunization of mice, made it possible to select antibodies that recognize epitopes on the native conformation of proteins N and P. Conformational epitopes, such as those present for "W1" and "Kfu2," exhibit particularly high binding constants for native BDV proteins (Bode, 2008), which are not achieved for linear epitopes of monoclonal antibodies produced using recombinant proteins.

[0052] The reporter molecule, also referred to as a marker or label, allows the direct or indirect generation of detectable signals. Suitable labels or markers are commonly known radioisotopes, but in particular also dyes such as fluorescent dyes (fluorophores) or chemiluminescent compounds (chromophores). Examples of these are fluorescein isothiocyanate (FITC), rhodamine, or luciferin. In one embodiment, the reporter molecules are enzymes, in particular alkaline phosphatase, β-galactosidase, or horseradish peroxidase, which convert substrates into signals with a color change. Imaging agents such as magnetic or paramagnetic marker molecules or metal ions are also included. In one embodiment, the reporter molecule is coupled to the secondary antibody via a binding partner of a binding pair, such as the streptavidin-biotin binding pair or the biotin-avidin binding pair.Suitable binding pairs are known to the person skilled in the art.

[0053] In a further third embodiment, in addition to the detection of the heterodimer, circulating immune complexes (CIC) composed of BDV antigens and specific antibodies bound thereto are also detected. Corresponding detection methods are known, for example, from DE 19758017 C2. The combination of the detection of the phosphorylated heterodimer according to the present invention with the detection of circulating immune complexes (CIC) and specific antibodies bound therein from the individual increases the specificity for detecting a BDV infection, in particular also for differentiating between acute BDV infection and chronic BDV infection. In a further embodiment, in addition to the detection of the heterodimer and the CIC, the detection method for BDV antibodies, also known from DE 19758017 C2, is carried out.The combination of the detection of the heterodimer according to the present invention with the CIC detection and the detection of free antibodies enables the differentiation of acute, chronic and dormant infection in the individual, see also . Figure 1 .

[0054] The method according to the invention is, in particular, one that enables the diagnosis and / or monitoring of the course of a BDV infection and / or is used to rule out a BDV infection, e.g., in psychiatric and / or neurological disorders. This means that the method according to the invention is one that diagnoses an acute BDV infection. In another aspect, it is a method that is suitable for monitoring the course of a BDV infection in combination with a CIC test and an antibody test, and in particular for differentiating between acute, chronic, and dormant BDV infection. It is also suitable, in the above combination, for detecting or ruling out BDV infections of various activity states in psychiatric and / or neurological disorders.

[0055] In a further aspect, the present invention is directed to the use of a diagnostic kit for a sandwich ELISA for detecting acute BDV infections as defined in the claims. This diagnostic kit comprises a first primary antibody and a second primary antibody. One of these two primary antibodies is directed against the p24 BDV phosphoprotein, the other primary antibody against the p40 BDV nucleoprotein. The diagnostic kit further comprises suitable secondary antibodies, but at least one secondary antibody labeled with a reporter molecule. Optionally, means for immobilizing a primary antibody to a surface and instructions for carrying out the method according to the invention using this kit are also provided.

[0056] The primary antibodies and secondary antibodies present in the kit are those described herein. The primary antibodies are those where one of these primary antibodies recognizes the phosphorylated protein of the p24 phosphorylated heterodimer, while the second primary antibody recognizes the p40 BDV nucleoprotein in the heterodimer. The secondary antibodies are those secondary antibodies as defined in the present application, in particular those originating from the same species and / or those recognizing different subclasses of primary antibodies.

[0057] In one embodiment, the diagnostic kit further comprises means for detecting CIC formed from BDV antigens and specific antibodies of the individual attached thereto. These means are, in particular, corresponding monoclonal antibodies.

[0058] Finally, the use of the method according to the invention and the diagnostic kit for the diagnosis and / or follow-up and / or exclusion of BDV infections in psychiatric and / or neurological diseases is provided.

[0059] The method according to the invention and the use of the method and the diagnostic kit allow a treatment plan for the BDV infection in the examined individual to be developed as an option for the treating physician. Accordingly, a method for treating acute BDV infections is provided, comprising the method for detecting an acute BDV infection according to the present invention and, upon diagnosis of an acute BDV infection, treating it with the antiviral drug amantadine sulfate (AS). This drug has proven highly effective against natural Borna viruses. AS has been approved for the antiviral treatment of viral influenza (influenza A) for over 40 years and can be prescribed for the treatment of Borna virus infection (off-label use). AS inhibits the replication of natural Borna viruses and thus the formation of harmful viral proteins (antigens) (Bode and Ludwig, 2003).The majority of infected patients with acute and / or chronic depression (70%) benefit sustainably from symptom improvement (Dietrich et al., 2000; Ferszt et al., 1999) parallel to the decline in viral markers in the blood. The antiviral effect of AS has also been demonstrated in infected patients whose depression was already in remission (no longer depressive symptoms). Follow-up in blood plasma using ELISA tests for determining CIC, antigen (pAG, previous test), and antibodies according to method DE 198 60 255 C2 showed a significant reduction in these test parameters after 14 weeks (Dietrich and Bode, 2008).

