JHM Virus Research Articles

Immune mediated clearance of JHM virus from the central nervous system.

The study of viral pathogenesis has revealed how animals respond to viral infection and the subsequent damage which results. It has become evident that the immune response plays the central role in determining the outcome of viral infection. Many studies have demonstrated the capacity of the immune system to combat viruses with a variety of weapons including interferons, T lymphocytes, natural killer cells, macrophages, antibody, and polymorphonuclear granulocytes (1). The T lymphocytes that respond to viral infection are traditionally recognized as helper, cytotoxic, or supressor cells. In the mouse, each of these groups posess cell surface molecules which confer distinct antigenic phenotypes to these various T cell subsets. Although recent evidence shows there are exceptions to this phenotypic classification of functional activity (2, 3), this approach remains useful in examining the population(s) responding to viral infection and their functions in clearance of the infectious agent from the host.KeywordsViral TiterThymidine KinasePeritoneal Exudate CellMouse Hepatitis VirusDelay Type Hypersensitivity ResponsivenessThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis and pathogenetic significance of class II MHC (Ia) antigen induction on astrocytes during JHM coronavirus infection in rats.

This laboratory has developed over the past several years a rat model for virus-induced1, 2, 3 central nervous system (CNS) disease involving inflammatory demyelinating lesions. The JHM strain of murine hepatitis coronavirus causes an array of neurological diseases differing in complexity depending on factors associated with the age and genetic background of the host4 and the particular JHM virus isolate utilized5. Factors related to the age and genetics of the host associated with susceptibility to disease appear to involve immunological function4, whereas factors related to the virus appear to depend on functional aspects of the E2 envelope glycoprotein4, 6, 7 (Wege et al., this volume).

Neuropathogenicity of mutant strains of mouse hepatitis virus, 1A and 2C, from DBT cells persistently infected with JHM strain.

Two plaque mutants 1a and 2c of the mouse hepatitis virus JHM strain (MHV-JHM) from persistently infected DBT cells2 were examined for neuropathogenicity in mice. The mutant strains had peplomer proteins different from those of the original MHV-JHM3 and they were plaque purified.

In vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication.

A lethal infection by neurotropic JHM strain of mouse hepatitis virus was prevented by the local adoptive transfer of three virus-specific Lyt-1+2-, L3T4+ T cell clones. The transfer of Lyt-1+ T cells specific for an unrelated antigen (hen egg lysozyme) did not protect. Protection required I region compatibility between the T cells and the recipients, and was reversed either by irradiation of the cells before transfer or by pretreatment of the recipients with cyclophosphamide. Adoptive transfer prevented death due to JHM virus infection but did not result in altered antiviral immunoglobulin synthesis or the suppression of viral replication in the central nervous system (CNS). The data presented implicate a local DTH response in the protection of the host from lethal infection of the CNS by a neurotropic virus, but clearly imply that other antiviral effector mechanisms are necessary for the suppression of viral replication.

In vivo and in vitro models of demyelinating disease: JHM virus in the rat central nervous system localized by in situ cDNA hybridization and immunofluorescent microscopy.

In situ probing of central nervous system (CNS) tissues has made it possible to associate the presence of JHM virus (JHMV) RNA with individual cells in the rat CNS. The presence of viral RNA was not always associated with antigen expression. The in situ hybridization revealed that cerebellar Purkinje cells and hippocampal neurons were highly susceptible to JHMV infection during either acute or paralytic disease. In the paralytic disease, Purkinje cell neurons frequently contained viral RNA. This observation suggests that these neurons, and perhaps others, may be repositories for JHMV in rats that undergo prolonged infections.

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.

Sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus.

cDNAs prepared from viral genomic RNA purified from two strains of infectious bronchitis virus (IBV) (Beaudette and M41) have been cloned into pBR322. Three of these clones, which contain the complete sequences of mRNA A for both strains, except for the leader sequences which are only present on the subgenomic messenger RNAs, have been sequenced using the dideoxy method. The sequences are similar for both strains, each containing a single long open reading frame of 1227 bases which predicts a polypeptide of molecular weight approximately 45 000. The genome position and size of this predicted polypeptide are consistent with it being the gene for the nucleocapsid protein. The amino acid sequence shows considerable homology with those of the nucleocapsids of murine hepatitis virus strains A59 and JHM. The major difference between the sequences determined for the two IBV strains is that the 3' non-coding region of the Beaudette strain contains a 184 base segment which is not present in the M41 strain.

