Laboratory for Clinical and Molecular Virology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK1
Author for correspondence: John Fazakerley. Fax +44 131 650 6511. e-mail John.Fazakerley{at}ed.ac.uk
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Abstract |
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Introduction |
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Intriguingly, MHV-68 was originally isolated from the tissues of a bank vole, Clethrionomys glareolus, in Slovakia by passage through neonatal mouse brain (Blaskovic et al., 1980 ) and was subsequently reisolated from the trigeminal ganglia of naturally and experimentally infected mice (Blaskovic et al., 1984
; Rajcani et al., 1985
). This and the virus growth characteristics led to a preliminary and incorrect classification of this virus as an alphaherpesvirus (Svobodova et al., 1982
). The virus is now known to be a gammaherpesvirus. By analogy with other gammaherpesviruses, the natural route of infection is likely to be oral or respiratory. How this virus gains access to the CNS and how frequently this occurs remain unknown. Other gammaherpesviruses have been associated with neurological disease. There is evidence to suggest that EBV is an important factor in the increased incidence of primary CNS lymphomas in both immunocompetent and immunosuppressed populations (Grant & Isaacson, 1992
; Itoyama et al., 1994
). There has also been a long-standing association of EBV with diverse neurological disorders including meningoencephalitis, multiple sclerosis, GuillainBarre syndrome, chronic fatigue syndrome and facial palsy (Archard & Bowles, 1988
; Hotchin et al., 1989
; Bray et al., 1992
; Roberg et al., 1991
; Martyn et al., 1993
; Imai et al., 1993
; Haahr et al., 1994
). In this study, we examine the ability of MHV-68 to gain access to, and to spread and persist within, the CNS.
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Methods |
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Cells.
A CNS-derived temperature-sensitive SV40 large T-antigen-transformed cell line, MGC7 (Terry et al., 1997 ), was infected with MHV-68 (m.o.i. 5 p.f.u.) and maintained in culture for several weeks in the presence of 4'-S-EtdU at a concentration of 1 µg/ml. Prior to inoculation, cells were removed from the flask using EDTA and washed in PBS at 4 °C.
Virus and virus administration.
MHV-68 was prepared by infection of BHK-21 cells at low multiplicity (0·001 p.f.u. per cell) as described previously (Sunil-Chandra et al., 1992a ; Usherwood et al., 1996
) and stored at -80 °C. Intranasal inoculations were performed under light halothane anaesthesia; 2x104 p.f.u. virus in 50 µl PBS was placed ont-o the nares and the mice were allowed to inhale the inoculum. For intracerebral inoculation, 2x104 p.f.u. of virus in a total volume of 20 µl was injected close to the midline using a 27 gauge needle. Cellular implantation of infected or control (uninfected) MGC7 cells was performed using a stereotaxic frame with co-ordinates for striatal and ventricular inoculation and confirmed by histological inspection post-mortem. Mice were anaesthetized using Hypnorm (Janssen Pharmaceuticals) and Hypnovel (Roche) injected into the peritoneum. Using a small drill, a hole was made in the skull and over 23 minutes the cell suspension (2x104) in 2 µl PBS was injected using a Hamilton syringe. The syringe was withdrawn slowly and the skin sutured. Mice were checked daily. No adverse reactions to the surgery were observed. Virus plaque assays were performed on BHK cell monolayers as previously described (Sunil-Chandra et al., 1992a
).
Immunocytochemistry.
