B-cell-mediated lysis of cells infected with the neurotropic JHM strain of mouse hepatitis virus.

Cells expressing the spike (S) glycoprotein of the neurotropic JHM strain (JHMV) of mouse hepatitis virus (MHV) are susceptible to lysis by B cells derived from na?ve mice, including B cells from perforin-deficient mice. Cytolysis requires interaction of the virus receptor and the viral S glycoprotein, is independent of other viral-induced components, and is not a unique property of B cells. Neutralizing anti-S-protein monoclonal antibodies (mAb) and a mAb specific for the viral receptor inhibit lysis. However, cells infected with an MHV strain unable to induce cell-cell fusion are resistant to lysis and lysis of JHMV-infected cells is inhibited by an anti-S-protein nonneutralizing mAb which prevents S-protein-mediated cell fusion. These data suggest that B cells may function as antibody-independent innate immune response during JHMV infection in vivo.     

Mouse hepatitis virus strain — Related patterns of tissue tropism in suckling mice

Summary The pattern of tissue tropism for several prototype and uncharacterized strains of mouse hepatitis virus (MHV) was studied by intranasal inoculation of each virus strain into groups of neonatal Swiss mice under otherwise identical conditions. Mice were killed at intervals up to 18 days after inoculation, and their tissues were examined for the presence of MHV antigen by indirect immunofluorescence. Two patterns of infection were apparent. Prototype MHV strains 1, 3, A59, JHM, S and uncharacterized MHV strains Tettnang and wt-1 produced a respiratory pattern, in which nose and lung were consistently involved with dissemination to other organs in a vascular distribution. Pulmonary vascular endothelium and alveolar septal cells, but not airway epithelium, were infected. An enteric pattern was observed with MHV-Y and wt-2 in which MHV antigen was largely restricted to the nose and bowel, with limited dissemination to other abdominal organs but not lung. Intestinal lesions in these mice were severe compared to those manifesting the respiratory pattern of infection. These results indicate that, like coronaviruses of other species, different strains of MHV possess different primary and secondary organotropisms following a natural route of inoculation in a susceptible host.With 3 Figures     

Stability of neurotropic mouse hepatitis virus (JHM strain) during chronic infection of neuroblastoma cells

Summary A line of mouse neuroblastoma cells which was chronically infected with the neurotropic strain (JHM) of MHV, a member of the coronavirus group, was established. These cells, designated N_J, exhibited typical MHV cytopathic effects (CPE) at all passage levels along with the continual production of infectious virus. Most cells were positive for viral antigen by immunofluorescence. Viral particles consistent with the morphology of MHV were found by electron microscopy. The uninfected neuroblastoma cell line did not contain a detectable population of cells resistant to JHM, and persistence did not elicit the production of interferon. No plaque morphology or temperature sensitive mutants were selected for in the N_J culture, and we were unable to detect the presence of either a defective or defective interfering virus population. The addition of low concentration antiviral antibody modulated the infection to a carrier culture with viral antigen in the cytoplasm of the cells, but no infectious virus was produced, and the cells lacked both surface viral antigen and CPE. Possible mechanisms of viral persistencein vitro are discussed.With 3 Figures     

Virus specificity of the antiviral state induced by IFN gamma correlates with resistance to MHV 3 infection

Summary A comparative study was carried out to investigate the correlation between the antiviral effect induced in macrophages by IFN gamma and the resistance of A/J and BALB/c mice to an experimental infection of MHV 3, MHV 4, and MHVA 59. Both mouse strains were resistant to intraperitoneal infection with MHV 4 or MHVA 59 and only the A/J mice showed resistance to MHV 3, the BALB/c mice being fully susceptible to this virus infection. Comparable growth kinetics, for all three viruses, were observed in both mouse strains, except for the MHV 3 growth in BALB/c mice, where the virus titre increased to a peak on day 2, remaining high until day 4 when the mice died of acute hepatitis. The IFN gamma titres in the peritoneum of mice preceded and correlated with the virus growth, higher titres being found in MHV 3 infected BALB/c mice. The highest titre was always observed 24 to 48 h after infection. Among viral strains grown in cultured macrophages, higher titres were always observed in cultures infected with MHVA 59, followed by MHV 3 and the lowest those infected with MHV 4. The macrophage activation by IFN gamma-induced a partial restriction of virus growth only in MHV 3 infected A/J mouse macrophages. A virus specificity of the IFN gamma-induced antiviral state was shown to be in direct correlation with the resistance of mice to MHV 3 infection.     

Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.

The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV-JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV-JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.     

Cell receptor-independent infection by a neurotropic murine coronavirus.

The cellular receptors for a coronavirus, mouse hepatitis virus (MHV), have been recently identified as one or more members of the carcinoembryonic antigen (CEA) family. The neurotropic JHM strain of MHV (MHV-JHM) possesses a highly fusogenic surface (S) glycoprotein. This protein is now shown to promote the spread of MHV into cells lacking the specific CEA-related MHV receptor. Resistant cells are recruited into MHV-induced syncytium with consequent production of progeny virus. Cell-to-cell spread of virus via membrane fusion without the requirement for specific cell surface receptor offers a novel way for virus to spread within infected hosts.     

