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Transverse section through the spinal cord 4 weeks after intracerebral infection with 105 p.f.u. of ts-342. There is extensive demyelination and some axonal degeneration in one dorsal column. Paraphenylenediamine staining was used. Bar marker represents 25 ~tm.
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The neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). T...
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... six animals manifested abnormal positional nystagmus starting at day 2 and persisting at least 6 months (Koolen et al., 1985). Histological abnormalities were determined at 4 weeks p.i. and were present in the CNS of mice infected with ts-43, ts-169 and ts-342. The caudal brain stem and spinal cord showed vacuolar disruption of the white matter (Fig. 2). Perivascular and leptomeningeal infiltration by mononuclear inflammatory cells was quite often observed (data not shown). Such lesions were present in one of four mice infected with l05 p.f.u, of ts-43, in all three mice infected with 105 p.f.u, of ts-169 and in all mice infected with 103 or 105 p.f.u, of ts-342. The animals infected ...
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Strains of the murine coronavirus mouse hepatitis virus type 4 (MHV-4) which contained a mutation in the E2 peplomer glycoprotein were obtained by selection for resistance to neutralization by monoclonal antibodies. Characterization of six variants representing two independent epitopes on E2, E2B and E2C, by in vitro neutralization and antibody-bin...
Citations
... Coronaviruses are positive-strand RNA viruses, characterized by the largest RNA genomes and a nested set of subgenomic mRNA during transcription [1,2,3]. MHV strain A59 is an extensively studied group II coronavirus [4,5] that causes enteritis, hepatitis, and central nervous system demyelination in susceptible mice [6,7]. Translation of the MHV-A59 genome yields 16 nonstructural proteins (nsps), which are encoded by two large overlapping open reading frames (ORF) termed ORF1a and ORF1b. ...
The replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins, nsp3, nsp4 and nsp6, are understood to be membrane anchors of the coronavirus replication complex. Nsp4 is localized to the ER membrane when expressed alone but is recruited into the replication complex in infected cells. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol.
We have determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and a C425S site-directed mutant. The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure features two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit. The monomers are arranged with their N- and C-termini in opposite orientations to form an "open" conformation. Mutation of Cys425 to Ser did not affect the monomer structure, although the mutant dimer adopts strikingly different conformations by crystal packing, with the cross-linked C-termini and parallel N-termini of two monomers forming a "closed" conformation. The WT nsp4C exists as a dimer in solution and can dissociate easily into monomers in a reducing environment.
As nsp4C is exposed in the reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4.
... In the field of virology ts mutants have been exploited extensively to study biological processes. For many plus-strand RNA viruses, including members of the Togaviridae, Picornaviridae, Coronaviridae, and Flaviviridae, a number of ts mutants have been described (references 11,14,19,23,30,38,41,45, and 47 and references therein). To our knowledge, only one other ts pestivirus (cp BVDV strain RIT 4350) has been reported so far (29). ...
For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33 degrees C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5 degrees C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5 degrees C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33 degrees C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.
... B cells and the antibodies they secrete could potentially also mediate demyelination. However, no correlation has been found in MHV-infected mice between antibody titers and severity of demyelination (Koolen et al, 1987). In some animal models of CNS demyelination, antibodies against self-antigens in the brain induce or exacerbate demyelination (Brehm et al, 1999; Genain et al, 1999 ) but there is no evidence for the induction of antibodies that recognize host proteins after MHV infection of the CNS. ...
Most murine hepatitis virus (MHV) strains, as their name suggests, infect the liver. However, several murine strains are tropic for the central nervous system (CNS) and cause encephalitis with subsequent CNS demyelination. The CNS demyelination shares pathological similarities with human CNS demyelinating diseases such as multiple sclerosis (MS). These viruses are, therefore, used to study the role of the immune system in viral clearance from the CNS, in CNS demyelination, and in remyelination. Nevertheless, it is still unclear exactly how MHV induces demyelination and to what extent the immune system plays a role in this pathology. Here we review this field in the context of the immune response to MHV in the liver and the CNS focusing on studies that have been published in the past 5 years.
