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Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.
Borna disease virus (BDV) is a neurotropic, enveloped virus with a nonsegmented negative-strand RNA genome (2). It naturally infects the central nervous system (CNS) of a broad range of mammalian species (14), where it efficiently establishes persistence in neuronal and nonneuronal cells (4). We recently reported the recovery of a recombinant BDV from cDNA (7, 13) that expresses the green fluorescent protein (GFP) from an additional transcription unit integrated near the 5′ end of its genome (12). The first-generation GFP vector originating from tissue culture-adapted BDV strain He/80 (13) was severely attenuated in the CNS of adult rats (12) and unable to productively infect mice (our unpublished data). Although mice are resistant to infections with primary BDV isolates, the virus can be adapted to gain replication competence in the CNS of these animals (6, 9). After serial passage of recombinant BDV strain He/80 in the brains of MRL mice, we identified five point mutations that confer replication competence in mice (1). Three of these mutations cause amino acid changes, two in the polymerase L and one in the phosphoprotein P. The other two mutations are silent and located within putative regulatory sequences flanking the initiation codon of the X gene. In a previous study we showed that incorporation of the two adaptive mutations into the L gene (LRD) improves the growth properties of the GFP-expressing virus in the CNS of rats (12), but the virus remained severely growth retarded in mice (our unpublished data). The mutation in the P gene was previously shown to reduce the sensitivity of the viral polymerase complex for the inhibitory activity of X (1). A recombinant virus carrying this P mutation in combination with the two mutations in L (BDV-PKLRD) showed strongly enhanced replication speed in mice, whereas the silent mutations within the X gene were found to reduce replication speed and pathogenicity of BDV-PKLRD in MRL mice (1) and rats (our unpublished data). In the present study we incorporated all five adaptive mutations into the BDV genome in order to generate a fully mouse-adapted GFP-expressing BDV vector, designated mBDV-GFP. We found that mBDV-GFP productively infected the central and the peripheral nervous systems of C57BL/6 mice and expressed easily detectable amounts of GFP during the entire observation period of up to 150 days.
To analyze viral dissemination in the mouse nervous system and to gain insight into the phenotypic identity of BDV-infected cells, we infected C57BL/6 mice intracranially with 10,000 FFU of mBDV-GFP. At the indicated time points postinfection (p.i.), the animals were transcardially perfused with 4% paraformaldehyde, vibratome sectioned, and immunostained for anticalbindin and antiparvalbumin to identify dentate granule cells and cerebellar Purkinje neurons, respectively (3). As early as 28 days p.i. we detected cells strongly resembling astrocytes in the subiculum (sub) that expressed significant amounts of GFP (Fig. (Fig.1A).1A). Only a few neurons appeared to be infected. Abundant GFP expression allowed the visualization of the dendritic processes of an individual pyramidal neuron (Fig. (Fig.1A).1A). At later time points we observed infected neurons in all hippocampal subfields, and GFP-expressing somata were also present in the calbindin-stained dentate granule cell layer (Fig. 1B and C). In the cerebellum GFP expression was detected in Purkinje cells (PC) and in cells of the internal granule layer at 65 days p.i. (Fig. (Fig.1D).1D). The endogenous GFP signal in infected PC was luminous enough to clearly visualize the characteristic dendritic arbor of individual PCs and their axonal processes protruding into the white matter (Fig. 1D to F). GFP-expressing PC and granule cells were still present in the cerebellum after extended periods of infection (Fig. 1E and F).
To check for viral dissemination throughout the CNS, we further examined cortical areas as well as cervical to lumbar segments of the spinal cord (SC). We detected abundant GFP expression in cortical neurons (Fig. (Fig.2A)2A) and found GFP-positive cell bodies representing myelinating oligodendrocytes in the corpus callosum (Fig. (Fig.2B).2B). From 65 days p.i. onwards we detected GFP expression in neurons and axons of the SC. GFP-positive neurons were mainly located in the cervical segment of the SC, and a single axon fiber was detected which extended into the lumbar part of the SC (Fig. 2C and D). At 120 days p.i., cross sections of the SC revealed a diffuse and punctate distribution of GFP signals. In the ventral horn of the cervical part we detected GFP expression in a typical motor neuron with dendritic processes and its axon directed toward the motor root (Fig. (Fig.2E).2E). Small GFP-labeled dots were found in all parts of the white matter of the SC, presumably representing cross-sectioned axonal processes (Fig. 2E and F). Only at 120 days p.i. were we able to trace BDV infection in a fiber of the peripheral sciatic nerve. Strong GFP signals were also detected in the perineural sheets, suggesting abundant infection of this nonneuronal tissue (Fig. (Fig.2G).2G). To the best of our knowledge, this is the first demonstration of BDV dissemination into the peripheral nervous system of mice, recapitulating similar findings in the nervous system of highly susceptible rats (5, 8, 15). Although persistently infected mice do not seem to transmit the virus, the slow progression of BDV infection from the central to the peripheral nervous system in mice is in accordance with reports showing that persistently infected rats secrete infectious BDV in the urine only after a prolonged period of infection (11). Our data thus support the idea that peripheral nerves provide a potential route for BDV to disseminate to secretory organs like the kidney and that the dissemination of BDV from the central to the peripheral nervous system is a critical step for a persistently infected rodent to become infectious.