[0060] AS is proven effective against natural Borna viruses of humans and horses in vitro in a dose-dependent manner. The titer of the human virus isolate Hu-H1 from a depressed bipolar patient (Bode et al., 1996) in human oligodendroglial cells is undetectable within 10 to 33 days, depending on amantadine sulfate concentrations ranging from 1.2 µg / ml to 0.4 µg / ml. The concentration of 0.4 µg / ml of AS correlates with the concentration achieved in the patient's plasma upon oral administration of 200 mg of AS per day (Bode et al., 1997).

[0061] Furthermore, a method for treating psychiatric and / or neurological disorders is provided, comprising the detection of a BDV infection, in particular an acute BDV infection, according to the present invention and, upon detection of a BDV infection, in particular detection of an acute BDV infection or detection of a chronically activated infection in combination with CIC. One treatment option for the individual can be treatment with 2 - 4 mg AS per kg of body weight daily orally. For a 75 kg patient, this is 150 to a maximum of 300 mg AS daily. Start with 1 mg AS per kg of body weight for the first three to four days. Dosage schedule: in the morning during the induction phase. Thereafter, the dosage schedule begins with 2 mg AS per kg of body weight, with half the daily dose in the morning and the other half of the daily dose at midday (1-1-0). In case of restlessness in the first one to two weeks or difficulty falling asleep, only take the daily dose in the morning (1-0-0).

[0062] Furthermore, a method for treating a BDV infection is provided, wherein the treatment is monitored as part of a follow-up using the method according to the invention. The treatment is carried out in such a way that if the treatment course is positive, characterized by clinical improvement on average after the first four to six weeks and reduced viral activity, characterized by a reduction in heterodimers and / or CIC in the blood plasma, the treatment can be completed after three months. A check of the blood values ​​for antigen (heterodimers) and CIC is mandatory after three months. Before discontinuing the medication, no heterodimers should be detectable and the CIC values ​​should be lower than before treatment. The medication should be tapered off with a dose reduction over four to seven days.

[0063] In the case of delayed treatment, characterized by slight clinical improvement after the first four to six weeks and only slight change or unchanged detection of these heterodimers, particularly the phosphorylated heterodimers, possibly in combination with CIC, treatment should be continued for a period of three months and the therapy modified if necessary. As a precautionary measure, in the case of slight clinical improvement, follow-up after 6 weeks using the method according to the invention in combination with CIC is mandatory and an adjustment of the therapeutic dose is indicated if antigen and / or CIC values ​​are low or unchanged. If the dose was previously 2 mg AS per kg body weight, an increase to 3 mg AS per kg body weight up to a maximum of 4 mg AS per kg body weight is indicated in the described constellation.

[0064] Common treatments for BDV infection in depressed psychiatric patients include the prescription of common antidepressants, such as tricyclic antidepressants or selective serotonin reuptake inhibitors (SSRIs). AS is excreted unchanged in the kidneys; there are no metabolites in the liver, and therefore no or negligible adverse interactions with these medications. Therefore, AS can be prescribed in addition to combat BDV infection without discontinuing the above-mentioned therapeutic agents. In patients with renal insufficiency requiring dialysis, an undesirable increase in AS blood levels may occur. Therefore, in such patients, the above-mentioned dosage should be taken only every second or third day, and blood levels of the drug should be monitored weekly if necessary.

[0065] Body fluids and body tissue in isolated form can be used as samples. Particularly suitable samples are blood samples, e.g., in the form of serum or plasma. Other suitable samples include cerebrospinal fluid (CSF) in neurological patients, but a negative result does not rule out infection, which is why a blood sample, either serum or plasma, is mandatory. In one embodiment, the sample is an isolated sample of blood plasma or blood serum. Description of the characters

