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M J Buchmeier

Publications and source records attributed to M J Buchmeier.

At least 91 records · Page 5Linked to original sources
PubMed1985

The S RNA segment of lymphocytic choriomeningitis virus codes for the nucleoprotein and glycoproteins 1 and 2.

The lymphocytic choriomeningitis virus (LCMV) genome consists of a large RNA segment and a small RNA segment. The three major structural proteins of this virus are an internal nucleoprotein and two surface glycoproteins. Intertypic reassortants between the Armstrong and WE strains of LCMV were made to map proteins encoded by the LCMV genome segments. Using monoclonal antibodies specific for the nucleoprotein and the glycoproteins of WE and Armstrong, we showed that the small RNA segment of LCMV codes for the three major structural polypeptides.

Animals↗
PubMed1985

Genetic mapping of lymphocytic choriomeningitis virus pathogenicity: virulence in guinea pigs is associated with the L RNA segment.

The Armstrong CA 1371 (ARM) and WE strains of lymphocytic choriomeningitis virus (LCMV) differ in the ability to produce disease in adult guinea pigs. Infection with the ARM strain is not lethal, even at high virus doses (greater than 10,000 PFU), whereas the WE strain causes 100% mortality even at low doses (less than 10 PFU). To determine the genetic basis of this virulence, intertypic reassortants were made between the ARM and WE strains of LCMV. The two reassortants with the genotypes WE/ARM (L segment of WE and S segment of ARM) and ARM/WE (L segment of ARM and S segment of WE) were tested for their pathogenicity in guinea pigs. The ARM/WE reassortant was avirulent like the ARM/ARM parental strain. Minimal viral replication was observed in organs of guinea pigs inoculated with 10(2) or 10(5) PFU of ARM/ARM or ARM/WE, and all animals survived. In contrast, the WE/ARM reassortant was highly virulent like the WE/WE parental strain and killed all of the infected animals. High levels of viral replication were observed in guinea pigs infected with the latter two strains. In contrast to these in vivo observations, both the parental strains and the ARM/WE or WE/ARM reassortants had similar growth potential in cultured guinea pig fibroblasts. Thus, the L RNA segment of LCMV WE is important for viral replication in vivo and is associated with fatal acute disease after infection of adult guinea pigs.

Animals↗
PubMed1985

Epitope-specific antibody response to murine hepatitis virus-4 (strain JHM).

Monoclonal hybridoma antibodies to the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) were used in a competition binding enzyme immunoassay to analyze at the epitope level the antibody response of mice after infection with MHV-4. Colonized mice often had pre-existing MHV antibodies directed against epitopes on the E2 glycoprotein, the E1 glycoprotein, and the nucleocapsid protein. These mice generated a secondary antibody response after virus inoculation, reaching peak levels 7 days after infection. In contrast, Nude/+ mice raised in a pathogen-free colony had no detectable circulating MHV antibodies and generated a primary antibody response which gradually increased to peak levels 14 to 28 days after infection. Kinetics of antibody responses against specific epitopes usually correlated well with measured total virus-specific antibody responses, but variation was observed. Mice injected with three antigenically distinct strains of MHV made antibody responses to conserved epitopes but not to an antigenic determinant absent in these strains. Measurement of epitope-specific responses in a polyclonal population of viral specific antibodies is feasible and a valuable adjunct in understanding viral immunity.

Animals↗
PubMed1984

Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus.

Viral proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and processing, as well as expression in animal tissues. Circulating polyclonal antibodies were also probed and characterized for their polypeptide specificities. Under appropriate conditions, this Western immunoblotting technique was quantitative. Finally, a highly sensitive dot immunoblotting assay was used to analyze the sensitivity to denaturation of various epitopes on the viral proteins. This assay detected picogram quantities of viral antigens and antibodies.

Antigens, Viral↗
PubMed1984

Topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain JHM): correlation with biological activities.

