【病毒外文文獻】1987 Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell culture

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Microbial Pathogenesis 1987 3 79 86 Coronavirus mouse hepatitis virus MHV A59 causes a persistent productive infection in primary glial cell cultures Ehud Lavi Akin Suzumura 2 Mikio Hirayama 2t Maureen K Highkin Donna M Dambach Donald H Silberberg2 and Susan R Weiss Departments of Microbiology and 2Neurology University of Pennsylvania School of Medicine Philadelphia Pennsylvania 19104 U S A Received November 12 1986 accepted in revised form February 4 1987 Lavi E Dept of Microbiology University of Pennsylvania PA 19104 6076 U S A A Suzumura M Hirayama M R Highkin D M Dambach D H Silberberg and S R Weiss Coronavirus mouse hepatitis virus MVH A59 causes a persistent productive infection in primary glial cell cultures Microbial Pathogenesis 1987 3 79 86 MHV A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter As part of the investigation into the mechanism of viral persistence infection of glial cells probable targets for chronic infection was studied by the use of mixed glial enriched oligodendrocyte and enriched astrocyte cultures Following MHV A59 infection in vitro approximately 10 of oligodendrocytes and 30 of astrocytes expressed viral antigens in the absence of overt cytopathic effect All cultures released infectious virus for the lifetime of the cultures for at least 45 days in the case of mixed glial cultures Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced These results show 1 that glial cells are early sites of infection in vivo as well as sites of infection in in vitro cultures and 2 that glial cells support a non lytic but productive infection in vitro and thus may contribute to viral persistence in vivo Key words Coronavirus mouse hepatitis virus MHV A59 oligodendrocytes astrocytes persistent infection Introduction Mouse hepatitis virus strain A59 MHV A59 a neurotropic murine coronavirus is an enveloped positive stranded RNA virus After intranasal or intracerebral inoculation of mice MHV A59 produces mild acute encephalitis with infection of selected regions of the central nervous system CNS and subsequent chronic demyelination MHV A59 infection of mice has been used as an experimental model system to study virus induced demyelination this may be important in the understanding of the pathogenesis of human demyelinating diseases such as multiple sclerosis MS and acute dis seminated encephalomyelitis ADEM Also at The Wistar Institute of Anatomy and Biology Philadelphia PA 19104 U S A tPresent address Department of Internal Medicine Fukui Medical College Fukui Japan Present address Department of Biological Science Monsanto St Louis MO U S A Address correspondence to Dr E Lavi Dept of Microbiology Johnson Pavilion University of Pennsylvania Philadelphia PA 19104 6076 U S A 0882 4010 87 080079 08 03 00 0 c 1987 Academic Press Ltd 80 E Lavi et al Studies with MHV JHM another neurotropic strain of MHV have suggested that MHV induced demyelination is the result of lytic infection of oligodendrocytes the myelin producing cel ls 5 6 We have previously shown that although infectious MHV A59 and viral antigens are extremely difficult if not impossible to detect after the first few weeks post infection viral genome persists in the white matter of infected mice during the chronic demyelinating stages of the disease These findings along with the observation that MHV A59 grows preferentially in non neuronal as opposed to neuronal cells in dissociated spinal cord cultures suggest that glial cells may be the site of MHV A59 persistence during chronic demyelination A recent report showed that MHV A59 can productively infect astrocytes in vitro 9 Thus as part of our approach to understanding the mechanism of MHV A59 persistence and its possible relationship to demyelination we studied the interaction between MHV A59 and oligodendrocytes and astrocytes in vitro using enriched cultures of each cell type derived from mice We have used these cultures to complement ongoing in vivo studies for several reasons 1 the interaction between virus and cell can be studied isolated from other host factors such as the immune system 2 unambiguous identification of cell type with the cell specific markers is more easily accomplished in vitro than in vivo and 3 a high percentage of a specific cell type as well as a large number of cells are obtained in each culture thus oligodendrocytes and astrocytes can be studied separately We report here that astrocytes and oligodendrocytes are early sites of MHV A59 infection in vivo and that glial cells support persistent productive MHV A59 infection in vitro Results Expression of viral antigen in glial cell cultures Mixed glial enriched oligodendrocyte and enriched astrocyte cultures were derived either from newborn or 3 week old C57BL 6 mice as described in Materials and methods Such cultures were infected with MHV A59 at multiplicity of