[0066] Figure 1shows the diagnostic scheme for acute, chronic, and dormant BDV infections. In a first test, the presence of the antigen is tested according to the method of the invention. If the antigen is present, an acute infection exists; if the antigen is negative, further testing is carried out to determine whether CIC is present. If CIC is present, a chronic infection is assumed; if CIC is negative, additional testing is carried out for antibodies. If no antibodies are present, the presence of an infection is ruled out; whereas if antibodies are detected exclusively (anti-p24 protein and / or anti-p40 protein antibodies), a dormant infection is assumed. Figure 2shows a scheme of the BDV antigen enzyme immunoassay (EIA) according to the method of the invention. The monoclonal mouse antibody Kfu2, directed against phosphorylated p24 protein, is immobilized on a microtiter plate by binding to a secondary antibody with anti-mouse IgG1 subclass specificity and then used to capture the heterodimers from the plasma / serum sample. These bound heterodimers are then detected with the monoclonal mouse antibody W1, directed against p40 protein. To visualize the reaction, a secondary antibody with anti-mouse IgG2a subclass specificity is used. Labeling of this antibody with reporter molecule biotin and subsequent streptavidin-mediated binding of alkaline phosphatase allows the corresponding color reaction to occur after addition of the substrate p-nitrophenyl phosphate. Figure 3shows a scheme of the BDV immune complex EIA, as described, for example, in DE 19758017 C2. There, both monoclonal mouse antibodies W1 and Kfu2 are immobilized on a microtiter plate via binding to a secondary antibody with anti-mouse IgG specificity and then used to capture the antigen-antibody complexes from the plasma / serum sample. These antigen-antibody complexes (BDV-CIC) are then incubated with enzyme-labeled secondary antibodies directed against the IgG of the host species in the sample (reporter molecule alkaline phosphatase) and a corresponding color reaction (as in Figure 2 ) proven. Figure 4shows a diagram of the BDV antibody EIA, as it can be used according to the invention. Here, the described monoclonal antibodies W1 and Kfu2 are also immobilized on a microtiter plate via binding to a secondary antibody with anti-mouse IgG specificity and then used as capture antibodies to initially bind native BDV antigens, which have previously been obtained in virus-free form from standardized infected cell culture. These bound BDV antigens then enable the detection of anti-BDV antibodies (anti-p24 or anti-p40 protein antibodies) from a plasma / serum sample. This is done after binding of anti-BDV antibodies present in the sample to the native antigen, then via enzyme-labeled secondary antibodies directed against the IgG of the host species of the sample (reporter molecule alkaline phosphatase) and a corresponding color reaction (as in Figure 2 ). Figure 5shows the results of antigen tests on individual samples from test persons, whereby BDV antigen is detected on the one hand with the method according to the invention (New pAg) and on the other hand with the earlier antigen detection method (Old pAg) described in DE 198 60 255 C2. Figure 6 shows the results of the combined application of the antigen test according to the invention with the CIC test and the antibody test, the latter two tests as described in DE 19758017 C2. The absorbance values ​​are shown cumulatively. Figure 7shows the complete amino acid sequence of the BDV N protein (Seq. ID. No. 1). The five binding sites N1 to N5 (Seq. ID Nos. 3 to 7) for the described monoclonal antibody W1, identified by epitope mapping, form the conformational epitope on the native BDV N protein, with N1, N3, and N5 being interactive binding sites according to comparison with the crystal structure (Bode, 2008). The binding sites are framed in black. The two domains that define the binding sites of the P protein on the N protein, amino acids 51–100 and 131–155, are framed in dashed lines (Berg et al., 1998). These show no overlap with the W1 binding sites. This allows N / P heterodimers to be detected. Figure 8shows the complete amino acid sequence of the BDV P protein (SEQ ID NO: 2). The three binding sites P1 to P3 (SEQ ID NO: 8 to 10) for the described monoclonal antibody Kfu2, identified by epitope mapping, form the conformational epitope on the native P protein. The binding sites are boxed in black. P1 is the main binding site and includes the main phosphorylation site (S26 / S28; Schwemmle et al., 1997) on the BDV P protein (boxed in dashed lines). Kfu2 exclusively recognizes the active, phosphorylated BDV P protein (Bode, 2008).

