Meta-analysis of putative human bornavirus sequences fails to provide evidence implicating Borna disease virus in mental illness

All Borna disease virus (BDV) sequences derived from human specimens published till date were thoroughly analysed and compared to sequences of BDV laboratory strains and to BDV sequences from animals which succumbed to classical Borna disease (BD). Despite high sequence conservation of the BDV genome, animal-derived BDV sequences clustered according to their geographic origin. However, in marked contrast, human-derived BDV sequences did not cluster according to their geographic origin but showed high sequence identities to BDV laboratory strains and animal-derived BDVs handled in the laboratories reporting the human strains. Japanese, US, Australian and French human-derived BDV sequences proved to be identical or very similar to animal-derived BDV sequences from Germany, although the human specimens were collected hundreds to thousands of miles away from the central European BD endemic regions. These findings suggest that previous studies linking BDV to human neuropsychiatric disease may have been compromised by inadvertent sample contamination.     

博尔纳病病毒感染的免疫发病机制

博尔纳病病毒(BDV)是单股负链RNA病毒,在体内和体外都缺乏细胞致病性。BDV可引起中枢神经系统持续感染并导致博尔纳病(BD),即免疫介导的脑脊髓炎。浸润的免疫细胞以CD4~ T细胞、CD8~ T细胞、巨噬细胞和B细胞为特征。CD8~ T细胞代表对核蛋白呈抗原特异性的效应细胞群。     

No evidence of endemic Borna disease virus infection in Australian horses in contrast with endemic infection in other continents

Summary. Borna disease virus (BDV) is a unique RNA virus that is a cause of neurological disease in horses, sheep and cats. The finding that BDV also infects humans has raised concern related to the impact of infection with this virus. The extent to which BDV may be endemic in geographical regions outside Europe is of interest in management of international movement of animals including horses. Sera from Australian horses (N = 553) sampled in Sydney, New South Wales (NSW), were analysed for BDV antigen, circulating immune complexes (CICs), and antibodies by monoclonal antibody-based ELISAs. One-tenth of the samples were investigated by further antibody tests, namely immunofluorescence (IFA) and a peptide ELISA, as well as for BDV RNA. The study revealed a very low frequency of serological markers that may be associated with exposure to BDV in Australian horses from NSW with a few sera (0.7%) displaying low range positive results in the CIC assay, and no detectable BDV RNA. This pattern is inconsistent with endemic BDV infection and strongly contrasts with the pattern of endemic infection, particularly in Europe.     

Serological evidence for Borna disease virus infection in humans, wild rodents and other vertebrates in Finland.

BACKGROUND: Borna disease virus (BDV) can infect many vertebrate species, including humans. BDV infection may lead to meningoencephalomyelitis in animals. An association with human neuropsychiatric diseases has been reported, but the causal relationship between BDV and human disease remains unclear. OBJECTIVES AND STUDY DESIGN: To find out whether BDV is present in Finland and to look for a potential reservoir, we examined a large panel of blood samples from different vertebrate species with immunofluorescence assay. Samples from horses, cats, dogs, sheep, cattle, large predators, grouse, wild rodents and humans were included. Most positive results were confirmed by other specific methods and in other laboratories. RESULTS AND CONCLUSIONS: BDV-specific antibodies were detected in 10 horses, 2 cats, as well as 2 horses and 1 dog from farms housing a previously detected seropositive horse. Interestingly, BDV-specific antibodies were further detected in three wild rodents. In humans, BDV-specific antibodies were detected in a veterinarian and in two patients suspected to have a Puumala hantavirus infection. Our serological analysis suggests that BDV infects various vertebrates in Finland, including humans. Furthermore, our data indicate for the first time that BDV infects also wild rodents.     

