Hybridoma cell lines producing monoclonal antibodies to the JHM strain of mouse hepatitis virus-4 (MHV-4) were established. By indirect immunofluorescence and immune precipitation, monoclonal antibodies of three viral polypeptide specificities were characterized. Monoclonal antibodies to nucleocapsid reacted in the cytoplasm of infected cells and precipitated the 60,OOOd nucleocapsid polypeptide (VP-4) of MHV-4. Other monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and were found to precipitate the 170,OOOd viral glycoprotein (GP-1). A third set of monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and precipitated the 25,OOOd viral glycoprotein (GP-5) and its precursor VP-6 (23,OOOd). Anti-GP-1 alone had direct neutralizing activity for MHV-4 virus, while in the presence of complement both anti-GP-1 and anti-GP-5 neutralized virus. Only anti-GP-1 had the ability to inhibit the spread of infection due to fusion in L241 cells. Thus, the viral glycoprotein GP-1 likely contains both the attachment and fusion activities of MHV-4.
A purified recombinant spike (S) protein was studied for its effect on stimulating human peripheral blood monocyte macrophages (PBMC). We examined inflammatory gene mRNA abundances found in S protein-treated PBMC using gene arrays. We identified differential mRNA abundances of genes with functional properties associated with antiviral (CXCL10) and inflammatory (IL-6, IL-8) responses. We confirmed cytokine mRNA increases by real time quantitative(q) RT-PCR or ELISA. We further analyzed the sensitivity and specificity of the prominent IL-8 response. By real time qRT-PCR, S protein was shown to stimulate IL-8 mRNA accumulation in a dose dependent manner while treatment with E protein did not. Also, titration of S protein-specific production and secretion of IL-8 by ELISA showed that the dose of 5.6nM of S produced a significant increase in IL-8 (p=0.003) compared to mock-treated controls. The increase in IL-8 stimulated by a concentration of 5.6 nM of S was comparable to concentrations seen for S protein binding to ACE2 or to neutralizing monoclonal antibody suggesting a physiological relevance. An NF-κB inhibitor, TPCK (N-Tosyl-L-Phenylalanine Chloromethyl Ketone) could suppress IL-8 production and secretion in response to S protein in PBMC and THP-1 cells and in HCoV-229E virus-infected PBMC. Activation and translocation of NF-κB was shown to occur rapidly following exposure of PBMC or THP-1 cells to S protein using a highly sensitive assay for active nuclear NF-κB p65 transcription factor. The results further suggested that released or secreted S protein could activate blood monocytes thru recognition by toll-like receptor (TLR)2 ligand.
ABSTRACT. We present a case in which human coronavirus was detected in the cerebrospinal fluid of a child presumed to have acute disseminated encephalomyelitis. In murine models, coronavirus has been found to cause a chronic demyelinating condition that resembles multiple sclerosis. Additionally, there is in vitro evidence of human coronavirus's ability to infect neural cells. This case report provides additional support for the hypothesis that coronavirus may be an important etiologic factor in the pathogenesis of demyelinating disease in humans. Pediatrics 2004;113:e73-e76. URL: http://www.pediatrics. org/cgi/content/full/113/1/e73; coronavirus, HCoV, acute disseminated encephalomyelitis, ADEM, postinfectious encephalitis, demyelination, child.ABBREVIATIONS. ADEM, acute disseminated encephalomyelitis; CNS, central nervous system; MRI, magnetic resonance imaging; CSF, cerebrospinal fluid; PCR, polymerase chain reaction; IgG, immunoglobulin G; RT, reverse transcription; HCoV, human coronavirus; MS, multiple sclerosis.A cute disseminated encephalomyelitis (ADEM) is a monophasic, demyelinating disease of the central nervous system (CNS) that primarily affects children and young adults. It is characterized by high-signal-intensity lesions in the white matter of the brain and spinal cord on T2-weighted magnetic resonance imaging (MRI). These lesions may be independent of the clinical findings. Children may present with diffuse encephalopathy, seizures, optic neuritis, hemiparesis, and/or symptoms suggestive of spinal cord transection.The epidemiology of the condition is unknown. A review of cases presenting to a children's hospital suggested a prevalence of ϳ4.5 cases per 10 000 pediatric hospital admissions, exclusive of newborns. 1 The etiology of the illness is cryptogenic, although the disorder is generally thought to be due to a paraor postinfectious process. Certainly, many case reports in the literature suggest an infectious process before the onset of CNS symptoms, and some have identified specific infectious agents in cases of ADEM. [2][3][4] However, in most cases, a specific etiologic infectious agent is not identified. For example, in a recent retrospective review of cases of ADEM, clinicians were able to identify an infectious agent in only 1 of 18 cases. 1 ADEM has also been described after immunizations. 5,6 Despite reports of the possible association between infection and ADEM, there is no clear understanding of the relationship between the infectious agent and the onset of demyelination. There is experimental evidence in mice for a relationship between coronavirus and CNS demyelination. [7][8][9] Little is known, however, about this virus's relationship to demyelinating disease in humans. Indeed, there have been no case reports of this virus in relation to ADEM. We report a case of demyelinating disease in a child in which cerebrospinal fluid (CSF) and nasopharyngeal specimens were positive for human coronavirus (HCoV) by polymerase chain reaction (PCR) and in whom a fourfold rise in antibody titer ...
Human coronavirus (HCoV) strain 229E infection, but not HCoV strain OC43 infection, of monocytes/macrophages from healthy donors and patients with multiple sclerosis in remission resulted in increased apoptosis, as measured by DNA changes and annexin V staining. Apoptosis correlated with the differential release of infectious virus. HCoV strain 229E titers were 10 3.5 to 10 6 50% tissue culture-infective doses (TCID 50 )/ml, and HCoV strain OC43 titers were only 10 1.2 to 10 2.7 TCID 50 /ml.
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