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Journal of Virology, May 2003, p. 6076-6081, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.6076-6081.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Two Major Histocompatibility Complex Class I-Restricted Epitopes of the Borna Disease Virus p10 Protein Identified by Cytotoxic T Lymphocytes Induced by DNA-Based Immunization

Yoshio Hashimoto,1,{dagger} Horng-Shen Chen,1 Cynthia Cunningham,2 Tahir H. Malik,3 and Patrick K. Lai1*

Department of Bioscience, Salem International University, Salem, West Virginia 26426-0500,1 Department of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9177 ,2 Laboratory of Pediatric and Respiratory Viral Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland 208923

Received 9 September 2002/ Accepted 18 February 2003


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ABSTRACT
 
Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8+ cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the L-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M1SSDLRLTLL10 and T8LLELVRRL16, associated with the RT1.Al major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.Al-restricted A230SYAQMTTY238 peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.


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TEXT
 
Borna disease (BD), often a lethal meningoencephalitis (24, 31, 39, 40), is caused by Borna disease virus (BDV), a neurotropic, enveloped virus with a single-stranded RNA genome of negative polarity (5, 11, 12, 23). This virus has a broad host range and has been detected in a wide variety of warm-blooded animals (6, 25, 29, 46). The footprint of this virus and its specific antibodies were detected in the blood of human patients with psychiatric disorders (3, 4, 13, 20, 30, 45), although its ability to cause human disease remains controversial.

Experimentally, BDV can be transmitted to many vertebrate species, with considerable variation in clinical outcome (reviewed in reference 17). The most investigated animal model for the pathogenesis of BDV infection is the Lewis rat. BDV is an example of a noncytolytic persistent virus. In naturally infected animals and experimentally infected adult rats, neurological disease and behavioral abnormalities appear to be immunopathologic in nature (reviewed in reference 42). Intracerebral infection of athymic or experimentally immunosuppressed adult rats does not produce BD (19, 43). In contrast, infection of immunocompetent adult rats results in encephalomyelitis, characterized by perivascular and parenchymal infiltrations of CD4+ and CD8+ T cells whose appearance is correlated with the onset of disease symptoms (33, 41). Recent studies showed that the immunopathology is mediated by CD8+ T cells that require help from the CD4+-T-cell subset (38, 39, 42). Rats treated with the OX8 monoclonal antibody (MAb) to CD8 did not develop BD, whereas treatment with OX68, the anti-CD4 MAb, was successful in inhibiting immunopathology and clinical disease in 50% of the treated animals (2). Lymphocyte preparations isolated from the diseased brains of the infected Lewis rats showed virus-specific cytotoxicity that could be blocked by an antibody (RT1.A) specific to major histocompatibility complex (MHC) class I of the rat (34), suggesting that the cytolytic activity was mediated by classical cytotoxic T lymphocytes (CTLs). Adoptive transfer of a BDV-specific, noncytolytic, CD4+-T-cell line that produced gamma interferon (IFN-{gamma}), interleukin-6, and interleukin-10 into virus-infected, immunosuppressed Lewis rats resulted in BD with CD8+ cell infiltration. In contrast, treatment of the recipients with the OX8 anti-CD8 MAb prior to the adoptive transfer abolished the expression of BD (33). Confirmation that the virus-specific CD8+ classical CTLs play a critical role in the immunopathogenesis of BD came from adoptive transfer of brain lymphocytes directly ex vivo from BDV-infected rats into immunosuppressed, BDV-infected recipients (41). The brain lymphocytes had a high level of RT1.A-restricted cytotoxicity in vitro and caused BD in the recipients between 7 and 10 days after adoptive transfer.

