key: cord-0899144-msxut0qc
authors: Perlman, S.; Schelper, R.; Bolger, E.; Ries, D.
title: Late onset, symptomatic, demyelinating encephalomyelitis in mice infected with MHV-JHM in the presence of maternal antibody
date: 1987-03-31
journal: Microbial Pathogenesis
DOI: 10.1016/0882-4010(87)90020-9
sha: 8afb280788ce0249c4be87370c9eac09a3d25f57
doc_id: 899144
cord_uid: msxut0qc

Abstract The presence of maternal antibodies protected suckling C57BL/6 mice from the clinical manifestations of the acute encephalomyelitis caused by mouse hepatitis virus, strain JHM (MHV-JHM), a coronavirus, even though histological evidence of encephalomyelitis was found at early times after inoculation. 100% of infected suckling mice developed a fatal disease in the absence of maternal antibody. By 14 days after inoculation, the brains of all antibody-protected mice examined were nearly normal on histological examination. At 3–8 weeks post-inoculation, approximately 40% of the antibody-protected mice developed a neurological disease characterized by hindlimb paralysis and wasting. Evidence of inflammation and demyelination was apparent in the spinal cord and brainstem. The mice that remained asymptomatic at this time showed few signs of inflammation and none developed clinical disease over the following 9 months. Viral antigen could be detected in most of the mice examined at all times after inoculation, whether symptomatic or not, and was particularly evident in the animals with hindlimb paralysis. MHV-JHM could be consistently cultured from the mice with hindlimb paralysis. These results show that maternal immune factors can completely protect susceptible mice from the acute, fatal, clinical encephalomyelitis caused by MHV-JHM, but cannot prevent the establishment of a latent state and subsequent development of virus-induced, clinically evident, demyelinating disease. This model will be useful for studying the virus and host factors important for the development of MHV-JHM latency and subsequent virus-induced demyelination.

Several strains of mouse hepatitis virus, a member of the coronavirus family, cause persistent neurological infections in rodents and as such serve as models of chronic neurological human diseases such as multiple sclerosis and subacute sclerosing panencephalitis .' , '

The JHM strain (MHV-JHM) causes either an acute encephalomyelitis or an acute or chronic demyelinating disease in mice and rats as well as a subacute demyelinating encephalomyelitis in rats . [3] [4] [5] [6] [7] [8] [9] [10] In all of these diseases, with the exception of the acute encephalomyelitis, oligodendrocytes are preferentially affected, with most of the virusinduced lesions located in the white matter of the brain and spinal cord . MHV-JHM Corresponding author . 0882-4010/87/030185+10 $03 .00/0 infects other tissues, such as the liver, but this is a relatively minor part of the disease process .

The outcome of a given infection is dependent upon the age and strain of rodent infected as well as upon the biological properties and route of inoculation of the virus . The administration of monoclonal antibody directed against the glycoprotein E2 of MHV-JHM prior to infection prevents the acute encephalomyelitis, but the mice still show evidence of demyelination on histological examination ." Similarly, the presence of maternal antibody protects suckling mice from the acute infection, when administered prior to inoculation of virus . 12 Maternal antibody crosses the neonatal gut until approximately 15 days postpartum ." Since MHV-JHM appears to persist in mice in the presence of maternal immune factors, 12 the immature mouse must be able to mount an immune response in order to prevent a recrudescence of the infection after the waning of passive antibody .

We have devised a model in which MHV-JHM-infected suckling mice developed a clinically asymptomatic encephalomyelitis within one week of inoculation ; virus could be isolated only infrequently from these mice . Subsequently, MHV-JHM could be cultured from about 40% of mice at 3-8 weeks post-inoculation with concomitant development of a clinically evident demyelinating encephalomyelitis . In this communication, we describe the virological, histological and serological characterization of this infection .