Monoclonal hybridoma antibodies (MAb) of defined polypeptide specificity and biological activity were used in a competition binding assay to identify antibody binding sites (epitopes) on the glycoproteins of murine hepatitis virus-4 strain JHM (MHV-4). Individual MAb were labeled with horseradish peroxidase (HRP) and used as probes in a competition enzyme immunoassay (EIA). Four topographically distinct antigenic sites were detected on the E2 glycoprotein of MHV-4. Antibodies reacting with these four determinants provisionally designated A(E2), B(E2), C(E2), and D(E2) had corresponding biological activities (M. J. Buchmeier, H. A. Lewicki, P. J. Talbot, and R. L. Knobler (1984) Virology 132, 261-270). Antibodies to sites A(E2) and B(E2) mediated virus neutralization in vitro and passively protected mice against lethal virus challenge in vivo. Antibody to site C(E2) neutralized virus efficiently in vitro but did not alter disease in vivo, while antibody to site D(E2) neither neutralized nor protected. Two major nonoverlapping antigenic sites were defined on the E1 glycoprotein. Overlapping epitopes A(E1) and B(E1) constituted one site and epitope C(E1) the other.

Animals↗
PubMed1984

Murine hepatitis virus-4 (strain JHM)-induced neurologic disease is modulated in vivo by monoclonal antibody.

Monoclonal hybridoma antibodies directed against the polypeptides of murine hepatitis virus-4 (JHM strain) were tested for their ability to alter the course of a normally lethal intracerebral virus challenge. Three monoclonal antibodies directed against two distinct epitopes on the E2 glycoprotein of MHV-4 protected mice against lethal virus challenge and converted the infection from fatal encephalomyelitis to demyelination. A single neutralizing antibody directed against a third epitope on E2 as well as seven nonneutralizing antibodies to E2, E1, and N polypeptides did not protect against challenge. In mice which received protective antibody, MHV-4 infection was not blocked, however, virus grew to lower titers in liver and brain, and virus replication in the CNS was more restricted than in unprotected mice. Decreased involvement of neurons in the brains of protected mice was observed, and no evidence of neuronal infection in the spinal cords was found. In contrast, oligodendrocytes were infected in the presence of protective antibody, and evidence of demylination associated with mononuclear cell infiltration was found. These studies demonstrate that antibody to a single epitope on a viral glycoprotein can substantially alter the course and phenotype of disease.

Animals↗
PubMed1983

A protein kinase activity in lymphocytic choriomeningitis virus and identification of the phosphorylated product using monoclonal antibody.

A cyclic AMP-independent protein kinase activity was found in purified preparations of the Armstrong CA 1371 strain of lymphocytic choriomeningitis virus (LCMV). Using the exquisite sensitivity of monoclonal antibodies to LCMV polypeptides, the internal nucleocapsid N protein was identified as the major virus-specific phosphorylated product of the endogenous protein kinase activity. This was accompanied by an increase in the electrophoretic mobility of N protein as detected by SDS-PAGE. After solubilization of the virus with 1% Nonidet P40 approximately 81% of the endogenous protein kinase activity remained associated with LCMV nucleocapsids recovered by equilibrium centrifugation at a density of 1.25 g/cm-3 in a linear renograffin gradient. Specific phosphorylation of N protein was reconfirmed in the purified nucleocapsid fraction and both phosphoserine and phosphothreonine found to be the phosphorylated products of the kinase reaction. Although the significance of this enzyme remains unclear, the presence of a protein kinase within LCMV may allow the regulation of LCMV replication and maturation by phosphorylation of virus-specific polypeptides. These events may in turn play a key role in determining the nature and outcome of LCMV infection.

Amino Acids↗
PubMed1983

Comparative analysis of the structural polypeptides of Ebola viruses from Sudan and Zaire.

Polypeptide structural analyses were performed by tryptic peptide mapping to assess the relationship between isolates of Ebola virus obtained in Sudan and Zaire. The results of these analyses indicate (1) that the Sudan and Zaire isolates are unique viral agents, (2) that multiple isolates within each group bear close resemblance to one another, (3) that suggestive evidence exists of conservation of homologous structure in the VP-2 (virion protein no. 2) nucleocapsid proteins of these viruses, and (4) that protein differences between the two Ebola virus subtypes appear to reside in the VP-1 and VP-3 virion polypeptides.

