Biomedical subjects
Publications and source records attributed to J S Wolinsky.
Mumps virus-infected and normal newborn hamster brains were imaged with magnetic resonance (MR) within 24 h after birth and serially scanned. Hydrocephalus developed explosively on Day 6 postinfection and progressed until death occurred soon after Day 10. This study demonstrates the utility of MR in the investigation of experimental viral encephalitis in very small laboratory animals.
The fusion protein (F) gene of mumps virus was cloned from a cDNA library constructed from infected cell mRNA. The F-specific plasmids were identified by hybridization to a degenerate oligonucleotide probe whose sequence was deduced from the N-terminal amino acid sequence of the F2 protein. The complete nucleotide sequence of the F gene was determined. The gene is 1786 nucleotides long and encodes one long open reading frame of 538 amino acids. The F protein has a 19-amino acid signal peptide cleaved between Cys and Val residues. The cleavage site for activation of the F0 protein into the mature F1,2 is Arg-Arg-His-Lys-Arg. A stretch of 30 hydrophobic amino acids near the C-terminus of the protein is followed by several charged amino acids and appears to serve as the anchoring domain for the protein in the lipid bilayer. The F gene of mumps virus is highly related to the F gene of the paramyxovirus SV-5.
Forty-five serum samples from 31 newborns and infants with the congenital rubella syndrome (CRS) were tested by immunoprecipitation to determine their antibody spectra to each of the structural proteins of rubella virus. Most sera (37/45) contained little or no E2 protein-specific antibody, but some (6/45) precipitated a greater amount of the E2 glycoprotein than the E1 glycoprotein. The relative E1/E2 ratio was found to decrease with time when serial serum samples from the same patient were tested. No correlation between the IgG class hemagglutination inhibition antibody titers and the E1/E2 ratio could be demonstrated. However, in some serum specimens relatively high neutralizing antibody titers were correlated with immunoprecipitation of the E2 glycopolypeptide. None of the CRS sera reacted well with the C protein. The immunoprecipitation patterns found in CRS sera were qualitatively different from those observed in a series of 25 sera from young adults with conventional serologic evidence of rubella immunity following natural infection. All of the natural immune sera recognized each of the three structural polypeptides of rubella virus.
An inverse relationship exists between the sialic acid content of a particle and its ability to activate the alternative complement pathway. The present studies were performed to determine if the neuraminidase (NANase) activities of different mumps virus strains could influence the ability of mumps virus infected cells to activate the alternative pathway. CV-1 cells were infected with three different mumps virus strains (RW, O'Take, and Kilham) and after 24 hours, 10 percent guinea pig serum (GPS) treated with EGTA/MgCl2 or GPS lacking the 4th component of complement (C4DGPS) was added to the cell monolayers. After 30 minutes, the percentage C3 consumed was determined by a functional hemolytic assay. Cells infected with RW (high NANase) consumed significantly more C3 (23.2 per cent) than cells infected with Kilham (5.7 percent, low NANase). Cells infected with O'Take were intermediate in their ability to activate C3. The degree of C3 deposition on the surface of infected cells, detected by fluorescence microscopy, was also greater for cells infected with the RW than the Kilham strain of mumps virus. These studies suggest that the NANase activity of mumps virus can influence the ability of infected cells to activate the alternative pathway and thereby, the ability of complement to participate in host defense against mumps virus infection.
A fusing variant of the nonfusing O'Take strain of mumps virus was obtained by growing virus under the selective pressure of the competitive neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The variant virus, O'Take(DANA)v1, causes extensive syncytial formation in CV-1 cell cultures in contrast to the relatively noncytopathic infection caused by the O'Take virus parent. Neuraminidase assays indicate that O'Take(DANA)v1 has no detectable neuraminidase activity using either fetuin or neuraminlactose as substrate. In addition, the O'Take(DANA)v1 virus can agglutinate red blood cells but cannot elute from these cells once adsorbed. No differences were detected in the biochemical or antigenic structure of the hemagglutinin-neuraminidase (HN) proteins from O'Take virus and O'Take(DANA)v1. These results indicate that the neuraminidase enzyme of the mumps virus HN glycoprotein is involved in modulating the cell fusion cytopathology of mumps virus infections.
Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.
The fusion (F) protein of mumps virus was purified by immunoaffinity chromatography using an anti-F monoclonal antibody. The F protein was reduced and alkylated, and the F1 and F2 chains were isolated by high-pressure size exclusion chromatography. Twenty-three amino acid residues from the amino terminus of each chain were identified following automated Edman degradation. The amino-terminal sequence of the F1 chain was homologous to previously reported F1 sequences from three other paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus). Secondary structure predictions suggest an alpha-helical conformation for the mumps virus F1 amino-terminal sequence. A helical wheel model of the paramyxovirus F1 NH2 terminus is presented which defines conserved and variable arcs of the helix and provides a spatial representation of this critical functional domain of the paramyxovirus fusion protein.