Unless stated otherwise, animals were sacrificed under anaesthesia by extensive perfusion with PBS alone or PBS followed by 2% paraformaldehydelysineperiodate (PLP) solution. Brains perfused with PBS only were removed and bisected down the midline. One half was immersion-fixed in 4% phosphate-buffered formal saline, processed and embedded in paraffin. The remaining half was either processed for DNA extraction or immersed in 20% sucrose solution, frozen in isopentane and then stored at -80 °C. PBS- and PLP-perfused brains were immersed in sucrose and cryopreserved as above. Paraffin-embedded sections were cut 5 µm thick and cryostat sections were cut 1015 µm thick, both sagittally, and mounted on Biobond- (British Biocell) coated slides, air-dried overnight at room temperature and stored at either 4 °C (paraffin-sections) or at -20 °C (cryosections). Immunostaining of paraffin-embedded sections for MHV-68 was performed as described previously (Sunil-Chandra et al., 1994 ). Briefly, sections were incubated for 2 h at room temperature with rabbit hyperimmune serum against MHV-68 (Sunil-Chandra et al., 1992a
) followed by incubation for 1 h with a secondary biotinylated goat anti-rabbit IgG (Vector laboratories). Signal was amplified using the Vector laboratories ABC kit and visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma fast). Non-specific staining was blocked with 5% normal goat serum prior to application of the primary antibody and all washes were done with PBS. No staining was observed on sections from the brains of uninfected control mice or the brains of mice infected with Semliki Forest virus. Immunostaining for lymphocyte subpopulations was carried out on cryosections cut from unfixed brains, perfused with PBS only. The sections were fixed in 100% ethanol at 4 °C for 10 min and rinsed immediately in PBS before incubation with antibodies to CD3 (KT3.1, a gift from S. Cobbold, Oxford, UK), CD4 (YTS 179.1) or CD8 (YTS 169.4) for 2 h following immersion in 0·3% hydrogen peroxide to block endogenous peroxidase activity. After washing in TBS0·1% Tween 20, cryosections were incubated for 1 h with biotinylated rabbit anti-rat antibody (Vector). All subsequent steps were as described above. Sections were counterstained in haematoxylin. Dual-colour fluorescent immunostaining to identify oligodendrocytes in the brain was performed on cryosections from PLP-fixed brains using rabbit polyclonal anti-bovine 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase, a gift from F. A. McMorris, Wistar Institute, USA; Raible & McMorris, 1989
) as primary antibody following the protocol described above. Sections were then incubated with FITC-labelled goat anti-rabbit (Serotec) for 1 h and remaining sites were blocked using 10% rabbit serum. Biotinylated MHV-68 rabbit serum diluted in PBS containing 10% normal rabbit serum was applied to slides for 1 h prior to washing and incubation with rhodamine-labelled streptavidin for 30 min. Confocal microscopy was done out using a Leica microscope attached to a Silicon Graphics work station.
In situ hybridization.
In situ hybridization on formalin-fixed, paraffin-embedded sections was done using either a DNA probe to the viral repeat region or an RNA probe to the virus-encoded tRNAs (Sunil-Chandra et al., 1994 ; Stewart et al., 1998
). Briefly, following pretreatment including microwaving in citrate buffer, hybridization was done with digoxigenin-labelled probes. Probes were detected with either an alkaline phosphatase-labelled or a biotin-labelled sheep anti-digoxigenin antibody and visualized using BCIPNBT substrate (Sigma) or following amplification with the Vector ABC system with DAB, as described previously (Fazakerley et al., 1993
; Stewart et al., 1998
). To control for specificity, the probes were hybridized to sections of Semliki Forest virus-infected mouse brain; no signal was detected.
DNA preparation and PCR analysis.
DNA from PBS-perfused brains frozen at -80 °C was prepared using Qiamp tissue kits (Qiagen). PCR analysis for MHV-68 DNA was performed using two sets of nested primers specific for the gp150 gene, as described previously (Stewart et al., 1998 ). The sensitivity of this nested PCR was found to be one copy of viral DNA in a background of 1 µg of cellular DNA.