Biological and macromolecular properties of murine cells persistently infected with MHV-JHM.

SummaryA persistently-infected neuroblastoma culture [Neuro-2A (JHMV)] was established with the murine hepatitis virus JHM [MHV-JHM]. After 100 days of passage, the endogenous virus [Neuro-2A (JHMV) end] released by this culture was unable to induce the syncytia typical of MHV-JHM and the endogenous virus was not temperature-sensitive. The Neuro-2A (JHMV) culture was cured of virus production by passage under neutralizing antibody [Neuro-2A (JHMV) Ab]. The Neuro-2A (JHMV) and the Neuro-2A (JHMV) Ab cultures were as susceptible to heterologous infection with mengovirus and vesicular stomatitis virus as the uninfected Neuro-2A culture. However, the Neuro-2A (JHMV) and Neuro-2A (JHMV) Ab cultures were partially resistant to homologous superinfection by MHV-JHM and the closely related MHV-A59. Virus related to MHV-JHM was rescued from the antibody-cured cells by cell fusion. The synthesis of MHV-JHM specific antigens by Neuro-2A (JHMV) cells, Neuro-2A (JHMV) Ab cells and 17 Cl-1 cells infected by Neuro-2A (JHMV) end was studied by SDS-PAGE. The genomic RNAs of MHV-JHM and Neuro-2A (JHMV) end were compared by oligonucleotide mapping. The results of the protein and RNA studies indicated that the genome of Neuro-2A (JHMV) end was substantially modified from the genome of MHV-JHM, but the modifications did not significantly alter the molecular size of the viral-specific proteins.

Hybridoma antibodies to the murine coronavirus JHM: characterization of epitopes on the peplomer protein (E2).

A panel of hybridoma antibodies that react with the surface peplomer glycoprotein (E2) of the murine coronavirus JHM were produced to characterize major antigenic domains associated with functions related to virulence. Three groups of hybridoma antibodies were differentiated by immunoprecipitation of lysates from JHM-infected cells. One group precipitated the virion structural proteins gp170 and gp98 together with the intracellular form of E2, gp150. A second group reacted with gp98 and gp150, and a third group precipitated gp150 only. Competition assays with biotinylated hybridoma antibodies allowed the definition of at least six different epitope groups. Only those antibodies which immunoprecipitated both gp170 and gp98 neutralized infectivity, inhibited cell fusion and protected infected rats against acute disease. Another class of antibodies binding to gp170 and gp98 also neutralized JHM virus, but did not inhibit fusion and did not protect against disease. Antibodies that immunoprecipitated gp150 and gp98 revealed only weak neutralization and did not inhibit cell fusion or protect animals. Four epitopes were defined by antibodies that immunoprecipitated gp150, but revealed no biological activity. These data indicate that the site responsible for cell fusion is associated with an epitope group carried by gp170 and gp98. Neutralizing antibodies bind to this and another epitope. Furthermore, protection of JHM-infected rats against acute disease requires both inhibition of cell fusion and neutralization of virus.

In vivo and in vitro models of demyelinating disease IX. Progression of JHM virus infection in the central nervous system of the rat during overt and asymptomatic phases

JHM virus, when inoculated into neonatal rats, can cause either a rapidly fatal acute encephalomyelitis or, after longer incubation periods, a paralytic disease. The cerebrospinal fluid (CSF) and serum anti-JHM virus IgG concentrations present in rats prior to onset of clinical symptoms or during the acute and paralytic phases of disease were compared. High CSF/serum ratios, indicative of local antibody production in the CNS, were noted only where disease was demonstrable suggesting that local antibody production accompanied the infection but did not prevent the neurological disease. Among animals in which neurologic symptoms had not become manifest, only those with elevated CSF/serum ratios were found to have histological CNS lesions. Immunofluorescent microscopy indicated that viral antigens were present in both glia and neurons. Antigen-positive cells were frequently present in histologically normal CNS tissue, while regions of necrosis were antigen negative. Testing for the presence of viral RNA with JHM cDNA probes revealed that the virus was rapidly disseminated throughout the CNS, presumably establishing centers of infection prior to the development of recognizable tissue damage. Viral RNA was also detected in the CNS following recovery from paralysis and as late as 5 months postinfection, where no disease occurred. These findings indicate that, although infection by JHM virus can spread rapidly throughout the CNS, formation of lesions during chronic disease is a slower process. The current data and previous observations suggest that JHM virus can remain in a latent state for periods of at least several months in rats without apparent neurologic disease despite the absence of any known provirus phase in the replicative strategy of coronaviruses.

Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis

When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1–2.4 × 10 6-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains' and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

Relapsing subacute demyelinating encephalomyelitis in rats during the course of coronavirus JHM infection

Temperature-sensitive mutants of the murine coronavirus JHM induced a subacute demyelinating encephalomyelitis (SDE) in young rats. Neurological symptoms were associated with marked lesions of primary demyelination in the white matter of the central nervous system (CNS), and developing after an incubation time of several weeks to months. Many rats survived this infection and recovered completely from this CNS disease. Among 43 survivors of SDE, 9 rats developed a relapse 27–153 days after onset of the first attack. Neuropathological examination of these animals revealed areas of fresh demyelination together with old remyelinated lesions. Viral antigens were detectable in the neighbourhood of fresh lesions and in some cases infectious virus was re-isolated from rats revealing low antibody titers to JHM virus. These results demonstrate that mutants of JHM virus can induce a relapsing demyelinating disease process, associated with a persistent infection, which possesses some similarities to chronic experimental allergic encephalomyelitis.

Adaptation of coronavirus JHM to persistent infection of murine sac(-) cells.

Coronaviruses can establish persistent infections in the central nervous system of rodents, and these are associated with demyelinating encephalomyelitis. The effects of persistence on the virus are difficult to study in vivo but may have a crucial influence on the course of infection. We therefore produced a persistent infection in vitro using the neurotropic coronavirus JHM, in order to investigate the events underlying the establishment of such an infection and the adaptation of the virus to persistence. The persistent infection was maintained for over 115 passages and continued to release high levels of infectious virus. During the 18 months of culture the number of cells expressing virus antigen detected by indirect immune fluorescence decreased to 40%. Analysis showed that the carried virus contained a significant proportion of heterogeneous temperature-sensitive mutants. All virus clones isolated possessed the capacity to induce a more productive growth cycle, a less pronounced cytopathic effect and showed a much reduced neurovirulence when inoculated into newborn and weanling rats. Evidence for structural changes involving the surface peplomer protein (E2) was obtained using hybridoma antibodies, which neutralized the parental JHM virus but not the JHM-Pi virus. Defective interfering particles and interferon activities have been excluded as possible agents instrumental in the establishment and maintenance of the chronic infection, and we suggest that the emergence of virus variants of lowered virulence is central to these processes.

Defective interfering particles of mouse hepatitis virus

After six to eight serial undiluted passages of mouse hepatitis virus (JHM strain) in DBT cell culture, a decrease in the yield of infectious virus occurred, and with further passages fluctuating yields of infectious virus were observed. The serially passaged virus interfered with the multiplication of the standard JHM virus, but not with vesicular stomatitis virus. After sucrose equilibrium centrifugation of high passage virus, a single peak contained both infectious virus and interfering activity. This virus population resembled the original JHM virus in its structural proteins, but it contained an increased proportion of a protein with a molecular weight of 65 × 10 3. Genomic RNA from standard JHM virus contained a single species of RNA with a molecular weight of 5.4 × 10 6. After five undiluted passages, however, the virion population contained two RNA species with molecular weights of 5.4 × 10 6 and 5.2 × 10 6. RNase T 1 resistant oligonucleotide finger-printing of these RNAs showed that the lower molecular weight RNA had lost several oligonucleotide spots that were present in the genomic RNA of the standard JHM virus. After several serial diluted passages of passage 10 virus, a single virus population was obtained which again had only standard virus RNA with a molecular weight of 5.4 × 10 6 and lacked interfering activity. These results indicated that defective interfering particles were generated by serial undiluted passages of JHM virus.

Fine specificity and genetic restriction of T cell clones specific for mouse hepatitis virus, strain JHM.

Mouse hepatitis viruses are members of the coronavirus group of animal viruses. Although named for their propensity to induce acute hepatitis in animals stressed by a variety of conditions, it has become clear that as a group they possess the ability to cause a diverse group of diseases in their natural host (Wege, et al, 1982). One strain of MHV, named JHM virus (JHMV), was isolated from mice found to have demyelinated lesions of the central nervous system (Bailey, et al, 1949). More recently it has been found that JHMV is not only capable of causing acute encephalomyelitis with demyelination but also chronic demyelination probably due to the establishment of a latent infection of oligodendroglia, the cells of myelin within the central nervous system (Herndon, et al, 1975; Stohlman and Weiner, 1981).