[0067] The invention is further explained with the aid of examples, without being limited to them. Examples

[0068] Isolated blood samples are provided from individuals demonstrating BDV infection, with the status of infection being either acute, chronic, dormant, or no infection. Table of medical personnel Chongqing study code Medical personnel (MP) Gender Old Antigen heterodimers CIC antibody Infection status sample Diluted 1:10 Diluted 1:20 Diluted 1:100 E3875 not specified female 50 0,066 0,055 no E4064 Administration masculine 51 0,073 0,027 no E3728 Ophthalmology female 29 0,059 0,033 no E3667 Gastroenterology masculine 28 0,074 0,067 no E3977 Infectious Diseases Department female 48 0,076 0,2835 chronic E4173 Breast surgery female 47 0,0815 0,2625 chronic E4272 Surgical preparation female 49 0,0605 0,6325 chronic E3731 Ophthalmology female 52 0,044 0,607 0,183 chronic E3693 Cardiac surgery female 59 0,0755 0,587 0,237 chronic E3838 not specified female 26 0,142 0,8975 acute E3727 Ophthalmology female 55 0,029 0,155 chronic E4265 Exp. Research female 41 0,2455 0,173 0,044 acute E3630 out of service female 35 0,214 0,1305 0,097 acute E4113 rehabilitation masculine 81 0,166 0,306 acute E4278 Surgical preparation masculine 29 0,236 0,715 acute E4123 rehabilitation masculine 30 0,2195 0,366 acute E3968 Positive control MP masculine 57 1,9835 0,3675 0,3025 acute Student Negative control 0,0495 0,068 no Evaluation of test results (standard for all BDV-EIA tests) Cut-off=0.100. Negative <=0.1; Borderline > 0.1-0.12; (+) weakly positive > 0.12-0.15; 1+ positive > 0.15-0.3 2+ positive > 0.3-0.6; 3+ positive >0.6-1.0; 4+ positive >1.0. The samples were obtained from a large study conducted at a university hospital in Chongqing, China, using the CIC and additional antibody tests and real-time RT-PCR (Liu et al. 2015). The tests were performed using the methods described in the DE 197 58 017 C2 used antibodies W1 and Kfu 2. W1 represents the primary antibody directed against the P40 BDV nucleoprotein, Kfu 2 represents the primary antibody directed against phosphorylated P-protein P24 phosphoprotein of BDV. The test setup for CIC detection and antibody detection follows the respective scheme in the Figures 3 and 4 The test setup of the method according to the invention for antigen detection (heterodimers) follows the scheme in Figure 2 . Material inventive method:

[0069] AffiniPure goat anti-mouse IgG1, Fc fragment-specific antibodies (concentration 1.3 mg antibody per ml), minimal cross-reaction with human, bovine, and rabbit serum proteins (code number 115-005-205, Jackson ImmunoResearch). Biotin-SP conjugated AffiniPure goat anti-mouse IgG2a, Fc fragment-specific antibodies, minimal cross-reaction with human, bovine, and rabbit serum proteins (code number 115-065-206, Jackson ImmunoResearch). Alkaline phosphatase-conjugated streptavidin (code number 016-050-084, Jackson ImmunoResearch). Alkaline phosphatase substrate kit (code number 172-1063, Biorad) or individual substances for substrate buffer and substrate (Bode et al.)., 2001) as follows: Substrate buffer, diethanolamine buffer pH 9.8 (diethanolamine 1.007 M + MgCl 2 6 H 2 O 0.5 mM + NaN 3 0.003 M); substrate, p-nitrophenyl phosphate hexahydrate (pNPP 6 H 2 O) (0.003 M); 5 mg tablets (code number N9389 Sigma Aldrich); always prepare fresh 1 mg pNPP per ml substrate buffer in the assay. Conjugate buffer (alkaline phosphatase) TBS-Tween pH 8.0 (TRIS 0.02 M + NaCl 0.14 M + KCl 0.003 M + 0.05% Tween 20). Wash buffer, NaCl-Tween (0.9% NaCl + 0.05% Tween 20 + NaN3 0.003 M). Sample dilution buffer (also for steps 2, 4, and 5 in . Figure 2 ), PBS-Tween pH 7.2 (KH 2 PO 4 0.0015 M + Na 2 HPO 4 · 12 H 2 O 0.008 M + NaCl 0.14 M + KCl 0.003 M + 0.05% Tween 20 + NaN 3 0.003 M). Coating buffer, 0.01 M sodium phosphate / 0.25 M NaCl pH 7.6 (NaH 2 PO 4 · 2 H 2 O 0.02 M [stock A], Na 2 HPO 4 0.02 M [stock B]; coating buffer contains 65 ml stock A + 435 ml stock B + 0.25 M NaCl in 1000 ml aqua bidest. Stop solution, 3 M NaOH

[0070] All chemical substances must be of ACS grade, preferably from Sigma-Aldrich. Nunc-Immuno 96-well microtiter plates for ELISA, MaxiSorp, F (flat bottom) (code number 442404, Nunc). BDV-specific key reagents

