Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections.

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.

miR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.

Borna disease virus 1 impairs DNA double-strand break repair through the ATR/Chk1 signalling pathway, resulting in learning and memory impairment in rats.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus that can establish persistent infection in the central nervous system and cause cognitive dysfunction in neonatally infected rats. However, the mechanisms that lead to this cognitive impairment remain unclear. DNA double-strand breaks (DSBs) and their repair are associated with brain development and cognition. If DNA repair in the brain is reduced or delayed and DNA damage accumulates, abnormal cognitive function may result. We generated a rat model of BoDV-1 infection during the neonatal period and assessed behavioural changes using the open field test and Morris water maze. The levels of DSBs were determined by immunofluorescence and comet assays. Western blotting assessed proteins associated with DNA repair pathways. The results showed that BoDV-1 downregulated the ATR/Chk1 signalling pathway in the brain, impairing DNA damage repair and increasing the number of DSBs, which ultimately leads to cognitive dysfunction. Our findings suggest a molecular mechanism by which BoDV-1 interferes with DNA damage repair to cause learning and memory impairment. This provides a theoretical basis for elucidating BoDV-1-induced neurodevelopmental impairment.

Borna disease virus phosphoprotein triggers the organization of viral inclusion bodies by liquid-liquid phase separation.

Inclusion bodies (IBs) are characteristic biomolecular condensates organized by the non-segmented negative-strand RNA viruses belonging to the order Mononegavirales. Although recent studies have revealed the characteristics of IBs formed by cytoplasmic mononegaviruses, that of Borna disease virus 1 (BoDV-1), a unique mononegavirus that forms IBs in the cell nucleus and establishes persistent infection remains elusive. Here, we characterize the IBs of BoDV-1 in terms of liquid-liquid phase separation (LLPS). The BoDV-1 phosphoprotein (P) alone induces LLPS and the nucleoprotein (N) is incorporated into the P droplets in vitro. In contrast, co-expression of N and P is required for the formation of IB-like structure in cells. Furthermore, while BoDV-1 P binds to RNA, an excess amount of RNA dissolves the liquid droplets formed by N and P in vitro. Notably, the intrinsically disordered N-terminal region of BoDV-1 P is essential to drive LLPS and to bind to RNA, suggesting that both abilities could compete with one another. These features are unique among mononegaviruses, and thus this study will contribute to a deeper understanding of LLPS-driven organization and RNA-mediated regulation of biomolecular condensates.

ICTV Virus Taxonomy Profile: Bornaviridae.

Members of the family Bornaviridae produce enveloped virions containing a linear negative-sense non-segmented RNA genome of about 9 kb. Bornaviruses are found in mammals, birds, reptiles and fish. The most-studied viruses with public health and veterinary impact are Borna disease virus 1 and variegated squirrel bornavirus 1, both of which cause fatal encephalitis in humans. Several orthobornaviruses cause neurological and intestinal disorders in birds, mostly parrots. Endogenous bornavirus-like sequences occur in the genomes of various animals. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bornaviridae, which is available at ictv.global/report/bornaviridae.

BoDV-1 infection induces neuroinflammation by activating the TLR4/MyD88/IRF5 signaling pathway, leading to learning and memory impairment in rats.

Borna disease virus (BoDV-1) can infect the hippocampus and limbic lobes of newborn rodents, causing cognitive deficits and abnormal behavior. Studies have found that neuroinflammation caused by viral infection in early life can affect brain development and impair learning and memory function, revealing the important role of neuroinflammation in cognitive impairment caused by viral infection. However, there is no research to explore the pathogenic mechanism of BoDV-1 in cognition from the direction of neuroinflammation. We established a BoDV-1 infection model in rats, and tested the learning and memory impairment by Morris water maze (MWM) experiment. RNAseq was introduced to detect changes in the gene expression profile of BoDV-1 infection, focusing on inflammation factors and related signaling pathways. BoDV-1 infection impairs the learning and memory of Sprague-Dawley rats in the MWM test and increases the expression of inflammatory cytokines in the hippocampus. RNAseq analysis found 986 differentially expressed genes (DEGs), of which 845 genes were upregulated and 141 genes were downregulated, and 28 genes were found to be enriched in the toll-like receptor (TLR) pathway. The expression of TLR4, MyD88, and IRF5 in the hippocampus was significantly changed in the BoDV-1 group. Our results indicate that BoDV-1 infection stimulates TLR4/MyD88/IRF5 pathway activation, causing the release of downstream inflammatory factors, which leads to neuroinflammation in rats. Neuroinflammation may play a significant role in learning and memory impairment caused by BoDV-1 infection.

