JHM Virus Research Articles

Neurovirulence of murine coronavirus JHM temperature-sensitive mutants in rats.

The murine coronavirus strain JHM is highly neurotropic in rats and has a marked tendency to cause demyelinating central nervous system diseases after intracerebral inoculation. The clinical diseases observed range from an acute encephalomyelitis occurring within 2 weeks postinfection to a subacute demyelinating encephalomyelitis developing several weeks or months postinfection. Uncloned wild-type virus induced both acute and subacute diseases, whereas cloned JHM virus grown in tissue culture caused only acute disease without the pronounced lesions of primary demyelination. In contrast, temperature-sensitive mutants selected from that clone were capable of inducing subacute demyelinating encephalomyelitis after prolonged incubation times. Viruses recovered from diseased animals were still temperature sensitive. Inoculation of temperature-sensitive mutants into suckling rats (age, 10 to 15 days) produced high rates of subacute demyelinating diseases running a more chronic course; these diseases often were not fatal. Those rats which did not show clinical signs frequently revealed inflammatory demyelinating lesions. These findings indicate that the rate and type of clinical disease are dependent on the neurovirulence of the virus mutant used for inoculation and the age of the animals at the time of infection.

Coronavirus JHM: nucleotide sequence of the mRNA that encodes nucleocapsid protein.

A DNA copy of the mRNA that encodes the nucleocapsid protein of Mouse Hepatitis Virus JHM has been cloned into pAT153. The DNA copy specifically inhibited the synthesis in vitro of the nucleocapsid protein. The cDNA was subcloned into M13 vectors and the entire sequence, 1767 bases including a 15 base terminal poly (A) tract, has been determined by chain-terminator sequencing. The sequence contained an open-reading frame that could encode a basic protein of mol.wt. 49700. From the predicted sequence it was apparent that the nucleocapsid protein has 5 basic regions, two of which are located near the middle of the sequence, a serine-rich region was also located, a feature which may be of functional importance as the nucleocapsid protein is phosphorylated at serine residues. The carboxy terminus of the nucleocapsid protein was found to be acidic. The 5' non-coding sequence contained a triple repeat of the pentamer AATCT, a structural feature which may play a significant role during the production of subgenomic viral mRNAs.

In Vivo and In Vitro Models of Demyelinating Disease: Endogenous Factors Influencing Demyelinating Disease Caused by Mouse Hepatitis Virus in Rats and Mice

Intracerebral inoculation of JHM virus (JHMV), the neuropathic strain of mouse hepatitis virus, into Wistar Furth, Wistar Lewis, and Fischer 344 rats at various ages indicated that Wistar Furth rats are more susceptible to the virus than are the other strains. Fischer 344 and Wistar Lewis rats were more resistant to inoculation at 2 and 5 days of age and completely resistant by 10 days of age. In contrast, Wistar Furth rats which were very susceptible at both 2 and 5 days of age remained susceptible until 21 days of age. Intracerebral challenge of an F1 cross between Wistar Furth and Wistar Lewis rats at 10 days of age indicated that resistance to JHMV infection is dominant. Cyclophosphamide treatment 28 days after intracerebral inoculation exacerbated an inapparent infection, leading to paralysis in eight of nine and death in six of nine Wistar Furth test rats. In such immunosuppressed animals, grey- and white-matter lesions were noted throughout the central nervous system, in contrast to the purely demyelinating lesions noted previously. Since rats, unlike mice, were not susceptible to disease after intracerebral injection with the serorelated viscerotropic strain MHV-3, we wished to extend our understanding of the neurological disease process elicited by the two viruses in rodents. For this reason, various mouse strains, including some with recognized immunodeficiencies, were challenged by different routes of inoculation. Intraperitoneal infection of nude and beige mice with JHMV indicated that lack of natural killer cell functions does not markedly enhance the susceptibility to virus, whereas T-cell activity appears to be essential for resisting infection. JHMV and MHV-3 replication in peritoneal macrophages from highly resistant A/J mice was reduced in comparison with that noted in macrophages from susceptible C57BL6/J mice. An initial intraperitoneal inoculation of JHMV was able to protect C57BL6/J mice against fatal intracerebral challenge within 3 days, whereas A/J mice remained susceptible beyond day 3. The protective effect did not appear to result from increased levels of circulating interferon, preceded elevation in serum JHMV-neutralizing antibody titers, and persisted for at least several weeks after intraperitoneal inoculation. Based on the combined studies described here and on previous work by us and others, it appears that the factors influencing the outcome of coronavirus disease in rodents are age at inoculation, route of challenge, genetic constitution of the virus and host, and competence of the immune system, particularly cellular immunity involving T-cells.

Analysis of age-dependent resistance to murine coronavirus JHM infection in mice

Resistance to intraperitoneal murine coronavirus JHM infection in mice develops with age. C3H mice were found to be fully susceptible up to the age of 20 days and resistant after 23 days of age. Protection of susceptible animals from death due to infection could be achieved by maternal antibodies or by transfer of spleen cells from immunized, but not from nonimmunized, donor mice. Lack of protection by transfer of unprimed adult spleen cells was not related to immunosuppression by the host. Moreover, resistance of adult mice could not be abrogated by application of lymphocytes from suckling mice, although immune suppression by other means did affect the resistance of adult animals. On the other hand, spleen cells from nonimmunized mice could be primed with inactivated JHM virus in suckling mice and protected these mice from death due to a subsequent virus infection. Thus, the outcome of infection with JHM virus in suckling and adult mice can be influenced by immunological events, but is not exclusively due to the different stages of immune competence.

Host cell nuclear function and murine hepatitis virus replication.