Induction of Natural Killer Cells and Interferon during Mouse Hepatitis Virus Infection of Resistant and Susceptible Inbred Mouse Strains

Following infection by mouse hepatitis virus (JHM strain), an induction of natural killer (NK) cell activity was observed in C3H mice, which are considered to be sensitive to JHM virus infection. In contrast, mice of the resistant SJL strain did not show any increase of NK cell activity after JHM virus infection. However, infection of both SJL and C3H mice with mouse hepatitis virus type 3 (MHV3) resulted in an increase of NK level, comparable to that observed with the JHM virus infection in the C3H strain. No significant differences were observed in the NK cell activity of the peritoneal exudate or spleen cells of infected mice. Low levels of interferon were detected in serum or peritoneal exudate of C3H mice infected with JHM virus 18 or 24 hours before, but no detectable early interferon production was found. Also no interferon could be detected in the resistant SJL mice. After JHM virus infection, the number of peritoneal exudate cells (PEC) was increased significantly in C3H mice but not in SJL mice. Macrophages obtained from the C3H mice supported virus replication, whereas SJL macrophages did not. Our data suggest that NK cells do not play a role in the resistance of SJL mice against JHM virus infection but may participate in the defence mechanisms against this virus in C3H mice.

In vivo and in vitro models of demyelinating diseases--VIII: Genetic, immunologic and cellular influences on JHM virus infection of rats.

A number of mouse hepatitis viruses, including MHV-A59, MHV-3, and JHM, have been used as in vivo models of neurotropic viral infections (Hirano et al., 1980; Knobler et al., 1981; Lampert et al., 1973; Le Prevost et al., 1975). The majority of experiments with these agents have involved mice as the host, a system which may be complicated by the viscerotropism of some of these coronaviruses. Previous work in our laboratory (Sorensen et al., 1980, 1982) and by others (Nagashima et al., 1978, 1979; Hirano et al., 1980) demonstrated that rats injected intracerebrally (IC) with JHM virus (JHMV) offer a challenging model for studies of neurotropic agents interacting with the central nervous system (CNS). The presence of anti-coronavirus antibodies in a high proportion of the human population (MacNaughton, 1982) give these models added relevance in regard to enquiries into the viral etiology of human diseases of the central nervous system (CNS), among them multiple sclerosis.

Antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus.

Monoclonal antibodies were produced to JHMV-DL, a neurotropic member of the mouse hepatitis virus (MHV) or murine coronavirus group. Of 23 antibodies isolated, 10 were specific for the major envelope glycoprotein, gp180/90, 10 for the nucleocapsid protein, pp60, and 3 for the minor envelope glycoprotein, gp25. Eleven different MHV isolates were used in antibody binding assays to study antigenic relationships among the viruses. Each MHV isolate tested had a unique pattern of antibody binding, indicating that each is a distinct strain. Conservation of JHMV-DL antigenic determinants varied among the three proteins, with pp60 showing intermediate conservation, gp180/90 little conservation, and gp25 marked conservation in the different MHV strains. Monoclonal antibodies to pp60 proved most useful in delineating antigenic relationships among MHV strains. These antigenic groups correlated with pathogenic types, indicating that pp60 may be one of the gene products which mediates the distinct disease patterns manifested by different murine coronaviruses.

Natural killer cell activity during mouse hepatitis virus infection: response in the absence of interferon.

The ability of the JHM3 strain of mouse hepatitis virus (MHV) to induce natural killer (NK) cells was examined. Infection of C57BL/6 (B6) mice with this virus resulted in the augmentation of natural cytotoxicity against YAC-I target cells in the absence of a detectable interferon response. The cells responsible for this increased cytotoxicity were sensitive to complement-mediated lysis with an anti-Q-5 reagent but not with a Thy 1.2 antiserum, indicating that they possess an NK-like surface phenotype. Although variation in the NK response of individual B6 mice following JHM virus infection was found, even the animal with the most responsive NK cell population had no detectable interferon in the spleen. This finding contrasted with observations with an unrelated virus (lymphocytic choriomeningitis virus) and a serologically related strain of MHV. Infection with both of these viruses induced augmented NK cell activity and interferon responses. In addition, we found that neither the ability to mount an augmented NK cell response nor preferential lysis of virus-infected targets correlated with resistance or susceptibility to JHM virus infection.