Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System.

Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.

The regulation of persistent Borna disease virus infection by RNA silencing factors in human cells.

Viral infection induces diverse cellular immune responses. Some viruses induce the production of antiviral cytokines, alterations of endogenous gene expression, and apoptosis; however, other viruses replicate without inducing such responses, enabling them to persistently infect cells. Infection by Borna disease virus type 1 (BoDV-1) can result in fatal immune-mediated encephalitis, including in humans, yet infection of cells in vitro is generally persistent. The regulatory mechanisms underlying this persistent infection remain unclear. Here, we show that an enhancer of RNA-silencing, TRBP, positively regulates BoDV RNA level in human cells. Knockdown of TRBP decreased BoDV RNA levels in persistently-infected cells, whereas overexpression of TRBP increased BoDV RNA levels. To investigate the mechanism underlying this phenomenon, we performed immunoprecipitation assays and found that TRBP interacts with BoDV RNA. Furthermore, we performed cell fractionation, which revealed that persistent infection with BoDV does not alter the localization of TRBP and other RNA silencing factors in cells. Our results showed the regulation of persistent BoDV infection by RNA-silencing factors in human cells.

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.

Modeling Borna Disease Virus In Vitro Spread Reveals the Mode of Antiviral Effect Conferred by an Endogenous Bornavirus-Like Element.

Endogenous retroviruses have demonstrated exaptation during long-term evolution with hosts, e.g., resulting in acquisition of antiviral effect on related extant viral infections. While empirical studies have found that an endogenous bornavirus-like element derived from viral nucleoprotein (itEBLN) in the ground squirrel genome shows antiviral effect on virus replication and de novo infection, the antiviral mechanism, dynamics, and quantitative effect of itEBLN remain unknown. In this study, we experimentally and theoretically investigated the dynamics of how an extant bornavirus, Borna disease virus 1 (BoDV-1), spreads and replicates in uninfected, BoDV-1-infected, and itEBLN-expressing cultured cells. Quantifying antiviral effect based on time course data sets, we found that the antiviral effects of itEBLN are estimated to be 75% and 34% on intercellular virus spread and intracellular virus replication, respectively. This discrepancy between intercellular virus spread and intracellular viral replication suggests that viral processes other than the replication of viral ribonucleoprotein complex (RNP) contributed to the suppression of virus spread in itEBLN-expressing cells. Because itEBLN binds to the BoDV-1 RNP, the suppression of viral RNP trafficking can be an attractive candidate explaining this discrepancy.IMPORTANCE Accumulating evidence suggests that some endogenous viral elements (EVEs), including endogenous retroviruses and endogenous nonretroviral virus elements, have acquired functions in the host as a result of long-term coevolution. Recently, an endogenous bornavirus-like element (itEBLN) found in the ground squirrel genome has been shown to have antiviral activity against exogenous bornavirus infection. In this study, we first quantified bornavirus spread in cultured cells and then calculated the antiviral activity of itEBLN on bornavirus infection. The calculated antiviral activity of itEBLN suggests its suppression of multiple processes in the viral life cycle. To our knowledge, this is the first study quantifying the antiviral activity of EVEs and speculating on a model of how some EVEs have acquired antiviral activity during host-virus arms races.

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.

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.

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.

Real-time qPCR identifies suitable reference genes for Borna disease virus-infected rat cortical neurons.

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.

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.

Borna disease virus nucleoprotein inhibits type I interferon induction through the interferon regulatory factor 7 pathway.

The expression of type I interferon (IFN) is one of the most potent innate defences against viral infection in higher vertebrates. Borna disease virus (BDV) establishes persistent, noncytolytic infections in animals and in cultured cells. Early studies have shown that the BDV phosphoprotein can inhibit the activation of type I IFN through the TBK1-IRF3 pathway. The function of the BDV nucleoprotein in the inhibition of IFN activity is not yet clear. In this study, we demonstrated IRF7 activation and increased IFN-α/ß expression in a BDV-persistently infected human oligodendroglia cell line following RNA interference-mediated BDV nucleoprotein silencing. Furthermore, we showed that BDV nucleoprotein prevented the nuclear localisation of IRF7 and inhibited endogenous IFN induction by poly(I:C), coxsackie virus B3 and IFN-ß. Our findings provide evidence for a previously undescribed mechanism by which the BDV nucleoprotein inhibits type I IFN expression by interfering with the IRF7 pathway.

Impact of Borna Disease Virus Infection on the Transcriptome of Differentiated Neuronal Cells and Its Modulation by Antiviral Treatment.