Murine hepatitis virus strains A59 and JHM replicated with equal efficiency in both nucleated and enucleated L2 cells. In addition, treatment of the host cell with either actinomycin D or alpha-amanitin, both inhibitors of host cell RNA synthesis, had no effect on virus replication. Therefore, the replication of murine hepatitis virus did not appear to depend upon either the presence of the host cell nucleus or continued host cell RNA synthesis.

Influence of the immune system on the course of infection with murine coronavirus JHM in suckling mice.

The course of infection with murine corona virus JHM in C3H mice depends on the age of the animals. Mice up to 20 days of age are fully susceptible while mice older than 23 days resist the infection. Protection of suckling mice from death of infection can be provided by intraperitoneal administration of immune spleen cells but not by non-immune spleen cells from adult mice. The immune spleen cells can be generated by priming adult mice, or by priming non-immune spleen cells from adult mice in baby mice with inactivated JHM virus. Thus the immune system might well be involved in the different outcome of infection with JHM-virus in suckling and adult mice, but it does not seem to be the exclusive factor responsible for the achievement of natural resistance.

Chronic central nervous system demyelination in mice after JHM virus infection.

The pathogenesis of murine hepatitis virus, strain JHM, was studied in 6- and 12-week-old C57/BL mice. There was 100% mortality in the 6-week-old mice after intracerebral inoculation. The lesions were characterized by necrotizing encephalomyelitis, without demyelination. Intracerebral inoculation of 12-week-old animals, however, resulted in no morbidity or mortality. The 12-week-old animals showed transient virus replication in the brain, spinal cord, and liver, which was cleared by day 14. Histologic examination showed evidence of ongoing demyelination, concomitant remyelination, and hydrocephalus ex vacuo. Although viral antigen was demonstrated by immunofluorescence in the central nervous system of these animals, no infectious virus was recovered, and immunosuppression regimens did not potentiate the disease.

Characterization of the cold-sensitive murine hepatitis virus mutants rescued from latently infected cells by cell fusion

Mouse hepatitis virus, a coronavirus, was rescued from latently infected mouse neuroblastoma cells by polyethylene glycol-induced fusion to permissive cells. The isolated viruses grew to equal or higher titers than parental JHM virus at 39° but were restricted in replication at 32° and hence were cold-sensitive mutants. Neither isolate synthesized RNA at the nonpermissive temperature (32°), and temperature shift experiments indicated that the restrictive cold-sensitive step in replication occurred during an early viral function. The isolates were unable to complement each other at 32°. Although coinfection between each mutant and parental JHM virus often resulted in decreased yields of both infecting viruses, interference could not be convincingly demonstrated. Both mutants were more thermostable than parental JHM virus and could be distinguished from each other and from parental JHM virus by their thermal inactivation kinetics. The isolates were obtained from single cell clones and therefore presumed to be homogeneous; however, thermal inactivation of one (S-3JHM) indicated the possible existence of a second population. Subclones of virus prepared from each isolate were also found to be thermally stable and to retain the cold sensitive defect. No indication of a second population of S-3JHM virus was detected.

Demyelinating encephalomyelitis induced by a long-term corona virus infection in rats

SummaryAbout 30% of weanling rats inoculated with JHM virus developed a subacute demyelinating encephalomyelitis (SDE) 3 weeks after inoculation (a.i.). From the remaining animals, 5% displayed overt neurological signs 3,6, and 8 months a.i. Animals with and without clinical signs 6–8 months a.i. were morphologically examined.Fresh demyelinating lesions could be demonstrated in paralyzed animals. Viral antigen was demonstrated and infectious JHM virus could be recovered from one animal which developed clinical signs at 3 months a.i. In one animal with clinical onset of 8 months a.i., completely remyelinated areas as well as recent demyelinating lesions were observed, suggesting a recurrence of the disease process. Remyelinated areas were also found in 40% of clinically silent animals. The morphology of the late onset of the demyelination was similar to that occurring in SDE. Remyelination consisted of both CNS and PNS-type. This animal model offers the possibility to investigate the virus-host relationship which is responsible for the induction of a demyelinating process after a long incubation period.

In vivo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV 3 strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV 3 is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells. This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.

Pathogenesis of demyelination induced by a mouse hepatitis.

Demyelinative lesions were produced in mice by inoculation of JHM virus, a neurotropic strain of mouse hepatitis virus. The production of these lesions was dependent on dosage of virus, age of mice, and route of inoculation. In animals with demyelination, fluorescent antibody staining showed viral antigen was most prominent in the cells of the white matter. No immunopathologic mechanism for demyelination was apparent. No fixation of immunoglobulins in the lesions was seen, nor did immunosuppression with cyclophosphamide prevent demyelination. Focal hepatitis, usually a concomitant finding with white-matter disease, could be prevented without altering the demyelinative lesions. Demyelination in JHM virus-infected mice appeared to be the direct result of infection of glial cells of the white matter.

Mechanism of demyelination in JHM virus encephalomyelitis. Electron microscopic studies.

SummaryWeanling mice were given intraperitoneal inoculations of the neurotropic, JHM strain of mouse hepatitis virus, the virulence of which had been altered by repeated mouse passages. Five to seven days later many animals developed hind leg paralysis. The pathology consisted of an acute encephalomyelitis with patchy demyelinating lesions in the brain stem and spinal cord. Virus particles, consistent with the appearance of corona viruses, were found in the cytoplasm of cells that were identified as oligondendrocytes by demonstrating connections of their plasma membranes with myelin lamellae. Following the degeneration of oligodendrocytes the myelin sheaths disintegrated or were stripped off intact axons by cytoplasmic tongues of polymorpho- and mononuclear leucocytes that intruded between myelin lamellae. The findings indicate that JHM virus has an affinity for oligodendrocytes in weanling mice and that demyelination occurs subsequently to the degeneration of the infected oligodendrocytes.