infection MOI of 1 tissue culture infectious dose TCID 50 cell and 3 days post infection were stained by double immunofluorescence for viral antigen and cell specific markers Approximately 30 of glial fibrillary acidic protein G FAP positive cells astrocytes 73 and 10 of galactocerebroside GaIC 14 positive cells oligodendrocytes expressed cytoplasmic viral antigens Figure 1 shows an example of cells that were positive for GaIC and viral antigens Viral antigen was not detected in control mock infected cultures or in cultures that were incubated with normal mouse serum There was no obvious cytopathic effect CPE in infected cultures observed with the light microscope during a period of 45 days as compared to uninfected cultures and there was no evidence of syncytia formation which is observed in lytic infections of MHV A59 in vitro in 17CI 1 mouse fibroblasts Thus at least some oligodendrocytes and some astrocytes can be infected by MHV A59 in vitro Because only 10 of the oligodendrocytes and 30 astrocytes contained viral antigen we wanted to determine whether all cells could be infected Thus mixed glial cultures were infected with MHV A59 at MOTs of 0 1 1 5 50 TCID50 cell The percentage of infected cells did not significantly increase with the higher multiplicities There was no obvious CPE during an observation period of 1 week To determine whether glial cells may be infected by MHV A59 in vivo as well as in vitro 5 day old mice were infected by intracerebral inoculation with 400 TCID50 of MHV A59 a lethal dose for suckling mice and sacrificed one day later Mixed glial 81 Fig 1 Expression of MHV A59 antigen in infected oligodendrocytes Oligodendrocyte enriched cultures were derived from 3 week old C57BL 6 mice by Percoll density gradient centrifugation and at 1 day after isolation cultures were infected with MHV A59 MOI 1 TCID 50 cell At 3 days post infection cells were washed and incubated with rabbit polyclonal anti galactocerebroside GaIC 1 100 dilution and rhodamine conjugated secondary antibody then fixed with acid alcohol and incubated with mouse polyclonal anti MHV A59 1 10 dilution and fluorescein conjugated secondary antibody When viewed with phase contrast a rhodamine b and fluorescein c optics positive cytoplasmic staining of MHV A59 antigen c was seen in cells that appear morphologically as oligodendrocytes a and stained with anti GaIC b Cells observed in a that are not stained in b and c are uninfected and not oligodendrocytes They are probably contaminating fibroblasts and astrocytes 82 E Lavi et al E 5 F 0 n2 4 U H 2 30 J 2 7 6 I I I I I I I I I 1 1 0 4 8 12 16 20 24 28 32 36 40 44 48 Hours post infection Fig 2 Growth kinetics of MHV A59 in glial cells as compared to 17CI 1 cells T75 flasks containing either 17CI 1 cells or mixed glial cells two weeks after extraction from newborn C57BL 6 mice were infected with MHV A59 at M01 1 TCID50 cell Samples of supernatant were removed at the indicated intervals and assayed for infectious virus in a TCID50assay in mouse fibroblasts All time points were assayed in duplicate cultures the values shown above are averages 17CI 1 cells 0 glial cells cultures were derived and one day later were stained by double immunofluorescence with viral antiserum and cell specific markers as described above Again both cell types were infected by the virus viral antigens were detected in approximately the same percentage of cells as in cultures that were infected in vitro Thus astrocyte and oligodendrocytes are early sites for MHV A59 infection in vivo Production of infectious virus by glial cell cultures Since both oligodendrocytes and astrocytes contained viral antigen in the absence of CPE we wanted to determine if infectious virus was produced by these cells Thus titers of infectious virus in the supernatants of infected cultures were determined using a tissue culture infectious dose TCID 50 assay These results are shown in Figs 2 and 3 The kinetics of infectious virus production were measured in the first 48 hours post infection in mixed glial cultures and compared with kinetics in 17CI 1 mouse fibroblasts the cell line used for growth of the virus In the 17C1 1 cells infected at MOI 1 TCID50 cell a productive lytic infection destroyed the cells by 20 hours post infection when virus production was maximal Fig 2 In glial cell cultures at both MOI 1 TCID50 cell Fig 2 and MO1 50 TCID50 cell data not shown the kinetics of production of infectious virus were similar to the kinetics in 17C1 cells However the glial cultures were strikingly different in that there was no CPE The fact that similar titers of virus were produced by cultures infected at MOI of 1 or 50 TCID50 cell is consistent with the fact that the percentage of cells expressing viral antigen also did not increase at higher MOIs see above Since the glial cells were not destroyed by infection we continued to culture these cells As shown in Fig 3 in the in vitro infected mixed glial cultures there