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Results |
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MHV-68 can generally spread to the CNS in the absence of type-I interferon responses
The cells observed to be infected by any virus are those in which the virus can replicate to levels detectable by the assay system in the presence of host responses. In the absence of the type-I interferon system some RNA viruses have been shown to infect a wider range of cell types than observed in the presence of interferon (Ryman et al., 2000 ). The type-I interferon system has been shown to be functionally important in controlling MHV-68 infection. Infection of mice deficient in the common type-I interferon receptor (IFN-
/
-Ro/o) results in a 100- to 1000-fold increase in lung virus titres and dissemination of the infection to other organs including the adrenal glands. The CNS was not examined in these studies (Dutia et al., 1999
). To determine whether these high virus titres and the absence of a functional interferon system result in spread of infection to the brain, parallel groups of six 45-week old 129 and 129IFN-
/
-Ro/o mice were infected intranasally with 2x104 p.f.u. of MHV-68 and studied for the presence of virus-infected cells in the brain at 6 days post-infection. All of the 129IFN-
/
-Ro/o mice demonstrated extensive infection of meningeal cells (Fig. 1J
) and more rarely infection of cells underlying the meninges. In addition there were perivascular foci of infection, indicative of virus spread from the blood. No virus-positive cells were observed in the main olfactory bulb, indicating that at least by day 6 following intranasal inoculation, even in the absence of interferon, virus was unable to enter the CNS along the olfactory nerve. Consistent with our first study on 129 mice (Table 1
), no virus-infected cells were observed in the brains of these mice following intranasal infection. We conclude that, although MHV-68 does not generally infect the CNS within 10 days of an intranasal inoculation, it has the ability to do so in the absence of a type-I interferon response and possibly therefore in other cases where high blood virus titres are established.
Infection of CNS cells following reactivation from a non-productive infection
Another route by which virus may gain access to the CNS during the natural course of an MHV-68 infection could be within infected leukocytes. MHV-68 infects B-lymphocytes and can establish a latent infection of these cells (Sunil-Chandra et al., 1992a ). To study the course of CNS infection following initiation from non-productively infected cells a transplantation model was adopted. We previously generated a temperature-sensitive SV40 large T-antigen-transformed glial cell line MGC7, from the brains of CBA mice (Terry et al., 1997
). MHV-68 infection of these cells in vitro is permissive and destructive (data not shown). 4'-S-EtdU is a compound which has been shown to inhibit replication of MHV-68 and other gammaherpesviruses (Barnes et al., 1999
). Continual treatment of MHV-68-infected MGC7 cells with 4'-S-EtdU results in their survival with persistence of virus in a non-productive state (Barnes et al., 1999
). MHV-68-infected MGC7 cells could be maintained and passaged in the presence of 4'-S-EtdU for several weeks. Following drug withdrawal, productive virus replication resumed with peak infectious virus titre on day 4 and death of all cells in the culture by day 7.
MHV-68-infected or uninfected MGC7 cells, which had been cultured with 4'-S-EtdU for 5 weeks, were stereotaxically implanted into either the striatum or the lateral ventricle of five, 45-week-old CBA mice. Mice receiving uninfected MGC7 cells remained healthy for 8 weeks after which time they were euthanized. Both groups of mice receiving MHV-68-infected cells showed signs of morbidity at day 7 and were euthanized. Histological analysis confirmed the site of inoculation. Following striatal implantation, neither meningeal nor ependymal cells showed signs of infection but widespread infection of the hippocampal pyramidal neurons was observed (Fig. 2A), as were foci of infected cells within the cortex. Following ventricular implantation the pattern of infection was similar to that observed following direct inoculation of virus with infection predominantly of meningeal, ependymal and underlying cells. We conclude that initiation of infection from a non-productive source within the CNS can result in a widespread CNS infection.
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Discussion |
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As a result of the relative inaccessibility of the CNS, many neurological diseases of viral aetiology are rare complications of relatively common systemic infections. However, viruses may gain access to the CNS by two principle routes, either by neuronal transport following infection of the peripheral nervous system or via the blood. The latter may provide entry either as cell-free virus in plasma or cell-associated virus in leukocytes. The most direct nerve entry route is the olfactory nerve, which provides a direct single cell pathway from the olfactory mucosa to the olfactory bulb within the CNS. In the studies presented here all inoculations were intranasal but no evidence of virus entry via the olfactory bulb was observed. The trigeminal nerve, which partly innervates the nasal epithelium, could also be involved in translocation of virus to the CNS via the trigeminal ganglia. Although not investigated in this study the trigeminal ganglia have been reported to be a reservoir of MHV-68 in the natural host (Blaskovic et al., 1984 ; Rajcani et al., 1985
). The second putative route of entry via the blood was investigated in mice lacking a functional type-I interferon system. MHV-68 is present at high titre in the blood (Dutia et al., 1999
) under these conditions and virus was able to enter the CNS resulting in widespread infection of the meninges and perivascular foci of infection.
