HAND2 suppresses favipiravir efficacy in treatment of Borna disease virus infection.

Borna disease virus (BoDV-1) is a bornavirus prototype that infects the central nervous system of various animal species and can cause fatal encephalitis in various animals including humans. Among the reported anti-BoDV-1 treatments, favipiravir (T-705) is one of the best candidates since it has been shown to be effective in reducing various bornavirus titers in cell culture. However, T-705 effectiveness on BoDV-1 is cell type-dependent, and the molecular mechanisms that explain this cell type-dependent difference remain unknown. In this study, we noticed a fact that T-705 efficiently suppressed BoDV-1 in infected 293T cells, but not in infected SH-SY5Y cells, and sought to identify protein(s) responsible for this cell-type-dependent difference in T-705 efficacy. By comparing the transcriptomes of BoDV-1-infected 293T and SH-SY5Y cells, we identified heart- and neural crest derivatives-expressed protein 2 (HAND2) as a candidate involved in T-705 interference. HAND2 overexpression partly attenuated the inhibitory effect of T-705, whereas HAND2 knockdown enhanced this effect. We also demonstrated an interaction between T-705 and HAND2. Furthermore, T-705 impaired HAND2-mediated host gene expression. Because HAND2 is an essential transcriptional regulator of embryogenesis, T-705 may exhibit its adverse effects such as teratogenicity and embryotoxicity through the impairment of HAND2 function. This study provides novel insights into the molecular mechanisms underlying T-705 interference in some cell types and inspires the development of improved T-705 derivatives for the treatment of RNA viruses.

Comparative study of virus and lymphocyte distribution with clinical data suggests early high dose immunosuppression as potential key factor for the therapy of patients with BoDV-1 infection.

ABSTRACTBorna disease virus 1 (BoDV-1) was just recently shown to cause predominantly fatal encephalitis in humans. Despite its rarity, bornavirus encephalitis (BVE) can be considered a model disease for encephalitic infections caused by neurotropic viruses and understanding its pathomechanism is of utmost relevance. Aim of this study was to compare the extent and distribution pattern of cerebral inflammation with the clinical course of disease, and individual therapeutic procedures. For this, autoptic brain material from seven patients with fatal BVE was included in this study. Tissue was stained immunohistochemically for pan-lymphocytic marker CD45, the nucleoprotein of BoDV-1, as well as glial marker GFAP and microglial marker Iba1. Sections were digitalized and counted for CD45-positive and BoDV-1-positive cells. For GFAP and Iba1, a semiquantitative score was determined. Furthermore, detailed information about the individual clinical course and therapy were retrieved and summarized in a standardized way. Analysis of the distribution of lymphocytes shows interindividual patterns. In contrast, when looking at the BoDV-1-positive glial cells and neurons, a massive viral involvement in the brain stem was noticeable. Three of the seven patients received early high-dose steroids, which led to a significantly lower lymphocytic infiltration of the central nervous tissue and a longer survival compared to the patients who were treated with steroids later in the course of disease. This study highlights the potential importance of early high-dose immunosuppressive therapy in BVE. Our findings hint at a promising treatment option which should be corroborated in future observational or prospective therapy studies.ABBREVIATIONS: BoDV-1: Borna disease virus 1; BVE: bornavirus encephalitis; Cb: cerebellum; CNS: central nervous system; FL: frontal lobe; GFAP: glial fibrillary acid protein; Hc: hippocampus; Iba1: ionized calcium-binding adapter molecule 1; Iba1act: general activation of microglial cells; Iba1nod: formation of microglial nodules; IL: insula; Me: mesencephalon; Mo: medulla oblongata; OL: occipital lobe; pASS: per average of 10 screenshots; patearly: patients treated with early high dose steroid shot; patlate: patients treated with late or none high dose steroid shot; Po: pons; So: stria olfactoria; Str: striatum.