Democratic Republic of the Congo↗
PubMed1983

Virus-induced immune complex disease: genetic control of C1q binding complexes in the circulation of mice persistently infected with lymphocytic choriomeningitis virus.

Virus-antibody immune complex formation, deposition, and disease is a common manifestation in most mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Although mice of several strains persistently infected with LCMV mount continuous anti-LCMV immune responses to the three virion structural polypeptides, the amount of antibody(s) made varies among strains. The formation of antibody to LCMV correlates with the detection of C1q binding materials (immune complexes) in the circulation; however, there is no correlation between the total amount of IgG and the C1q binding material. SWR/J mice (H2qq) are high responders (high levels of anti-LCMV antibodies, high C1q binding responses), whereas BALB/W mice (H-2dd) are low responders (low levels of anti-LCMV antibodies, low C1q binding responses). Sera from 37 SWR/J mice, 12 wk of age, bound 55.1% of offered 125I C1q compared to 8.4% for 37 age and sex-matched BALB/W mice. SWR/J mice made approximately 60-fold more antibody to LCMV than did BALB/W mice. To define the genetic factors involved, we used the C1q binding assay, high responder and low responder mice, their hybrid offspring, backcrosses of the hybrids to both high and low responder parents, and selected recombinant inbred mouse strains. From studies with BALB/W mice persistently infected with LCMV, we defined low C1q responder mice as those having binding activity of 18.4% or less (BALB/W mean C1q binding value + 2 SD). By using this criteria for 12-wk-old mice, 55/57 (96%) of SWR/J, 27/37 (73%) of F1(SxB or BxS), 13/21 (62%) of qq or 11/20 (55%) qd from F1 x SWR/J, and 6/13 (45%) qd from F1 x BALB/W were high C1q responders. In contrast, none of the 17 dd mice from the F1 x BALB/W cross were high responders. Thus, F1 hybrid mice are high responders (dominant) and data from backcrossing F1 hybrids to either high or low responder parents suggested the complexes associated with C1q binding was controlled by gene(s) in the H-2 complex. Support for the role of Ir gene(s) was provided by experiments showing LCMV persistently infected BALB/kae (H-2 KkIkDk) and recombinant inbred strains B10.A (KkIkDd) and A.TL (KsIkDd) were high C1q responders, whereas B10.A(5R) (KbIbDd) and BALB/cby (KdIdDd) were not. The wide scatter in C1q binding levels observed among C1q high responder mice and the production of C1q binding levels in F1 mice that are intermediate between high and low responder strains suggested that, in addition to H-2-linked genes, other non-H-2-linked genes also play a role in this response. The amount of C1q binding complexes in LCMV persistently infected mouse strains and their crosses was unrelated to the amounts of infectious virus carried in their sera. Despite the low C1q binding by sera of H-2dd mice that originated from mating F1 hybrid (qd) x BALB/WEHI (dd), these mice carried equivalent amounts of infectious virus as their qd littermates or qd and qq mice originated from F1 x SWR/J mice.

Animals↗
PubMed1983

Ultrastructural localization of viral antigens in the CNS of mice persistently infected with lymphocytic choriomeningitis virus (LCMV).

Lymphocytic choriomeningitis virus (LCMV) produces a persistent infection of the nervous system in susceptible mice. To map the localization of viral antigens in the central nervous system (CNS), the authors studied, by means of ultrastructural immune peroxidase techniques, 4-6-month-old mice persistently infected with LCMV following an intracerebral inoculation at birth. The greatest number of infected neurons was observed in the cortex, particularly of the limbic system, and certain nuclei of the hypothalamus. In the cerebellum, Purkinje cells selectively expressed viral antigens. Moderate numbers of infected neurons were found in the anterior horns of the spinal cord, basal ganglia, and thalamus. The immunoperoxidase technique using monoclonal antibodies showed that persistently infected neurons primarily expressed the nucleocapsid protein antigens of LCMV. Glycopeptide antigens were minimally expressed. Electron-microscopic examination of selected individual infected neurons showed viral antigens exclusively associated with ribosomes. No staining was seen on cell surfaces. Glutaraldehyde-fixed CNS tissue studied by electron microscopy did not reveal morphologic abnormalities or mature viral particles. This study demonstrates that LCMV persistently infects specific neuronal populations. Infected neurons express viral antigens in association with host ribosomes and show no significant morphologic alterations.