... Although the Golgi complex has been identified as a target of autoantibodies for almost a decade, little is known about the antigenic targets or the events that lead to the induction of anti-Golgi antibodies. An attractive feature to the study of Golgi antigens is the knowledge that viruses, including HIV (29), coronaviruses (30), rubella (31), and various others (reviewed in reference 32), are processed in, and disrupt (33,34), the Golgi complex. Observations that certain viruses induce anti-Golgi antibodies in mice (35,36), and that individuals infected with EBV (12) and the HIV-I virus (13) bear anti-Golgi antibodies, add incentive to the study of the molecular characteristics of the Golgi autoantigens. ...
Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.
... Likewise, the strain HSV-1 BAN from a patient with multiple sclerosis, which displayed weak neurovirulence in vivo, gave a markedly low replication yield in both cell types. Restricted viral replication as a pathogenetic factor in demyelination was found in a study of mice infected with mutants of the coronavirus mouse hepatitis virus (35), and has earlier been suggested for HSV in humans in a hypothesis on multiple sclerosis (36). However, we have not been able to test this hypothesis properly, due to the lack of additional viral isolates from the CNS of patients with multiple sclerosis. ...
Herpes simplex virus (HSV) isolates derived from the central nervous system of ten patients with HSV-1-induced encephalitis, one patient with multiple sclerosis, and 14 patients with HSV-2-induced meningitis were investigated for neurovirulence by assaying the LD50 after nose and intracerebral (i.c.) inoculation of mice. HSV-1 encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with HSV-1 isolates from patients with oral lesions only, whereas HSV-2 meningitis strains were significantly more virulent after i.c. inoculation when compared with HSV-2 isolates from patients with genital lesions only. No correlation between high neurovirulence (defined as low LD50 for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neurovirulent HSV-1 strain isolated from a patient with multiple sclerosis gave low replication yields. After nose inoculation, a highly neuroinvasive HSV-1 laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainstem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brainstem. Neuroinvasiveness of the virus strain might be one factor of relevance in the pathogenesis of HSV-1 encephalitis in man.
... Low temperature adapted ts PPV mutants were shown to be effective when employed as modified live vaccines [9,13]. Revertants of ts mutants have also been described and a possible mechanism for revertants has been suggested in various viruses [1,15,17,24]. ...
... KBSH appeared to gain the ability to replicate in swine fetuses following serial cell culture passages at 39 °C evidenced by fetal death and virus detection in fetal tissues. This result can be supported by previous reports that the replication at near the body temperatures were correlated with virulence in poliovirus [17] and mouse corona virus [15]. Compared to experimental fetal infection with NADL-8 or Kresse isolate [3,16], fetal abnormalities were less severe. ...
Previous studies have shown that replication in vitro of the porcine parvovirus (PPV) isolate, KBSH, was restricted at 39 degrees C but not at 37 degrees C. In contrast, replication of the Kresse isolate was restricted at 37 degrees C but not at 39 degrees C. In this study, Kresse and KBSH isolates were passaged up to ten times in swine testicle (ST) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral DNA synthesis, and progeny virus were evaluated. KBSH became adapted for replication at 39 degrees C upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral DNA concentration. This finding was also observed with Kresse virus isolate continuously passaged at 37 degrees C. Neither isolate became adapted for replication at 32 degrees C. In an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, KBSH passaged 10 times either at 37 degrees C or 39 degrees C was inoculated into swine fetuses. Two of four fetuses inoculated with 39 degrees C-passaged KBSH were dead and hemorrhagic or mummified. All four fetuses inoculated with 39 degrees C-KBSH contained viral antigen and viral DNA. In contrast, fetuses inoculated with 37 degrees C-passaged KBSH, or with cell culture fluid were normal in appearance. Viral antigen and viral DNA were not demonstrated in fetuses inoculated with 37 degrees C-KBSH or cell culture fluids. These findings suggest the possibility that the ability to replicate at 39 degrees C is associated with virulence in swine fetuses.
