CHAPTER 22 Borna Disease
Borna disease (BD) is a naturally occurring, infectious, usually fatal, progressive meningopolioencephalitis, predominantly affecting horses and sheep; less often other Equidae, cattle, goats, and rabbits; and occasionally a variety of other animal species and possibly humans.* Synonyms used in the past, such as “hot-headed disease” (Hitzige Kopfkrankheit), “brain fever,” “subacute meningoencephalitis,” and “hypersomnia of horses,” reflect the restriction of the disease to the nervous system. The current name of BD originated from a devastating epidemic among horses of a cavalry regiment between 1894 and 1896 near the town of Borna in Saxonia/Germany. More sporadic occurrence of equine BD has since been described in different areas of Germany,52 Switzerland,81 Liechtenstein, and Austria.128,139 There are reports of equine BD cases in Japan,129 but these could not be confirmed by others.138 It is uncertain whether clinically similar cases of equine encephalitis in France, Romania, Libya, and the Near East were in fact BD. However, good reason exists to assume that BDV infections are more widely distributed because virus-specific antibodies have been demonstrated in horses from several additional countries.†
During the first decade of the twentieth century, studies of BD focused primarily on defining the etiology, pathology, and pathophysiology of the disease. Final proof for a viral etiology was presented in 1927 by Zwick et al.147 by reproducing the disease with bacteria-free filtrates of brain homogenates from affected horses. Histopathologic studies of the brain, described in detail by Joest and Degen,59 Seifried and Spatz,120 and Gosztonyi and Ludwig,33 revealed massive perivascular infiltrations, reactive astrogliosis, and intranuclear eosinophilic “Joest-Degen” inclusion bodies. The pathologic changes were preferentially localized in the limbic system, which may be connected with behavioral alterations observed in some animals.107
Detailed studies have been performed on the spectrum of susceptible host species and on the manifestation of the disease.48,107,122 More recently, the causative virus, Borna disease virus (BDV), has been cultivated in cell cultures,56,80 aspects of pathogenesis of the disease have been elucidated,85,86,102–104,124 and the molecular characterization of BDV‡ and its replication strategy21,22,115,116,130 were achieved. With the advent of a reverse-genetics system to produce infectious complementary deoxyribonucleic acid (cDNA) clones, future studies will enable detailed molecular analysis of the genome organization and gene products encoded by BDV, as well as investigation of the regulation of BDV genome expression.88,116,118 The organization of the viral genome led to the classification of BDV in the order Mononegavirales as the new family Bornaviridae with the genus Bornavirus.
More detail on the historical background and various aspects of BD are available from Koprowski and Lipkin68 and Richt et al.101,102
ETIOLOGY
Borna disease virus is an enveloped virus with a nonsegmented, negative-sense, single-stranded (ss) ribonucleic acid (RNA) genome of 8.9 kilobases. It has the characteristic genomic organization of members of the order Mononegavirales. Some genotypic and phenotypic distinctions of BDV required the classification in the new virus family, the Bornaviridae. Thus, several features of BDV replication are unique compared with other members of this order, such as the nuclear site of replication and transcription, RNA splicing, and the overlap of transcription units.21,22,115,130 In addition, there is a high degree of genetic stability and homology among wild-type and experimentally host-adapted viruses.54,96,122 The complete sequence of the genome from several BDV isolates has been determined.13,19,96 Phylogenetic analysis of wild-type and laboratory strains indicate distinct virus clusters corresponding to geographically endemic areas in Central Europe.67 BDV particles are spherical, enveloped, and approximately 130 nm in diameter, with spikes 7 nm in length and a nucleocapsid 4 nm in diameter. BDV particles are released by budding on the cell surface.66
On the complementary positive-strand RNA (cRNA), at least six open reading frames (ORFs) can be identified. ORF I, at the 5′ end of the cRNA, encodes the 357/370–amino acid (aa) nucleoprotein, NP (p38/p39); ORF II encodes the 201-aa phosphoprotein, P (p24); ORF III encodes the 142-aa matrix protein, M; ORF IV encodes the 503-aa glycoprotein, GP; and ORF V (at 3′ end of cRNA) encodes the RNA-dependent RNA–polymerase (RdRp) of BDV, the L protein, which is more than 1600 aa long.22,115,130,135 Overlapping with ORF II, the ORF x1 encodes the 87-aa BDV p10 protein.136 All these virus-specific proteins have been detected in BDV-infected material. Detailed studies on the function of individual BDV proteins are in progress.