BUD23-TRMT112 interacts with the L protein of Borna disease virus and mediates the chromosomal tethering of viral ribonucleoproteins.

Persistent intranuclear infection is an uncommon infection strategy among RNA viruses. However, Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, maintains viral infection in the cell nucleus by forming structured aggregates of viral ribonucleoproteins (vRNPs), and by tethering these vRNPs onto the host chromosomes. To better understand the nuclear infection strategy of BoDV-1, we determined the host protein interactors of the BoDV-1 large (L) protein. By proximity-dependent biotinylation, we identified several nuclear host proteins interacting with BoDV-1 L, one of which is TRMT112, a partner of several methyltransferases (MTases). TRMT112 binds with BoDV-1 L at the RNA-dependent RNA polymerase domain, together with BUD23, an 18S ribosomal RNA MTase and 40S ribosomal maturation factor. We then discovered that BUD23-TRMT112 mediates the chromosomal tethering of BoDV-1 vRNPs, and that the MTase activity is necessary in the tethering process. These findings provide us a better understanding on how nuclear host proteins assist the chromosomal tethering of BoDV-1, as well as new prospects of host-viral interactions for intranuclear infection strategy of orthobornaviruses.

Downregulation of SOCS gene expression can inhibit the formation of acute and persistent BDV infections.

High expression of suppressors of cytokine signalling (SOCS) has been detected during various viral infections. As a negative feedback regulator, SOCS participates in the regulation of multiple signalling pathways. In this study, to study the related mechanism between SOCS and BDV and to explore the effect of SOCS on IFN pathways in nerve cells, downregulated of SOCS1/3 in oligodendroglial (OL) cells and OL cells persistently infected with BDV (OL/BDV) were constructed with RNA interference technology. An interferon inducer (poly I:C, PIC) and an IFN-α/ß R1 antibody were used as stimulation in the SOCS1/3 low-expression cell models, qRT-PCR was used to detect type I IFN and BDV nucleic acid expression, Western blot was used to detect the expression of BDV P40 protein. After BDV acute infection with OL cells which with downregulated SOCS expression, the virus accounting was not detected, and the viral protein expression was lower than that of OL/BDV cells; the OL/BDV cells with downregulated SOCS expression had lower virus nucleic acid and protein expression than OL/BDV cells. Stimulated by IFN-α/ß R1 antibody, the expression of type I interferon in OL/BDV cells decreased, and the content of BDV nucleic acid and protein increased, which was higher than that of OL/BDV cells. From the results, it was concluded that downregulating SOCS1/3 can inhibit the formation of acute BDV infection and virus replication in persistent BDV infection by promoting the expression of IFN-α/ß and that SOCS can be used as a new target for antiviral therapy.

Obesity induced by Borna disease virus in rats: key roles of hypothalamic fast-acting neurotransmitters and inflammatory infiltrates.