Neurological diseases and viral dynamics in the brains of neonatally borna disease virus-infected gerbils

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that causes a chronic neurological disease in a wide variety of animal species. To develop a better understanding of the correlation between neurological disorders caused by BDV infection and virus distribution in the brain, we investigated viral dynamics in the central nervous system (CNS) of neonatally BDV-infected gerbils during the late stage of infection. Despite the severe symptoms and aggressive proliferation of BDV in the infected gerbils, no apparent neuroanatomical abnormalities or neuronal cell loss was observed in the infected gerbil brain. Furthermore, no or only minimal infiltration was observed in the infected gerbil brain. By in situ hybridization and real-time PCR analyses, we demonstrated that the predominant area of expression of BDV mRNA, as well as the protein, was shifted in the brain in association with progression of disease. In nondiseased gerbils, the virus replication was predominantly detected in the cerebral cortex and hippocampus of the CNS. On the other hand, diseased animals showed a high level of expression in the lower brain stem and cerebellum, especially in Purkinje cell neurons. These observations suggested that significant replication of the virus in specific areas of the CNS is critical for development of the neurological disorders in BDV-infected neonatal gerbils.     

Analysis of virus-specific RNA species and proteins in Freon-113 preparations of the Borna disease virus

Treatment of homogenates from Borna disease virus (BDV)-infected brain tissue or cell cultures with Freon-113 yielded infectious particles with a buoyant density of 1.16–1.22 g/ml. Positive- and negative-stranded BDV-specific RNA species as well as three virus-specific proteins, known to be present in BDV-infected cell extracts, were demonstrated in these Freon-treated fractions. When the Freon-purified virus preparations were treated with RNase A prior to RNA extraction, only negative-stranded, genomic RNA was detected in Northern blot hybridizations using sense and antisense RNA probes. These data substantiate that BDV is a negative-stranded RNA virus.     

Demonstration of Borna disease virus (BDV) in specific regions of the brain from horses positive for serum antibodies to BDV but negative for BDV RNA in the blood and internal organs

Sero- and molecular-epidemiological studies on Borna disease virus (BDV) infection show that BDV RNA is not always detected in the peripheral blood mononuclear cells (PBMCs) from serum anti-BDV antibody-positive individuals such as horses, sheep, cattle, cats, and humans. In this study we demonstrated BDV RNA signals by polymerase chain reaction only in restricted regions of the brain from horses with locomotor disease. Four of six horses examined showed apparently positive reactions for anti-BDV antibodies. Specific regions of the brain of these four horses were positive for BDV RNA but the internal organs, lymph nodes, and PBMCs were negative. Histological studies of their brains revealed no apparent histological abnormalities such as inflammatory reactions. These results suggest that BDV chronically infects certain restricted regions of brain in seropositive horses. Received: 6 January 1997     

A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle

A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5′-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104 bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID₅₀ of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep.     

Borna disease virus infection in racing horses with behavioral and movement disorders

Summary.  Borna disease virus (BDV) is a neurotropic agent with capacity to infect and cause neurological disease in a broad range of warmblooded hosts including horses, sheep, cattle, cats, and possibly also humans. The epidemiology of BDV is largely unknown. However, it is likely that subclinically infected animals may represent potential virus reservoirs. In two groups of Swedish racing horses, one clinically healthy and one consisting of horses with diffuse neurological signs, the BDV seroprevalence was 24.5% and 57.7%, respectively. BDV RNA was detected in peripheral blood mononuclear cells in 8 out of 28 (28.6%) investigated horses, the majority of the BDV RNA-positive horses belonging to the group with neurological signs. There was a close relationship between the Swedish equine BDV isolates and previously reported equine BDVs in Europe. Our results point to an association of BDV infection with atypical disease patterns in horses such as diffuse mental and gait disturbances. These findings may be of importance for the understanding of the epidemiology of BDV infections in animals and man. Accepted September 4, 1998 Received May 27, 1998     

Borna disease virus interference with neuronal plasticity

Viruses able to infect the central nervous system (CNS) are increasingly being recognized as important factors that can cause mental diseases by interfering with neuronal plasticity. The mechanisms whereby such infections disturb brain functions are beginning to emerge. Borna disease virus (BDV), which causes a persistent infection of neurons without direct cytolysis in several mammalian hosts, has recently gained interest as a unique model to study the mechanisms of viral interference with neuronal plasticity. This review will summarize several hypotheses that have been put forward to explain possible levels of BDV interference with brain function.     