Correlation between growth potential of mouse hepatitis viruses in macrophages and their virulence for mice.

Correlation between the virulence of mouse hepatitis virus (MHV) for mice and the growth potential of the virus in peritoneal adherent cells was observed for highly virulent MHV-2 and avirulent MHV-1, JHM, and MHV-S strains. However, this phenomenon was not observed in strain MHV-3, which multiplied to almost the same degree in peritoneal adherent cells from susceptible and resistant mouse strains.     

Genetic heterogeneity of murine coronaviruses

Several mouse hepatitis viruses (MHV) with different pathogenicity were studied by oligonucleotide fingerprinting. Two strains, MHV-K and MHV-D, which were isolated in Japan and, which cause anaplasia and necrosis of bone marrow and diarrhea, respectively, were found to be closely related to MHV-A59, the prototype MHV. Two other MHV strains, isolated from nude mice, were found to have diverged extensively from the known MHV strains. The MHVs isolated from separate cloned neuroblastoma cell lines persistently infected with JHM strain were also found to have diverged more markedly than the corresponding virus maintained under the conditions of lytic infection. Genetic divergence during persistent infection may be one of the mechanisms by which the MHV diverges.     

Difference in Bgp-independent fusion activity among mouse hepatitis viruses

Summary.  Mouse hepatitis virus (MHV) utilizes a mouse biliary glycoprotein (Bgp) as a receptor. Co-cultivation of MHV-nonpermissive hamster BHK cells devoid of mouse Bgp with mouse DBT cells infected with MHV-A59 or JHMV induces syncytia formation on BHK cells (Bgp-independent fusion). This study shows the difference in Bgp-independent fusion activity among various MHV strains. Under a phase contrast microscopy, JHMV (cl-2, sp-4) induced the Bgp-independent syncytia on BHK cells similar to those observed on DBT cells, while such syncytia were not seen with the infection of other MHV strains (MHV-1, MHV-3, MHV-A59, MHV-S, srr7, srr11 and srr18). Tiny syncytia detectable only by immunofluorescence were produced with the latter MHV strains except for srr7 which failed to produce syncytia. MHVs except for srr7 grew in BHK cells after Bgp-independent infection. The Bgp-independent fusion by JHMV was inhibited either by anti-S1 or anti-S2 antibodies. These results showed that the JHMV spike protein had a remarkably high Bgp-independent fusion activity. Accepted May 18, 1999 Received February 15, 1999     

RNA and polypeptide homology among murine coronaviruses

Using a ³²P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [³²P]cDNA and infected cell RNA from the other four MHV strains demonstrated 70–80% homology. By gel transfer analysis, the A59 [³²P]cDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virus-specific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981)Virology, 112, 596–604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [³⁵S]methionine-labeled viral proteins by SDS-polyacrylamide gel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEI polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [³⁵Sknethionine-labeled N proteins was found to coelute by high-performance liquid chromotography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV-3, JHM, and A59.     

Characterization of coronavirus JHM variants isolated from Wistar Furth rats with a viral-induced demyelinating disease

Murine hepatitis virus (MHV) can cause neurological disease when inoculated intracerebrally (ic) into mice and rats. Specifically the JHM strain of MHV (MHV-JHM) generally causes an acute encephalitis when inoculated ic into 2-day-old Wistar Furth rats. In contrast, JHM generally produces a chronic demyelinating disease with resulting posterior paralysis when inoculated ic into 10-day-old Wistar Furth rats. In addition, while JHM readily produces a productive infection in a mouse fibroblast cell line (L-2), it does not form syncytia or replicate well in a tissue cell line of glial origin (G26-24). We have isolated and characterized three MHV-JHM viral variants from the central nervous system of two Wistar Furth rats with a MHV-JHM-induced demyelinating disease. The pattern of viral-specific mRNA for all three of these variants differed from what was observed for the wild-type parental MHV-JHM that had been passaged only in tissue culture. One of these variants, ATllf cord virus, which induced a chronic demyelinating disease in 2- or 10-day-old intracerebrally inoculated Wistar Furth rats, had a deletion in the coding region of the peplomer glycoprotein mRNA. In addition, this variant formed massive syncytia and replicated well in G26-24 cells. We have not detected this deletion in the other two JHM variants, ATllf brain virus and ATlle brain virus. ATllf brain virus and ATlle brain virus primarily produced an acute encephalitis when reinoculated into 2- or 10-day-old Wistar Furth rats. In addition, these two variants did not form syncytia and had a reduced ability to replicate in G26-24 cells.     

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.     

Comparison of polypeptides of two strains of murine hepatitis virus.