Several lines of evidence, including the location on the genome, indicate that the nucleoprotein (NP) and the phosphoprotein (P), together with the L–protein, are part of the ribonucleoprotein (RNP) and therefore part of the functional BDV replication complex. BDV RNPs are infectious after transfection into susceptible cell lines.16 BDV-p10 can also bind to the RNP complex, and it is speculated that it may act as a negative regulator of the BDV polymerase activity.90,116,119,143 Associated with the BDV envelope are the matrix (M) and the glycoprotein (GP). The BDV-M was thought to be glycosylated, but recent studies demonstrate that it is a nonglycosylated matrix protein, similar to that found in other viruses of the order Mononegavirales.70 Similar to the Filoviridae and Rhabdoviridae, BDV possesses a single surface glycoprotein, BDV-GP,31,98,117 which is posttranslationally modified by N-glycosylation and cleavage by a subtilisin-like protease into two fragments; this is a prerequisite for the invasion of BDV into cells.31,89,98
RNA transcripts encoding BDV proteins are initiated at three transcriptional start sites and terminated at five transcriptional termination sites. RNA from the first transcriptional unit codes for the NP.21,22,115,130 Alternative initiation at two in-frame AUGs results in the p38 and p39 isoforms of the NP. The RNA from the second transcription unit is bicistronic and codes for the P and p10 proteins. Similar to NP, two isoforms of P have been detected.64 Translation of P and p10 is most likely accomplished by “leaky scanning” because no spliced products of this messenger RNA (mRNA) have been described so far.136 RNA transcripts originating from the third transcriptional start site can be terminated at two different termination sites. In both cases, the transcripts may contain up to three introns, and depending on whether intron 1 and/or intron 2 is spliced, the respective mature mRNA can code for the M, GP, or L protein.* It can be assumed that RNA splicing may play an important role in the regulation of BDV genome expression by increasing the versatility of its primary transcripts and by providing the possibility for controlled synthesis of new BDV polypeptides.
The induction of a persistent, noncytopathic infection in cell cultures and in brain cells of a variety of animal species, the low replication rate, and therefore the difficulty in detection of virions in infected material might be the consequences of the tight control of protein expression from the third transcriptional unit. Recently, a novel strategy for viral replication-control has been postulated. BDV seems to restrict its propagation efficacy by defined 5′-terminal trimming of genomic and antigenomic RNA molecules.105,116,118
EPIDEMIOLOGY
To date, BD in horses has been recognized only in Germany, Switzerland, Liechtenstein, and Austria. However, the diagnosis may have been overlooked in other areas because of a lack of diagnostic effort. Seroprevalence studies demonstrate that BDV infections can occur worldwide. BDV-specific antibodies were detected in horses from many countries, including several European countries, Turkey, Israel, Japan, Iran, China, Australia, and the United States.†
Extensive studies of the seroprevalence of BDV are only reported from Germany, where BD is the most important viral CNS disease of horses. These studies demonstrate that the disease occurs predominantly in endemic regions in the central and southern parts of Germany. In contrast to the epidemic course of BD at the end of the nineteenth century, a significant reduction of BD incidence to about 0.3% was noted in an endemic region in central Germany in 1960 and from 1989 to 1996.131 At present the incidence in endemic regions in Germany is even lower, approximately 0.02% to 0.04% in Bavaria.36 A seasonal accumulation of cases is observed in April, May, and June, with a significant decrease in late fall and winter.‡
Most BDV infections appear to be inapparent infections, as indicated by various studies determining BDV seroprevalence. The average seroprevalence of BDV-specific antibodies in clinically healthy German horses is approximately 11.5%,52,99 which increases significantly to 22.5% in endemic regions. In stables with diseased horses, the prevalence of BDV-specific antibodies is approximately 50%.36 In 72% of horse stables with cases of BD, only individual animals showed clinical signs of BD. Repeated outbreaks of BD in stables are possible, although these are usually observed some time (2 months to several years) after the initial outbreak of BD.36,99 There is no explanation for the discrepancy between the high seroprevalence and the low incidence of disease so far. The development of Borna disease after BDV infection may depend on the genetic factors, age, and immune status of the host and the genetic characteristics of the virus.