The loss of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) was compared by quantitative immunocytochemistry in demyelinating lesions of measles encephalomyelitis (ME), multiple sclerosis (MS), and progressive multifocal leukoencephalopathy (PML). Serial sections from paraffin-embedded tissue were reacted with antisera for MAG and MBP, and areas of staining loss were compared morphometrically. Lesions in ME showed MAG loss equal to that of MBP, lesions of PML showed MAG loss greater than that of MBP, and MS lesions showed a mixture of patterns. These data demonstrate distinctive patterns of MAG and MBP loss in these three diseases.
A panel of murine monoclonal antibodies prepared against rubella virus is described. Fourteen of these monoclonal antibodies react with the E1 glycoprotein of rubella virus and define a total of six spacially separate epitopes in competitive inhibition assays. Antibodies binding to epitopes E1(a), E1(b), E1(c), or E1(e) inhibit the hemagglutinin function of the virus, while antibodies binding to epitopes E1(d) or E1(f) do not. Monoclonal antibodies binding to epitopes E1(c) or E1(d) prevent virus infectivity and identify antigen in distinct intracytoplasmic vacuoles of rubella virus-infected Vero cells by indirect immunofluorescence. Monoclonal antibody to epitope E1(f) localizes antigen primarily to the plasma membrane of infected cells, while antibodies binding to epitopes E1(a), E1(b), or E1(e) localize antigen throughout the infected cell's cytoplasm. A single monoclonal antibody is described which only reacts with the mature form of the virion E2 glycoprotein after rubella virus is treated with a disulfide-bond reducing agent. This antibody immunoprecipitates a 43,000 MW precursor to the E2 glycoprotein from lysates of infected cells and localizes its antigen throughout the cytoplasm of infected cells. The five remaining monoclonal antibodies react with the rubella virus C polypeptide. They define four topographically separate epitopes on the C polypeptide, C(a), C(b), C(c), and C(d), each of which is diffusely distributed throughout the cytoplasm of rubella virus-infected cells.
Newborn Syrian hamsters were challanged with an intracerebral inoculum containing 128 50% lethal doses of the Kilham strain of mumps virus and treated 24 h later with a single intraperitoneal injection of mouse monoclonal antibody. Monoclonal antibodies reactive with epitopes on the fusion glycoprotein of mumps virus could not inhibit hemagglutination or neutralize infectivity in vitro and failed to provide biologically important protection against the in vivo infection. In contrast, monoclonal antibodies reactive with epitopes on the hemagglutinin-neuraminidase glycoprotein of mumps virus inhibited hemagglutination and neutralized infectivity in vitro and protected infected animals from the otherwise lethal central nervous system virus infection. Similar protection was provided by both purified immunoglobulin and F(ab')2 fragments. Immuno-cytochemical and virological studies showed diminished virus antigen and virus titers in the brains of successfully treated animals. It appears that a topographically restricted region of the hemagglutinin-neuraminidase molecule of the Kilham strain of mumps virus is of critical importance for immune recognition by the infected host.
We investigated the hypothesis that a persistent viral infection of the thymus gland might trigger the autoimmune disease myasthenia gravis (MG). Thymus glands of nine patients with recent onset of MG were studied by a variety of techniques to detect the presence of occult viruses. No evidence of viral infection was found.
We studied 19 patients with postinfectious encephalomyelitis complicating natural measles-virus infections, and our results support the hypothesis that this demyelinating disease has a pathogenesis similar to that of experimental allergic encephalomyelitis. Early myelin destruction was demonstrated by the presence of myelin basic protein in cerebrospinal fluid, and lymphocyte proliferative responses to myelin basic protein were found in 8 of 17 patients tested. A lack of intrathecal synthesis of antibody against measles virus suggests that measles encephalomyelitis may not be dependent on virus replication within the central nervous system. Similar lymphoproliferative responses to myelin basic protein of lymphocytes from single patients with encephalomyelitis after rabies vaccine or after varicella or rubella virus infections suggest a common immune-mediated pathogenesis for the perivenular demyelinating disease that can follow the injection of neural tissues or infection by a variety of viruses.