Antiviral treatment perspective against Borna disease virus 1 infection in major depression: a double-blind placebo-controlled randomized clinical trial.

BACKGROUND: Whether Borna disease virus (BDV-1) is a human pathogen remained controversial until recent encephalitis cases showed BDV-1 infection could even be deadly. This called to mind previous evidence for an infectious contribution of BDV-1 to mental disorders. Pilot open trials suggested that BDV-1 infected depressed patients benefitted from antiviral therapy with a licensed drug (amantadine) which also tested sensitive in vitro. Here, we designed a double-blind placebo-controlled randomized clinical trial (RCT) which cross-linked depression and BDV-1 infection, addressing both the antidepressant and antiviral efficacy of amantadine. METHODS: The interventional phase II RCT (two 7-weeks-treatment periods and a 12-months follow-up) at the Hannover Medical School (MHH), Germany, assigned currently depressed BDV-1 infected patients with either major depression (MD; N = 23) or bipolar disorder (BD; N = 13) to amantadine sulphate (PK-Merz®; twice 100 mg orally daily) or placebo treatment, and contrariwise, respectively. Clinical changes were assessed every 2-3 weeks by the 21-item Hamilton rating scale for depression (HAMD) (total, single, and combined scores). BDV-1 activity was determined accordingly in blood plasma by enzyme immune assays for antigens (PAG), antibodies (AB) and circulating immune complexes (CIC). RESULTS: Primary outcomes (≥25% HAMD reduction, week 7) were 81.3% amantadine vs. 35.3% placebo responder (p = 0.003), a large clinical effect size (ES; Cohen's d) of 1.046, and excellent drug tolerance. Amantadine was safe reducing suicidal behaviour in the first 2 weeks. Pre-treatment maximum infection levels were predictive of clinical improvement (AB, p = 0.001; PAG, p = 0.026; HAMD week 7). Respective PAG and CIC levels correlated with AB reduction (p = 0,001 and p = 0.034, respectively). Follow-up benefits (12 months) correlated with dropped cumulative infection measures over time (p < 0.001). In vitro, amantadine concentrations as low as 2.4-10 ng/mL (50% infection-inhibitory dose) prevented infection with human BDV Hu-H1, while closely related memantine failed up to 100,000-fold higher concentration (200 µg/mL). CONCLUSIONS: Our findings indicate profound antidepressant efficacy of safe oral amantadine treatment, paralleling antiviral effects at various infection levels. This not only supports the paradigm of a link of BDV-1 infection and depression. It provides a novel possibly practice-changing low cost mental health care perspective for depressed BDV-1-infected patients addressing global needs. TRIAL REGISTRATION: The trial was retrospectively registered in the German Clinical Trials Registry on 04th of March 2015. The trial ID is DRKS00007649; https://www.drks.de/drks_web/setLocale_EN.do.

Cannabinoid rescue of striatal progenitor cells in chronic Borna disease viral encephalitis in rats.

A growing number of environmental and pharmacologic manipulations have been shown to influence adult neurogenesis. Borna disease virus (BDV) in rats causes cortical and subcortical infection with extrapyramidal motor symptoms, and hippocampal infection suppresses neurogenesis. Given the known effects of cannabinoids in promoting neural progenitor cell survival, the authors examined in vivo effects of chronic BDV infection in rats on BrdU-positive progenitor cells in striatum, together with neuroprotective actions of cannabinoids. Birth and survival of BrdU-positive progenitor cells in striatum of BDV-infected rats treated with a general cannabinoid agonist (WIN 55,212 1 mg/kg i.p. b.i.d. x 7 days) were examined, as well as anti-inflammatory, antiviral, and nutritional effects of cannabinoids. Cannabinoid treatment protected BrdU-positive progenitor cells in striatum that were susceptible to virus-induced injury (p < .01) through suppression of microglia activation (p < .001). As a consequence of their anti-inflammatory actions and support of neural progenitor cell survival, cannabinoids may be adjunctive treatment for encephalitides with microglial inflammation and neurodegeneration.