was a continual but variable release of virus at moderate levels for at least 45 days post infection Similarly moderate levels of infectious virus were measured in supernatants from oligodendrocyte cultures during a 17 day period and in the supernatants from astrocyte cultures during a 14 day period As shown in Fig 4 the continual virus release was also observed in mixed glial cell cultures that had been derived from pre infected mice 1 day after infection Thus in vitro glial cultures either infected in vitro or derived from pre infected mice produced infectious virus as long as the cultures Coronavirus MHV A59 infection of glial cells 8 7 a 0 Mixed glial E 6 5 g v 4 f s 3 0 2 1 I l I 1 1 l I 5 10 15 20 25 30 3540 45 Days post infection 8 7 E 6 n 5 4 0 311111rn J 2 a VI c J U Oligodendrocyte 81 7 0 Astrocyte 60 5r 4 30 2 c I l I I I 0 5 10 15 20 25 30 35 40 45 Days post infection Fig 3 Titers of MHV A59 in glial cell cultures infected in vitro Mixed glial cell cultures as well as oligodendrocyte and astrocyte enriched cultures derived from newborn C57BL 6 mice were infected with MHV A59 MOl 1 TCID50 cell At the indicated intervals post infection supernatants were removed and tested for infectious virus by a TCID50 assay on 17CI 1 mouse fibroblasts a Mixed glial cell cultures b Enriched astrocyte cultures c Enriched oligodendrocyte cultures Mixed glial cell cultures and astrocytes cultures contained approximately 5 fold more cells per ml supernatant than oligodendrocyte cultures Thus the amount of virus produced per cell for oligodendrocyte cultures was similar to that for the astrocyte cultures were maintained There was no CPE observed in any of the cultures for the time courses monitored in Figs 3 and 4 that is up to 45 days for the mixed glial cultures There were no morphological changes in the cultures that could be correlated with fluctuations in viral titers released Discussion and conclusions Oligodendrocytes and astrocytes are not only susceptible to MHV A59 infection in vitro but are also early sites of virus infection in vivo Mixed glial cultures enriched astrocyte cultures and enriched oligodendrocyte cultures derived from newborn mice all support persistent productive infection with MHV A59 which occurs in the absence of CPE We were unable to examine viral antigens and virus production in glial cells extracted from weanling mice after in vivo infection because of technical difficulties in deriving such cultures Thus the interaction of MHV A59 with glial cells is different from that of the virus with 17CI 1 mouse fibroblasts in that the latter cells undergo syncytia formation and subsequent cell lysis within the first day post infection Our results suggest that astrocytes are sites of viral persistence in the cultures The astrocytes cultured from mouse brain divide in culture and the cultures remain 90 pure for the duration of the experiment as assayed by staining with GFAP 10 1 1 It b 1 1 1 5 10 15 20 25 30 35 40 45 Days post infection 83 84 E Lavi et al 7 6 E 0 5 UI 0 4a J 3 20 5 10 15 Days in culture Fig 4 Titer of MHV A59 in glial cell cultures derived from mice that were infected in vivo Mixed glial cultures were derived from 5 day old mice 1 day after intracerebral infection with MHV A59 400 TCID50 inoculum At the times indicated supernatants were removed and titered for infectious virus by TCID50assay on L 2 mouse fibroblasts is more difficult to be sure of the role of oligodendrocytes in viral persistence Oligodendrocytes do not divide in culture thus enriched oligodendrocyte cultures 70 80 pure at 3 days after plating are only 20 40 GaIC positive at 2 weeks after plating Although virus production continued for the 2 weeks that the enriched oligodendrocyte cultures were maintained in vitro Fig 3 it is difficult to be sure that infectious virus was continually being produced by the oligodendrocytes as the cultures became contaminated with virus producing fibroblasts and astrocytes However some of the remaining oligodendrocytes GaIC positivite cells did remain positive for viral antigens The non lytic nature of the interaction between MHV A59 and glial cells may be either due to host cell factors that modify viral gene expression or to selection of variant virus defective in expression of one or more viral genes The latter has been shown to be the case in persistent paramyxovirus infections The selection of a viral mutant is less likely in MHV A59 infected glial cells as the virus produced by these cells is lytic in 17CI 1 cells It is more likely that an interaction of glial host cell factors with virus results in a non lytic infection We are investigating viral RNA and protein expression during MHV persistence in glial cells The outcome of infection of MHV in primary glial cells is dependent on the virus strain and the age and strain of the mice from which the cultures are derived In another study of MHV A59 in primary glial cell cultures infection was productive and lytic and