Six open reading frames (ORFs) have been identified in the BDV genome, coding for proteins N, X, P, M, G, and L (5, 11, 27). Of these, four have been tested for the ability to induce CTLs or to serve as CTL targets. By use of recombinant vaccinia virus-expressing BDV N (p40), G (gp94), P (p24), or M (gp18) to immunize Lewis rats, CTLs have been found against N and G, but not against P and M (35). There is also some evidence that CTLs against N may be important to the immunopathogenesis of BD (22, 35, 36). Brain CTLs from diseased rats were cytolytic to N-positive targets but not to G-expressing targets. The peptide A230SYAQMTTY238 of N has been identified as an RT1.Al-restricted target of these brain-derived CTLs from infected Lewis rats (15, 36). Since CTL responses to BDV X (p10) have never been studied, we made use of plasmid DNA-based vaccination and examined their presence in Lewis rats.

We first validated the fact that plasmid DNA-based vaccination can induce the production of CTLs against N. Four-week-old Lewis rats (Harlan, Indianapolis, Ind.) were given four doses (one dose every 3 weeks) of the eukaryotic vector pDL-N.Wild (21), which expresses BDV p40. Each dose, containing 30 µg of the plasmid DNA in a 100-µl volume, was injected intramuscularly into the quadriceps. The animals were cared for according to the 1997 Guidelines of the NIH for Care and Use of Laboratory Animals, and all experimental protocols were approved by the Institutional Animal Care and Use Committee. Three weeks after the last injection, the rats were sacrificed. Blood, spleens, and drainage lymph nodes were collected. The mononuclear cells were tested for CTL activity directly ex vivo or after restimulation (107 cells/ml) in vitro for 5 days with the recombinant p40 protein (75 µg/ml). Table 1 shows that mononuclear cells from the spleens of Lewis rats primed by N cDNA did not show any cytotoxic activity directly ex vivo. Restimulation of the primed mononuclear cells was needed. Mononuclear cells from the spleens of Lewis rats receiving four doses of the pDL-N.Wild plasmid DNA did not lyse the BDV-infected and noninfected targets. In contrast, mononuclear cells (107 cells/ml) restimulated in vitro for 5 days with the p40 protein (75 µg/ml) lysed the BDV-infected F10/BV targets, but not the noninfected F10 targets (kindly provided by Lothar Stitz, Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tübingen, Germany). The cytotoxicity of the in vivo primed and boosted and in vitro restimulated mononuclear cells to the BDV-infected targets was restricted by MHC class I molecules. Treatment of the targets with OX18, the anti-RT.1A (class I MHC of rats) MAb, at 37°C in a humidified atmosphere of 5% CO2 for 90 min before the addition of the effectors abolished the cytotoxicity altogether (Table 1), suggesting that the anti-BDV lysis was mediated by classical CTLs that are RT.1A restricted. Mononuclear cells from the peripheral blood essentially gave comparable results (data not shown).


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  		TABLE 1. Induction of a cytotoxic response to BDV in Lewis rats by intramuscular immunization with four doses of the pDL-N.Wild plasmid DNA

To study CTLs against BDV X, 4-week-old Lewis rats (Harlan) were immunized against BDV p10 with 30 µg of pcX-FLAG plasmid DNA (27) in a 100-µl volume injected intramuscularly into the quadriceps. Three weeks later, the priming dose was followed by three boosters of the same dose, injected at the same site, at 3 weeks apart. The animals were bled via the tail vein immediately prior to and 2 weeks after each immunization. In addition to blood sampling, groups of animals were sacrificed at various time points after each injection, and splenic and lymph node mononuclear cells were tested for BDV-specific CTL effector functions directly ex vivo and after restimulation with recombinant p10. Like plasmid DNA-based vaccination against p40, mononuclear cells from the spleens of Lewis rats primed by four doses of the pcX-FLAG plasmid did not show any cytotoxic activity directly ex vivo (Table 2). After restimulation in vitro for 5 days with the recombinant p10 protein (75 µg/ml), these mononuclear cells effectively lysed the BDV-infected F10/BV targets (Table 2 and Fig. 1B), but not the noninfected F10 targets (Table 2 and Fig. 1A). This level of cytotoxicity to BDV-infected cells after priming and restimulation with BDV X (Table 2 and Fig. 1B) was comparable to that seen after priming and restimulation with BDV N (Table 1). Restimulation of the X-primed mononuclear cells with the irrelevant p40 antigen gave no cytotoxicity to the F10/BV targets (Table 2). Mononuclear cells from naïve Lewis rats stimulated in vitro with the recombinant p40 or p10 protein had no cytotoxicity (data not shown).