Activators of potassium M currents have anticonvulsant actions in two rat models of encephalitis.

Opioid systems in hippocampus regulate excitability and kappa opioids have a role in anticonvulsant protection, but their mechanisms of action are incompletely understood. We examined the ability of opioid and nonopioid agents with overlapping ionic mechanisms and actions similar to kappa opioid agonists, to block seizures in rat models of encephalitis due to Borna Disease virus and Herpes Simplex Virus Type-1. Naltrindole, a delta antagonist and thus a kappa opioid sparing agent, (10 mg/kg s.c.) blocked spontaneous and naloxone (opioid antagonist)-induced seizures in the models, but produced somatic signs similar to opioid withdrawal. Given that delta antagonists as well as kappa opioid agonists in hippocampus enhance potassium M currents (I(M)), we tested the effect of the I(M) augmenter flupirtine. Flupirtine (20 mg/kg i.p.) prevented seizures in Borna and herpes infected rats, without signs of withdrawal, hypotonia or sedation. The results support the efficacy of opioid and nonopioid drugs in modulating naloxone-induced seizures in critical illness due to viral encephalitis and by analogy, opioid withdrawal seizures.

Antiviral activity of favipiravir (T-705) against mammalian and avian bornaviruses.

Bornaviruses, non-segmented, negative-strand RNA viruses, are emerging agents with the potential for causing various types of neurological symptoms. Previous studies have shown that ribavirin, a nucleic acid analog with broad-spectrum antiviral activity, has a potent antiviral effect on infections with a mammalian bornavirus, Borna disease virus (BoDV-1), as well as avian bornaviruses. However, ribavirin-based treatment does not eliminate bornaviruses from persistently infected cells and viral replication resumes after treatment cessation. Therefore, the development of a novel effective anti-bornavirus treatment is needed. To identify such agents, we screened nucleoside/nucleotide mimetics for agents with anti-bornavirus activity. We used Vero cells infected with recombinant BoDV-1 carrying Gaussia luciferase to monitor BoDV-1 replication and found that favipiravir (T-705) is a potent inhibitor of BoDV-1 replication. T-705 suppressed BoDV-1 replication in a dose- and time-dependent manner during the observation period of 4 weeks. Notably, no increase in luciferase activity or in the number of BoDV-1-positive cells was detected in the at least 4 weeks following T-705 removal. Finally, we demonstrated that T-705 effectively suppressed viral replication of both BoDV-1 and an avian bornavirus, suggesting that T-705 may have a strong antiviral activity against a broad range of bornaviruses. Our findings provide a novel and effective option for treating persistent bornavirus infection.

Naloxone-induced seizures in rats infected with Borna disease virus.

The opioid antagonist naloxone is widely used in the emergency treatment of nontraumatic coma. Although it is uncommon for serious side effects to result from administration of opiate antagonists, we report that naloxone can have epileptogenic effects in the context of encephalitis. In an experimental model of viral encephalitis, rats infected with Borna disease virus developed myoclonic, generalized clonic, or atonic seizures; behavior arrest; and staring spells when treated with naloxone. These findings suggest a novel neuropharmacologic link, through opioid peptide systems, between epilepsy and encephalitis and disclose a potential contraindication to use of opioid antagonists in nontraumatic coma.

Borna disease virus--does it infect humans and cause psychiatric disorders?

Antibodies recognizing Borna disease virus (BDV) antigens were first demonstrated in the blood of psychiatric patients approximately 15 years ago. Since that time, a highly controversial debate arose whether BDV infects humans and whether it causes psychiatric disorders. In this review, we critically discuss the results of numerous studies that assessed this possibility by using virological and serological methods. We conclude that there is presently no strong experimental evidence supporting the notion that BDV is a human pathogen. The possibility remains, however, that an antigenically related agent is associated with human psychiatric disorders.

