Horses

Naturally occurring Borna disease has been recognized in horses in eastern Germany since the 18th Century. However, it gained notoriety (and its name) when in 1894–1896 it immobilized a regiment of cavalry based in the town of Borna in Saxony (Richt et al., Reference Richt, Grabner and Herzog2000). Equine BoDV-1 infection occurs naturally only in Germany, Switzerland, Lichtenstein and Austria where on average about 12% of horses are seropositive.

The typical disease course in horses is characterized by an acute encephalitis that develops following an incubation period of 4 weeks to 3 months (Richt et al., Reference Richt, Pfeuffer, Christ, Frese, Bechter and Herzog1997). Non-specific clinical findings such as depression (apathy, somnolence, stupor), fever and anorexia precede ataxia. Horses spread their legs or cross them, and support themselves by pressing their head against the manger or wall. Repetitive behaviors including circling, vacuous slow motion chewing and severe tooth grinding may occur. Eventually, the horses become paretic, develop neurogenic torticollis, or blindness, coma and death. Lethality exceeds 80% (Ludwig and Bode, Reference Ludwig and Bode2000; Lipkin et al., Reference Lipkin, Briese and Hornig2011). Death usually occurs 1–4 weeks after the onset of clinical signs. A chronic recurring form of the disease may develop in up to 10% of cases and thus some horses may become persistently infected carriers. Serologic surveys indicate that infection without obvious clinical disease is common (Richt et al., Reference Richt, Grabner and Herzog2000).

On necropsy, horses show no gross lesions. Histopathology shows a non-purulent meningoencephalomyelitis with randomly scattered inflammatory foci in the hippocampus and along the central axis including the mesencephalon and the hypothalamus (Fig. 2). These foci consist of extensive lymphocytic perivascular infiltrates involving primarily the gray matter (Rott and Becht, Reference Rott and Becht1995; Bilzer et al., Reference Bilzer, Grabner and Stitz1996). Most of the inflammatory cells in these foci are CD3+ T cells (Caplazi and Ehrensperger, Reference Caplazi and Ehrensperger1998). Of these, CD4+ cells outnumber CD8+ T cells. Natural killer (NK) cells are likely present in the infiltrates as well (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998). Macrophages are fewer and B cells/plasma cells fewer still. Polymorphonuclear leukocytes are rarely present. The highest viral titers occur in the hippocampus and the piriform cortex reflecting viral invasion from the olfactory bulbs. The lowest viral titers are in the cerebellum (Gosztonyi, Reference Gosztonyi2008). The virus is primarily located within neuronal nuclei (Richt et al., Reference Richt, Grabner and Herzog2000). Less consistently, smaller amounts of viral antigen may be detected in the neuronal perikaryon, dendrites and axons (Bilzer et al., Reference Bilzer, Grabner and Stitz1996). There is usually a correlation between viral titer and the severity of the brain lesions and the inflammatory exudate is associated with the presence of viral antigen, but the inflammatory cells never appear to contain viral antigens (Gosztonyi and Ludwig, Reference Gosztonyi and Ludwig1984). In chronic infections, viral antigen is demonstrable in astrocytes and the meningeal surface is infiltrated with mononuclear cells (Richt et al., Reference Richt, Grabner and Herzog2000). Inflammation may spread from the gray matter into the adjacent white matter and involve nerve roots and the spinal ganglia (Johnson, Reference Johnson1980).

Fig. 2. The major sites within the equine brain that BoDV-1 appears to favor. Infected neurons are also scattered diffusely throughout the cerebrum. The arrows indicate what is believed to be the ‘natural’ route of viral invasion, originating in the olfactory bulb.

Sheep

Natural Borna disease in sheep resembles that in horses although large numbers of sheep may be affected within a flock unlike horses where the disease usually affects few animals (Richt et al., Reference Richt, Pfeuffer, Christ, Frese, Bechter and Herzog1997). Clinical signs vary from minor behavioral changes to severe encephalomyelitis reflecting the intensity of the inflammatory response in the brain (Vahlenkamp et al., Reference Vahlenkamp, Konrath, Weber and Muller2002). A short period of depression precedes overt disease including somnolence, ataxia and multiple deficits (Metzler et al., Reference Metzler, Frei and Danner1976; Waelchli et al., Reference Waelchli, Ehrensperger, Metzler and Winder1985). As in horses, the majority of BoDV-1 infections in sheep remain asymptomatic. The location of the virus within the brain is identical to that in horses. Lethality in clinically affected animals is at least 50%. Some sheep recover completely while others survive but fail to thrive (Metzler et al., Reference Metzler, Minder, Wegmann and Zindel1979).

Cats

BoDV-1 causes staggering disease in cats (Lundgren et al., Reference Lundgren, Zimmermann, Bode, Czech, Gosztonyi, Lindberg and Ludwig1995b; Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014; Lutz et al., Reference Lutz, Addie, Boucraut-Baralon, Egberink, Frymus, Gruffydd-Jones, Hartmann, Horzinek, Hosie, Lloret, Marsilio, Pennisi, Radford, Thiry, Truyen and Mostl2015). This begins with fever, apathy and a reduced appetite. Cats eventually develop ataxia, gait disturbances, blindness, lower back pain, behavioral changes, loss of postural reactions and hind-leg paralysis (Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014). Some may be unable to retract their claws (Lutz et al., Reference Lutz, Addie, Boucraut-Baralon, Egberink, Frymus, Gruffydd-Jones, Hartmann, Horzinek, Hosie, Lloret, Marsilio, Pennisi, Radford, Thiry, Truyen and Mostl2015). A similar disease can be induced by experimental challenge with BoDV-1 (Lundgren et al., Reference Lundgren, Johannisson, Zimmermann, Bode, Rozell, Muluneh, Lindberg and Ludwig1997). Some cats recover. There are anecdotal reports of cats surviving the acute infection and later developing extreme obesity (Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014). The lesions in the cat brain are similar to those observed in horses although plasma cells may be more prominent (Lundgren et al., Reference Lundgren, Johannisson, Zimmermann, Bode, Rozell, Muluneh, Lindberg and Ludwig1997). While it is possible that cats become infected by eating small infected mammals or birds (Berg et al., Reference Berg, Johansson, Montell and Berg2001) it is just as likely that it results from exposure to the urine of infected shrews (see below). It has been suggested that as in horses and sheep, the virus probably enters the cat through the olfactory epithelium and the oropharyngeal mucosa and then gains access to the brain by intra-axonal spread (Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014).