Human obesity epidemic is increasing worldwide with major adverse consequences on health. Among other possible causes, the hypothesis of an infectious contribution is worth it to be considered. Here, we report on an animal model of virus-induced obesity which might help to better understand underlying processes in human obesity. Eighty Wistar rats, between 30 and 60 days of age, were intracerebrally inoculated with Borna disease virus (BDV-1), a neurotropic negative-strand RNA virus infecting an unusually broad host spectrum including humans. Half of the rats developed fatal encephalitis, while the other half, after 3-4 months, continuously gained weight. At tripled weights, rats were sacrificed by trans-cardial fixative perfusion. Neuropathology revealed prevailing inflammatory infiltrates in the median eminence (ME), progressive degeneration of neurons of the paraventricular nucleus, the entorhinal cortex and the amygdala, and a strikingly high-grade involution of the hippocampus with hydrocephalus. Immune histology revealed that major BDV-1 antigens were preferentially present at glutamatergic receptor sites, while GABAergic areas remained free from BDV-1. Virus-induced suppression of the glutamatergic system caused GABAergic predominance. In the hypothalamus, this shifted the energy balance to the anabolic appetite-stimulating side governed by GABA, allowing for excessive fat accumulation in obese rats. Furthermore, inflammatory infiltrates in the ME and ventro-medial arcuate nucleus hindered free access of appetite-suppressing hormones leptin and insulin. The hormone transport system in hypothalamic areas outside the ME became blocked by excessively produced leptin, leading to leptin resistance. The resulting hyperleptinemic milieu combined with suppressed glutamatergic mechanisms was a characteristic feature of the found metabolic pathology. In conclusion, the study provided clear evidence that BDV-1 induced obesity in the rat model is the result of interdependent structural and functional metabolic changes. They can be explained by an immunologically induced hypothalamic microcirculation-defect, combined with a disturbance of neurotransmitter regulatory systems. The proposed mechanism may also have implications for human health. BDV-1 infection has been frequently found in depressive patients. Independently, comorbidity between depression and obesity has been reported, either. Future studies should address the exciting question of whether BDV-1 infection could be a link, whatsoever, between these two conditions.

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.

ADAR2 Is Involved in Self and Nonself Recognition of Borna Disease Virus Genomic RNA in the Nucleus.

Cells sense pathogen-derived double-stranded RNA (dsRNA) as nonself. To avoid autoimmune activation by self dsRNA, cells utilize A-to-I editing by adenosine deaminase acting on RNA 1 (ADAR1) to disrupt dsRNA structures. Considering that viruses have evolved to exploit host machinery, A-to-I editing could benefit innate immune evasion by viruses. Borna disease virus (BoDV), a nuclear-replicating RNA virus, may require escape from nonself RNA-sensing and immune responses to establish persistent infection in the nucleus; however, the strategy by which BoDV evades nonself recognition is unclear. Here, we evaluated the involvement of ADARs in BoDV infection. The infection efficiency of BoDV was markedly decreased in both ADAR1 and ADAR2 knockdown cells at the early phase of infection. Microarray analysis using ADAR2 knockdown cells revealed that ADAR2 reduces immune responses even in the absence of infection. Knockdown of ADAR2 but not ADAR1 significantly reduced the spread and titer of BoDV in infected cells. Furthermore, ADAR2 knockout decreased the infection efficiency of BoDV, and overexpression of ADAR2 rescued the reduced infectivity in ADAR2 knockdown cells. However, the growth of influenza A virus, which causes acute infection in the nucleus, was not affected by ADAR2 knockdown. Moreover, ADAR2 bound to BoDV genomic RNA and induced A-to-G mutations in the genomes of persistently infected cells. We finally demonstrated that BoDV produced in ADAR2 knockdown cells induces stronger innate immune responses than those produced in wild-type cells. Taken together, our results suggest that BoDV utilizes ADAR2 to edit its genome to appear as "self" RNA in order to maintain persistent infection in the nucleus.IMPORTANCE Cells use the editing activity of adenosine deaminase acting on RNA proteins (ADARs) to prevent autoimmune responses induced by self dsRNA, but viruses can exploit this process to their advantage. Borna disease virus (BoDV), a nuclear-replicating RNA virus, must escape nonself RNA sensing by the host to establish persistent infection in the nucleus. We evaluated whether BoDV utilizes ADARs to prevent innate immune induction. ADAR2 plays a key role throughout the BoDV life cycle. ADAR2 knockdown reduced A-to-I editing of BoDV genomic RNA, leading to the induction of a strong innate immune response. These data suggest that BoDV exploits ADAR2 to edit nonself genomic RNA to appear as self RNA for innate immune evasion and establishment of persistent infection.

miR-146a promotes Borna disease virus 1 replication through IRAK1/TRAF6/NF-κB signaling pathway.