Neuropharmacological sequelae of persistent CNS viral infections: lessons from Borna disease virus.

Borna Disease Virus (BDV) is a neurotropic RNA virus that is worldwide in distribution, causing movement and behavior disorders in a wide range of animal species. BDV has also been reported to be associated with neuropsychiatric diseases of humans by serologic study and by recovery of nucleic acid or virus from blood or brain. Natural infections of horses and sheep produce encephalitis with erratic excited behaviors, hyperkinetic movement or gait abnormalities; naturally infected cats have ataxic "staggering disease." Experimentally infected primates develop hyperactivity, aggression, disinhibition, then apathy; prosimians (lower primates) have hyperactivity, circadian disruption, abnormal social and dominance behaviors, and postural disorders. However, the neuropharmacological determinants of BD phenotypes in laboratory and natural hosts are incompletely understood. Here we review how experimentally infected rodents have provided models for examining behavioral, pharmacologic, and biochemical responses to viral challenge, and how rodents experimentally infected as neonates or as adolescents are providing models for examining age-specific neuropharmacological adaptations to viral injury.

Infections in horses: diagnosis and therapy.

Borna Disease Virus (BDV) is a unique RNA virus, whose organs of manifestation are the brain and blood of animals as well as humans. The infection disrupts certain cell functions, but does not damage the cell structure. The infection with BDV can exist without associated clinical symptoms. Furthermore the majority of natural BDV-infections occur unnoticed without causing symptoms particularly those in connection with only a slight BDV-infection. BDV-infected horses can be detected by an extremely practicable ELISA based on blood samples and developed by the Berlin Working Group under guidance of Ludwig and Bode. All three serological Borna-Disease (BD) parameters antigen-, immune complex- and antibody-titer can be measured with this ELISA. However a single testing can not lead to a final evaluation of the infection so that progressive investigations are unavoidable. Blood tests in intervals of 4-6 weeks show the course of infection and help to adjust the specific treatment. After an infection an examination of the antigen- and immune complex-titer will show whether this occurrence is acute and activated or not. Therefore we examined 3481 blood samples of different horses by ELISA. 1841 (50%) were BDV-infected. Approximately 40% of the infected horses were clinically healthy and approximately 43% were clinically ill. The relatively high infection rate could be justified by the fact that these subjects had more or less direct contact with clinically ill horses. Furthermore, it is possible that the highly Borna positive, but not clinically ill horses were tested shortly before the symptoms of disease would appear. Moreover there were also horses that have had a high BDV-titer without showing any sign of the BDV-disease. These animals were thus able to live with the infection. Our investigations focused on highly seropositive BDV-infected horses (1) (Fig. 1). The results can not be linked to BD typical endemic regions due to the fact of today's far more sophisticated testing methods. Horses are more than ever used for leisure activity and become subjects to a worldwide marketing and movement. Any stress situation, especially in competitions as shown in long-term monitoring of sick horses, leads to worsening of symptoms. In this context it should be noted that a test for activated BDV-infection is still not common. EU-wide regulations should therefore be considered.

Human Borna disease virus-infection and its therapy in affective disorders.

Patients with affective disorders show an enhanced prevalence of Borna disease virus (BDV)-infection. Furthermore, BDV causes latent infection preferably in limbic central nervous structures and is suggested to be causally related to subtypes of affective disorders, especially with melancholic clinical features or bipolarity. Such a possible link was highlighted by the first report of amantadine showing an antidepressive and an antiviral efficacy against BDV in a patient with a bipolar disorder. This article summarizes clinical studies which followed this first report on the use of amantadine in BDV-infected patients with an affective disorder. A special focus is given on an open clinical study in patients with depression (n = 25), a study in remitted patients with affective disorders (n = 16), and the effect of amantadine on severe hypomanic or moderately manic patients with a bipolar disorder in an on-off-on study. In these studies amantadine reduced clinical symptoms paralleled by a reduction of BDV-infection in depressive patients, it also reduced all three BDV-parameters (BDV-Ab, -AG, and -CICs) in remitted patients, and it even reduced severe hypomania and moderate mania in bipolar patients. These data suggest the existence of an etiopathogenetic link between BDV and subtypes of affective disorders.