The route of transmission of BDV is uncertain. Investigations in rats showed that BDV can be shed in nasal and lacrimal secretions as well as in urine, and transmission most likely occurs through open nerve endings in the nasal and pharyngeal mucosa.82,111 In a few cases, infectious virus could be isolated from the lacrimal and parotid glands of horses with BD (S. Herzog, unpublished results), and virus-specific RNA could be demonstrated in nasal and lacrimal secretions and saliva of such animals73; this was also possible in a few seropositive, inapparently infected horses.100 However, there is no evidence of virus replication in lacrimal or parotid gland of inapparently BDV-infected horses (S. Herzog, unpublished results). This indicates that horses infected with BDV, especially with clinical signs of BD, could play a role in the transmission of BDV. To date, no evidence suggests that other animal species (e.g., rodents) play a role in the transmission of BDV.
PATHOGENESIS
Most of the currently available information regarding the pathogenesis of BDV infection is derived from studies of experimentally infected Lewis rats and recently, also from BDV-infected mice.23,41,51,85,86 The use of genetically altered animals provides new opportunities to study the immunopathogenesis of BD in more detail. In general, it can be assumed that the virus enters the body by intranasal infection through olfactory nerve endings.82,73,100,111 Another possible route is orally, through the trigeminal nerve.5 After the virus enters the nervous system, it migrates along the axons of the olfactory system to the brain, where it replicates in neurons and glial cells, preferentially in the limbic system. Over time the virus disseminates throughout the central nervous system (CNS), then spreads to the peripheral nervous system and the neuronal cells of the retina.71,72,85 Axonal transport, with consequent protection from recognition of foreign antigens by the humoral immune system, may explain the lack of neutralizing antibodies until late in infection.
In adult rats the virus demonstrates a strict neurotropism. Viral antigens and infectivity persist throughout the life of the infected animals exclusively in neural tissues; that is, the animals develop a persistent CNS infection. The occurrence of clinical signs correlates with the appearance of inflammatory lesions in the brain. In general, the inflammatory reaction consists of mononuclear cells and is centered in the limbic system, but it spreads to other areas of the brain during the course of infection. Interestingly, late after infection, the inflammation decreases despite the presence of viral antigen and infectious virus.23,51,85,86 This might be caused by a switch from a T helper cell 1 (Th1) to a T helper cell 2 (Th2) immune response in later stages of the disease.43 In contrast, neonatally infected rats harbor infectious virus not only in the CNS, but also in parenchymal cells of peripheral organs.53 Although these animals do not manifest clinical signs and inflammatory infiltrates, they shed virus in various secretions and excretions and are therefore virus carriers.53,82
When diseased horses and sheep were analyzed for the presence of BDV-specific RNA by the reverse transcriptase– polymerase chain reaction (RT-PCR) technique, the bulbus olfactorius, nucleus caudatus, hippocampus, and cerebral cortex of all animals were positive.73 BDV RNA was also present in the spinal cord, eye, nasal mucosa, parotid salivary gland, lung, heart, liver, kidney, bladder, and ovaries in some animals. Presence of BDV RNA in tissues of nonneural origin could be caused by virus replication in nerve endings within these organs. In addition, BDV-specific RNA was detected in conjunctival fluid, nasal secretions, and saliva of two infected animals.73 Interestingly, viral dissemination and persistence in the brain of BDV-infected mice is not affected by overexpression or deletion of various cytokines or chemokine receptors.27,46,69
Both naturally and experimentally, BDV infects a broad spectrum of warm-blooded animals, inducing persistent infection without cytolytic destruction of cells. Sequence analysis of virus isolates obtained from various animal species or tissue culture reveals a remarkable conservation of the genome. The highly conserved viral genome suggests that BDV adapts easily to various animal species without significant genetic change. The rate of production and release of infectious virus in vitro and in vivo is extremely low despite the presence of relatively high amounts of viral antigen in infected cells. This indicates an abortive cycle of viral reproduction in most cell types infected. Evidence suggests that BDV replication in brain cells and probably other cell types may be controlled by the virus through various strategies, such as direct modification of the viral genome, control of transcription, regulation of viral protein expression (e.g., ratios of BDV-N vs. BDV-P), or abrogation of BDV glycoprotein synthesis.22,98,116,130,140 BDV can interfere with various cellular proteins or signaling cascades (e.g., neurite outgrowth factor HMG-1, neurotropin signaling, Raf/MEK/ERK pathway, activation of NF-κB) or the induction of the antiviral interferon (IFN) response.12,32,60,93,132 This interaction with host cell function might represent additional viral strategies to spread, replicate, and persist in the CNS of its host.