BACKGROUND/AIMS: Borna disease virus 1 (BoDV-1) is a negative single-stranded RNA virus that is highly neurotropic. BoDV-1 infection can damage the central nervous system and cause inflammation. To survive in host cells, BoDV-1 must evade the host innate immune response. A previous study showed that miR-146a expression increased in neonatal rats infected with BoDV-1. miR-146a is a microRNA suggested to negatively regulate innate immune and inflammatory responses and antiviral pathways. Many groups have reported that its overexpression facilitates viral replication. However, it is unclear whether miR-146a is involved in escape from the host immune response during BoDV-1 infection. METHODS: In this study, BoDV-1 was used to infect neonatal rats within 24 h of birth intracranially, as well as to infect human microglial cells (HMC3). miR-146a expression was analyzed by RT-qPCR. The TargetScanHuman database was used to find the target genes of miR-146a. A search of the binding sites of miR-146a and its target gene's 3'-untranslated region (3'UTR) was also performed using RNAhybrid software. The binding sites of miR-146a and the target gene's 3'UTR were detected by dual luciferase reporter assays. Overexpression and suppression studies of miR-146a were performed to determine its effect on BoDV-1 replication. The relative protein expression of members of the IRAK1/TRAF6/NF-κB signaling pathway was also evaluated by western blotting in HMC3. RESULTS: After BoDV-1 infection of neurons in vivo and of HMC3 cells, miR-146a expression was significantly upregulated. miR-146a overexpression in HMC3 cells promoted viral replication, while its inhibition inhibited it. Through the TargetScanHuman database, we identified the target genes of anti-inflammatory miR-146a: IRAK1 and TRAF6. We also found that BoDV-1 could inhibit IRAK1 and TRAF6 expression in HMC3 cells. Moreover, we showed that the inhibition of IRAK1 and TRAF6 also led to decreases in the expression of P65 and phosphorylated P65 in the downstream NF-κB pathway. Subsequently, we confirmed the interaction of miR-146a with IRAK1 and TRAF6 by luciferase assay. CONCLUSION: Our results suggest that miR-146a inhibits the IRAK1/TRAF6/NF-κB signaling pathway to facilitate BoDV-1 survival in host cells.

Intracellular dynamics of actin affects Borna disease virus replication in the nucleus.

Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that uniquely replicates and establishes persistent infection in cell nucleus. Recent studies have demonstrated the presence of actin in the nucleus and its role in intranuclear phenomena such as transcription and DNA repair. Although nuclear actin is involved in the life cycle of some intranuclear DNA viruses, the interaction between BoDV and nuclear actin has not been reported. In this study, we show that the inhibition of the nucleocytoplasmic transport of actin affects the replication of BoDV in the nucleus. The knockdown of a nuclear export factor of actin, exportin 6, results in the induction of structural aberration in intranuclear viral factories of BoDV. Furthermore, the inhibition of the nuclear export of actin promotes accumulation of viral matrix protein in the cytoplasm and periphery of the infected cells. These results suggest that the dynamics of actin affect the replication of BoDV by disturbing the structure of viral factories in the nucleus.

Memory Impairment Induced by Borna Disease Virus 1 Infection is Associated with Reduced H3K9 Acetylation.