[0071] W1 (mouse subclass IgG2a) specifically directed against the BDV nuclear protein (N protein); Kfu2 (mouse subclass IgG1) specifically directed against the BDV phosphoprotein (P protein); W1 and Kfu2 are monoclonal mouse antibodies, first described by Ludwig et al., 1993. The inventors have already produced hybridoma supernatants of these two antibodies, which are suitable for industrial-scale test kit production. The inventors also have hybridoma cells that allow for the unlimited production of additional batches of the monoclonal antibodies W1 and Kfu2. Further characterization of W1 and Kfu2 (Bode, 2008)

[0072] Short name W1 species Mouse Subclone TL9 IgG subclass IgG 2a Specificity N-protein p40 of BDV (reference strain V) Dissociation constant (K D ) 2.31×10 -9< +- 0.26×10 -9< M (native N protein) Epitope type Conformational epitope (discontinuous) Hybridoma supernatant (=ready-to-use) 1:500 working dilution in BDV ELISAs Detection of recombinant (r)N protein Yes (test with previous antigen ELISA) Detection limit of purified rN 1.5 - 3 nanograms per mL Short name Kfu2 species Mouse Subclone 57-4-33 IgG subclass IgG1 Specificity P-protein p24 of BDV (reference strain V) Dissociation constant (K D ) 3.33×10 -9< +- 0.34×10 -9< M (native P protein) Epitope type Conformational epitope (discontinuous) Hybridoma supernatant (=ready-to-use) 1:500 working dilution in BDV ELISAs Detection of recombinant (r)P protein non-phosphorylated No (test with previous antigen ELISA) Recognition of rP, phosphorylated in vitro Yes (test with previous antigen ELISA) First description of the epitope sequences of W1 and Kfu2

[0073] Epitope on N protein p40 of BDV SEQ ID No. W1 Epitope type Conformational epitope (discontinuous) Number of binding sites Five 5mer peptides each (N1-N5) N1 3 Amino acid sequence 32_KFLQY_36 N2 4 Amino acid sequence 116_AKFYG_120 N3 5 Amino acid sequence 168 TMMAA 172 N4 6 Amino acid sequence 294 LAPRS 298 N5 7 Amino acid sequence 307_FYWSK_311 See also Figure 7

[0074] Epitope on P-protein p24 of BDV SEQ ID No. Kfu2 Epitope type Conformational epitope (discontinuous) Number of binding sites Three (15mer, 12mer, 5mer peptides), P1-P3 P1, main domain, phosphorylation site serine 26 / 28 8 Amino acid sequence 22_RRERSGSPRPRKVPR-36 P2 9 Amino acid sequence 46_LLKDLRKNPSMI_57 P3 10 Amino acid sequence 142_DRSMK_146 See also Figure 8 Protocol of the inventive method:

[0075] 100 µl of a 1:1000 dilution of the goat anti-mouse IgG1 secondary antibody diluted with coating buffer is pipetted into the MaxiSorp F microtiter plates and incubated for one hour at 37°C. Three wash cycles with the wash buffer (120 ml total) are then performed. The monoclonal antibody Kfu2, diluted 1:500 (hybridoma supernatant) in PBS-Tween, is then incubated at 37°C for one hour with 100 µl of the monoclonal antibody. The samples (diluted 1:10 with PBS-Tween) are pipetted into the microtiter plates at 100 µl per well and incubated for one hour at 37°C, followed by three wash cycles with the wash buffer. Subsequently, the monoclonal antibody W1 is added at a dilution of 1:500 as hybridoma supernatant diluted in PBS-Tween at 100 µl per well, incubated for one hour at 37°C, followed by three wash cycles.Subsequently, the goat anti-mouse IgG2a secondary antibody labeled with biotin at a dilution of 1:1000 in PBS-Tween is added in an amount of 100 µl per well, incubated for one hour at 37°C and then washed with three wash cycles.

[0076] Then, alkaline phosphatase-labeled streptavidin, diluted 1:1000 in TBS-Tween pH 8.0 conjugate buffer, is added at a volume of 100 µl per well and incubated for 30 min at 37°C. This is followed by three wash cycles with wash buffer.

[0077] The detection reaction is then carried out with p-nitrophenyl phosphate (1 mg / ml) of p-NPP in substrate buffer (pH 9.8). 100 µl of the corresponding p-NPP solution is added, followed by incubation at room temperature for 2–5 minutes. Continuous visual control is required; the negative control must remain colorless, while the positive control changes color. The color reaction is stopped by adding 50 µl of the stop solution (3 M NaOH) to each well. The absorbance is immediately determined at 405 nm using a suitable multichannel photometer. A "blank" measurement is performed against unreacted substrate. Determination of the CIC according to the procedures described in DE 19758017 C2

[0078] The determination of CIC in the samples was carried out according to the procedures described in DE 19758017 C2 (see scheme above in Figure 3To optimize the diagnostic kit for CIC determination for practical use, the incubation times and temperatures were standardized to 1 h at 37°C for all steps, except for the visualization steps, i.e., substrate incubation (C3), stop solution addition (C4), and measurement (C5). The procedure for monitoring the development of the color reaction was the same as for the invention described above. The initial dilution of the samples to be tested of 1:20 in individual determinations with validation against positive and negative controls offers the same level of security as the inclusion of further dilutions.