Borna disease virus infection,a human mental-health risk

This article focuses on human Borna disease virus (BDV) infections, most notably on the development of valid diagnostic systems, which have arisen as a major research issue in the past decade. The significance of a novel modular triple enzyme-linked immunosorbent assay that is capable of specifically measuring anti-BDV antibodies as well as major structural proteins N (p40) and P (p24) in the blood, either as free antigens in the plasma or as antibody-bound circulating immune complexes (CICs), is explained. The impact of CICs and plasma antigen, which indicate periods of antigenemia in the course of BDV infection, along with other infection markers that are still in use is discussed. The review further provides new insight into possible links of BDV to human diseases, summarizing cross-sectional and longitudinal data which correlate acute depression with the presence and amount of antigen and CICs. Moreover, BDV prevalence in healthy people is reevaluated, suggesting that this was previously underestimated. Antiviral efficacy of amantadine, in vivo and in vitro, is outlined as well, with emphasis on wild-type (human and equine) versus laboratory strains. Finally, the pros and cons of the association of BDV with human disease, as detailed in the literature, are critically discussed and related to our data and concepts. This article supports existing correlative evidence for a pathogenic role of BDV infection in particular human mental disorders, in analogy to what has been proven for a variety of animal species.     

新疆伊犁地区动物脑组织博尔纳病病毒p24基因片段的检测

目的 了解新疆伊犁地区动物脑博尔纳病病毒(Borna disease virus,BDV)感染现状,分析该病毒的种系来源,预防我国BDV大规模暴发流行.方法 采用巢式逆转录实时荧光定量聚合酶链反应(FQ-RT-PCR)的方法,对新疆地区200匹马、75头驴和100只牧羊犬脑组织标本进行BDVp24基因片段的检测.并对阳性标本进行BDV p40基因片段扩增电泳,同时排除GFP-p24和pMD-19质粒污染,最后克隆测序,进行基因同源性和氨基酸序列分析并分析BDV种系发生.结果 3例伊犁马、4例伊犁驴和9例牧羊犬的脑组织标本BDV p24检测阳性,阳性率分别为1.5%、5.3%、9.0%;BDV p40基因片段检测和质粒标准品GFP-p24、pMD-19检测均为阴性;BDV p24扩增产物序列与He/80株的同源性为100%.结论 新疆伊犁地区马、驴和狗可能存在BDV脑内的自然感染,该地区BDV流行株与He/80株存在同源性.     

Non-suppurative meningoencephalitis of unknown origin in cats and dogs: an immunohistochemical study

Non-suppurative meningoencephalitis of unknown cause is a frequent finding in dogs and cats. Fifty-three dogs and 33 cats with non-suppurative meningoencephalitis of unknown aetiology were examined immunohistochemically for 18 different infectious agents, including viruses, bacteria and prion protein(Sc). In 14 (26%) of the dogs and 13 (39%) of the cats a causative agent was identified in the central nervous system (CNS), two dogs and one cat giving positive results for two infectious agents simultaneously. The study revealed infections with known causative agents (porcine herpes virus 1, feline infectious peritonitis virus, Escherichia coli) and a new disease pattern of parvovirus infection in the CNS of dogs and cats. Infection of the CNS with feline leukaemia virus was found in a cat. Five dogs and four cats gave positive results for West Nile virus (WNV) antigen. In one dog, canine parainfluenza virus antigen was detected in the brain. Four dogs and four cats gave positive results for encephalomyocarditis virus (EMCV). The significance of the detection of WNV and EMCV antigen requires further study. The aetiology remained undetermined in 39 dogs (74%) and 20 cats (61%). Although it is possible that non-infectious causes play a more important role than previously thought, infections with hitherto unrecognized agents cannot be ruled out.     

Failure to detect Borna disease virus antigen and RNA in human blood.

BACKGROUND: Borna disease virus (BDV) is the etiological agent of a rare progressive meningoencephalitis that affects mostly horses and sheep. There is an unresolved debate whether also humans are susceptible to infection with BDV and if so, whether this might be associated with neuropsychiatric diseases. One recent key publication employing an ELISA-based sandwich assay reported prevalences of BDV-specific circulating immune complexes in human blood as high as 30% in the normal population and up to 100% in psychiatric patients [Bode L, Reckwald P, Severus WE, Stoyloff R, Ferszt R, Dietrich DE, et al. Borna disease virus-specific circulating immune complexes, antigenemia, and free antibodies--the key marker triplet determining infection and prevailing in severe mood disorders. Mol Psychiatry 2001;6(4):481-91]. However, this report did not examine for the physical presence of BDV antigens in human blood, and therefore, these seemingly high prevalences may not reflect Borna virus-specific signals. OBJECTIVES: We attempted to correlate string plasma signals in the particular sandwich ELISA system with the presence of BDV antigens. STUDY DESIGN: Four preselected plasma samples with high reactivity in the described assay were analysed by immunoaffinity purification and highly sensitive real-time RT-PCR. RESULTS: Neither method did provide any evidence for the presence of viral proteins or nucleic acids. CONCLUSIONS: Our findings argue against the concept that the described sandwich ELISA reliably detects BDV-specific antigens in human blood, therefore do not support the hypothesis that BDV is a pathogen of humans.     