Acute encephalomyelitis Suckling mice born to unimmunized mothers infected intranasally with MHV-JHM (5 x 10' PFU) at 10 days of age developed an acute encephalomyelitis with all mice dying by 7 days post-inoculation (Fig . 1) . Clinical signs including hunching, ruffled fur, irritability and lethargy. Three of 12 mice developed these signs within 3 days of inoculation, whereas the remaining 9/12 did so within the following 4 days . Virus titers in the brains of infected animals at 3 and 5 days post-inoculation were approximately 4 x 10 3 and 2 x 10' PFU/g respectively (Table 1 ) , and viral antigen could be found throughout the brain parenchyma (Table 2 ), in both the grey and white matter, particularly at day 5 post-inoculation . Histological examination of the brain at 3 days post-inoculation revealed a small amount of leptomeningeal infiltration whereas at 5 days post-inoculation it showed an acute encephalitis, including perivascular, parenchymal and leptomeningeal inflammatory infiltrates, and proliferating astrocytes and microglia surrounding neurons (glial stars) . Antibody protected mice : clinical studies If female mice were immunized prior to mating, the offspring were completely protected against the acute, fatal encephalomyelitis in agreement with published results ." In our experiments, female mice were immunized with MHV-JHM in Freund's adjuvant five times . Resulting antibody titers ranged from 1 :700 to 1 :4300 (mean 1 :2700) by plaque-reduction neutralization assay (Fig . 2) . The offspring were infected intranasally at 10 days of age with the same dose of virus (5x10' PFU) that caused the acute encephalomyelitis in the offspring of unimmunized mothers . As shown in Fig . 1 , 100% of the suckling mice survived the infection, and none developed any signs of clinical disease during the first few weeks following virus inoculation . In the period 3-8 weeks after inoculation (mean : 35 d, range 23-60 d), 40% (12/30) of the mice developed a neurological illness characterized by hindlimb paralysis . The clinical disease was variable in the rapidity with which hindlimb paresis progressed to complete paralysis . In some degeneration . Virus could be cultured from brains and spinal cords but not livers at this time (Table 1 ) . Using an immunoperoxidase assay, viral antigen was detected throughout the brain in both the grey and white matter in the cerebrum, cerebellum and brainstem . No viral antigen could be detected in the spinal cord at this time ( Table  2) Day 7 : On histological examination, the brains of mice 7 days after inoculation showed evidence of meningoencephalitis (Fig . 3) , although the mice were totally asymptomatic . There were perivascular lymphocytic infiltrates and widespread, focal petechiae throughout the grey and white matter . Subependymal and meningeal infiltrates were also present . The cerebrum was heavily involved, with fewer inflammatory cells present in the hippocampus . Glial stars, similar to those seen in the acutely-infected mice, were present in the cerebral cortex adjacent to injured neurons . Lymphocytic infiltrates were also apparent in the diencephalon and pons, with minimal involvement of the cerebellum . The liver showed evidence of viral infection also, with a few areas of lymphocytic infiltration in a nodular pattern apparent, although infectious virus was not present . Viral antigen could be readily detected throughout the grey and white matter of the brain by immunocytochemical analysis of frozen sections and was also observed in scattered foci in a few of the spinal cords of these mice (Table 2 ) . Virus could be cultured from the brain at seven days after inoculation in 1 of 6 mice examined (Table 1) .

Day 14 : By 14 days after inoculation, the brains showed much less evidence of disease on histological examination . No meningeal infiltration was present and only a small amount of lymphocytic proliferation was apparent in the grey and white matter of the parenchyma . Viral antigen could be detected by immunoperoxidase assay in scattered cells of the brains and spinal cords of two of three mice examined ( Table 2) . Many of the cells containing antigen were not in areas of cellular infiltration . Virus could be cultured at low titer from the brains of one of six mice assayed at this time (Table 1 ), but not from the liver or spinal cord . Day 30 post-inoculation and longer in mice without neurological disease : Histological examination of the brains of mice without neurological disease at 30 d postinoculation and longer showed no meningeal infiltration, but did show a few areas of lymphocytic infiltration of the parenchyma . Viral antigen could be detected by immunocytochemical methods in scattered cells in most of the mice examined (Table  2) . Occasionally the antigen-containing cells were surrounded by a lymphocytic infiltrate, but more commonly, these cells did not invoke an inflammatory response (Fig . 4) . Hematoxylin-eosin stained sections of the spinal cord showed no abnormalities except for a few lymphocytic nodules in one of the five mice examined . Examination of the livers of the older mice revealed a few focal parenchymal infiltrates with lymphocytes and eosinophils present . Viral antigen was detected in only 1 /8 of the livers examined and in marked contrast to the results obtained in the antigen studies, no virus could be cultured from the brains, spinal cords or livers of these older mice (Table 1) .