Measles encephalomyelitis appears to be an immune-mediated parainfectious disorder, but it is unclear whether viral invasion of brain is an obligate step in its development. Immunocytochemical methods were used to search for virus antigen in formalin-fixed, paraffin-embedded central nervous system (CNS) tissues from 10 patients with measles encephalomyelitis and 12 patients who had died of measles without CNS involvement. All the CNS tissues studied were viral antigen negative. Similarly fixed CNS tissues from all of 6 patients with subacute sclerosing panencephalitis were viral antigen positive and served as controls. The pattern of perivenular demyelination was also determined in 4 cases of measles encephalomyelitis using antibodies to myelin associated glycoprotein and myelin basic protein and a Luxol fast blue stain. Areas of demyelination in serial sections were quantitated, and no morphometrical differences were found among tissues stained with the three stains. The data suggest the lack of virus replication in the CNS during encephalomyelitis or fatal measles without CNS symptoms. The pattern of loss of myelin associated glycoprotein and myelin basic protein in regions of perivenular demyelination resembles that reported in experimental allergic encephalomyelitis. This pattern of demyelination has been proposed to result from a primary attack on the myelin sheath rather than from direct involvement of the oligodendroglial cell.
Rubella virus appears to have few rivals in the variety of its pathologic expressions in man. The authors explore some of these, address the virus's ability to establish persistent infections, discuss the introduction of an effective vaccine for rubella, and touch on future research into rubella virus.
Purified rubella virus contains three major structural polypeptides whose apparent molecular weights are 62,000, a 47,000-54,000 complex, and 38,000 when analyzed by polyacrylamide gel electrophoresis. Both the 62,000 and 47,000-54,000 dalton polypeptides are glycosylated, but they vary in their relative [3H]glucosamine and [3H]mannose content. Limited-digest peptide maps confirm that each polypeptide is distinct and that the 47,000-54,000 dalton complex is a series of three glycopolypeptides with extensive similarities. Under nonreducing conditions, both the 62,000 dalton glycopolypeptide and the 47,000-54,000 dalton complex exist as monomers and also as disulfide-linked complexes. A complex of 105,000 daltons is a dimer of the 62,000 dalton glycopolypeptide and a second, at 95,000 daltons, is a mixed disulfide-bonded dimer of the 62,000 dalton glycopolypeptide and 47,000-54,000 dalton complex. The 38,000 dalton polypeptide migrated exclusively as a dimer of 78,000 daltons when unreduced. Two monoclonal antibodies which inhibit the hemagglutinin function of the virus were shown to be directed against the 62,000 dalton glycopolypeptide. This glycoprotein is therefore responsible at least in part for the hemagglutinin function of rubella virus.
Neuropathological findings in a patient with fatal neurological complications due to infection with Mycoplasma pneumoniae were similar to those seen in postinfectious encephalitis and acute hemorrhagic leukoencephalitis. This case supports the hypothesis that immune mechanisms play a role in the pathogenesis of neurological symptoms during mycoplasmal infections.
Four patients with herpes zoster ophthalmicus and delayed contralateral hemiparesis are described, and their findings are compared with those in patients previously reported in the English language literature. The current patients evidenced multifocal ipsilateral cerebral angiitis by angiography and multifocal infarcts in the distribution of the ipsilateral middle cerebral artery by computed tomographic scanning. Cerebrospinal fluid showed mononuclear pleocytosis, positive oligoclonal bands, and an elevated immunoglobulin G index. Two patients were treated with corticosteroids and acyclovir, and 1 with corticosteroids alone, all without apparent response. Necrotizing angiitis ipsilateral to the herpes zoster ophthalmicus was demonstrated postmortem in 1 patient with multifocal cerebral infarction and progressive leukoencephalopathy. Neither herpes varicella zoster immunocytochemical reactivity nor viral inclusions were seen. The leukoencephalopathy associated with herpes varicella zoster either may be caused by cerebral angiitis or, as previously reported, may be a temporally remote manifestation of persistent herpes varicella zoster infection. The cerebral angiitis associated with herpes varicella zoster is histologically similar to granulomatous angiitis, and both may be related to herpes varicella zoster infection of the cerebral vasculature.
Focal central nervous system demyelination is a prominent feature of Ross River virus encephalitis in mice. The present ultrastructural study shows that oligodendrocytes are a primary site of viral replication. The earliest myelin disruption occurs in association with an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes, which are later replaced by macrophages. Viral particles are found in oligodendrocytes, selected neuronal populations, macrophages, and polymorphonuclear leukocytes through the end of the first week of infection as macrophages remove myelin from normal-appearing axons. Between the second and third weeks of infection, axons within foci of demyelination partially remyelinate with central myelin. Schwann cells are not found within regions of central remyelination. Cyclophosphamide treatment does not prevent or delay demyelination or remyelination. Results of this and previous studies strongly suggest that Ross River virus--induced demyelination is not immune mediated but rather the direct result of viral infection of oligodendrocytes.