Viral polypeptides labeled with [³⁵S]methionine in mouse L fibroblasts infected with either of two strains (JHM or MHV3) of mouse hepatitis virus (MHV) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four major polypeptides of apparent molecular weights, 180,000 (p′180′), 56,000 (p′56′), 24,000 (p′24′), and 22,000 (p′22′) were detected after a 15-min pulse of methionine. During a 2-hr chase performed late in infection (5.5 hr PI) a polypeptide of molecular weight 50,000 (p′50) was observed which appears to be derived from p′56′ by proteolytic processing. Both p′56′ and p′50′ were enriched in the amino acid arginine. Of the five major viral polypeptides only p′180′ could be labeled by growing infected cells in the presence of [¹⁴C]glucosamine; moreover, it was the only polypeptide whose mobility of PAGE was altered after labeling infected cells with [³⁵S]methionine in the presence of glycosylation-inhibiting drugs. Despite the extreme similarity in polypeptide patterns specified by JHM and MHV3, the apparent molecular weights of each of the five major JHM polypeptides were consistently lower (by about 500–1000) daltons than the corresponding ones of MHV3.     

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.     

SJL/J resistance to mouse hepatitis virus-JHM-induced neurologic disease can be partially overcome by viral variants of S and host immunosuppression.

The basis of the resistance of SJL/J mice to various strains of mouse hepatitis virus (MHV) has been the subject of some debate, especially as it relates to the number and nature of the determinants involved. Our previous work demonstrated that resistance by primary SJL/J glial cultures may involve events subsequent to viral gene expression, possibly due to a defect in cell-to-cell spread of the infection. Since S, the virion's major spike glycoprotein, is known to facilitate the spread of infection due to its syncytiogenic properties, we decided to investigate the role of this viral structural protein in resistance by primary SJL/J glial cells. Variants possessing deletions within the S coding region were able to grow in SJL/J glial cells 10-100 times easier and fuse five-times more efficiently than wt virus. Induction of neurologic disease in SJL/J mice following intracranial inoculation with either wt JHMV or the S deletion variant, AT11f cord, was age-dependent, occurring only in animals inoculated under 4 weeks of age. Resistance in older animals to wt and variant viruses could be abrogated by immunosuppression with cyclosporin A. However, both disease incidence and viral brain titers were higher in animals receiving the JHM variant AT11f cord virus, suggesting that SJL/J resistance to neurologic disease may manifest itself through interactions between inefficient cell-to-cell spread of the infection and protective aspects of the immune response.     

The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection

The spike (S) and hemagglutinin/esterase (HE) of bovine coronavirus (BCV) are the two envelope proteins that recognize the same receptor-determinant of 9-O-acetylneuraminic acid on host cells. However, the precise and relative roles of the two proteins in BCV infectivity remain elusive. To unequivocally determine their roles in viral cytopathogenicity, we developed a system in which phenotypically chimeric viruses were generated by infecting a closely related mouse hepatitis virus (MHV) in cells that stably express an individual BCV protein (S or HE). The chimeric viruses were then used to infect human rectal tumor (HRT)-18 cells that are permissive to BCV but are nonsusceptible to MHV. Using this approach, we found that the chimeric virus containing the BCV S protein on the virion surface entered and replicated in HRT-18 cells; this was specifically blocked by prior treatment of the virus with a neutralizing antibody specific to the BCV S protein, indicating that the BCV S protein is responsible for initiating chimeric virus infection. In contrast, chimeric viruses that contain biologically active and functional BCV HE protein on the surface failed to enter HRT-18 cells, indicating that the BCV HE protein alone is not sufficient for BCV infection. Taken together, these results demonstrate that the S protein but not the HE protein of BCV is necessary and sufficient for infection of the chimeric viruses in HRT-18 cells, suggesting that BCV likely uses the S protein as a primary vehicle to infect permissive cells.     

Down-regulation of Bgp1(a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus 3 infection

Together with the evidence that the reduced virus growth and the antiviral state induced by interferon (IFN)-gamma, occurring only in macrophages from resistant animals, correlated with the decrease of MHV3 binding to macrophage membrane proteins, we show here the expression of cellular and viral genes in resistant (A/J) and susceptible (BALB/c) mouse macrophages after IFN-gamma activation/infection. The expression of interferon response gene 47 and interferon regulatory factor 1 genes takes place after IFN-gamma activation in both macrophages, indicating their activation. The expression of the biliary glycoprotein 1(a) (Bgp1(a), the main virus receptor) decreased only in IFN-gamma-activated A/J mouse macrophages, in contrast to the expression of the Bgp2 (alternative receptor), which was not influenced by IFN-gamma activation. The synthesis of both viral mRNA and virus particles was delayed only in IFN-gamma-activated A/J mouse macrophages compared with susceptible BALB/c macrophages. Besides the evidence that IFN-gamma may modulate the expression of the Bgp1(a) isoform of carcinoembryonic antigen family, these data show that IFN-gamma, which induces resistance against MHV3 infection, may be involved in the down-regulation of the main viral receptor expression, a key step forward in our understanding of the molecular basis of resistance against virus infection.