Many viral encephalitides are highly neuroinvasive: alphaviruses such as the equine encephalitis viruses, Sindbis virus and Semliki Forest virus efficiently gain access to the CNS early in infection when virus titres in the blood are high and before they are brought under control by immune responses. The studies reported here indicate that MHV-68 does not generally establish a CNS infection during the first 10 days of infection. MHV-68 establishes a lifelong latent infection in B-lymphocytes (Sunil-Chandra et al., 1992b ; Weck et al., 1999
). This provides a long period of time over which rare events that give rise to CNS infection could occur and accumulate. Activated but not resting lymphocytes are able to enter the CNS (Knopf et al., 1998
; Hickey, 1999
) and spread of infection following entry of an MHV-68-infected B-lymphocyte would seem a likely scenario for seeding a CNS infection. Though we were unable to parallel this experimentally using infected B-lymphocytes, widespread infection of the CNS was demonstrated following reactivation of MHV-68 from implanted non-productively infected glial cells. Perhaps with time, there is occasional spread of the infection to the CNS, or perhaps with time the cumulative effect of rare events giving rise to CNS infection establish this in most animals. Our limited study of five mice, 1 year after intranasal inoculation, suggests that CNS infection is not generally established. Even the finding of viral DNA by PCR in one of five brains at 1 year post-infection must be interpreted with caution since the blood from this mouse was also PCR-positive and although the mice were extensively perfused it cannot be ruled out that residual blood remained, perhaps in a blocked vessel.
Whereas these studies provide no clear evidence that MHV-68 generally establishes a CNS infection at early time-points or over the long-term, it is clear from our studies on the IFN-Ro/o mice that this virus can under some circumstances gain access to the CNS and our implantation studies demonstrate that MHV-68 can persist in the brain for at least 12 months. These mice in which long-term persistence was observed had received implants of non-productively infected glial cells and were treated with an antiviral drug for 10 days after implantation. Mice that received implanted cells and no drug developed a widespread infection and died. The survival of the drug-treated mice after drug withdrawal could well reflect priming of immune responses by low levels of virus. This is also likely to be the case in any leukocyte-mediated spread of MHV-68 to the CNS from persistently infected cells at any time after establishment of antiviral immune responses.
DNA of other gammaherpesviruses including equine herpesvirus-2 (EHV-2) and bovine herpesvirus-4 (BHV-4) has been demonstrated by PCR in the olfactory bulbs and other regions of the CNS of ponies, calves and mice following experimental intranasal infections (Borchers et al., 1998 ; Egyed & Bartha, 1998
; Rizvi et al., 1997a
, b
). In HIV-infected human individuals, EBV-positive primary CNS lymphomas are a major and growing problem (Flinn & Ambinder, 1996
) and there has been a long-standing association of EBV with a number of diverse neurological disorders (Archard & Bowles, 1988
; Hotchin et al., 1989
; Bray et al., 1992
; Roberg et al., 1991
; Martyn et al., 1993
; Imai et al., 1993
; Haahr et al., 1994
). A recent PCR-based survey has indicated a high incidence (63%) of KHSV nucleic acid in post-mortem brains of normal healthy individuals in the Chinese population in Hong Kong, suggesting that this virus is neuroinvasive and has the ability to persist in the human CNS (Chan et al., 2000
). The development of MHV-68 infection in the mouse provides a good small-animal model to study gammaherpesvirus biology, particularly human EBV and KSHV infections where studies have been restricted by the limited host range of these viruses and where direct studies on humans are complicated or in many cases not possible. So far the MHV-68 model has been used to understand the pathology and immunology of this virus with respect to its peripheral tropism and transforming characteristics. The study presented here extends the model by characterizing events in the CNS.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 July 2000;
accepted 7 August 2000.