[0079] When describing the materials, the code numbers are added below: AffiniPure goat anti-mouse IgG, Fc fragment-specific antibody (code no. 115-005-071; Jackson ImmunoResearch). Alkaline phosphatase-conjugated AffiniPure goat anti-human IgG, Fc fragment-specific antibody, minimal cross-reactivity with serum proteins from cattle, horses, and mice (code no. 109-055-098; Jackson ImmunoResearch) for human samples. Alkaline phosphatase-conjugated AffiniPure goat anti-horse IgG, Fc fragment-specific antibody (code no. 108-055-008; Jackson ImmunoResearch) for horse samples. Key reagents W1 and Kfu2, substrate kit or substrate buffer and substrate tablets, buffer, and Nunc MaxiSorp F ELISA plates as described above for the new invention. Determination of antibodies in plasma / serum according to the procedures described in DE 19758017 C2

[0080] The determination of antibodies against the p24 protein and / or p40 protein in plasma / serum samples was carried out in principle according to the procedure described in DE 19758017 C2 (see scheme above in Figure 4). Analogous to the CIC test, to optimize the diagnostic kit for antibody determination for practical use, the incubation times and temperatures were standardized to 1 h at 37°C for all steps, except for the visualization steps. Furthermore, instead of the described initial dilution of 1:50, the dilution of the samples was adjusted to 1:100. It was shown that the initial dilution of 1:100 for the samples to be tested for antibodies in individual determinations with validation against positive and negative controls offers the same level of reliability as the inclusion of additional dilutions.

[0081] To perform the BDV antibody EIA described in DE 19758017 C2, detection antigen from BDV-infected cells is required. The working dilution of the detection antigens from infected cell cultures is between 1:100 and 1:300, depending on the batch.

[0082] On December 12, 1997, a human oligodendroglial cell line (OLIGO / TL) persistently infected with BDV strain V (Briese et al., 1994) was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig under the number DSM ACC2334, which enables the unlimited production of detection antigen.

[0083] For the description of the other materials, see CIC test. Results:

[0084] In the Figure 5 A comparison of the method according to the invention with the previous antigen test is shown. Since the previous antigen test uses a sample dilution of 1:2, its results are included both for this dilution and for the 1:10 dilution applicable to the test according to the invention.

[0085] The Figure 5shows that sample E4265 was only positive with the test according to the invention, while it was not detected with the previous antigen test, neither at 1:10 nor at 1:2 sample dilution. Sample E3630 was weakly positive with the test according to the invention. The positive signal was confirmed in a further investigation (Table and Figure 6 ). The previous antigen test also showed a weakly positive to borderline positive signal, but only at a 1:2 dilution. The next dilution, 1:4, was already clearly negative with an absorbance value of 0.034 (not shown), resulting in a negative result. A repeat test was no longer possible due to the high sample volume for a dilution series starting at 1:2, i.e., 100 µl.

[0086] Samples E3727 and E3731 were negative in both antigen tests, including the 1:2 dilution in the previous antigen test. Both samples came from the doctor / nurse team in the clinic's ophthalmology department. However, the combination with the CIC test shows that both were chronically infected with BDV (see table). Team member E3731 had high CIC values ​​and additional antibodies, which suggests a recent acute viral flare.

[0087] For samples E4265 and E3630, which were only positive with the new test at a sample dilution of 1:10, it should be emphasized that the previous test was not able to detect BDV antigens, or at least not clearly, even at its standard five-fold lower sample dilution of 1:2. The test according to the invention thus exhibited at least five times higher sensitivity than the previous test and was able to demonstrate that the medical staff (E4265 female, research; E3630 female, off-duty) were experiencing an acute BDV infection at the time of sample collection. If only the previous antigen test had been available, the diagnosis of "chronic infection" would have had to be made due to the simultaneous detection of CIC, and the acute exposure would have been overlooked (see table).

[0088] Results of the combined application of the antigen test according to the invention with the CIC test and the antibody (AK) test according to DE 19758017 C2 show Figure 6The values ​​from Table 1 are thus clearly represented by cumulative columns.

