James A. Robb

Pathogenic murine coronaviruses. II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V.

Abstract We have identified nine intracellular virus-specific proteins in cells infected with JHMV or A59V. Seven virus-specific proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two additional virus-specific proteins were detected by two-dimensional gel electrophoresis. The A59V- and JHMV-specific proteins differ slightly in molecular weight. Four of the nine proteins are structural proteins. The synthesis of the nine virus-specific proteins is noncoordinate with respect to time.

Pathogenic murine coronaviruses I. Characterization of biological behavior in vitro and virus-specific intracellular RNA of strongly neurotropic JHMV and weakly neurotropic A59V viruses

Abstract JHM virus (JHMV) and A59 virus (A59V) are neurotropic members of the hepatoencephalitis group of murine coronaviridae. JHMV has a markedly greater neurotropism for weanling BALB/c mice than does A59V. Both viruses display one-hit kinetics when grown in vitro in 17CL-16 cells, a clone of BALB/c3T3 cells. Virus-specific intranuclear, cytoplasmic, and surface antigens have been observed for both viruses by immunofluorescence. The intranuclear antigen appears first at about 2 hr after infection (hpi) followed by the development of the cytoplasmic and surface antigens at 3 hpi at 38.5°. Most, if not all cells that develop the intranuclear antigen, produce cytoplasmic antigen and presumably progeny virus. Progeny virus production is independent of cell fusion and formation of syncytia. Virus-specific ribonucleoprotein is synthesized in the presence of 1 μg/ml actinomycin D, a concentration sufficient to inhibit the synthesis of cellular ribonucleoprotein species that have sedimentation properties similar to the virus-specific species. The virus-specific ribonucleoprotein species that is resistant to 10 mM EDTA, presumptive virion ribonucleoprotein, has a sedimentation value in sucrose of about 230 S for JHMV and 200 S for A59V. The species of virus-specific ribonucleoprotein that are sensitive to 10 mM EDTA presumptive messenger ribonucleoprotein, are about 40–100 S in sucrose for both viruses. The purified presumptive virion RNA is about 50 S in sucrose for both viruses. The major species of presumptive mRNA of both viruses is about 18 S with secondary species of about 28 S in sucrose. Denaturation of the virus-specific RNA with heat and dimethylsulfoxide does not appreciably alter the sedimentation profiles of either the presumptive virion RNA or mRNA species.

Pathogenic murine coronaviruses. III. Biological and biochemical characterization of temperature-sensitive mutants of JHMV.

Abstract JHMV is a neurotropic member of the hepatoencephalitis group of murine coronaviridae. The characteristics of the biology and intracellular viral RNA synthesis and the intracellular viral protein synthesis of JHMV are discussed in the two previous papers, respectively. This paper describes the neuropathogenesis of JHMV and the isolation and characterization of 34 temperature-sensitive mutants of JHMV. These mutants were selected for their inability to induce syncytia formation after low multiplicity infection (m.o.i. = 0.1 iU) in BALB/c 17CL-1 cells at 38.5° as compared to the induction of syncytia at 33°. N-Methyl-N′-nitrosoguanidine (14 mutants) and 5-fluorouridine (20 mutants) were used as mutagens at a concentration that reduced infectivity by 90–95%. Characterization of these mutants included: induction of syncytia; synthesis of JHMV-specific intracellular RNA; progeny yields at 33, 37, and 38.5°; synthesis of JHMV-specific antigens as determined by indirect immunofluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; virion thermostability; neuropathogenesis including isolation of virus from infected brain, immunofluorescence of infected brain, and histopathology of brain and spinal cord by light and transmission electron microscopy; ability to protect mice from a lethal JHMV infection; and complementation. RNA-minus ( 17 34 ), RNA-intermediate ( 14 34 ), and RNA-plus ( 3 34 ) groups were defined. One mutant, N3, produces chronic meningitis and demyelination without typical JHMV encephalitis in spite of the fact that neurons are infected as detected by immunofluorescence. This altered neuropathogenesis cannot be explained by “leakiness” or reversion. In addition, non-temperature-sensitive variants of JHMV have been selected for altered neuropathogenesis and are described.