The history and treatment of a bipolar patient diagnosed with Borna disease virus infection. Case report.

A description of Bipolar Disorder and its treatment costs. The prevalence of various psychiatric disorders in the United States in which Borna Disease Virus (BDV) may play a role. My personal history of Bipolar Disorder including: diagnoses and treatment of Borna Disease Virus infection.

Ribavirin inhibits Borna disease virus proliferation and fatal neurological diseases in neonatally infected gerbils.

By using neonatal gerbils, we assessed the effect of ribavirin on the proliferation of Borna disease virus (BDV) in the brain. The intracranial inoculation of ribavirin reduced viral propagation in the acutely infected brain, resulting in protection from fatal neurological disorders. We found that the treatment with ribavirin markedly reduces the numbers of OX-42-positive microglial cells, but does not activate expression of Th1 cytokines, in BDV-infected gerbil brains. Our results suggested that ribavirin directly inhibits BDV replication and might be a potential tool for the treatment of BDV infection.

A role for endocannabinoids in viral-induced dyskinetic and convulsive phenomena.

Dyskinesias and seizures are both medically refractory disorders for which cannabinoid-based treatments have shown early promise as primary or adjunctive therapy. Using the Borna disease (BD) virus rat, an animal model of viral encephalopathy with spontaneous hyperkinetic movements and seizure susceptibility, we identified a key role for endocannabinoids in the maintenance of a balanced tone of activity in extrapyramidal and limbic circuits. BD rats showed significant elevations of the endocannabinoid anandamide in subthalamic nucleus, a relay nucleus compromised in hyperkinetic disorders. While direct and indirect cannabinoid agonists had limited motor effects in BD rats, abrupt reductions of endocannabinoid tone by the CB1 antagonist SR141716A (0.3 mg/kg, i.p.) caused seizures characterized by myoclonic jerks time-locked to periodic spike/sharp wave discharges on hippocampal electroencephalography. The general opiate antagonist naloxone (NLX) (1 mg/kg, s.c.), another pharmacologic treatment with potential efficacy in dyskinesias or L-DOPA motor complications, produced similar seizures. No changes in anandamide levels in hippocampus and amygdala were found in convulsing NLX-treated BD rats. In contrast, NLX significantly increased anandamide levels in the same areas of normal uninfected animals, possibly protecting against seizures. Pretreatment with the anandamide transport blocker AM404 (20 mg/kg, i.p.) prevented NLX-induced seizures. These findings are consistent with an anticonvulsant role for endocannabinoids, counteracting aberrant firing produced by convulsive agents, and with a functional or reciprocal relation between opioid and cannabinoid tone with respect to limbic convulsive phenomena.

Borna disease virus.

Borna disease virus, a negative-strand RNA virus, infects a wide variety of warm-blooded animals. Depending on the age of the host and the integrity of its immune response, infection may be asymptomatic or cause a broad spectrum of behavioral disorders. Unusual features of Borna disease virus biology include nuclear localization of replication and transcription; diverse strategies for regulation of gene expression; and interaction with signaling pathways resulting in subtle neuropathology. Although the question of human infection remains unresolved, burgeoning interest in this unique pathogen has provided tools for exploring the pharmacology and neurochemistry of neuropsychiatric disorders potentially linked to infection. Analysis of rodent models of infection has yielded insights into mechanisms by which neurotropic agents and/or immune factors may impact developing or mature central nervous system circuitry to effect complex disturbances in movement and behavior.

Neurological disease and encephalitis in cats experimentally infected with Borna disease virus.