[0089] The Figure 6 illustrates that the test according to the invention was able to identify six acute infections in the example panel (E4123, E4278, E4113, E3630, E4265, and E3838). Negative results from the test according to the invention, in combination with the CIC test, were able to identify six chronic infections (E3727, E3693, E3731, E4272, E4173, E3977). Negative CIC and antibody tests were able to exclude BDV infection in four cases from the panel (E3667, E3728, E4064, E3875).

[0090] The sample panel of medical personnel, who, according to structured interviews, had no history of mental illness, either past or present, was chosen to demonstrate that the test according to the invention, alone and in combination with CIC and antibody tests, is capable of differentiating between acute, chronic, and dormant infections, regardless of clinical symptoms, and of ruling out BDV infection with a single blood sample. Chronic stress is common in everyday medical practice, so an increased risk of activation does not seem unusual. In addition, there is an increased risk of infection in the daily care of sick people.

[0091] The significantly higher sample volume of 100 µl for the previous antigen test, with an initial 1:2 dilution and subsequent dilution series, was only available for selective parallel testing, as in Figure 5shown, available for the study samples that have already been examined many times elsewhere.

[0092] The advantages of the novel method for antigen detection according to the invention are diverse and practical even without direct comparison.

[0093] On the one hand, it allows for broad applicability for a wide variety of sample materials, meaning any type of body or tissue fluid. BDV activity is measured independently of the host species. Furthermore, the determination of phosphorylated heterodimers of p24 and p40 allows the verification of acute BDV infections, since the primary antibody Kfu2 binds only the active phosphorylated form of the BDV P protein. This is a unique feature of the monoclonal antibody Kfu2 used here.

[0094] In contrast to the test described in DE 19758017 C2, the heterodimer is determined here. In contrast, the previous method is based on the use of two capture antibodies, i.e., immobilized primary antibodies against both p24 and p40, in order to also determine monomeric forms of these. Accordingly, however, differentiation of the phosphorylated heterodimers present could not be achieved. In one embodiment, the method according to the invention uses secondary antibodies, which allow for high reproducibility. In the embodiment with the binding pair biotin-streptavidin coupled to an enzyme such as alkaline phosphatase, signal amplification and thus improved detectability are achieved compared to the previous visualization of the signal solely using enzyme-labeled anti-antibodies.Furthermore, significantly less patient sample is required (10 µl for a single determination, 20 µl for dilution series starting at 1:10), and the sensitivity of the method is higher due to the fivefold higher initial sample dilution of 1:10. This was particularly evident in the fact that acute BDV infection could be identified in more individuals than with the previous antigen test.

[0095] The present invention provides a completely new test method for acute BDV infections that exhibits higher sensitivity than the previous antigen test and is independent of limited resources, such as rabbit antibodies. Combined with the CIC test and antibody test, the new method is capable of reliably distinguishing between acute, chronic, and dormant infections (in humans and animals), thus raising BDV diagnostics to a previously unattained level of quality.

[0096] The method according to the invention is therefore particularly suitable for diagnosing and monitoring the progression of a BDV infection, particularly for determining the treatment plan. Furthermore, the method according to the invention can be part of a treatment procedure.

[0097] The previously unpublished amino acid sequences of the binding sites that form the respective conformational epitopes are identified here for the first time. They represent essential molecular components of the properties of the monoclonal antibodies W1 and Kfu2.

[0098] The present invention provides a completely new test method for acute BDV infections that is independent of limited resources and can be used across species in humans and animals. By combining the new diagnostic kit with the use of known methods or kits for the detection of circulating immune complexes (CIC) and antibodies in plasma or serum, the new method is capable of reliably distinguishing between acute, chronic, and dormant infections (in humans and animals) and achieving previously unattained diagnostic accuracy. Cited literature (alphabetical)

[0099] Berg M, et al., J Gen Virol. 1998; 79 (Pt 12):2957-2963. Bode L. Human Bornavirus infection - towards a valid diagnostic system. APMIS Suppl 124, 2008; 116: 21-39. Bode L, et al. The Lancet 1997; 349: 178-179. Bode L, et al. Mol Psychiatry 1996; 1: 200-212. Bode L, Ludwig H. Borna disease virus infection, a human mental-health risk. Clin Microbiol Rev. 2003; 16(3):534-545. Review. Bode L, et al. Mol Psychiatry 2001; 6: 481-491. Briese T, et al. Proc Natl Acad Sci U S A. 1994; 91(10):4362-4366. Cubitt B, et al. J Virol. 1994; 68(3):1382-1396. Dietrich DE, Bode L. Human Borna disease virus infection and its therapy in affective disorders. APMIS Suppl 124, 2008; 116: 61-65. Dietrich DE, et al. Bipolar Disorder 2000; 2: 65-70. Ferszt R, et al. Pharmacopsychiatry 1999; 32 (4):142-147 Liu X, et al. Virol J. 2015; 12: 39. Ludwig H, et al. Arch Virol 1993; Suppl 7:111-133. Rudolph M, et al. Structure 2003;11: 1219-1226. Schwemmle M, et al. J Biol Chem. 1997; 272(35):21818-21823.