Animals↗
PubMed1982

In vivo and in vitro models of demyelinating diseases. V. Comparison of the assembly of mouse hepatitis virus, strain JHM, in two murine cell lines.

The developmental sequence of a neurotropic strain (JHM) of mouse hepatitis virus was examined by transmission electron microscopy and immunocytology. The nucleoprotein core of this coronavirus, which contains RNA of positive polarity and is helical in configuration, becomes incorporated into enveloped particles in the same manner as the nucleocapsids of the orthomyxo- and paramyxoviruses. However, JHM virus is assembled intracellularly by budding at surfaces of smooth membranous vacuoles. A comparison of JHM virus replication in L2 and 17Cl-1 cell lines revealed that L2 cells undergo more rapid cytopathology and cease virus production much sooner than 17Cl-l cells. In L2 cells the accumulation of core material appears to continue after the abrupt cessation of virus assembly. This is evident by the massive cytoplasmic accumulation of structure resembling nucleocapsids, which react with hybridoma antibody to the nucleocapsid antigen as demonstrated by the immunoperoxidase procedure. The current findings are consistent with our previously published demonstration, using cells of neural and other deviation, of the fundamental role of the host cell type in regulating the replication and expression of coronaviruses.

Animals↗
PubMed1981

In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon.

Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

Antigens, Viral↗
PubMed1980

Monoclonal antibodies to lymphocytic choriomeningitis virus react with pathogenic arenaviruses.

Certain arenaviruses have become widely recognized as important human pathogens, the most notable among these being Lassa virus, the causative agent of Lassa fever. Two other members of the group, Junin and Machupo virus, are the aetiological agents of Argentine and Bolivian haemorrhagic fevers, respectively. All these agents share both morphological features and to varying degrees serological cross-reactivity with other non-pathogenic arenaviruses. Despite the evident clinical importance of these viruses, work to define their physiochemical structure and to develop rapid and precise diagnostic techniques has been slow. Consequently, the definitive relationships among West African Lassa strains, strains of a related agent from Mozambique and of an Old World arenavirus, lymphocytic choriomeningitis (LCM), have not been established. This problem is of more than simple taxonomic importance in view of the fact that a Mozambique virus strain produced subclinical infection in experimental monkeys which were then resistant to challenge with monkey and human virulent Lassa virus from Sierra Leone. We have explored the use of monoclonal hybridoma antibodies generated against relatively less hazardous arenaviruses to define antigens cross-reactive with the important human pathogens of the group. Here we describe the use of monoclonal antibodies directed against LCM, virus to define antigenic specificities shared among LCM, Lassa and Mozambique viruses.

Antibodies, Viral↗
PubMed1980

Virus-induced immune complex disease: specific anti-viral antibody and C1q binding material in the circulation during persistent lymphocytic choriomeningitis virus infection.

Mice of several strains persistently infected with lymphocytic choriomeningitis virus (LCMV) mount continuous anti-LCMV immune responses leading to the formation and tissue deposition of immune complexes. Such mice carry infectious virus-immunoglobulin (presumably anti-LCMV antibody) complexes in the circulation. We have now determined that anti-LCMV antibody both complexed and free is found in the circulation of mice persistently infected with LCMV. This antibody reacts specifically against the three main LCMV structural polypeptides: nucleoprotein, 63,000 m.w. and two glycopeptides, GP-1 and GP-2 with m.w. of 45,000 and 35,000, respectively. A C1q binding assay was developed and found to be effective in measuring C1Q binding substances (presumably virus-anti-viral Ig complexes) in the circulations of several strains of mice persistently infected with LCMV. With different strains of mice, the levels, time of formation, and fate of C1q binding materials varied markedly. Formation of antibodies to LCMV was correlated with the detection of C1q binding materials. Mice (SWR/J) persistently infected with lactic dehydrogenase virus also form infectious virus-Ig in their sera but deposit minimal amounts of complexes in their tissues. In such mice, C1q binding substances did not form in the circulation.

Animals↗