... Temperature dependent replication has been reported for various families of viruses including members of the orthomyxoviridae [24], herpesviridae [2,7], picornaviridae [1,9], paramyxoviridae [6], and coronavirus [16]. Temperature-sensitive (ts) mutants have also been described for parvovirus, H 1 [27], Kilham rat virus [28], and porcine parvovirus [8,12]. ...
The replication of four porcine parvovirus isolates, NADL-8, NADL-2, KBSH, and Kresse, in swine testes cells were compared at temperatures of 32, 37, and 39 degrees C. Replication of the Kresse isolate was restricted at 32 and 37 degrees C as evidenced by progeny virus, virus polypeptide and viral DNA synthesis, but not at 39 degrees C. In contrast, replication of KBSH was restricted at 39 degrees C, but not at 37 or 32 degrees C. Findings from this study support the contention that replication of KBSH and Kresse isolates are temperature dependent.
Murine hepatitis virus (MHV) infections exhibit remarkable variability in cytopathology ranging from acutely cytolytic to essentially asymptomatic levels, In this report, we assess the role of the MHV receptor (MHVR) in controlling this variable virus-induced cytopathology, We developed human (HeLa) cell lines in which the MHVR was produced in a regulated fashion by placing MHVR cDNA under the control of an inducible promoter, Depending on the extent of induction, MHVR levels ranged from less than similar to 1,500 molecules per cell (designated R-lo) to similar to 300,000 molecules per cell (designated R-hi). Throughout this range, the otherwise MHV-resistant HeLa cells were rendered susceptible to infection. However, infection in the R-lo cells occurred without any overt evidence of cytopathology, while the corresponding R-hi cells died within 14 h after infection. When the HeLa-MHVR cells were infected with vaccinia virus recombinants encoding MHV spike (S) proteins, the R-hi cells succumbed within 12 h postinfection; R-lo cells infected in parallel were intact, as judged by trypan blue exclusion. This acute cytopathology was not due solely to syncytium formation between the cells producing S and MHVR, because fusion-blocking antiviral antibodies did not prevent it. These findings raised the possibility of an intracellular interaction between S and MHVR in the acute cell death. Indeed, we identified intracellular complexes of S and MHVR via coimmunoprecipitation of endoglycosidase H-sensitive forms of the two proteins. We suggest that MHV infections can become acutely cytopathic once these intracellular complexes rise above a critical threshold level.
Hepatitis caused by mouse hepatitis virus (MHV-A59), a murine coronavirus, is accompanied by direct infection and replication of virus within the liver. We demonstrate here that the aminoglycoside hygromycin B is able to eliminate MHV-A59 infection from mouse peritoneal macrophages and cultured liver cells in vitro and is also able to reduce levels of virus replication and necrotic liver foci in vivo.
Infection of mixed glial cell cultures with mouse hepatitis virus (MHV)-A59 results in an approximately six-fold increase in the level of major histocompatibility complex (MHC) class I mRNA. In situ hybridization of glial cell cultures infected with MHV-A59 again showed enhanced MHC mRNA expression, both in infected and uninfected cells. These results extend our earlier finding that MHC surface antigens are enhanced on astrocytes and oligodendrocytes after MHV-A59 infection and suggest that this enhancement is a result of an increase in the steady-state level of MHC mRNA. We further demonstrate that increases in MHC mRNA occur in the murine central nervous system (CNS) following infection in vivo. Northern blot analysis of RNA from the brains of infected animals showed transient expression of both MHC class I and class II mRNA over the first 14 days of infection. Expression coincided with viral replication and clearance. In situ hybridization of brain sections from infected animals showed that class I and class II expression was widespread throughout all portions of the brain and in uninfected as well as infected cells. Viral RNA, in contrast, was observed in small foci of cells and mostly within the limbic system. Thus enhancement of MHC mRNA was not restricted either to areas of infection or inflammation. The spatial relationship between viral and MHC expression supports our hypothesis that a soluble mediator is involved in the mechanism of the increase in MHC levels. The fact that MHC induction occurs in vivo as well as in vitro suggests MHC may be important in the mechanism of MHV-induced disease.