Claims

1. A method for detecting acute Borna disease virus (BDV) infections, comprising the step of determining the presence of heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein in a sample using a sandwich ELISA, wherein a first primary antibody is present immobilized and a second primary antibody is added non-immobilized after incubation of the sample with the immobilized first antibody and these primary antibodies are monoclonal antibodies, characterized in that the first primary antibody is directed against the phosphorylated p24 BDV phosphoprotein and the second primary antibody is directed against p40 BDV nucleoprotein epitopes, those which do not overlap with the binding domain of the BDV phosphoprotein on the BDV nucleoprotein, for the determination of p24 phosphorylated heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein.

2. Method according to one of the preceding claims, characterized in that the immobilized primary antibody is present immobilized on a surface via a first secondary antibody or is immobilized directly on the surface.

3. Method according to one of the preceding claims, characterized in that the non-immobilized primary second antibody is determined via a second secondary antibody comprising a reporter molecule.

4. The method according to any one of the preceding claims, wherein the first primary antibody and the second primary antibody are from different species.

5. The method according to any one of the preceding claims, wherein the secondary antibodies are of different species or are derived from the same species.

6. Method according to one of the preceding claims, characterized in that circulating immune complexes (CIC) of BDV antigens, in particular of p24 protein and / or p40 protein and specific antibodies attached thereto, are additionally detected.

7. Method according to one of the preceding claims, characterized in that additionally circulating free antibodies against p24 protein and / or the p40 protein, in particular against phosphorylated p24 protein and against p40 protein, are detected.

8. Method according to one of the preceding claims, characterized in that this method is used for diagnosis and / or follow-up, in particular for differentiating acute BDV infections from chronic and dormant infections and / or their follow-up and for excluding a BDV infection, in particular in psychiatric and / or neurological diseases, as well as for determining the prevalence of infection in epidemiological studies on the mental and physical health of the population, in particular that this method, including the differentiation of acute BDV infections from chronic and dormant infections and / or their follow-up, is used for the differential diagnosis and exclusion of a BDV infection in other chronic diseases and chronic infections with other pathogens which are associated with reduced immune defense, as well as in diseases which require treatment with drugs to reduce or suppress the immune system.

9. Use of a diagnostic kit for a sandwich ELISA for the detection of acute BDV infections according to a method according to any one of claims 1 to 8, comprising a first primary antibody and a second primary antibody, wherein one of said primary antibodies is directed against the p24 BDV phosphoprotein and the other primary antibody is directed against the p40 BDV nucleoprotein; at least one secondary antibody labeled with a reporter molecule; optionally means for immobilizing a primary antibody to a surface and instructions for carrying out a method according to any one of claims 1 to 8, wherein said primary antibodies are monoclonal antibodies and wherein the first primary antibody is directed against the phosphorylated p24 BDV phosphoprotein and the second primary antibody is directed against p40 BDV nucleoprotein epitopes, those which do not overlap with the binding domain of the BDV phosphoprotein on the BDV nucleoprotein, for the determination of p24 phosphorylated heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein.

10. The use of a diagnostic kit for a sandwich ELISA for the detection of acute BDV infections according to claim 9, wherein the primary antibodies are as defined in claim 1 or 4 and / or wherein the secondary antibodies are as defined in any one of claims 3 and 5.

11. Use of a diagnostic kit according to any one of claims 9 or 10, further comprising means for detecting CIC from BDV antigens and specific antibodies attached thereto.

12. Use of a diagnostic kit according to any one of claims 9 to 11, further comprising means for detecting circulating free antibodies against the p24 protein and / or p40 protein, in particular the phosphorylated p24 protein and the p40 protein in plasma / serum.

13. Use of the method according to any one of claims 1 to 8 or of the diagnostic kit according to any one of claims 9 to 12 for the diagnosis and / or follow-up and / or exclusion of BDV infections, in particular in psychiatric and / or neurological diseases, for the differential diagnosis of chronic diseases and / or chronic infections of other genesis which are associated with a weakened immune defense and / or diseases in which a drug-induced weakened / suppressed immune defense is present or for determining the prevalence of infection in the healthy population.