Borna disease is caused by a virus-induced immunopathologic reaction.123 This was convincingly shown in rodent systems. Infection of adult immunocompetent rats results in encephalitis and disease, whereas infection of newborn, athymic, or immunosuppressed animals leads to neither encephalitis nor disease, despite persistent high levels of virus in the CNS of these virus carriers.* Newborn infected animals appear clinically normal;53 however, some physical, behavioral, and pathologic abnormalities are observed. Learning deficits; elevated cytokine or chemokine expression, even in the absence of inflammation; and degeneration of postnatally developing brain areas have been described.1,95,97,110 These changes might be caused by direct effects of the virus on cell and organ functions. BDV-specific antibodies adoptively transferred into immunosuppressed, virus-infected recipients do not induce pathologic alterations or disease.86 Neutralizing antibodies to BDV are only detectable late, if ever, after infection and might play a role in preventing generalized infection with BDV. Transfer of immune serum into immune-incompetent newborn rats could not prevent persistent CNS infection but did prevent dissemination of the virus from neural tissues to peripheral organs.125 However, adoptive transfer of immune cells from spleen or lymph nodes of BDV-infected animals is effective in the induction of BD in immunosuppressed virus carriers.85,86
Mice develop a nonpurulent meningoencephalitis with a typical neurologic disorder only when they are infected as newborns. In contrast, adult BDV-infected mice show neither obvious clinical signs nor significant inflammatory alterations in the brain.41,109 The clinical signs of neonatal BDV-infected mice range from ataxia to paralysis of the hindlimbs. The incidence and severity of the BDV-induced clinical manifestations vary considerably between the different mouse strains used for infection. MRL/+ mice develop strong neurologic signs,109 whereas C57BL/6 mice were clinically inconspicuous.41 The reason for the different course of experimental BDV infection in adult versus newborn rats and mice remains unclear.
The pathogenesis of BD is somehow similar to that observed for lymphocytic choriomeningitis virus (LCMV) infection,145 in which a virus-induced cell-mediated immune response causes disease. Whereas CD8+ cells are responsible for induction of LCM, CD4+ and CD8+ cells are apparently responsible for development of BD. The role of virus-specific T cells in the pathogenesis of BD was demonstrated by passive transfer of in vitro established homogenous BDV-specific CD4+ T-cell lines into immunosuppressed virus carriers. The recipients consistently developed clinical signs characteristic of acute BD.103,104 However, evidence indicates that in addition to virus-specific CD4+ T cells, CD8+ cells are also involved in pathologic alterations.91,95,126 These are thought to be a main effector cell in BDV-infection of mice, although there is no lysis of BDV-infected neurons observed in infected mice or rat brains. In rats as well as in mice, BDV-specific CD8+ cytotoxic T lymphocytes (CTLs) are mainly directed against the viral nucleoprotein.45,92,112 The kinetics of CTL induction and subsequent recruitment of these cells to the brain determine the severity of BDV-induced neurologic disease. Downregulation of the functional avidity of virus-specific CD8+ T cells in experimentally infected mice seems to be involved in controlling the inflammatory reaction and facilitating viral persistence.26 However, immune control of BDV infection could generally be achieved only by antigen-specific immune priming or adoptive transfer of BDV-specific T cells.44,49,74,104 Taken together, these data indicate that T cells play a crucial role in the pathogenesis of BD. The severe meningoencephalitis with mononuclear infiltrates observed after BDV infection most likely represents a delayed-type hypersensitivity reaction.
As observed in the experimental rat model, brain tissues from various Equidae with natural BD have mononuclear immune cell infiltration and increased expressions of major histocompatibility complex (MHC) class I and class II antigen were described.6,14,50 The composition of the inflammatory cell infiltrates is similar to that observed in experimentally BDV-infected rats, and therefore a similar pathogenesis of disease in naturally infected Equidae and experimentally infected rats is presumed.
CLINICAL FINDINGS
Natural BDV infection can result in peracute, acute, or subacute Borna disease with meningoencephalitis, which leads to death 1 to 4 weeks after onset of initial signs in more than 80% of animals.* In less severe cases, spontaneous recovery is occasionally observed despite a persistent CNS infection.20,36,80,113 In up to 10% of animals with BD, a chronic, sometimes recurrent course of disease is observed.20,36,37,80,113 A bland encephalitis without obvious clinical signs is possible.36
The incubation period for BD after natural infection is variable, ranging from 2 weeks to several months.113,114 In a large number of experimentally infected animals, the average incubation period was 2 to 3 months. More recently, experimental intracerebral infection of three ponies with various doses of BDV resulted in an incubation period of 15 to 26 days.63
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