Although GFP-positive cells were present throughout the observation period, we were surprised by the rather low abundance of these cells at late times of infection (Fig. 1B to F). To analyze whether downregulation of GFP expression was responsible for the low abundance of GFP-positive cells, we sacrificed two animals each at 6 weeks and 8 weeks p.i. and extracted protein from the CNS. Western blot analyses using antisera against BDV-N and GFP, respectively, showed an increase of the BDV-N signal between weeks 6 and 8, whereas the GFP signal was substantially weaker at the later time point (Fig. (Fig.3A).3A). We further analyzed transcription of the authentic BDV mRNAs encoding the N, P, and X proteins and the GFP mRNA in the brains of two animals sacrificed 10 weeks p.i. (Fig. (Fig.3B).3B). The authentic BDV mRNAs were readily detected on a blot loaded with 5 μg of total RNA per lane. In contrast, incubation of a blot containing 20 μg of total RNA per lane with a radiolabeled GFP probe resulted only in very weak GFP signals, indicating strong reduction of GFP transcription compared to that detected in infected Vero cells (12). Finally, immunofluorescent labeling of BDV-N in the dentate gyrus of a mouse sacrificed at 150 days p.i. revealed that the majority of BDV-infected dentate granular cells (red) did not contain detectable amounts of GFP (Fig. (Fig.3C).3C). Only a minor population of cell somata showed a clear costaining for GFP and nucleoprotein N (yellow cell body and green dendrites), indicating the existence of a population of mBDV-GFP viruses with severely reduced GFP expression capacities. We recently demonstrated that BDV is able to downregulate transcription of an ectopic X gene by modification of the transcription termination/initiation signals directly upstream of the inserted gene (10). To analyze whether similar mechanisms were responsible for the downregulation of GFP expression in mice, we isolated RNA from the brains of mice sacrificed 4 and 10 weeks after infection with mBDV-GFP and amplified by reverse transcription-PCR (RT-PCR) the complete GFP gene, including the upstream sequences that regulate termination and reinitiation of transcription (Fig. (Fig.3D).3D). Bulk and single clone analyses demonstrated that the termination signal upstream of the GFP gene was modified by insertion of one or two additional A residues (Fig. (Fig.3E),3E), whereas the coding sequence of the GFP gene remained unaltered (data not shown). These findings clearly suggest that downregulation of GFP expression occurred at the level of transcription. Insertion of additional A nucleotides into the UA7 termination motif might interfere with efficient recognition of the termination signal as previously postulated (10). Northern blot analysis of GFP expression 10 weeks p.i. (Fig. (Fig.3B)3B) provided no evidence for polymerase read-through at the termination signal (data not shown), which would result in the synthesis of a bicistronic L-GFP mRNA. We therefore favor the possibility that the additional A residues downregulate the efficiency of transcription reinitiation at the downstream initiation signal, thereby reducing the abundance of GFP mRNA. Further analyses will be required to distinguish between the two possibilities. It also remains unclear why it is apparently advantageous for the virus to downregulate GFP expression. We cannot exclude the possibility that the long-term expression of large quantities of GFP is toxic for infected neuronal cells, thereby inhibiting efficient virus spread. Alternatively, abundant transcription of the GFP mRNA by the viral RNA synthesis machinery might interfere with the balanced expression of viral proteins, slowing down BDV replication in the restrictive mouse system.
Our study demonstrates that foreign genes can be expressed efficiently and permanently in the mouse nervous system using a mouse-adapted BDV vector. Such vectors may represent a good alternative to classical transgenic approaches to study the effect of therapeutic genes in the mouse nervous system, especially in situations where transgene expression in only a fraction of neurons and glial cells is desired. Our data further demonstrate that rodent-adapted BDV-GFP vectors represent a valuable tool to further analyze viral dissemination from the central nervous system to the periphery during persistent infection. Detailed analysis of these processes will be instrumental to further elucidate the mechanisms underlying BDV transmission and epidemiology.
We thank Heidi Banse and Rosita Frank for excellent technical assistance and Otto Haller and Sandra Wille for helpful comments on the manuscript.
This work was supported by grants SCHN 765/1-5 and STA 338/8-1 to U.S. and P.S. and grant HE 1520 to B.H. from the Deutsche Forschungsgemeinschaft.
Published ahead of print on 10 March 2010.