BACKGROUND/AIMS: Borna disease virus 1 (BoDV-1) infection induces cognitive impairment in rodents. Emerging evidence has demonstrated that Chromatin remodeling through histone acetylation can regulate cognitive function. In the present study, we investigated the epigenetic regulation of chromatin that underlies BoDV-1-induced cognitive changes in the hippocampus. METHODS: Immunofluorescence assay was applied to detect BoDV-1 infection in hippocampal neurons and Sprague-Dawley rats models. The histone acetylation levels both in vivo and vitro were assessed by western blots. The acetylation-regulated genes were identified by ChIP-seq and verified by RT-qPCR. Cognitive functions were evaluated with Morris Water Maze test. In addition, Golgi staining, and electrophysiology were used to study changes in synaptic structure and function. RESULTS: BoDV-1 infection of hippocampal neurons significantly decreased H3K9 histone acetylation level and inhibited transcription of several synaptic genes, including postsynaptic density 95 (PSD95) and brain-derived neurotrophic factor (BDNF). Furthermore, BoDV-1 infection of Sprague Dawley rats disrupted synaptic plasticity and caused spatial memory impairment. These rats also exhibited dysregulated hippocampal H3K9 acetylation and decreased PSD95 and BDNF protein expression. Treatment with the HDAC inhibitor, suberanilohydroxamic acid (SAHA), attenuated the negative effects of BoDV-1. CONCLUSION: Our results demonstrate that regulation of H3K9 histone acetylation may play an important role in BoDV-1-induced memory impairment, whereas SAHA may confer protection against BoDV-1-induced cognitive impairments. This study finds important mechanism of BoDV-1 infection disturbing neuronal synaptic plasticity and inducing cognitive dysfunction from the perspective of histone modification.

Hippocampal expression of a virus-derived protein impairs memory in mice.

The analysis of the biology of neurotropic viruses, notably of their interference with cellular signaling, provides a useful tool to get further insight into the role of specific pathways in the control of behavioral functions. Here, we exploited the natural property of a viral protein identified as a major effector of behavioral disorders during infection. We used the phosphoprotein (P) of Borna disease virus, which acts as a decoy substrate for protein kinase C (PKC) when expressed in neurons and disrupts synaptic plasticity. By a lentiviral-based strategy, we directed the singled-out expression of P in the dentate gyrus of the hippocampus and we examined its impact on mouse behavior. Mice expressing the P protein displayed increased anxiety and impaired long-term memory in contextual and spatial memory tasks. Interestingly, these effects were dependent on P protein phosphorylation by PKC, as expression of a mutant form of P devoid of its PKC phosphorylation sites had no effect on these behaviors. We also revealed features of behavioral impairment induced by P protein expression but that were independent of its phosphorylation by PKC. Altogether, our findings provide insight into the behavioral correlates of viral infection, as well as into the impact of virus-mediated alterations of the PKC pathway on behavioral functions.

Different inhibitory effects on the proliferation and apoptosis of human and laboratory Borna disease virus­infected human neuroblastoma SH­SY5Y cells in vitro.

Borna disease virus (BDV) is a neurotropic and non­cytolytic virus, which causes behavioral disorders in a wide range of warm­blooded species. It is well established that BDV induces neurodegeneration by impairing neurogenesis and interfering with neuronal functioning in the limbic system. In the present study, the potential role of BDV infection in SH­SY5Y cells was identified, and comparisons of two original BDV strains (the human Hu­H1 and the laboratory Strain V) were performed to further elucidate the phenotypes of BDV pathogenesis with strain differences. Cell Counting Kit­8 and flow cytometric analyses revealed that the two BDV strain­infected groups exhibited marked anti­proliferation and cell cycle arrest compared with the control group, and the Hu­H1 strain caused more evident effects. However, the Hu­H1 strain did not exert effects on the apoptosis of SH­SH5Y cells, while Strain V led to a marked increase in apoptosis upon initial infection. Western blot analysis confirmed the upregulation of apoptosis regulator BAX protein and the downregulation of apoptosis regulator Bcl­2 protein caused by the two BDV strains. The results of the present study provided evidence that infection with BDV suppressed SH­SY5Y cellular functioning and exhibited divergent antiproliferative and apoptotic roles in cells between the two strains. The present study provided an insight for future investigation of strain differences and underlying pathomechanisms.

Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein.