BoDV-1 triggers an intense T cell reaction in the cat brain. Lundgren et al. observed elevated T cell numbers in the blood and brains of experimentally challenged cats (Lundgren et al., Reference Lundgren, Lindberg, Ludwig and Gosztonyi1995a, Reference Lundgren, Johannisson, Zimmermann, Bode, Rozell, Muluneh, Lindberg and Ludwig1997). Berg et al. used flow cytometry to demonstrate that the CD8+ T cells in the brain of infected cats belong to a non-major histocompatibility complex (MHC) restricted population and suggested that they could be important in viral clearance from neurons (Berg et al., Reference Berg, Johannisson, Johansson, Hein, Berg and Dorries1999). Some cats may develop disease in the absence of gross encephalitis, a disease process similar to that seen in neonatal rats (See below). Infected cats express high levels of interferon (IFN)-γ in their brains and this may promote NK and T cell cytotoxicity and neuronal destruction (Wensman et al., Reference Wensman, Ilback, Hjertstrom, Blomstrom, Gustavsson, Jaderlund, Strom-Holst, Belak, Berg and Berg2011, Reference Wensman, Jaderlund, Holst and Berg2014). However BoDV-1 can survive in the presence of IFN-γ (Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014). CD8+ cells stimulated by BoDV-1 are found in blood, spleen and brain. Cats may also develop inflammatory changes in their intra-abdominal ganglia and the adrenal medulla (Wensman et al., Reference Wensman, Jaderlund, Gustavsson, Hansson-Hamlin, Karlstam, Lilliehook, Ostrom, Belak, Berg and Holst2012).

Humans

At least one bornaviral species can infect humans. In 2011–2013, three breeders of variegated squirrels (Sciurus variegatoides) in Germany developed a progressive meningoencephalitis and died within 2–4 months (Hoffmann et al., Reference Hoffmann, Tappe, Hoper, Herden, Boldt, Mawrin, Niederstrasser, Muller, Jenckel, Van Der Grinten, Lutter, Abendroth, Teifke, Cadar, Schmidt-Chanasit, Ulrich and Beer2015). Their clinical disease progressed from a fever, to progressive psychomotor slowing, confusion, ocular paresis, coma and death. On autopsy, their brains showed edema, gliosis, lymphocyte infiltration with perivascular cuffing, and necrosis. Subsequently a bornavirus was isolated from their brains as well as from the brains of one of their squirrels. Sequencing of this virus genome revealed that it was a previously unidentified mammalian bornavirus (VSBV-1). The distribution of virus in the brains of these patients and in the squirrel resembled that seen in equine Borna disease. There was a high viral RNA load in an oropharyngeal swab from the squirrel suggesting that squirrels may act as carriers and that the virus may have been accidentally transmitted by squirrel bites (Hoffmann et al., Reference Hoffmann, Tappe, Hoper, Herden, Boldt, Mawrin, Niederstrasser, Muller, Jenckel, Van Der Grinten, Lutter, Abendroth, Teifke, Cadar, Schmidt-Chanasit, Ulrich and Beer2015). One of these patients had high levels of autoantibodies to the Yo autoantigen, an antigen located within Purkinje cells. However other authors have considered the presence of these autoantibodies in other viral encephalitides to be a clinically irrelevant epiphenomenon (Jarius and Wildemann, Reference Jarius and Wildemann2015).

In 1985, it was suggested that Borna disease virus could cause mental illness in humans (Amsterdam et al., Reference Amsterdam, Winokur, Dyson, Herzog, Gonzalez, Rott and Koprowski1985). Thus Rott et al. using an indirect immunofluorescent focus assay, detected antibodies to BoDV-1 in 16 out of 979 psychiatric patients but none were found in 200 normal volunteers (Rott et al., Reference Rott, Herzog, Fleischer, Winokur, Amsterdam, Dyson and Koprowski1985). Fu et al. also reported that patients with mental illness had antibody titers against BoDV-1 (Fu et al., Reference Fu, Amsterdam, Kao, Shankar, Koprowski and Dietzschold1993). These, and subsequent reports stimulated extensive investigations into possible links between bornaviral infection and mental health. It was suggested that BoDV-1 was associated with human neuropsychiatric diseases including bipolar disorder, chronic fatigue syndrome, schizophrenia and unipolar depression. However, isolation of BoDV-1 from humans is rare, and the serologic results obtained are possibly mis- or over-interpreted (Lipkin et al., Reference Lipkin, Briese and Hornig2011). Positive reverse-transcriptase polymerase chain reaction results may have been a result of inadvertent laboratory contamination, because the human-derived sequences showed marked similarity to animal-derived laboratory strains (Durrwald et al., Reference Durrwald, Kolodziejek, Herzog and Nowotny2007). It is now generally accepted that bornaviruses are not a significant cause of human neuropsychiatric disease (Lipkin et al., Reference Lipkin, Briese and Hornig2011; Hornig et al., Reference Hornig, Briese, Licinio, Khabbaz, Altshuler, Potkin, Schwemmle, Siemetzki, Mintz, Honkavuori, Kraemer, Egan, Whybrow, Bunney and Lipkin2012).

Rodents

Experimental BoDV-1 infection of laboratory rats and mice has revealed many key features of bornaviral pathogenesis. One of the most significant features of these infections is the difference between the nature of the disease in adult and newborn rats.

