Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-06-29
2002-06-11
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C422S067000, C424S159100, C424S141100
Reexamination Certificate
active
06403301
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a process for detecting BDV infections via detecting immune complexes which circulate in body fluids (CICs), a process for detecting CICs in general, in particular BDV-specific CICs, and to a diagnostic kit which is suitable for these detection processes.
BACKGROUND OF THE INVENTION
BDV infections are found in a large number of livestock and domestic animals (horses, sheep, cattle, cats) and in humans. BDV is a coated virus of 90 nm diameter with a genome of unsegmented single-stranded RNA of negative polarity which encodes 5 genes (genome size: 8.9 kilobases). Related viruses include, for example, the rabies virus and the measles virus. Owing to genetic particularities (replication in the nucleus of the host cell), BDV is classified as the prototype of a separate family of viruses (Bornaviridae). BDV has a particular preference for the nerve cells in the limbic system of the brain, a functional unit in which behavior, emotions and memory functions are controlled. However, other cell types are also attacked by BDV. Peripheral mononuclear leukocytes (PBMCs) are of particular diagnostic importance. BDV does not destroy the host cells (no cytopathogenic effect), neither in the host (in vivo) nor in cell culture (in vitro). The primary pathogenic effect of BDV is based on a functional disturbance in the infected brain cells which is probably induced by interaction with neurotransmitter receptors. Experimental data obtained on animals suggest that glutamate receptors are (reversibly) blocked. The exact mechanism and the receptor type are as yet unknown.
In animals, BDV infections are associated with periodic behavioral disorders with the indicating signs listlessness, hypoprosexia and ataxia. In humans, infectious human Bornaviruses have been isolated from patients with recurrent endogenous affective disorders, and it is highly likely that they are involved in these disorders and possibly other disorders of the cerebral function (also presumably via neurotransmitter functions). Endogenous recurrent depressions, including the manic-depressive form, account for 1-5% of the important psychiatric diseases and are a considerable health problem owing to the severity of the disablement, not only for the sufferers, but, from the socioeconomical angle, also for society.
BDV infections persist in animals and humans, probably for life. The sources of the infection remain entirely unknown. The course of the infection is distinguished by latent and activated phases. During the activated phases, clinical symptoms can occur. In animals (horses are the most investigated species), a number of infections without disease exist (asymptomatic carriers). This percentage can amount to up to 50% in a group of horses in which an illness was identified. The danger of an intense, disease-associated activation phase of the infection depends on genetic factors and the stress of the individual (stress factors, immunosuppression).
In humans, there is probably no general risk of illness for healthy infected individuals which are not predisposed to affective disorders. In contrast, an increased risk of disease by BDV infection in the narrower sense exists in individuals with a predisposition of developing an affective disorder, in individuals where an affective disorder is already clinically apparent, and in as yet healthy first-degree relations of these individuals.
There is an indisputable increasing demand for reliable diagnostic systems for identifying BDV infection, not only in asymptomatic carriers (for epidemiological reasons), but also for monitoring the infection phases in diseased individuals.
Conventional antibody tests are known since the BDV virus has already been studied thoroughly and is already fully sequenced. Until 1993, an antibody test in the serum was the only possibility. As a rule, antibodies are directed against the BDV nucleoprotein p40 (relative molecular weight 40 kDa) and against the phosphoprotein p24 (24 kDa). These antibodies do not neutralize the virus. It soon emerged that the lack of specific antibodies did not exclude infection with BDV. This means that the antibodies do not persist in the serum of the infected individual, or not for a very long time. Moreover, the Ab concentrations (titers) in naturally infected humans and animals are only low in the first place and cannot always be identified with less sensitive test systems.
Important progress, in particular for assessing disease-relevant activations of the BDV infection, was made by the discovery that PBMCs express viral proteins which can be detected in cells after disrupting the latter. These proteins are occasionally also found in the plasma. Protein (AG) expression has proved to be the decisive activity parameter. It correlates well with clinical disease (humans and animals), can be quantified and is of great importance for assessing the further pathogenesis (chances of recovery, response to antidepressants and the like). Also, AG determination cannot be replaced by detecting viral nucleic acid in the PBMCs via amplification with nested RT PCR, since it is only the virus itself which is detected by this method, but no information can be obtained on its current or, possibly, future activity.
As a rule, the AG marker is limited to part (2-3 weeks) of the acute disease phase and can no longer be detected during convalescence. Thereafter, the Ab may be detectable intermittently, depending on the severity of the activation episode, but they may also be completely absent.
This current state of BDV diagnostics fails especially during convalescence, during the symptom-free interval (which may last for years) of a patient (humans and animals) and in infected individuals which are as yet disease-free, i.e., in brief, in the case of BDV infections in the latent stage. Both test parameters, with AG and Ab, can give erroneous negative results during the latent phase, even though the infection continues to exist.
Journal of Clinical Microbiology,
1997, vol. 35, no. 7, pp. 1661-1666, Horimoto, T. et al. (via MEDLINE TO 97339670) describes an ELISA test for detecting BDV-specific antibodies in which microwell plates were coated with BDV p40 antigen in order to catch and detect antibodies specific for this BDV nucleoprotein. It was found that many specimens which had previously been assayed differently and were found to be seropositive gave a negative result with regard to the antibodies. It was concluded that the other studies would have contained a high percentage of erroneously positive results. However, the results also allowed the conclusion that BDV infections cannot be detected in each individual case via the antibody titer. Thus, an overall diagnostic approach seems to be required.
It was therefore the object of the invention to develop a novel detection process for the infection to allow as complete a detection as possible of BDV infections, which, owing to their persistence, can last for very long periods, with acute and latent phases alternating. Also, it was intended to provide a diagnostic kit which can be used for such a detection.
SUMMARY OF THE INVENTION
To achieve this object, the invention provides a process for detecting Borna disease virus (BDV) infection, in which immune complexes of BDV antigens and specific antibodies attached thereto, which circulate freely in the body fluid, are detected in a body fluid specimen to be tested by means of a suitable immunological assay. An essential aspect of the invention is therefore based on the finding that high concentrations of circulating immune complexes composed of BDV antigens and antibodies against them which are formed specifically by the body occur in body fluids, for example in blood serum, during certain phases of the disease. These immune complexes can also be present in particular during precisely those disease phases during which antigens and/or antibodies cannot be detected.
In principle, the existence of immune complexes, i.e. complexes composed of antigens and antibodies and which are freely mobile in the blood,
Bode Liv
Ludwig Hans
Brown Stacy S.
Foley & Lardner
Park Hankyel T.
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