Animals with hindlimb paralysis : 40% of the antibody-protected mice developed neurological disease from 23 to 60 days post-inoculation . Histological examination of the central nervous system of these mice revealed multiple abnormalities, especially in the spinal cord . Areas of destruction were apparent in the mesencephalon and pons, primarily in the white matter, although lymphocytic infiltrates were also present in the adjacent grey matter . The cerebrum showed a few areas of lymphocytic infiltration, and a basilar meningeal infiltrate was apparent . The spinal cord had many areas of focal damage with lymphocytic infiltration . After staining with Luxol Fast Blue, extensive white matter destruction was apparent with many foamy histiocytes present (Fig . 5) . Many degenerating pyknotic cells were also observed in the white matter (Fig . 5 ) . Although these cells could not be definitively identified by electron microscopy, their small size and presence in white matter suggested that they were most likely oligodendrocytes . A few coronavirus-like particles were tentatively identified by electron microscopy but they were not present in large clusters, as seen in the acute infection s (data not shown) .

Viral antigen could be detected throughout the brain and spinal cord, principally in the white matter (Table 2) . Virus was cultured from the brains and spinal cords of mice with hindlimb paralysis at titers approaching those found in acutely infected mice (Table 1) . Serological studies Mean neutralization titers, as determined by a constant virus-variable serum plaque reduction assay was 1 :2700 in immunized female mice (Fig . 2) . Antibody titers in the offspring rapidly decreased over the first few weeks of life, and became undetectable in all mice which developed hindlimb paralysis . Many of the mice which remained asymptomatic also had undetectable titers, although low levels of neutralizing antibody were present in some . Thus, the majority of mice, whether symptomatic or not, did not develop significant levels of neutralizing antibody to MHV-JHM, suggesting that the level of antibody did not correlate with protection from the delayed onset neurological disease .

Sera from immunized females and from mice which developed hindlimb paralysis were tested for their ability to neutralize virus isolated from infected, symptomatic mice . The antibody titers in each case were approximately the same as when tested against the original strain of JHM, so that maternal sera had high neutralizing titers whereas those of the symptomatic offspring had titers < 1 :100 .

Maternal immune factors fully protect suckling mice from the acute, fatal encephalomyelitis induced by the murine coronavirus JHM even if administered one day after inoculation ." In the present study, these observations were extended to a longer period of follow-up . We found that suckling mice were asymptomatic for several weeks after inoculation with the virus, although at one week after inoculation, all the mice had histological evidence of acute encephalitis which was indistinguishable from that seen in unprotected mice dying from the acute encephalomyelitis . Over the next few weeks, evidence of inflammatory response subsided in the antibody-protected mice, although virus was still present since viral antigen, but not infectious virus, could be detected in most mice . In 60%, infectious virus could never be detected and the mice remained asymptomatic . In the other 40%, however, virus could be cultured from the brain and spinal cord, in association with neurological disease characterized by hindlimb paralysis and histological evidence of myelin destruction .

Several factors might have contributed to the appearance of clinical disease in only a fraction of the infected mice . A cell-associated variant of the virus, better able to evade the host immune system may have been selected during the period of clinical latency, as has been postulated to occur with measles virus in subacute sclerosing panencephalitis ."-" In chronically infected neuroblastoma cells, MHV-JHM has been shown to evolve in such a manner in the presence of neutralizing antibody and could only be cultured after cell fusion with susceptible cells ." The host immune system may also have contributed to the persistence of the virus by its inability to eliminate the virus. Maternal antibody is readily absorbed by the neonatal mouse until approximately 15 days postpartum ." After this time, no new maternal antibody is absorbed, and the maternal antibody present in the suckling mouse decays with an approximate halflife of 5-7 days . 18 The mice in our experiments developed hindlimb paralysis at a time when maternal antibody should have decreased by several half lives. None of the clinically affected mice produced neutralizing antibody, but this deficiency was not limited to symptomatic mice, since only 50% of the asymptomatic mice developed a low, but detectable antibody response (Fig . 2) .

The pathogenesis of the delayed neurological disease observed in mice protected with maternal antibody is very similar to the subacute demyelinating encephalomyelitis seen in either weanling or suckling rats inoculated intracerebrally with MHV-JHM . Dependent on the strain of rat, a fraction of animals will develop a subacute neurological disease characterized by hindlimb paralysis, after a variable latency period . Virus can be cultured from animals only during the initial stages of the infection and from animals which develop clinically evident disease ." Histological examination of brains from clinically ill rats reveals chronic demyelination and remyelination, and a small proportion of asymptomatic animals show evidence of white matter disease as well .' Intracellular viral RNA isolated both from rats with acute encephalomyelitis and from those with hindlimb paralysis has been reported to have different electrophoretic mobilities than does viral RNA isolated from infected tissue culture cells, 19 '20 suggesting that a variant of the virus has arisen during the rat infection . Whether mice with the chronic neurological disease show the same or similar changes in viral RNA remains to be determined .