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  		TABLE 2. Induction of cytotoxic response to BDV by plasmid immunization is antigen specific



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  		FIG. 1. A single dose of priming with pcX-FLAG plasmid DNA is sufficient to induce an RT1.Al-restricted, BDV-specific cytotoxic response. Lewis rats were immunized intramuscularly by one ({circ}), two ({triangleup}), or three ({square}) doses of pcX-FLAG plasmid DNA (30 µg/dose) as indicated. Three weeks after the injections, splenic mononuclear cells were restimulated in vitro with recombinant His6-p10 protein (75 µg/ml) for 5 days. Cytotoxicity was measured by use of the nonradiometric CytoTox 96 assay (Promega, Madison, Wis.), with Lewis F10 cells (A) and their BDV-infected counterpart, F10/BV cells (B), as targets. The anti-RT1.A MAb OX18 was used to treat the target cells (•) for 90 min at 37°C, prior to exposure to the effectors, to determine the class I MHC restriction of the antigen-specific cytotoxicity.

Intramuscular immunization with a single dose of X cDNA was able to induce a healthy anti-BDV cytotoxic response 3 weeks postinoculation, giving more than 33% lysis of the F10/BV targets at an effector-to-target cell (E:T) ratio of 30:1. A second injection enhanced this cytotoxicity, but additional boosting with the pcX-FLAG plasmid did not significantly improve the cytotoxic activity (Fig. 1). The BDV-specific cytotoxicity induced by in vivo priming and in vitro restimulation with BDV X was restricted by the RT.1Al molecules. Treatment of the targets with the OX18 MAb abolished the cytotoxicity altogether (Fig. 1).

Two-color fluorescein-activated cell sorter (FACS) analysis was performed to study the CD8+ effectors. The mononuclear cells (106 cells) from rats primed with three doses of pcX-FLAG plasmid and restimulated in vitro with the recombinant p10 protein were mixed with an R-phycoerythrin (PE)-labeled OX8 MAb (anti-rat CD8; Serotec, Raleigh, N.C.) in the presence of 0.1% (wt/vol) sodium azide for 30 min on ice. After two washes, the cells were fixed on ice for 20 min with 250 µl of BD Cytofix/Cytoperm (BD Bioscience, San Jose, Calif.) containing 4% (wt/vol) paraformaldehyde. After two washes with BD Perm/Wash solution (BD Bioscience), the permeabilized cells were stained with fluorescein isothiocyanate (FITC)-labeled DB-1 (anti-rat IFN-{gamma}; Serotec) on ice for 30 min. FACS analysis was performed at the West Virginia University Flow Cytometry Core Facility with a FACScalibur device (Becton Dickinson) using the CellQuest software package (Becton Dickinson). Argon laser output was 15 mW at 488 nm, and the band filter for FITC was 519 nm and for PE was 578 nm. Data were collected on at least 15,000 cells, as determined by forward (size) and side (complexity) light scatter intensity. The results were expressed as percentages of specific populations of cells that stained positively for each marker. Figure 2 shows the results of a representative experiment. About 79% of the restimulated cells in culture were CD8- but were activated, as determined by intracellular IFN-{gamma} staining (lower right quadrant), in contrast to 15% of the total cells that were activated CD8+ effectors, expressing cell surface CD8 and intracellular IFN-{gamma} (upper right quadrant).



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  		FIG. 2. Only a small proportion of the in vitro restimulated cells are activated CD8+ cells. Lewis rats were immunized intramuscularly by three doses of pcX-FLAG plasmid DNA (30 µg/dose) at 3-week intervals. Three weeks after the last boost, splenic mononuclear cells were restimulated in vitro with recombinant His6-p10 protein (75 µg/ml) for 5 days. The percentage of activated CD8+ cells was determined by two-color FACS after staining with the PE-conjugated OX8 (Serotec) and FITC-conjugated DB1 (Serotec) MAbs.

Based on previously published peptide motifs and the anchor residues of the RT1.Al molecule (37) of the Lewis rats, the SYFPEITHI database has been used to correctly predict the p40 peptide, A230SYAQMTTY238, recognized by BDV-specific CTLs in association with RT1.Al molecules (36). To perform epitope prediction for p10, the codon sequence was first predicted from the nucleotide sequence of BDV ORF X (GenBank accession number AF030353) by use of the Omiga software package (Oxford Molecular, Madison, Wis.). Nonamer peptides that may be presented by RT1.Al to T lymphocytes were determined by epitope selection by use of the SYFPEITHI database as previously described (36). Peptides M1SSDLRLTL9, S2SDLRLTLL10, and T8LLELVRRL16 of p10 were predicted as most likely to be presented by the RT1.A1 molecules to the CTLs of Lewis rats. The p10 peptides M1SSDLRLTLL10 and T8LLELVRRL16 and the previously reported p40 peptide A230SYAQMTTY238 (15, 36), with an N-terminal H+ and a C-terminal OH-, were synthesized and purified by a commercial source (GenoSys; Sigma). Purification was done by reverse-phase high-performance liquid chromatography through an ODS-AQ (YMC Co. Ltd., Kyoto, Japan) C-18, 3-µm particle, 12-nm-pore-size, 4.0- by 50-mm column and detected by measuring the absorption at 214 nm in continuous flow elution. The purified peptides were determined to be >90% pure by mass spectral analyses with the Applied Biosystems Voyager System 1105 and were loaded onto F10 cells (5 x 103 cells per peptide concentration) for 90 min at 37°C in an atmosphere of 5% CO2. The peptide-loaded F10 cells and F10 cells mock treated the same way were used as CTL targets to identify the p10 epitopes recognized by the CTLs.

Peripheral blood mononuclear cells from a Lewis rat, primed in vivo to the pDL-N.Wild plasmid DNA and restimulated in vitro with the recombinant p40 protein, gave 36.7% lysis of the F10 cells loaded with the A230SYAQMTTY238 peptide (5.0 µg/5 x 103 F10 cells), at an E:T ratio of 30:1. The same effectors did not lyse F10 cells that were not loaded with the peptide (-5.1% lysis). Figure 3 shows the lysis by plasmid-primed p10-specific CTLs of F10 cells loaded with the synthesized p10 peptides M1SSDLRLTTL10 and T8LLELVRRL16. F10 cells loaded with the unrelated BDV p40 peptide A230SYAQMTTY238 were used as controls. Effectors from Lewis rats primed in vivo with three doses of the pcX-FLAG plasmid and restimulated in vitro with the recombinant p10 protein lysed the F10 cells loaded with the p10 peptide M1SSDLRLTTL10 or T8LLELVRRL16. They did not lyse the F10 cells pulsed with the unrelated A230SYAQMTTY238 peptide. As expected, the lysis of these M1SSDLRLTTL10- and T8LLELVRRL16-loaded F10 cells was RT1.Al restricted (Table 3), and the reaction could be blocked by treating these targets with the OX18 MAb prior to exposure to the effectors.



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  		FIG. 3. Two p10 epitopes are recognized by antigen-specific CTLs induced by plasmid immunization. Lewis rats were immunized intramuscularly with three doses of pcX-FLAG plasmid DNA (30 µg/dose) at 3-week intervals. Three weeks after the last boost, splenic mononuclear cells were restimulated in vitro with recombinant His6-p10 protein (75 µg/ml) for 5 days. Cytotoxicity was measured by use of the nonradiometric CytoTox 96 assay (Promega), with Lewis F10 cells or F10 cells loaded with peptide 1 (M1SSDLRLTTL10) or 2 (T8LLELVRRL16) of p10 as targets. F10 cells loaded with the unrelated peptide 3 (A230SYAQMTTY238) of p40 served as control targets.


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  		TABLE 3. Epitope-specific cytotoxcity to BDV X is class I MHC restricted

To test whether plasmid DNA-based vaccination is as effective in generating a BDV-specific antibody response as it is for a CTL response, BDV-specific antibodies in serum and/or plasma of the in vivo primed Lewis rats were monitored by Western blotting with recombinant glutathione S-transferase (GST)-p10 and GST-p40 purified from Escherichia coli transformed by the prokaryotic expression vectors pGEX-X (27) and pGEX-N.Wild (27), respectively. Antibody isotypes were detected by use of an alkaline phosphatase-conjugated rabbit antibody to rat immunoglobulin G (IgG) (Sigma) and then horseradish peroxidase-conjugated MAb MARM-4 (anti-rat IgM), MARG1-2 (anti-rat IgG1), MARG2a-1 (anti-rat IgG2a), or MARG2b-8 (anti-rat IgG2b) (all from Zymed), followed by color development with an alkaline phosphatase conjugate substrate kit (Bio-Rad) or an Opti-4CN substrate kit (Bio-Rad) for horseradish peroxidase. Primary injection of the pDL-N.Wild plasmid DNA induced an anti-p40 titer of 1:80 in Lewis rats. In contrast, Table 4 shows that no anti-p10 antibody was detected until after the third injection of the pcX-FLAG plasmid DNA. Only IgM was detected, and that was at a low titer. However, this poor antibody response to p10 could be optimized by protein boosting after the primary or second cDNA injection. Boosting with a single injection of recombinant His6-p10 purified from E. coli transformed by the pET-X-FLAG expression plasmid gave IgM and IgG antibodies when tested against the GST-p10 fusion protein (Table 4). The absence of the p10-specific IgG in sera without a protein boost suggests the absence of T-cell help, especially by Th2 cells, and antibody switching.


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  		TABLE 4. Antibody response to BDV p10 induced by plasmid immunization

The most studied experimental model of BD is the infection of Lewis rats. T cells represent the major pathway of immunopathology of BD in Lewis rats, and CD8+ T cells in particular play a major role in inducing severe encephalitis and degenerative encephalopathy (2, 33-36, 41, 42). Previous studies have shown that the CD8+ T cells infiltrating diseased brains are specific to BDV N (35, 36). The p40 peptide A230SYAQMTTY238 had been identified as an RT1.Al-restricted target of these brain-derived CTLs from infected Lewis rats (15, 36). Although immunization with a recombinant vaccinia virus containing a BDV N cDNA insert successfully induced N-specific, MHC class I-restricted CTLs, the present report represents the first study to describe successful induction of CTL responses against BDV by use of plasmid DNA-based immunization. Mononuclear cells from Lewis rats immunized with the pDL-N.Wild plasmid were cytotoxic to F10 targets loaded with the A230SYAQMTTY238 peptide of N after restimulation in vitro with the recombinant N protein.

Among the six known BDV ORFs, CTL responses to N and G have been found (35). The present communication reports the CTL response to BDV p10. Priming with the pcX-FLAG plasmid in vivo, followed by in vitro restimulation with a recombinant p10 protein, induced a BDV-specific, MHC class I-restricted CTL response. Lysis of the BDV-infected F10/BV cells could be blocked by treating the targets with the OX18 MAb prior to exposure to the effectors. After peptide epitope prediction, the peptides M1SSDLRLTTL10 and T8LLELVRRL16 were recognized in combination with the RT1.Al molecules of Lewis rats by the p10-specific CTLs. Mononuclear cells from Lewis rats immunized with the pcX-FLAG plasmid, followed by restimulation in vitro with the recombinant p10 protein, gave comparable lysis of the F10 targets loaded with the M1SSDLRLTTL10 or T8LLELVRRL16 peptide, suggesting that these two p10 epitopes may be codominant. As expected, treatment with the OX18 MAb completely blocked lysis of the peptide-loaded targets.