The replication of murine coronaviruses in enucleated cells

Abstract Coronaviruses JHMV and A59V have been shown to replicate, produce viral-specific antigens and cytopathic effects (CPE) in enucleated 17CL-1 cells.

Immunologically mediated and nonspecific inflammatory responses in the middle ear.

Abstract The immune response to antigens presented to the middle ear has been postulated as a factor in the pathogenesis of serous otitis media. We have previously described the immune response to keyhole limpet hemocyanin (KLH) presented to the middle ear in guinea pigs. In this report we describe the histologic and cytologic response in the middle ear cavity. KLH with or without alum was introduced into the middle ear. The nonspecific inflammation caused by alum alone served as comparison. No effusion was observed after any of these interventions. Large numbers of polymorphonuclear neuropils (PMNs) infiltrated the middle ear cavities of all subjects within 24 hours; this PMN response was gradually supplanted by lymphocytic and mononuclear cell infiltration. Eosinophils and basophils were noted only after introduction of antigen, with or without alum. All subjects showed submucosal edema and some degree of epithelial hyperplasia. Proliferation of respiratory epithelium was only seen after injection of alum with or without antigen. The appearance of plasma cells in the middle ear mucosa, indicating the development of a local immune response, was noted only after injection of antigen with alum adjuvant. The results indicate that both the development of a potent local immune response and the elicitation of a strong inflammatory response can occur in the middle ear without development of effusion.

Integration of SV40 DNA and induction of cellular DNA synthesis after a ts SV40 infection.

Abstract The early temperature-sensitive mutant of simian virus 40 (SV40), ts∗101 , was characterized in nonpermissive Chinese hamster (ChH) and permissive monkey CV-1 cells for integration of viral DNA and induction of cellular DNA synthesis. At 42° in CV-1 cells, this mutant did not induce the synthesis of T antigen or cellular and viral DNA. The viral DNA did not integrate into the CV-1 DNA, although nuclear penetration occurred. In ChH cells the mutant induced T antigen at 40° and 42°, and the viral DNA integrated into ChH DNA, but ChH DNA synthesis was not induced. These findings suggest that the integration of SV40 DNA does not depend on the replication of cellular DNA and that, at least functionally, integration precedes the induction of cellular DNA synthesis.

Adaptive immunity of the tympanic membrane

In a previous experiment an antiserum was developed in the rabbit from the lamina propria of the tympanic membrane of the guinea pig. In the present study 18 Hartley guinea pigs were used in an in vivo experiment in which the antiserum (RAGTM IgG) served passively to immunize the animals subjected to various forms of trauma to the right tympanic membrane. Two groups (18 animals) immunized with normal rabbit IgG or normal saline served as controls. The left tympanic membrane remained untouched in all groups and served as an internal control. Various forms of trauma (infection, cauterization, section), various times (one to 21 days), and diverse techniques of staining (immunofluorescence, complement, and immunoperoxidase) were studied. The results indicate that the combination of trauma and sensitization in the first group evokes a particular response in the lamina propria of the tympanic membrane in immunized animals (RAGTM IgG). The form of trauma does not influence the results, but time seems to be an important factor. This work addresses several questions concerning the possible role of the combination of trauma and sensitization in conditions that involve the tympanic membrane and middle ear clinically.

Antigenicity of the Guinea Pig's Tympanic Membrane

The immunogenicity of the middle ear constituents is a matter of prime importance. In this work, the authors were interested in the antigenicity of the guinea pigs tympanic membrane. They reached the conclusion, after developing an antiserum (IgG), that the tympanic membrane of the guinea pig can be antigenic in the rabbit. After purification and absorption, a cross-reactivity remains between the tympanic membrane and the mucosae of the upper respiratory system.

Immune response of guinea pig tympanic membrane.

The authors present the results of their experiment on the immune response of the guinea pig tympanic membraneTM.

Altered Nuclear Structure during Productive and Abortive Infections with SV40 DNA

A striking alteration in nuclear morphology, detected by simian virus 40 (SV40) large-T antigen-specific immunofluorescence, occurred in cells infected by form I SV40 DNA but not by SV40 virions. This alteration did not require viral or cellular DNA synthesis.