Neonatal rats

In neonatal rats, experimental infection with BoDV-1 results in transient mild inflammation, and there are no immediate clinical signs of disease. However the virus does cause glial activation that eventually leads to significant changes in brain development, behavioral abnormalities and a life-long persistent infection (Hornig et al., Reference Hornig, Weissenbock, Horscroft and Lipkin1999). This infection is initiated at a time when the rat brain is continuing to develop and its neuronal connections are being tuned and adapted to environmental influences (Gonzalez-Dunia et al., Reference Gonzalez-Dunia, Volmer, Mayer and Schwemmle2005). Herzog et al. compared BoDV-1 distribution following challenge in adult and neonatal rats. In adults, the virus was restricted to the central nervous system, but in neonates it was also found in the heart, adrenal, stomach and intestine but not blood (Herzog et al., Reference Herzog, Kompter, Frese and Rott1984).

This persistent infection of newborn rats results in selective injury to those areas of the brain undergoing significant post-natal development (de la Torre, Reference de la Torre2002). Thus, there is neuronal loss in the cortex, hippocampus and cerebellum (Fig. 3). The lesions in the hippocampus are concentrated in the granule cells of the dentate gyrus. This neuronal degeneration results from progressive granule cell apoptosis (Hornig et al., Reference Hornig, Weissenbock, Horscroft and Lipkin1999). Neonatal neuronal plasticity is accompanied by formation of new synapses and an increase in dendritic arborization (Engert and Bonhoeffer, Reference Engert and Bonhoeffer1999). Synaptic density is altered in BoDV-1-infected neonatal rats and in BoDV-1-P transgenic mice (Gonzalez-Dunia et al., Reference Gonzalez-Dunia, Watanabe, Syan, Mallory, Masliah and de la Torre2000; Kamitani et al., Reference Kamitani, Ono, Yoshino, Kobayashi, Taharaguchi, Lee, Yamashita, Kobayashi, Okamoto, Taniyama, Tomonaga and Ikuta2003)

Fig. 3. A schematic diagram showing the multiple alterations in neurogenesis induced by BoDV-1 in neonatal mice.

Eisenman et al. showed reduced cerebellar size but normal lamellar organization in BoDV-1-infected neonatal rats (Eisenman et al., Reference Eisenman, Brothers, Tran, Kean, Dickson, Dietzschold and Hooper1999). Gaps eventually develop in the Purkinje cell layer of the cerebellum and it has been estimated that up to 75% of Purkinje cells may be lost in these animals (Bautista et al., Reference Bautista, Rubin, Moran, Schwartz and Carbone1995; Eisenman et al., Reference Eisenman, Brothers, Tran, Kean, Dickson, Dietzschold and Hooper1999).

Persistently infected neonatal rats also develop an astrocytosis (Ovanesov et al., Reference Ovanesov, Ayhan, Wolbert, Moldovan, Sauder and Pletnikov2008a). These astrocytes control homeostasis around synapses and have a key role in removing excess neurotransmitters such as glutamate (Coulter and Eid, Reference Coulter and Eid2012). Bornavirus-infected astrocytes have a reduced ability to take up glutamate (de la Torre, Reference de la Torre2002) (see below).

Persistently infected neonatal rats have impaired cognitive functions, and deficiencies in fear conditioning (Carbone et al., Reference Carbone, Rubin, Nishino and Pletnikov2001; Pletnikov et al., Reference Pletnikov, Rubin, Vogel, Moran and Carbone2002). Despite their cerebellar abnormalities, they do not show ataxia, but they do exhibit novelty-induced hyperactivity, chronic anxiety and have abnormal sleep-wake cycles, as well as decreased play behavior (Hornig et al., Reference Hornig, Solbrig, Horscroft, Weissenbock and Lipkin2001; Pletnikov et al., Reference Pletnikov, Rubin, Vogel, Moran and Carbone2002). These neonatal rat behaviors resembles some human neuropsychiatric disorders especially autism spectrum disorder (Lancaster et al., Reference Lancaster, Dietz, Moran and Pletnikov2007).

Other species

BoDV-1 has been used to induce experimental infections in many other mammals. The clinical disease and outcome vary between species. Rabbits, for example, develop a fatal paralytic disease similar to that seen in horses (Richt and Rott, Reference Richt and Rott2001); tree shrews (Tupaia glis) develop aberrant neurologic behaviors (Richt et al., Reference Richt, Vandewoude, Zink, Clements, Herzog, Stitz, Rott and Narayan1992); and rhesus monkeys develop severe paralytic disease with retinopathy (Stitz et al., Reference Stitz, Krey and Ludwig1981; Richt et al., Reference Richt, Vandewoude, Zink, Clements, Herzog, Stitz, Rott and Narayan1992). BoDV-1 has been detected in two dogs with neurologic symptoms (Weissenbock et al., Reference Weissenbock, Nowotny, Caplazi, Kolodziejek and Ehrensperger1998; Okamoto et al., Reference Okamoto, Kagawa, Kamitani, Hagiwara, Kirisawa, Iwai, Ikuta and Taniyama2002). Cattle can also develop Borna disease but it is uncommon and sporadic (Bode et al., Reference Bode, Durrwald and Ludwig1994; Caplazi et al., Reference Caplazi, Waldvogel, Stitz, Braun and Ehrensperger1994). Bornaviral encephalitis has also been recorded in captive alpacas and wild deer (Jacobsen et al., Reference Jacobsen, Algermissen, Schaudien, Venner, Herzog, Wentz, Hewicker-Trautwein, Baumgartner and Herden2010).