Borna disease virus (BoDV-1) is a highly neurotropic RNA virus that causes neurobehavioral disturbances such as abnormal social activities and memory impairment. Although impairments in the neural circuits caused by BoDV-1 infection induce these disturbances, the molecular basis remains unclear. Furthermore, it is unknown whether anti-BoDV-1 treatments can attenuate BoDV-1-mediated transcriptomic changes in neuronal cells. In this study, we investigated the effects of BoDV-1 infection on neuronal differentiation and the transcriptome of differentiated neuronal cells using persistently BoDV-1-infected cells. Although BoDV-1 infection did not have a detectable effect on intracellular neuronal differentiation processes, differentiated neuronal cells exhibited transcriptomic changes in differentiation-related genes. Some of these transcriptomic changes, such as the decrease in the expression of apoptosis-related genes, were recovered by anti-BoDV-1 treatment, while alterations in the expression of other genes remained after treatment. We further demonstrated that a decrease in cell viability induced by differentiation processes in BoDV-1-infected cells can be relieved with anti-BoDV-1 treatment. This study provides fundamental information regarding transcriptomic changes after BoDV-1 infection and the treatment in neuronal cells.

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

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.

PrPSc level and incubation time in a transgenic mouse model expressing Borna disease virus phosphoprotein after intracerebral prion infection.

Our previous studies have shown that the persistent expression of Borna disease virus phosphoprotein (BDV P) in mice leads to behavioral abnormalities resembling those in BDV-infected animals. In this study, we investigated whether the neurobehavioral abnormalities genetically induced by BDV P influence experimental prion disease. The effect of the phosphoprotein on prion diseases was evaluated based on the incubation time and survival curve, as well as the abnormal isoform of prion protein (PrP(Sc)) levels in brains of BDV P Tg mice treated with proteinase K (PK) treatment and subjected to western blotting. Increased expression of the BDV P transgene had no effect on the PrP(Sc) level, incubation time, or survival curve. The abnormalities induced by BDV P are different from those induced by prion diseases, indicating that the signaling cascades induced by the phosphoprotein differ from those induced by prion diseases.

RNA from Borna disease virus in patients with schizophrenia, schizoaffective patients, and in their biological relatives.

Numerous interactions of the immune system with the central nervous system have been described recently. Mood and psychotic disorders, such as severe depression and schizophrenia, are both heterogeneous disorders regarding clinical symptomatology, the acuity of symptoms, the clinical course, the treatment response, and probably also the etiology. Detection of p24 RNA from Borna disease virus (BDV) by the reverse transcriptase polymerase chain reaction in patients with schizophrenia, schizoaffective disorder, and in their biological relatives was evaluated. The subjects were 27 schizophrenic and schizoaffective patients, 27 healthy controls, 20 relatives without psychiatric disease, and 24 relatives with mood disorder, who attended the Psychiatric Ambulatory of Londrina State University, Paraná, Brazil. The subjects were interviewed by structured diagnostic criteria categorized according to the Diagnostic and Statistical Manual of Mental Disorders-IV, axis I, (SCID-IV). The mean duration of illness in schizophrenic and schizoaffective patients was 15.341+/-1.494 years and the median age at onset was 22.4+/-7.371 years. There were no significant differences in gender (P=0.297), age (P=0.99), albumin (P=0.26), and body mass index (kg/m(2)) (p=0.28), among patients, controls, and relatives. Patients and biological relatives had significantly higher positive p24 RNA BDV detection than controls (P=0.04); however, the clinical significance of BDV remains to be clarified.

Detection of Borna disease virus p24 RNA in peripheral blood cells from Brazilian mood and psychotic disorder patients.

BACKGROUND: Borna disease virus (BDV) is a virus that naturally infects a broad range of warm-blooded animals. BDV is an enveloped virus, non-segmented, negative-stranded RNA genome and has an organization characteristic of a member of Bornaviridae in the order of Mononegavirale. In the present work we investigated the presence of BDV p24 RNA in peripheral blood cells from 30 psychiatric patients (19 with mood disorder and 11 with psychotic disorder) and 30 healthy volunteers as the control group. METHODS: All subjects were interviewed by structured diagnostic criteria categorized according to the DSM-IV, Axis I (SCID-V). The presence of BDV p24 RNA was investigated by nested reverse transcriptase PCR (RT-PCR) using specific primers to p24 from BDV. The specificity of the detection was analyzed by the sequencing of PCR products. RESULTS: The mean duration of illness in mood and psychotic patients with p24 RNA of BDV was 25 (+/-12.3) years and the median age was 43.77 (+/-15.2) years. There were no significant differences in gender and age among patients and control group, neither duration of illness among patients with mood and psychotic disorders in the presence or absence of p24 RNA of BDV. We found a frequency of 33.33% (10/30) of BDV-RNA on patient's group and 13.33% (4/30) on control group. The given sequences revealed identity with GenBank database sequence for BDV. CONCLUSION: The detection of a higher level of BDV-RNA in the peripheral blood cells of patients than on control group should help our understanding of the pathogenesis in the disease.