Similarly, MHV-A59, a related strain of mouse hepatitis virus, also causes a subacute demyelinating disease in mice . 21 .22 Demyelination was apparent for several months after infection and viral antigen and RNA could be detected 3 and 10 months, respectively, post-inoculation . Unlike the antibody-modulated MHV-JHM infection, however, virus could only be isolated from mice during the first 25 days postinoculation and neutralizing antibody was present in all surviving mice .

MHV-JHM was first isolated from a mouse that developed hindlimb paralysis .' After several passages of the virus in mice, the period between inoculation of virus and onset of clinical disease became progressively shorter . In addition, an increasingly larger percentage of the mice developed acute encephalomyelitis with a decrease in the number of mice that developed hindlimb paralysis . Since then it has been difficult to obtain reproducibly a delayed, symptomatic, demyelinating encephalomyelitis in mice . The presence of maternal antibody facilitates this process and this model will allow determination of the factors important for viral latency and virus-induced demyelination in the mouse central nervous system .

Cells and viruses . The JHM strain of mouse hepatitis virus (MHV-JHM), kindly supplied by Dr S . Weiss, was used in all studies . The virus was plaque-purified twice and grown in the 17CL-1 line of BALB/c 3T3 cells (also obtained from Dr S . Weiss) in Dulbecco's MEM supplemented with 10% fetal calf serum . Virus titer was 2-6x 106 PFU/ml as determined by plaque assay on 17CL-1 or L-2 cells . L-2 cells, generously supplied by Dr J . Leibowitz, were also grown in Dulbecco's MEM with 10% fetal calf serum .

Animals . Pathogen-free C57BL/6 mice (Jackson Laboratories) were used in all studies . All mice were screened for prior MHV-JHM infection using an indirect immunofluorescence assay and all were antibody-negative prior to experimental manipulation . Female mice were immunized weekly for 5 weeks with MHV-JHM in Freund's adjuvant prior to mating . At 10 days of age, the offspring of immunized mice were inoculated intranasally with 2-6x10°PFU in 10 µl . To titer virus from infected animals, each organ of interest was placed in Dulbecco's MEM and sonicated to near homogeneity . Large particles were removed by centrifugation for 10 min at 1000 g. Virus was titered on 17CL-1 or L-2 cells, with nearly equivalent results . The virus neutralization titer of mice sera was determined by a constant virus-variable serum plaque reduction assay . 23 Immunoperoxidase studies . Animals were perfused with phosphate-buffered saline (PBS) via intracardiac puncture . Isolated organs were embedded in Tissue-Tek II O .C .T . medium (Miles Laboratory) and frozen in liquid nitrogen . Sections were prepared and fixed with methanol containing 0 .3% hydrogen peroxide. The sections were treated sequentially with normal horse serum, with rabbit polyclonal antivirus antibody at a dilution of 1 :50 or 1 :100, then with biotinylated goat antirabbit antibody at a dilution of 1 :100 or 1 :200 and finally with Vectastain ABC (Vector Laboratories) . The slides were developed by exposure to diaminobenzidine (0 .8 mg/ml) in hydrogen peroxide (0 .01%) . The slides were air-dried, mounted and examined under the light microscope . Slides were briefly counterstained with hematoxylin prior to examination . No staining was observed if normal rabbit serum was substituted for the antivirus antibody or if brains from uninfected mice were used instead of infected brains .

Immunofluorescence assay . 17CL-1 cells were infected on coverslips with MHV-JHM at a m .o .i . of 1 . After 12 hours, the cells were fixed with methanol and reacted with antisera from unimmunized or immunized mice at various dilutions . After washing, the coverslips were treated with fluoroscein conjugated goat anti-mouse IgG diluted 1 :200 and examined using a fluorescent microscope .

Light and electron microscopy . For light or electron microscopy, animals were perfused with 10% formalin or 3% glutaraldehyde via intracardiac injection . For light microscopy, the isolated organs were embedded in paraffin, cut into 4-10 pm sections and stained with either hematoxylin and eosin or Luxol Fast Blue . For transmission electron microscopy, the organs were fixed with 3% glutaraldehyde, post fixed with osmium tetroxide, dehydrated and embedded in Epon . Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and examined using a Hitachi H-600 electron microscope .

We thank Drs James Bale, Jr, C . Martin Stoltzfus, Richard Andersen and William Bell for S . Perl man et alt helpful discussion . S .P . was supported by the Medical Foundation in the initial phases of this work . The investigation was supported in part by N .I .H . Biomedical Research Grant RR 05372 .

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