Encephalitis

In BoDV-1 infected rats, the most severely affected brain regions include the olfactory bulb, the dentate gyrus, the caudate nucleus and the hippocampus as well as adjacent structures such as the mesencephalon, central gray matter, substantia nigra and hypothalamus (Tomonaga et al., Reference Tomonaga, Kobayashi and Ikuta2002). BoDV-1 thus exhibits a preferential tropism for the rodent limbic system (de la Torre, Reference de la Torre2002). Unfortunately, the viral cellular receptor(s) have yet to be identified, so the biochemical basis of this tropism is unknown.

BoDV-1 enters cells by receptor-mediated endocytosis (Gonzalez-Dunia et al., Reference Gonzalez-Dunia, Cubitt and de la Torre1998). The primary route of natural invasion in most mammals is most likely through the nasal epithelium (Morales et al., Reference Morales, Herzog, Kompter, Frese and Rott1988; Sauder and Staeheli, Reference Sauder and Staeheli2003). The olfactory bulbs of experimentally infected horses show inflammation and edema early in disease (Solbrig and Koob, Reference Solbrig and Koob2003). The virus replicates in the neurons at the initial entry site and then migrates intra-axonally towards the brain (Carbone et al., Reference Carbone, Duchala, Griffin, Kincaid and Narayan1987; Salinas et al., Reference Salinas, Schiavo and Kremer2010). The viral surface glycoprotein is required for cell to cell transfer (Bajramovic et al., Reference Bajramovic, Munter, Syan, Nehrbass, Brahic and Gonzalez-Dunia2003; Lennartz et al., Reference Lennartz, Bayer, Czerwonka, Lu, Kehr, Hirz, Steinmetzer, Garten and Herden2016). The viral RNP complex spreads by axonal and polysynaptic neuronal transmission. Once within the axon, intracellular microtubules transport the viral RNP (Clemente et al., Reference Clemente, Sisman, Aza-Blanc and de la Torre2010). The protein dynein forms motor complexes with BoDV-1 RNP and transport it from the nerve terminal to the cell body (Gosztonyi et al., Reference Gosztonyi, Dietzschold, Kao, Rupprecht, Ludwig and Koprowski1993; Clemente et al., Reference Clemente, Sisman, Aza-Blanc and de la Torre2010). Subsequently, RNP spreads to other cells such as astrocytes, oligodendroglia, ependymal cells and possibly Schwann cells. Eventually viral RNA can be detected in all peripheral nerves (Enbergs et al., Reference Enbergs, Vahlenkamp, Kipar and Muller2001). As inflammation develops, perivascular cuffs form. These cuffs contain CD4+ and CD8+ T cells and macrophages (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998).

BoDV-1 is not cytotoxic in cultured cells (Stitz et al., Reference Stitz, Bilzer and Planz2002; Matsumoto et al., Reference Matsumoto, Hayashi, Omori, Honda, Daito, Horie, Ikuta, Fujino, Nakamura, Schneider, Chase, Yoshimori, Schwemmle and Tomonaga2012). The virus is strongly cell associated and produces very few infectious virions per cell even though large quantities of RNP are present (de la Torre, Reference de la Torre2002; Tomonaga et al., Reference Tomonaga, Kobayashi and Ikuta2002). Virions are not detected in infected brain either, suggesting that virus spreads as an RNP complex (Zimmermann et al., Reference Zimmermann, Breter, Rudolph and Ludwig1994). Transgenic mice expressing BoDV P-protein in their astrocytes show major behavioral abnormalities such as aggressiveness, hyperexcitability and special reference memory deficit resembling those seen in bornavirus-infected mice (Honda et al., Reference Honda, Fujino, Okuzaki, Ohtaki, Matsumoto, Horie, Daito, Itoh and Tomonaga2011). These behaviors are associated with alterations in the expression of genes associated with the transforming growth factor (TGF)-β pathway (Kamitani et al., Reference Kamitani, Shoya, Kobayashi, Watanabe, Lee, Zhang, Tomonaga and Ikuta2001; Nishino et al., Reference Nishino, Murakami and Funaba2015).

Wu et al. infected hippocampal slice cultures of several rat strains with BoDV-1 (Wu et al., Reference Wu, Schulz, Lin, Saar, Patone, Fischer, Hubner, Heimrich and Schwemmle2013). Cultures from some strains such as Lewis (LEW) showed disrupted architecture while others, such as Sprague Dawley (SD) did not. The efficacy of viral replication was however identical in cultures from different rat strains. These strain differences also occur in vivo. Media harvested from uninfected LEW or SD cultures could prevent BoDV-1-induced damage in LEW cultures. Infection with BoDV-1 reduced the availability of this inhibitory factor in LEW but not SD cultures. Genetic analysis indicated that a bornaviral resistance locus is present on rat chr6q16 and a susceptibility locus on chr3q21–23 (Wu et al., Reference Wu, Schulz, Lin, Saar, Patone, Fischer, Hubner, Heimrich and Schwemmle2013).

Retinitis

BoDV-1 spreads to the retina from the brain along the optic nerve. Krey et al. blocked the optic nerves of rabbits by xenon coagulation and then infected them intracerebrally with BoDV-1 (Krey et al., Reference Krey, Ludwig and Rott1979). Retinopathy did not develop, and viral antigen could not be detected in animals with blocked nerves. Blindness is regularly observed in equine Borna disease as a result of retinal degeneration with lymphoplasmacytic infiltration (Bilzer et al., Reference Bilzer, Grabner and Stitz1996). Lymphocytic infiltrates may also be observed within the optic nerve. There is a great diversity in its severity, and not all horses have detectable bornavirus in their retinas (Dietzel et al., Reference Dietzel, Kuhrt, Stahl, Kacza, Seeger, Weber, Uhlig, Reichenbach, Grosche and Pannicke2007).