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.

Effects of genetic background on neonatal Borna disease virus infection-induced neurodevelopmental damage. I. Brain pathology and behavioral deficits.

The pathogenic mechanisms of gene-environment interactions determining variability of human neurodevelopmental disorders remain unclear. In the two consecutive papers, we used the neonatal Borna disease virus (BDV) infection rat model of neurodevelopmental damage to evaluate brain pathology, monoamine alterations, behavioral deficits, and responses to pharmacological treatments in two inbred rat strains, Lewis and Fisher344. The first paper reports that despite comparable virus replication and distribution in the brain of both rat strains, neonatal BDV infection produced significantly greater thinning of the neocortex in BDV-infected Fisher344 rats compared to BDV-infected Lewis rats, while no strain-related differences were found in BDV-induced granule cell loss in the dentate gyrus of the hippocampus and cerebellar hypoplasia. Unlike BDV-infected Lewis rats, more severe BDV-induced brain pathology in Fisher344 rats was associated with (1) greater locomotor activity to novelty and (2) impairment of habituation and prepulse inhibition of the acoustic startle response. The present data demonstrate that the same environmental insult can produce differential neuroanatomical and behavioral abnormalities in genetically different inbred rat strains.

Effects of genetic background on neonatal Borna disease virus infection-induced neurodevelopmental damage. II. Neurochemical alterations and responses to pharmacological treatments.

The gene-environment interplay is thought to determine variability in clinical conditions and responses to therapy in human neurodevelopmental disorders. Studying abnormal brain and behavior development in inbred strains of rodents can help in the identification of the complex pathogenic mechanisms of the host-environment interaction. This paper is the second one in a series of the two reports of the use of the Borna disease virus (BDV) infection model of neurodevelopmental damage to characterize effects of genetic background on virus-induced neurodevelopmental damage in inbred rat strains, Lewis and Fisher344. The present data demonstrate that neonatal BDV infection produced regional and strain-related alterations in levels of serotonin, norepinephrine and in levels of serotonin turnover at postnatal day 120. Neonatal BDV infection also induced upregulation of hippocampal 5-HT(1a) and cortical 5-HT(2a) receptors in Lewis rats and downregulation of cortical 5-HT(2a) receptors in Fisher344 rats. BDV-associated regional downregulation of D(2) receptors and dopamine transporter sites were noted in Fisher344 rats. In addition to the neurochemical disturbances, neonatal BDV infection induced differential responses to serotonin compounds. While 8-OH-DPAT suppressed virus-enhanced ambulation in BDV-infected Fisher344, fluoxetine inhibited virus-induced hyperactivity in BDV-infected Lewis rats only. The present data provide new insights into the pathogenic events that lead to differential responses to pharmacological treatments in genetically different animals following exposure to the same environmental challenge.

Experimental and natural borna disease virus infections: presence of viral RNA in cells of the peripheral blood.

Cells of the peripheral blood of experimentally and naturally borna disease virus (BDV)-infected animals and of human psychiatric patients and healthy individuals were analyzed for the presence of viral RNA using a BDV-p40-specific nested reverse transcription-polymerase chain reaction (RT-PCR). The assay proved to be highly sensitive as 10 RNA molecules were reproducibly amplified. BDV RNA was detected in blood cells of experimentally infected immunocompetent mice and rats. Mice were persistently infected without showing clinical signs of borna disease (BD), whereas the rats suffered from acute BD. Among 19 horses examined, five were positive for viral RNA in the blood. In a flock of sheep with a history of BD, 1 out of 25 clinically healthy animals was positive. BDV RNA was also detected in cells of the peripheral blood of 10 out of 27 selected humans with psychiatric disorders, and in 2 out of 13 healthy individuals. Remarkably, BDV-specific RNA was present in some cases in the absence of BDV-specific antibodies. Sequence analysis of PCR products confirmed the specificity of the amplification system. The presence of BDV RNA in the blood of naturally and experimentally BDV-infected individuals may point to an incidental but relevant role of blood for the spread of BDV in the infected organism, as well as for the transmission of BDV to other individuals.