Barrier-bred cats were inoculated intracerebrally with either the rabbit-adapted Borna disease virus (BDV) strain V or a newly isolated feline BDV, obtained from a cat with natural staggering disease (SD). Three out of eight inoculated cats developed neurological signs and non-suppurative encephalitis; all three recovered from the acute stage of disease. Sero-conversion and the development of neutralizing antibodies occurred in all of the virus-inoculated cats. In addition, cats inoculated with feline BDV showed an early peripheral T cell response not present in cats inoculated with BDV strain V, suggesting that the feline virus exerted a more vigorous effect on the immune system. Using immunohistochemistry and a reverse transcriptase-polymerase chain reaction assay, BDV-specific antigen and nucleic acid could be demonstrated in brain samples from each cat with encephalitis, showing that incomplete viral clearance was probably responsible for the maintenance of inflammation. The successful induction of neurological signs and encephalitis in one cat infected with feline BDV, together with the detection of BDV-specific antigen and nucleic acid in the brain, provides strong evidence for the notion that BDV is the etiological agent behind feline SD.

Influence of immunosuppressive treatment of Borna Disease in rabbits.

A total of 72 Borna Disease virus infected rabbits were treated with different concentrations of cyclophosphamide, glucocorticoids or both in combination. Comparison with untreated, infected rabbits showed a drastic alteration in the clinical picture, a considerable prolongation of the survival time, and differences in weight and body temperature during the course of the disease. The immunosuppressed animals had no or low amounts of antibodies in the serum and cerebrospinal fluid, but they harbored infectious virus and high amounts of specific antigen in the brain. A quotient of the relative amount of antibodies and antigen never exceeded 0.20 and was significantly lower than in untreated rabbits. Immunohistologically, differences in location and distribution of antigen as compared to positive untreated animals could not be detected. In the immunosuppressed animals perivascular infiltrates were not observed in the different regions of the brain.

Borna disease virus-induced retinouveitis treated with immunosuppressive drugs.

Borna disease occurs naturally in horses and sheep and causes an encephalomyelitis which is fatal. Little is known about the etiologic agent. There is evidence, however, that this neutrotropic virus belongs to the conventional enveloped RNA viruses. Experimentally infected rabbits exhibited a highly reproducible multifocal retinochoroidopathy. Clinical, histologic, as well as virologic results suggested that immunologic events gave rise to the characteristic clinical, histologic, as well as virologic results suggested that immunologic events gave rise to the characteristic clinical expression of the disease. To investigate possible immunpathologic factors, infected rabbits were treated with immunosuppressive drugs. As compared with controls three outstanding features were observed in the treated group: (1) The time interval between infection and the occurrence of inflammatory ocular foci was considerably prolonged; (2) confluency of retinal lesions was not noted in the early course of the disease; and (3) a lack of ocular lesions or a nonprogression over several days, never observed in controls, occurred in a small percentage of treated animals. These observations indicate that the clinical course of virus-induced inflammatory lesions of the retina and choroid can be changed by treatment with immunosuppressive drugs. It can be assumed that the appearance of the individual fundus lesion depends on the immuno-logic status of the infected host.

Amantadine revisited: an open trial of amantadinesulfate treatment in chronically depressed patients with Borna disease virus infection.

Amantadinesulfate is a well known substance which has proven useful in the treatment and prophylaxis of viral infections, in treating symptoms of Parkinson's disease, cocaine dependence, and apathy in multiple sclerosis. It has also been reported as having mild antidepressive effects not sufficient to warrant its use as an antidepressant. Striking antidepressive effects in some patients have been attributed to its antiviral activity against human Borna disease virus (BDV) infection which is frequently seen in patients with depressive episodes. In this 8 to 12 week open study of oral amantadine in 30 depressed patients with various states of BDV infection we found a significant antidepressive response in 19 of 30. Peripheral BDV antigen indicating acute infection was cleared in both responders and non-responders, but only in responders peripheral infection was significantly reduced.

Borna disease virus is not sensitive to amantadine.

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.