Muller cells are retinal glial cells (Kacza et al., Reference Kacza, Vahlenkamp, Enbergs, Richt, Germer, Kuhrt, Reichenbach, Muller, Herden, Stahl and Seeger2000, Reference Kacza, Mohr, Pannicke, Kuhrt, Dietzel, Fluss, Richt, Vahlenkamp, Stahl, Reichenbach and Seeger2001). The neuron:Muller cell ratio in the retina is significantly reduced in diseased horses as compared with controls. This appears to be due to a concomitant loss of neurons and an increase in glia. The neuronal degeneration begins in the outer retinal layer where the photoreceptors are located but all retinal layers show reduced thickness (Kacza et al., Reference Kacza, Vahlenkamp, Enbergs, Richt, Germer, Kuhrt, Reichenbach, Muller, Herden, Stahl and Seeger2000).

Neurons are also lost in the retinas of BoDV-1 infected rats and rabbits (Narayan et al., Reference Narayan, Herzog, Frese, Scheefers and Rott1983b). Four weeks after intracerebral inoculation of rats there is a significant thinning of the retina due to a loss of photoreceptor segments and ganglion cells. At the same time, there is a great increase in the number of glial cells in the ganglion cell and inner plexiform layers (Kacza et al., Reference Kacza, Vahlenkamp, Enbergs, Richt, Germer, Kuhrt, Reichenbach, Muller, Herden, Stahl and Seeger2000). Microglia and macrophages are involved in the neuronophagocytosis that accompanies this neurodegeneration (Kacza et al., Reference Kacza, Vahlenkamp, Enbergs, Richt, Germer, Kuhrt, Reichenbach, Muller, Herden, Stahl and Seeger2000). Muller cells show moderate changes (Iandiev et al., Reference Iandiev, Biedermann, Bringmann, Reichel, Reichenbach and Pannicke2006). Kacza et al. infected Lewis rats with BoDV-1 intracerebrally (Kacza et al., Reference Kacza, Mohr, Pannicke, Kuhrt, Dietzel, Fluss, Richt, Vahlenkamp, Stahl, Reichenbach and Seeger2001). Within months their retinal thickness had declined to a third of that in control animals. Photoreceptor segments were completely destroyed and the number of neurons reduced. There were many active microglia and macrophages undertaking neuronophagocytosis. Muller cells showed signs of gliosis, alterations in glutamate synthetase, altered K⁺ currents and thickened stem processes (Kacza et al., Reference Kacza, Mohr, Pannicke, Kuhrt, Dietzel, Fluss, Richt, Vahlenkamp, Stahl, Reichenbach and Seeger2001).

Narayan et al. attributed bornaviral retinitis to a transient attack by cytotoxic T cells (Narayan et al., Reference Narayan, Herzog, Frese, Scheefers and Rott1983b). For example, Krey et al. treated BoDV-1 infected rabbits with immunosuppressive drugs that delayed the onset of retinitis (Krey et al., Reference Krey, Ludwig and Gierend1981). The treatment reduced the confluency of the retinal lesions and some treated animals either lacked eye lesions or showed nonprogression. Subsequently, Stahl et al. showed that the retinal T cell infiltration consisted of αβTCR+, CD4+, CD8+ cells (Stahl et al., Reference Stahl, Mohr, Kacza, Reimers, Pannicke, Sauder, Reichenbach and Seeger2003). B cells were rarely found by Stahl et al. but Hatalski et al. found them to be plentiful (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998). This may reflect the use of different reagents. Cytokine transcripts in affected retinas included raised IL-1β, IL-6, IFNγ and tumor necrosis factor (TNF)-α, as well as CXCL10, chemokine ligand (CCL)2 and CCL4. By day 36 the levels of these transcripts had returned to normal. These cytokines were probably derived from the infiltrating T cells and NK cells (Sauder and de la Torre, Reference Sauder and de la Torre1999). Chronic bornaviral infections may result from a switch in the brain-infiltrating T cells from generating a Th1 response to a Th2 response (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998).

Alterations in neurogenesis

In the developing rodent brain, neurogenesis occurs predominantly in the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus (Katsumoto et al., Reference Katsumoto, Lu, Miranda and Ransohoff2014). It is unclear how neuronal loss occurs in persistently infected neonatal rats but it is probably due to induced apoptosis (Hornig et al., Reference Hornig, Weissenbock, Horscroft and Lipkin1999; Zocher et al., Reference Zocher, Czub, Schulte-Monting, de la Torre and Sauder2000; Ovanesov et al., Reference Ovanesov, Moldovan, Smith, Vogel and Pletnikov2008b). Damage to synaptic structures precedes neuronal loss in persistently infected rats and this may impair trafficking of growth factors (Volmer et al., Reference Volmer, Prat, Le Masson, Garenne and Gonzalez-Dunia2007). Bornaviral-induced disturbances in the neurotrophin system also contribute to this neurodegeneration. Thus Zocher et al. found reduced levels of neurotrophin 3, brain-derived neurotropic factor (BDNF) and nerve growth factor in the hippocampus 14-days post-infection in newborn rats and in the cerebellum by 21 days post-infection (Zocher et al., Reference Zocher, Czub, Schulte-Monting, de la Torre and Sauder2000) (Fig. 3). They also detected reduced levels of neurotrophin receptors in both the hippocampus and cerebellum. Neonatal persistently-infected rats fail to gain weight but lack of nourishment does not account for the brain lesions or the lack of weight gain (Dietz and Pletnikov, Reference Dietz and Pletnikov2003). There are increased concentrations of serotonin in the hippocampus of bornavirus- infected neonates (Dietz and Pletnikov, Reference Dietz and Pletnikov2003). Hans et al. examined the effect of bornaviral infection on the response of hippocampal neurons to neurotrophin BDNF. Persistent infection blocked BDNF-induced extracellular signal-regulated kinases-1/2 (ERK) phosphorylation even although the expression of the BDNF receptor was normal (Hans et al., Reference Hans, Bajramovic, Syan, Perret, Dunia, Brahic and Gonzalez-Dunia2004). As a result, BDNF-induced expression of synaptic vesicle proteins is blocked, potentially causing defective synaptic organization. Persistently-infected newborn rats show a progressive decline in the expression of synaptic markers (growth-associated protein 43, a presynaptic membrane phosphoprotein and synaptophysin, a calcium-binding protein found in presynaptic vesicles) followed by a loss of up to 30% of cortical neurons (Gonzalez-Dunia et al., Reference Gonzalez-Dunia, Watanabe, Syan, Mallory, Masliah and de la Torre2000). This decline in synaptic density and neuronal plasticity occurs primarily within the cerebrum and the hippocampus.

Obesity

Some adult rats that survive acute BoDV-1 disease begin to overeat and as a result, become obese ((Narayan et al., Reference Narayan, Herzog, Frese, Scheefers and Rott1983a; Wensman et al., Reference Wensman, Jaderlund, Holst and Berg2014). Obesity also develops in some persistently infected neonatal rats (Lyons et al., Reference Lyons, Nagashima and Zabriskie2002). This uncontrolled appetite may result from damage to hunger control centers in the brain (Nagashima et al., Reference Nagashima, Zabriskie and Lyons1992; Gosztonyi and Ludwig, Reference Gosztonyi and Ludwig1995). The neonatal rats develop inflammation in their pituitary stalk (Gosztonyi and Ludwig, Reference Gosztonyi and Ludwig1995). As the obesity syndrome develops, the number of virus-infected cells grows and leads to progressive involution of the hypothalamus as well as vacuolar degeneration of neurons in the hypothalamic paraventricular nucleus. Herden et al. compared the brain lesions of a pathogenic BoDV-1 strain with that of an obesity-inducing strain (Herden et al., Reference Herden, Herzog, Richt, Nesseler, Christ, Failing and Frese2000). The obesity-inducing-strain lesions were restricted to the septum, hippocampus, ventromedial hypothalamus and amygdala. Herden et al. also examined the levels of neuropeptides in BoDV-1-infected brains and found that expression of melanocyte-stimulating hormone (MSH) was reduced in infected animals (Herden et al., Reference Herden, Herzog, Richt, Nesseler, Christ, Failing and Frese2000; Herden et al., Reference Herden, Schluesener and Richt2005). α-MSH reduces appetite so its deficiency may have an opposite effect and bornaviral obesity could therefore be due to lesions in the melanocortin feeding center within the hypothalamus.

Immune-mediated T cell attack

The primary mechanism of neuronal damage in bornaviral encephalitis is T cell-mediated neuronal cytotoxicity. These T cells are activated by viral infection and as a result, mount a type 1 response characterized by the production of both IFN-γ and TNF-α (Baruch and Schwartz, Reference Baruch and Schwartz2013). The neurotransmitters glutamine and acetylcholine also favor T cell activation (Pacheco et al., Reference Pacheco, Riquelme and Kalergis2010; Pikor et al., Reference Pikor, Astarita, Summers-Deluca, Galicia, Qu, Ward, Armstrong, Dominguez, Malhotra, Heiden, Kay, Castanov, Touil, Boon, O'Connor, Bar-Or, Prat, Ramaglia, Ludwin, Turley and Gommerman2015) as do activated Th17 cells (Nouri et al., Reference Nouri, Bredberg, Westrom and Lavasani2014).

In bornaviral lesions, the perivascular cellular infiltrates predominantly consist of CD4+ T cells while CD8+ cells predominate within the brain parenchyma. The development of neuronal lesions is specifically associated with invasion by CD8+ cells (Sobbe et al., Reference Sobbe, Bilzer, Gommel, Noske, Planz and Stitz1997). The importance of these T cell infiltrates can be demonstrated by adoptive transfer of lymphocytes from infected to uninfected rats. For example, Narayan et al. could transmit the disease using spleen cells from 4-week-old rats transferred into cyclosporine-treated recipients (Narayan et al., Reference Narayan, Herzog, Frese, Scheefers and Rott1983a). Rott et al. were able to establish a virus-specific, CD4+ T cell line that induced typical bornaviral lesions when administered to recipient rats of the corresponding MHC class II haplotype (Rott et al., Reference Rott, Herzog, Richt and Stitz1988). Sobbe et al. induced typical bornaviral brain lesions in rats by adoptive transfer of CD8+ brain T cells (Sobbe et al., Reference Sobbe, Bilzer, Gommel, Noske, Planz and Stitz1997). Additionally, lymphocytes obtained from rats early in bornaviral infection could transfer the disease but not late in infection implying that the cytotoxic cells were generated early in the disease process (Narayan et al., Reference Narayan, Herzog, Frese, Scheefers and Rott1983a). Planz et al. identified a peptide, ASYAQMTTY, from BoDV-1 nucleoprotein that was recognized by CD8+ T cells in association with the rat MHC class I molecule, RT1.A, and, as a result, triggered T cell cytotoxicity (Planz et al., Reference Planz, Dumrese, Hulpusch, Schirle, Stevanovic and Stitz2001). Transgenic mice expressing the nucleoprotein are resistant to disease, presumably because they are immunologically tolerant to it (Schwemmle et al., Reference Schwemmle, Salvatore, Shi, Richt, Lee and Lipkin1998). Lymphocytes from the brains of rats with acute bornaviral disease show MHC class I restricted cytotoxic T cell activity. MHC class I expression occurs on astrocytes and on some neurons in BoDV-1-infected brains. MHC class I production by neurons is normally minimal but can be induced (Planz et al., Reference Planz, Bilzer, Sobbe and Stitz1993).

Support for the essential role of T cells is also provided by immunosuppressive studies. For example, Rott et al. demonstrated that T cell elimination prevented inflammation and the development of clinical disease. A single intraperitoneal injection of cyclophosphamide given to adult rats prevented the development of bornaviral encephalitis and clinical disease. Stitz et al. were able to prevent the development of bornaviral disease by administering cyclosporine prior to infection (Stitz et al., Reference Stitz, Soeder, Deschl, Frese and Rott1989). Stitz et al. also treated bornavirus-infected adult rats with monoclonal antibodies against N-protein-specific CD4+ and CD8+ T cells (Stitz et al., Reference Stitz, Sobbe and Bilzer1992). Both types of antibody suppressed the encephalitis but anti-CD8 was more effective than anti-CD4. These monoclonal antibodies worked best if given before or shortly after infection and they did not prevent encephalitis or disease when given more than 4 days after infection. BoDV-1 infection will not result in disease in athymic nude rats (Herzog et al., Reference Herzog, Wonigeit, Frese, Hedrich and Rott1985). Thus the lesions of bornaviral encephalitis in rats are mediated primarily by N-protein-specific CD8+ T cells.

In persistently infected neonatal rats, unlike adult rats, T cells may be protective. Thus persistent BoDV-1 infection could be prevented by prior administration of a virus-specific CD4+ T cell line. Recipient rats developed a transient, mild encephalitis that lasted for only a few days. This T cell line had no cytotoxic properties but virus clearance was accompanied by the appearance of CD8+ cytotoxic T cells in the recipients (Noske et al., Reference Noske, Bilzer, Planz and Stitz1998). It is interesting to note however, that in adult rats infected with BoDV-1, the virus is found exclusively in the brain. In cyclosporine-treated rats the virus can be detected in peripheral nerve fibers and adjacent tissues such as lung, liver and spleen (Stitz et al., Reference Stitz, Schilken and Frese1991). This suggests that T cells are responsible for containing BoDV-1 within the central nervous system (CNS). In subsequent studies, Stitz et al. examined the role of antibodies in restricting BoDV-1 to the CNS. Immune serum transfer into cyclosporine-treated or newborn rats resulted in restriction of the virus to the CNS (Stitz et al., Reference Stitz, Noske, Planz, Furrer, Lipkin and Bilzer1998).

Hatalski et al. followed the evolution of the immune response in the brain of BoDV-1-infected Lewis rats (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998). Thus at the peak of the acute infection, the perivascular infiltrates contained both CD4+ and CD8+ T cells as well as a significant population of NK cells. The NK cells could be identified in the brain lesions 3.5 weeks post-infection before the onset of clinical disease. As the disease progressed and became chronic, the numbers of all cell types dropped and the authors suggested that this reflected a switch from a type 1 to a type 2 immune response. This suggestion was supported by a rise in serum IgE levels. Hatalski et al. also measured cytokine mRNA expression in infected brains. They showed a significant increase in the levels of the proinflammatory cytokines, IL-1α, IL-2, IL-6, TNF-α and IFN-γ that peaked at 5 weeks post-infection when acute inflammation was maximal. On the other hand, the anti-inflammatory cytokine, IL-4 increased to reach maximal levels at the end of the study at 15 weeks. The regulatory cytokine, TGF-β in contrast, peaked at 5 weeks and then stabilized (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998). It is possible that the decline in inflammation seen in long-term bornaviral infections may also be due to the development of neuroprotective Treg cells (Walsh et al., Reference Walsh, Watson and Kipnis2014). Nishino et al. investigated this upregulation of TGF-β in BoDV-1-infected rats. They found that signal receptors for TGF-β1 were also upregulated as were inhibitin/activin βC, two components of the TGF-β pathway (Nishino et al., Reference Nishino, Ooishi, Kurokawa, Fujino, Murakami, Madarame, Hashimoto, Sugiyama and Funaba2009). This may reflect the ongoing immunosuppressive effects of bornaviral P-protein (Nishino et al., Reference Nishino, Murakami and Funaba2015).

Microglia and the role of chronic inflammation

The second important pathogenic process in bornaviral disease results from prolonged activation of microglia (Weissenbock et al., Reference Weissenbock, Hornig, Hickey and Lipkin2000; Ovanesov et al., Reference Ovanesov, Sauder, Rubin, Richt, Nath, Carbone and Pletnikov2006, Reference Ovanesov, Vogel, Moran and Pletnikov2007, Reference Ovanesov, Ayhan, Wolbert, Moldovan, Sauder and Pletnikov2008a, Reference Ovanesov, Moldovan, Smith, Vogel and Pletnikovb). In the healthy brain, microglia remodel neuronal synapses and secrete neurotrophic proteins that help maintain effective neuronal network functions (Zocher et al., Reference Zocher, Czub, Schulte-Monting, de la Torre and Sauder2000). When activated however, microglia flood the brain with the inflammatory cytokines, TNF-α, IFN-β and IL-10 as well as induced nitric oxide synthase (iNOS), and reactive oxygen and nitrogen species (ROS and RNS) (Gonzalez et al., Reference Gonzalez, Elgueta, Montoya and Pacheco2014). These molecules induce neuronal dysfunction and death (Heneka et al., Reference Heneka, Kummer and Latz2014; Papageorgiou et al., Reference Papageorgiou, Lewen, Galow, Cesetti, Scheffel, Regen, Hanisch and Kann2015). Zheng et al. have demonstrated that the severity of the neurologic signs and of the corresponding encephalitis correlated well with iNOS and cNOS mRNA expression in rat bornaviral disease and that the distribution of iNOS-positive cells in the basolateral cortex and the hippocampus correlated with sites of BoDV-1 infected cells (Zheng et al., Reference Zheng, Schafer, Weihe, Sheng, Corisdeo, Fu, Koprowski and Dietzschold1993). This cannot however be the whole explanation for bornaviral pathogenicity since Hausmann et al. demonstrated that BoDV-1 caused neurologic disease in mice that lacked IFN-γ, Fas, iNOS or the chemokine receptor, CXCR3 (Hausmann et al., Reference Hausmann, Sauder, Wasmer, Lu and Staeheli2004).

Microglia are activated by BoDV-1 (Plata-Salaman et al., Reference Plata-Salaman, Ilyin, Gayle, Romanovitch and Carbone1999; Ovanesov et al., Reference Ovanesov, Sauder, Rubin, Richt, Nath, Carbone and Pletnikov2006) (Fig. 5). In BoDV-1-infected adult rats, these activated microglia can be detected in the dentate gyrus at 10 days post-infection but detectable loss of granule cells is not seen until 30 days. Thus the virus activates the microglia long before neuronal loss and it is therefore unlikely that the activated microglia alone trigger neuronal loss and dysfunction (Ovanesov et al., Reference Ovanesov, Moldovan, Smith, Vogel and Pletnikov2008b). Nevertheless, increased levels of brain IL-6, TNF-α and iNOS mRNAs do correlate with the severity of the inflammatory lesions in infected brains (de la Torre, Reference de la Torre2002).

Fig. 5. The role of microglial activation in the pathogenesis of bornaviral encephalitis. M1 activation of the microglia results in the flooding of the brain with multiple cytokines as well as potent oxidants resulting in neuronal death. Should the microglial phenotype change to M2, the resulting cytokines will reduce inflammation and cell destruction and promote repair although destroyed neurons unlikely to be replaced.

Classically activated microglia (M1 cells) have two alternative fates. They may differentiate into regulatory cells (M2 cells) and so reduce inflammation and promote tissue repair or alternatively, they can undergo uncontrolled activation and trigger chronic inflammation resulting in the production of neurotoxic factors and progressive neural loss (Gonzalez et al., Reference Gonzalez, Elgueta, Montoya and Pacheco2014). M2 activation of microglia enhances their release of neurotrophic factors, proteases, IL-4, TGFβ and arginase 1 and stimulates their phagocytic activity. M2 activation thus reduces inflammation and promotes tissue repair. The anti-inflammatory cytokines, IL-4 and TGFβ are produced during the later stages of bornaviral encephalitis (Hatalski et al., Reference Hatalski, Hickey and Lipkin1998). While they are mainly derived from T cells, they may also be produced by microglia (Heneka et al., Reference Heneka, Kummer and Latz2014).

Morimoto et al. used dexamethasone to inhibit bornavirus-induced inflammation in rats. They suggested that bornaviral disease involved an early inflammatory reaction mediated by resident microglia leading to sensitization (antigen processing and presentation), and an influx of primed T cells. Restimulation of these infiltrating T cells led to the local production of a cytokine mixture amplifying the reaction. Subsequently, recruitment and activation of microglia continued this process. Morimoto et al. suggested that the initial expression of pro-inflammatory cytokines was directly mediated by microglial BoDV-1 (Morimoto et al., Reference Morimoto, Hooper, Bornhorst, Corisdeo, Bette, Fu, Schafer, Koprowski, Weihe and Dietzschold1996).

Astrocytes and glutamate toxicity

The third major mechanism of bornaviral-induced neuronal destruction results from astrocyte-mediated disturbances in glutamate levels.

Astrocytes are the major microglial cell population in the brain (Rossi and Volterra, Reference Rossi and Volterra2009). Under normal conditions, they supply glucose to neurons and regulate the composition of extracellular fluid. Importantly, they remove excess potassium ions and neurotransmitters, especially glutamate (Coulter and Eid, Reference Coulter and Eid2012). Astrocytes take up extracellular glutamate, transform this into glutamine using glutamine synthetase and subsequently shuttle the glutamine back to neurons using transporters. Between 80 and 90% of extracellular glutamate uptake in the brain is through these astrocytic glutamine transporters (Vesce et al., Reference Vesce, Rossi, Brambilla and Volterra2007; Coulter and Eid, Reference Coulter and Eid2012) (Fig. 6). Once within neurons glutamine is reconverted to glutamate. This glutamate may act as a neurotransmitter directly or it is decarboxylated to form γ-amino butyric acid (GABA), which also acts as a neurotransmitter.

Fig. 6. The role of glutamate in BoDV-1 encephalitis. The normal glutamate cycle requires that astrocytes remove excess glutamate from the extracellular fluid, convert it to glutamine and return it to the neurons. If astrocytes are damaged glutamate accumulates and kills neurons. GABAergic neurons release GABA that inhibits glutamate excitotoxicity and microglial activation. If these neurons are also damaged by BoDV-1 then additional neuronal loss will be expected.

Other effects on neurotransmission

Bornaviral encephalitis in Lewis rats is associated with a decline in the cholinergic activity of the brain (Gies et al., Reference Gies, Bilzer, Stitz and Staiger1998, Reference Gies, Gorcs, Mulder, Planz, Stitz, Bilzer, Luiten and Harkany2001). This decline appears to be due to a loss of choline acetyltransferase activity in the cerebral cortex and hippocampus and a reduction in acetylcholinesterase in these regions. These declines parallel the loss of neurons in these areas. The decline in choline acetyltransferase occurs in the pre-encephalitic stage of bornaviral infection prior to T cell infiltration into the brains (Gies et al., Reference Gies, Bilzer, Stitz and Staiger1998).

BoDV-1 also affects the dopamine system. Neonatal rats experimentally infected with BoDV-1 develop a hyperactive movement disorder (Solbrig et al., Reference Solbrig, Koob, Joyce and Lipkin1996). Since locomotor activity is regulated through the dopamine system, Solbrig et al. examined the dopamine receptors in these rats (Solbrig et al., Reference Solbrig, Koob, Joyce and Lipkin1996). They found that there was reduced binding to several dopamine receptors in the nucleus accumbens. The nucleus accumbens is the site where the limbic system and motor information interact. It plays a role in motivational, appetite and locomotor behaviors. Bornaviral-induced alterations have also been recorded in other neurotransmitters such as cholecystokinin and somatostatin (de la Torre, Reference de la Torre2002).