virtually all the cells contained viral antigen This study differed from ours in that the MHV A59 strain used was at least 30 fold more lethal in mice and a different strain and age of mice was used to derive the cultures In studies with JHM a more neuropathogenic MHV strain chronic productive infections were established in primary glial cells or enriched oligodendrocyte cultures similar to that which we described It was shown that resistance to JHM infection in in vitro rat oligodendrocyte cultures was related to the developmental state of the cells Perhaps the fact that even at high MOIs in our studies only 10 30 of glial cells expressed viral antigen is related to the physiological or developmental state of the cells The facts that viral nucleic acids are restricted to white matter during chronic MHV A59 infection in mice and that glial cells undergo persistent productive infection in vitro support the hypothesis that virus persists in glial cells during chronic demyelination Persistent infection of oligodendrocytes may cause a malfunction of the myelin I I I I I I I I I I I I I I I I I I 1 I 1 I I I 20 25 Coronavirus MHV A59 infection of glial cells 85 synthesis or may alter myelination by a more indirect perhaps immune mediated mechanism Persistent MHV infection of primary glial cells is accompanied by enhancement of H 2 class 1 antigen expression 20 This could potentially lead to the recognition of infected oligodendrocytes by cytotoxic T lymphocytes that could lyse the oligodendrocytes to cause demyelination We are currently investigating this idea Materials and methods Glial cell cultures All cultures were derived from C57BL 6 mice obtained from Jackson Laboratories designated MHV free Mixed glial cell cultures were derived from 3 week old mice by mincing and trypsinization of brains followed by filtration through nylon mesh and plating onto tissue culture flasks Oligodendrocytes were purified from these brains by Percoll gradient centrifugation Alternatively mixed glial cultures were derived from newborm mice and after 2 weeks in culture were subcultured into enriched astrocytes and enriched oligodendrocyte cultures by the procedure of McCarthy and DeVellis 10 designed for rat glial cells as modified for use with mouse cells Briefly this involves shaking off of the less adherent oligodendrocytes and replating of these cells onto poly L lysine coated cover slips at a concentration of approximately 5x 104 cells per cm2 while the astrocytes remain adherent to the tissue culture plastic In both cases infections were performed on cells at 3 4 weeks of development In both cases cultures were similar in terms of percentage of cells containing viral antigen and amount of infectious virus produced Because the cells were difficult to obtain routinely from the older mice most of the experiments were performed on cultures derived from the newborn mice All cells were grown in Dulbecco s medium with 10 fetal calf serum at 37 C in the presence of 5 CO2 The purity of the cultures was determined by indirect immunofluorescence Cells were stained either with antiserum directed against galac tocerebroside GaIC an oligodendrocyte specific marker 14 or antiserum against glial fibrillary acidic protein GFAP an astrocyte specific marker 13 followed by a secondary antibody conjugated with fluorescein or rhodamine As defined by staining with these antisera 3 days after plating oligodendrocyte enriched cultures consisted of more than 80 oligodendrocytes whereas astrocyte enriched cultures consisted of more than 90 astrocytes Infection of cultures with MHV A59 Plaque purified MHV A59 grown in 17CI 1 cells was used in all infections 3 17Cl 1 cells were infected at MOI 1 TCID50 cell and glial cultures were infected 1 3 days after plating with MOIs as indicated At the time of infection 17Cl 1 mixed glial and astrocyte cultures contained approximately 1 5x105 cells per cm 2 Enriched oligodendrocytes contained approximately 5x 104 cells per cm 2 After adsorption for 1 hour at room temperature cultures were washed with phosphate buffered saline three times and fed with warm media At the indicated intervals post infection supernatants were collected replaced with fresh media and tested for infectious virus by a TCIDS assay in 17CI 1 or L 2 mouse fibroblasts as indicated as previously described Titers were approximately the same in both cell types Infection in mice Five day old C57BL 6 mice were infected intracerebrally with 400 TCID50 of MHV A59 One day later brains were removed and mixed glial cultures derived by the techniques of McCarthy and DeVellis16 as described above Antigen dection by immunofluorescence Cultures were stained by immunofluorescence When staining for oligodendrocytes expressing viral antigens cells were washed and incubated with rabbit polyclonal anti galactocerebroside serum 1 100 dilution and rhodamine con jugated secondary antibody then fixed with a acetic acid ethanol and incubated with mou