BACKGROUND: Borna disease virus (BoDV), which has a negative-sense, single-stranded RNA genome, causes persistent infection in the cell nucleus. The nuclear export signal (NES) of the viral nucleoprotein (N) consisting of leucine at positions 128 and 131 and isoleucine at positions 133 and 136 overlaps with one of two predicted binding sites for the viral phosphoprotein (P). A previous study demonstrated that higher expression of BoDV-P inhibits nuclear export of N; however, the function of N NES in the interaction with P remains unclear. We examined the subcellular localization, viral polymerase activity, and P-binding ability of BoDV-N NES mutants. We also characterized a recombinant BoDV (rBoDV) harboring an NES mutation of N. RESULTS: BoDV-N with four alanine-substitutions in the leucine and isoleucine residues of the NES impaired its cytoplasmic localization and abolished polymerase activity and P-binding ability. Although an alanine-substitution at position 131 markedly enhanced viral polymerase activity as determined by a minigenome assay, rBoDV harboring this mutation showed expression of viral RNAs and proteins relative to that of wild-type rBoDV. CONCLUSIONS: Our results demonstrate that BoDV-N NES has a dual function in BoDV replication, i.e., nuclear export of N and an interaction with P, affecting viral polymerase activity in the nucleus.

Change in the responsiveness of interferon-stimulated genes during early pregnancy in cows with Borna virus-1 infection.

BACKGROUND: Borna disease virus is a neurotropic pathogen and infects the central nervous system. This virus infected a variety of animal species including cows. The most of cows infected with Borna disease virus 1 (BoDV-1) exhibit subclinical infection without any neurological symptoms throughout their lifetime. We previously reported on the low conception rates in-seropositive cows. Interferon-τ (IFN-τ) plays an important role in stable fertilization, and is produced from the fetal side following embryo growth at 15-40 days of pregnancy. IFN-τ induces the expression of interferon-stimulated gene (ISG) 15 and Mx2 in peripheral blood mononuclear cells (PBMCs). To understand the embryo growth and maternal reaction during early pregnancy in cows with BoDV-1 infection, we aimed to assess the gene expression of ISG15 and Mx2 from PBMCs in BoDV-1-seropositive cows. RESULTS: None of the cows showed any clinical and neurological symptoms. Among the cows that conceived, the expressions of the ISG15 and Mx2 genes were greater in the BoDV-1-seropositive cows than in the BoDV-1-seronegative cows; the difference was significant between the cows that conceived and those that did not (P < 0.05). CONCLUSIONS: The expression of ISG15 and Mx2 genes during early pregnancy significantly increased in the BoDV-1-seropositive cows and may be important for the maintenance of stable pregnancy in BoDV-1-infected cows. In contrast, the gene expression levels of ISG15 and Mx2 did not significantly increase during early pregnancy in BoDV-1-seronegative cows. Thus, BoDV-1 infection may lead to instability in the maintenance of early pregnancy by interfering with INF-τ production.

Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons.

Borna disease virus (BDV) is a neurotropic RNA virus that infects the limbic system of mammals and results in behavioral disorders. The hippocampus is a core region in the limbic system, which contributes to memory and learning and is important in the regulation of emotion. However, no validated microRNA housekeeping genes have yet been identified in BDV­infected rat primary hippocampal neurons. Proper normalization is key in accurate miRNA expression analysis. The present study used reverse transcription­quantitative polymerase chain reaction (RT­qPCR) to evaluate the expression stability of 10 commonly used reference genes [miR­92a, 5S, U6, miR­103, miR­101a, miR-let-7a, miR­16, E2 small nucleolar RNA (snoRNA), U87 and miR­191] in BDV­infected rat hippocampal neurons and non­infected controls across 12 days post­infection. The data was analyzed by four statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ­Ct method. Subsequently, the most suitable reference genes (miR­101a and U87) and the least suitable (snoRNA) were determined by the RankAggreg package. miR­155 was selected as a standard by which to evaluate the most and least suitable reference genes. When normalized to the most stable reference gene there were significant differences between the two groups. However, when the data were normalized to the less stably expressed gene, the results were not significant. miR­101a was recommended as a suitable reference gene for BDV-infected rat primary hippocampal neurons.