用蛋白印迹试验检测精神分裂症患者血清中博尔纳病病毒-p24抗体的研究

目的：探讨博尔纳病病毒（BDV）感染在我国的流行情况及其与精神分裂症的相关性。方法：在对优化表达的GST－BDV－p24融合蛋白进行特异性鉴定的基础上，通过确定融合蛋白与第一抗体、第二抗体间的最佳反应条件，建立可行的检测BDV－p24特异抗体的蛋白印迹（Western-blot）方法，进而对黑龙江地区精神分裂症患者和正常人对照血清中BDV－p24特异性抗体进行了检测，并通过血清－GST蛋白吸收后W estern-blot实验对阳性血清进行了确认。结果：116例精神分裂症患者中检出BDV－p24阳性血清10例，阳性检出率为8.6%，而正常人血清标本中未检出阳性。结论：我国存在博尔纳病病毒的感染，博尔纳病病毒的感染可能与精神分裂症的发生有关。     

Borna disease virus is not sensitive to amantadine

Summary.  Successful inhibition of Borna disease virus (BDV) by amantadine in cultured cells and in an infected human individual has been reported [Bode et al. (1997) Lancet 349: 178–179]. We now found that infection of monkey Vero cells by laboratory strains of BDV was not influenced by amantadine under conditions that reduced the yields of influenza A virus by about 400 fold. Amantadine treatment of Vero cells persistently infected with BDV did not result in reduced viral RNA levels, and application of the drug to persistently infected BALB/c mice had no effect on the concentration of BDV in their brains. Thus, susceptibility to amantadine is not a characteristic of BDV, although it may be observed with certain primary virus isolates. Received May 21, 1997 Accepted June 19, 1997     

Lack of antiviral effect of amantadine in Borna disease virus infection

The antiviral effect of amantadine (1-aminoadamantane) was tested in vitro as well as in vivo. Treatment of persistently Borna disease virus (BDV)-infected cell lines of different origin and for various length of time did not result in a general reduction of virus titer or clearance of virus from infected cells. In vivo, rats were treated with amantadine by daily oral application or by use of osmotic pumps, and in both cases treatment was started before infection. Neither route of application of the drug had any influence on the time of onset of disease, on antiviral antibody titers, on virus titer in the brain, on the severity of the inflammatory reaction in the brain, or on the severity of neurological symptoms. These experiments, although revealing negative results and obtained using a virus from a natural case of Borna disease grown after isolation in vitro for a long period of time, should caution from the general use of amantadine as a curative agent against BDV infection as has been implicated recently [Bode et al. (1997) Lancet 349:178–179]. Received: 26 November 1997     

Borna Disease Virus and Human Disease

The biology of Borna disease virus (BDV) strongly supports the likelihood of human infection with BDV or a variant of BDV. Thus far, the evidence supporting BDV infection in humans has initiated much controversy among basic and clinical scientists; only time and additional research will support or refute the hypothesis of human BDV infection. Until an assay of acceptable specificity and sensitivity has been developed, validated, and used to document human BDV infection, scientists cannot reasonably begin to associate BDV infection with specific disease syndromes. Clinical studies seeking causal associations between BDV infection and specific diseases must ensure the proper identification of the BDV infection status of patients and control subjects by using a validated, highly sensitive, and highly specific assay (or series of assays). For clinical studies, a highly sensitive "screening" test followed by a highly specific confirmatory test will be of significant benefit. Although it is possible to formulate hypotheses about the clinical outcomes of human BDV infection based on animal model work, to date no human disease has been causally linked to human BDV infection. Scientists all over the world are actively pursuing these issues, and with continuing advances in clinical and basic BDV research, the answers cannot be far away.     

Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-kappaB) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes.