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Short Communication |
The Roslin Institute, Royal School of Veterinary Studies, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH9 1QH, UK
Correspondence
John K. Fazakerley
John.Fazakerley{at}ed.ac.uk
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ABSTRACT |
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A colour version of Fig. 2 is available with the online version of this paper.
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). This system has the advantage over many other models of encephalitis in that SFV is efficiently neuroinvasive allowing the study of central nervous system (CNS) events without direct intracerebral inoculation and consequent disturbance of the blood–brain barrier; it also produces lesions of inflammatory demyelination. Virus inoculated intraperitoneally first replicates in several tissues, resulting in a high titre plasma viraemia from which virus crosses the blood–brain barrier to establish small perivascular foci of CNS infection, and infectious virus is detectable in the brain from day 2 to 10 (Fazakerley et al., 1993). SFV predominantly infects neurons and oligodendrocytes, but replication is restricted in the mature neurons of the adult mouse brain (Amor et al., 1996; Fazakerley et al., 1993, 2006). Clearance of infectious virus is followed, days 10–21, by the appearance of lesions of inflammatory demyelination that are dependent upon the presence of CD8+ T lymphocytes (Subak-Sharpe et al., 1993). In athymic nu/nu mice, which lack T lymphocytes and produce only antiviral IgM, titres of infectious virus in the blood are reduced to undetectable levels, but high titres of infectious virus remain in the brain (Fazakerley & Webb, 1987). In mice with severe combined immunodeficiency (SCID), which have no antibody and no functional T or B lymphocytes, high titres of infectious virus are detectable in both the blood and the brain for several weeks (Amor et al., 1996). These two experimental systems are complex to interpret in that defects exist in both the antibody and the cell-mediated arms of the immune response. To determine the role of antibodies in clearance of infectious brain virus and generation of lesions of demyelination, the course of infection was investigated in antibody deficient µMT mice; µMT mice lack B lymphocytes and antibodies, but otherwise have an intact immune system (Kitamura et al., 1991).
Studies using in situ hybridization and PCR have detected alphavirus RNA in the brains of immunocompetent mice many weeks after infection (Donnelly et al., 1997; Levine & Griffin, 1992). Infectivity assays only determine levels of infectious virus above a limit of detection and only detect infectious virus in excess of the neutralizing capacity of the homogenized tissue sample. Absence of infectious virus, as detected in an infectivity assay, therefore does not necessarily equate to elimination of all infectious virus or of virus material capable of giving rise to infectious virus. In this study, virus clearance was determined both by infectivity assay and by quantitative RT-PCR (q-PCR) to assess virus RNA load.
Groups (n=12) of 129xC57BL/6 µMT (µMT) and 129xC57BL/6 (wt) mice were inoculated intraperitoneally with 5000 p.f.u. of the avirulent A7(74) strain of SFV. All mice were bred and maintained in the Centre for Infectious Diseases Animal Unit, College of Medicine & Veterinary Medicine, University of Edinburgh, UK. Animals were kept in HEPA-filtered boxes in specific pathogen-free and environmentally enriched conditions with a 12 h light–dark cycle and food and water supplied ad libitum. All breeding and experimental studies were agreed by the University of Edinburgh Ethical review Committee and were carried out under the authority of a UK Home Office license. All animals were used between 4 and 5 weeks of age. Three mice were sampled at 4 days and 2, 4 and 8 weeks post-infection. Serum samples and one half of each brain were used to titre infectivity. The other half of each brain was divided into two and used to extract RNA and to study neuropathological changes.
At 4 days and 2, 4 and 8 weeks post-infection, infectious virus was detectable by standard plaque assay (Fazakerley et al., 1993) in the sera of all µMT mice, indicating a persistent plasma viraemia (Fig. 1a). The serum virus infectivity titres ranged from 2.6 to 4.8 log10 p.f.u. ml–1. The range was similar at 2, 4 and 8 weeks. In contrast, by 4 days and all times thereafter all wt mice had infectivity titres below the limit of detection (Fig. 1a); this is consistent with previous studies (Fazakerley et al., 1993). In the brain, high levels of infectious virus were detectable at 4 days in both µMT and wt mice (Fig. 1b). In µMT mice, infectious virus remained detectable for 8 weeks, albeit at considerably reduced levels; all mice sampled between 2 and 8 weeks post-infection had detectable levels of infectious virus (Fig. 1b). Titres ranged from 2.4 to 5.4 log10 p.f.u. g–1 brain. In contrast, from 2 weeks post-infection, infectious virus in the brains of all wt mice were below the limit of detection (Fig. 1b). We conclude that antibodies are required to clear infectious virus from both the blood and the brain.
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). At each time point, µMT mice had higher levels of viral RNA than wt mice. The difference between the two mouse strains was 20-fold at 2 weeks and 50-fold at 8 weeks. Virus loads were compared using the Mann–Whitney test and were significantly different (P<0.05) at all time points. In µMT mice there was no correlation between infectious virus titres (serum or brain) and levels of brain virus RNA. Levels of virus RNA in the brain declined with time in both mouse strains. At 8 weeks post-infection, >6 weeks after infectious virus was no-longer detectable, 3/4 wt mice still had virus RNA detectable in the brain.
Previous studies have shown that between 2 and 4 weeks post-infection SFV A7(74)-infected mice have lesions of inflammatory demyelination in the brain and that these lesions require adaptive immune responses, including CD8 T cells (Subak-Sharpe et al., 1993). To determine whether the lesions of demyelination also require antibodies, the brains of the three µMT and three wt mice were sampled at 2 weeks post-infection. Brains were fixed by immersion in 10 % phosphate buffered formal saline for at least 48 h, processed through paraffin wax, stained with luxol fast blue (LFB) and cresyl fast violet (Kluver & Barrera, 1953) and coded prior to examination. At least three sections from three separate areas of each brain were studied and scored for the extent of inflammation and demyelination (Table 1). The brains of mice from both mouse strains showed microcystic changes, mononuclear cell inflammatory infiltrates, which were mostly perivascular, and lesions of demyelination (Fig. 2). We conclude that antibodies are not required to generate lesions of demyelination.
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). The present study shows, for the first time, the temporal course of virus RNA levels in the brain. In the brains of immunocompetent (129xC57BL/6) mice, as expected, infectious virus was cleared within 2 weeks; however, levels of virus RNA declined slowly. The levels of virus RNA were not quantified; however, the presence of alphavirus RNA months after infection in the brains of immunocompetent mice has also been reported for the M9 strain of SFV and the AR339 strain of Sindbis virus (Donnelly et al., 1997; Levine & Griffin, 1992).
In the brains of SFV-infected µMT mice, following an early reduction in levels of infectious virus, infectious virus and virus RNA remained detectable throughout the 12 weeks of the study. There was considerable variation in titres between mice. Persistence of infectious virus for months in the brain is also observed in athymic nu/nu and SCID mice infected with SFV A7(74) and, as in the current study, levels of infectious virus show considerable variation between individual mice (Amor et al., 1996; Fazakerley et al., 1993). The TE strain of Sindbis virus has also been observed to persist for weeks in the brains of SCID and µMT mice (Burdeinick-Kerr et al., 2007; Levine et al., 1991). Clearly, at least for alphaviruses, antibody is required to clear infectious virus from the CNS. The explanation for the high variability in SFV infectivity titres between mice and for the variability between levels of infectious virus and virus RNA is not clear. One factor may be persistence of virus RNA that cannot give rise to infectious virus; another may be that levels of infectious virus, but not virus RNA, rise and fall in response to some defence system, perhaps interferons.
Both immunocompetent and µMT mice sampled at 2 weeks post-infection had CNS inflammation and demyelination. The extent of both was less than that observed in our previous studies with the same strain of SFV in BALB/c mice (Fazakerley et al., 1983; Subak-Sharpe et al., 1993). This is most likely to reflect genetic differences between the mouse strains, which for C57BL/6 and BALB/c have been demonstrated to affect immune responses in many systems and which can affect the course of SFV encephalitis (Suckling et al., 1980). Interestingly, inflammation was more extensive in the µMT mice than in wt mice. Most likely, this is because in the absence of antibodies more virus-infected cells resulted in more antigen and more chemokines driving the inflammation; demyelination also had a slightly higher score consistent with more infected oligodendrocyte targets of CD8+ T cells. Our previous studies showed that demyelination requires CD8+ T cells; however, these studies did not establish whether antibodies were also required (Subak-Sharpe et al., 1993). Studies by others (Mokhtarian et al., 2003) suggest that B cells and anti-myelin antibodies also contribute to myelin pathology. The current study makes it clear that T-cell responses are sufficient and that antibodies are not required.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Burdeinick-Kerr, R., Wind, J. & Griffin, D. E. (2007). Synergistic roles of antibody and interferon in noncytolytic clearance of Sindbis virus from different regions of the central nervous system. J Virol 81, 5628–5636.
Donnelly, S. M., Sheahan, B. J. & Atkins, G. J. (1997). Long-term effects of Semliki Forest virus infection in the mouse central nervous system. Neuropathol Appl Neurobiol 23, 235–241.[CrossRef][Medline]
Fazakerley, J. K. (2004). Semliki Forest virus infection of laboratory mice: a model to study the pathogenesis of viral encephalitis. Arch Virol Suppl 18, 179–190.[Medline]
Fazakerley, J. K. & Webb, H. E. (1987). Semliki Forest virus-induced, immune-mediated demyelination: adoptive transfer studies and viral persistence in nude mice. J Gen Virol 68, 377–385.
Fazakerley, J. K., Amor, S. & Webb, H. E. (1983). Reconstitution of Semliki Forest virus infected mice, induces immune mediated pathological changes in the CNS. Clin Exp Immunol 52, 115–120.[Medline]
Fazakerley, J. K., Pathak, S., Scallan, M., Amor, S. & Dyson, H. (1993). Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons. Virology 195, 627–637.[CrossRef][Medline]
Fazakerley, J. K., Cotterill, C. L., Lee, G. & Graham, A. (2006). Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus-infected mouse brain: a novel system to study virus-oligodendrocyte interactions. Neuropathol Appl Neurobiol 32, 397–409.[CrossRef][Medline]
Kitamura, D., Roes, J., Kuhn, R. & Rajewsky, K. (1991). A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 350, 423–426.[CrossRef][Medline]
Kluver, H. & Barrera, E. (1953). A method for the combined staining of cells and fibres in the nervous system. J Neuropathol Exp Neurol 12, 400–403.[Medline]
Levine, B. & Griffin, D. E. (1992). Persistence of viral RNA in mouse brains after recovery from acute alphavirus encephalitis. J Virol 66, 6429–6435.
Levine, B., Hardwick, J. M., Trapp, B. D., Crawford, T. O., Bollinger, R. C. & Griffin, D. E. (1991). Antibody-mediated clearance of alphavirus infection from neurons. Science 254, 856–860.
Mokhtarian, F., Huan, C. M., Roman, C. & Raine, C. S. (2003). Semliki Forest virus-induced demyelination and remyelination – involvement of B cells and anti-myelin antibodies. J Neuroimmunol 137, 19–31.[CrossRef][Medline]
Subak-Sharpe, I., Dyson, H. & Fazakerley, J. (1993). In vivo depletion of CD8+ T cells prevents lesions of demyelination in Semliki Forest virus infection. J Virol 67, 7629–7633.
Suckling, A. J., Jagelman, S., Illavia, S. J. & Webb, H. E. (1980). The effect of mouse strain on the pathogenesis of the encephalitis and demyelination induced by avirulent Semliki Forest virus infections. Br J Exp Pathol 61, 281–284.[Medline]
Received 26 March 2008;
accepted 4 June 2008.
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) and 129xC57BL/6 (
) mice in serum (a) and brain (b). Titres were measured by plaque assay in BHK-21 cells. SFV virus RNA titres in brain tissue were measured by q-PCR (c). Each point represents the mean of two replicates. The horizontal bars for each group represent the mean value for all samples. The limit of detection is indicated by the dashed line. In (c) at each time point there was a significant difference between the two animal groups using Mann–Whitney test (P<0.05). Comparison of brain virus titres and RNA levels in individual mice demonstrated no correlation; for the µMT mice r2=0.16 and for the 129xC57BL/6 mice r2=0.26.