Plasmid-based immunization was a poor inducer of an anti-p10 humoral response in Lewis rats. In contrast, it primed a strong BDV-specific CTL response. Previous studies have shown that DNA injection primes naive CTLs by two mechanisms. The first involves the uptake of DNA by neighboring cells after injection of the naked plasmid DNA into the skin or muscle (i.e., transfection of dendritic cells [10]). The second involves secretion or leakage of the expressed antigen by the injected cells which is taken up by dendritic cells that present them in association with class I MHC (i.e., cross-priming [14]). Priming of CTLs by the antigen-expressing transfected dendritic cells is likely to be less dependent on CD4+-T-cell help (9, 44), while cross-priming likely requires T-cell help (8, 26). One and two injections of pcX-FLAG selectively primed BDV-specific CTLs without generating an antibody response, which suggests that priming by the transfected dendritic cells is the major mechanism. However, cross-priming cannot be completely ruled out, because dendritic cells may phagocytose apoptotic bodies derived from the transfected tissue cells—as they do for virally infected cells (1)—to give cross-priming. The CpG motifs present in the pcX-FLAG plasmid can also bind to Toll-like receptors and are potent adjuvants of Th1-like responses that favor CTL instead of antibody responses. Spleen and blood mononuclear cells collected directly ex vivo had no detected CTL effector functions unless they were restimulated in vitro with antigen-presenting cells loaded with p10 and not with p40. Stimulation in vitro of naïve mononuclear cells did not give any cytotoxic activity. These observations suggest that T-cell help is absolutely required to give a robust CTL response and are consistent with previous reports suggesting that help from CD4+ T cells is needed to give CTL responses to BDV (38, 39, 42). Priming in vivo with pcX-FLAG, followed by in vitro restimulation with p10, gave 30 to 50% lysis of F10/BV targets at an E:T ratio of 30 to 1. Considering that only 15% of the restimulated effectors were CD8+ and IFN-{gamma}+, i.e., activated CD8+ effectors, the percentage of antigen-specific, RT1.Al-restricted, lysis of the targets was quite high.

In experimental BD, as exemplified by BDV infection of adult Lewis rats, CD8+ T cells play a major role in immunopathogenesis (2, 33-36, 41, 42). Yet these CD8+ T cells also mediate immunologic elimination of the virus and provide immunoprotection (15). These CD8+ T cells are directed to BDV N and most likely to the A230SYAQMTTY238 peptide restricted by the RT1.Al molecules of Lewis rats. CTL responses to BDV X have never been studied. Our findings that CTLs to BDV X prevail in Lewis rats and that these CTLs are directed to two codominant, RT1.Al-restricted epitopes, M1SSDLRLTTL10 and T8LLELVRRL16, are novel. It will be important to study whether these BDV X-specific CTLs infiltrate the Lewis rat brains in BD and whether they play a role in immunopathogenesis, immunoprotection, or both.


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ACKNOWLEDGMENTS
 
This work was supported in part by Public Health Service grant 1P20RR16477, grant P20RR16440 from the National Center for Research Resources, and a seed grant from the Salem International University.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioscience, Salem International University, 223 West Main St., Salem, WV 26426-0500. Phone: (304) 782-5575. Fax: (304) 782-5579. E-mail: lai{at}salemiu.edu. Back

{dagger} Present address: Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Back


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Journal of Virology, May 2003, p. 6076-6081, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.6076-6081.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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  • Henkel, M., Planz, O., Fischer, T., Stitz, L., Rziha, H.-J. (2005). Prevention of Virus Persistence and Protection against Immunopathology after Borna Disease Virus Infection of the Brain by a Novel Orf Virus Recombinant. J. Virol. 79: 314-325 [Abstract] [Full Text]  

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