Manfred Mussgay

Bunyaviruses and Bunyaviridae

A new family is described, the Bunyaviridae, which contains a single genus, Bunyavirus. The main characteristics of the family are as follows: single-stranded RNA, total molecular weight about 7 X 10(6) daltons, probably in three segments. Virions spherical, enveloped particles 90-100 nm in diameter. Envelope contains at least one virus-specified glycopeptide. Develop in the cytoplasm, mature by budding into smooth-surfaced vesicles in the Golgi region or nearby. Internal ribonucleoprotein composed of long strands 2-2.5 nm broad. There are at least 150 members, 87 serologically related bunyaviruses and other probable bunyaviruses.

Electron microscopic and biological studies on the growth of Venezuelan equine encephalitis virus in KB cells

Abstract The development of Venezuelan equine encephalitis virus was investigated by biological assay methods combined with electron microscopic observations. KB cells were exposed to 260 plaque-forming units per cell for 1 hour at 37°. During this time, about 90% of the cells became infected and an increase of virus titer could be detected 4–5 hours after infection. The highest titer of extracellular virus was observed 11 hours after infection. Each cell produced 2700 plaque-forming units. After passing the surface membrane of the cell, the virus was released in about 20 seconds. On the basis of electron microscopic observations, it is suggested that the virus matures on membranes which surround cytoplasmic vacuoles. From these membranes, the spherical virus particles with diameters of 40–45 mμ detach and migrate to the surface membrane of the cell, and from there they are extruded. Besides the virus particles, other spherical particles with a diameter of 25 mμ were observed. These particles were found attached to membranes on which the virus matures, or clusters of them were found distributed in the cytoplasm. It could not be decided whether these 25-mμ particles are virus precursors or are a by-product of virus development. Virus particles were seen before an increase of virus titer could be detected. Although VEE virus was observed maturing intracytoplasmically, most of the cell-associated virus was neutralized by specific antibodies. These discrepancies between morphological findings and biological results are discussed.

Isolation of a p15 polypeptide from bovine leukemia virus and detection of specific antibodies in leukemic cattle

Abstract Bovine leukemia virus (BLV) was isolated and purified from cultivated leukocytes of leukemic cattle and from long-term cultures of BLV-infected fetal lamb kidney cells. Because of their morphological characteristics and the detection of virion-associated reverse transcriptase activity, the particles were considered to be oncornaviruses. From purified BLV preparations, a precipitating basic protein designated BLV p15 with an apparent molecular weight of 14,800 was isolated. The antigen was heterogeneous in charge with an isoelectric point of 8.3 (8.2–8.5). The antigen cross-reacted with antisera from leukotic cattle and sheep in both agar-gel immunodiffusion and complement fixation tests. No serological relationship was detected, however, either to murine and feline leukemia viruses or to bovine syncytial and Maedi-Visna viruses by either technique. A total of 564 sera from different cattle farms with a history of enzootic bovine leukosis was investigated in the double immunodiffusion test using the purified BLV p15 antigen. In addition, serologically positive reagents were detected among hematologically positive (51.5%), suspect (14.5%), and negative (0.9%) animals.

Detection of cytotoxic lymphoid spleen cells from stu-mice with moloney sarcoma by a 3h-proline microcytotoxicity assay.

A microcytotoxicity assay with3H-proline prelabeled target cells was used for the detection of sensitized lymphoid spleen cells from STU inbred mice inoculated with Moloney sarcoma virus (MSV-M) or ascitic MSV-M tumor cells. The target cell line was derived from ascitic MSV-M tumor cells. With regard to the specificity of the assay nonimmune spleen cells displayed no or only a weak cytotoxicity against these cells, and this was also the case when3H-proline-labeled secondary cultures of syngeneic mouse embryo cells were exposed to both sensitized and nonimmune spleen cells. The time-course pattern of the development of cytotoxic lymphoid spleen cells in STU mice inoculated intramuscularly either with MSV-M or ascitic MSV-M tumor cells was studied. At the stages of tumor development, peak tumor size, and tumor regression the lymphoid spleen cell preparations were found to have relatively strong cytotoxic activity independent of whether the tumor was induced by MSV-M inoculation or tumor cell transplantation. However, in the latter case effector cells appeared earlier and were demonstrable for a longer period than in MSV-M-inoculated mice. Antitheta serum treatment of lymphoid spleen cells taken at the stage of peak tumor size abrogated the cytotoxic activity or diminished it considerably indicating a T-lymphocyte response.

Properties of components obtained by treatment of Semliki Forest virus with Tween 80 and tri(n-butyl)phosphate.

Summary Purified Semliki Forest virus was analysed by analytical sedimentation, rate zonal sedimentation, isopycnic sedimentation, polyacrylamide gel electrophoresis and electron microscopy. It was found that the spherical virus particles had a sedimentation coefficient of 241 S (249 S determined by rate zonal sedimentation), and a buoyant density in sucrose of 1.190 g/ml; one protein and one glycoprotein were present in the virus; after fixation and negative staining with phosphotungstic acid the diameter of the particles was measured to be 50 to 56 nm. Treatment of Semliki Forest virus with 0.1% Tween 80 and 1% tri(n-butyl)phosphate (TNBP) for 1 h at 4 °C destroyed infectivity and resulted in the appearance of the following haemagglutinating and immunizing virus components. (1) A spherical 211 S (217 S) component, 69 to 75 nm in diameter, consisting of an envelope and a core; one protein and one glycoprotein were present; the [3H]-uridine:[14C)-protein ratio was 2.8-fold higher than that of double-labelled untreated virus. (2) A spherical component, 70 to 73 nm in diameter, consisting of the virus envelope lacking intact cores; s 20,w = 140 S (153 S); one glycoprotein was detected; the [3H]-uridine: [14C]-protein ratio was about 1/3 of untreated virus. (3) A 135 S component. (4) Structures assumed to represent envelope fragments; much of them had a sedimentation coefficient of 49 S. It was concluded from experiments with [3H]-uridine-[14C]-amino acids double-labelled and [14C]-stearic acid-labelled virus that Tween 80-TNBP dissociates lipids and some protein components from the virus.

Preparation of Inactivated Vaccines against Alphaviruses Using Semliki Forest Virus-White Mouse as a Model. I. Inactivation Experiments and Evaluation of Double Inactivated Subunit Vaccines

Inactivation of Semliki Forest virus (SFV) with formalin, β-propiolactone, hydroxylamine and 2-ethyl ethylenimine was studied. Immunogenicity of SFV was best retained after formalin inactivation. Vacc

Investigations on complement-fixing subunits of a group A arbo virus (Sindbis)

Abstract Sindbis virus grown in chick embryo tissue cultures was concentrated by ultracentrifugation and subsequently subjected to CsCl density gradient centrifugations. Fractions containing “complete” Sindbis virus were collected. These “complete” virus particles carry infectivity, hemagglutinating and complement-fixing activity. After treatment with Tween 80 and ether a noninfectious form, more dense (1.277) than “complete” virus (1.233) and detectable by its hemagglutinating and complement -fixing activity, was released from the “complete” virus particles. In addition to this hemagglutinin, noninfectious complement-fixing components with buoyant densities equal to or smaller (1.234-1.164) than that of “complete” virus and associated with a very weak hemagglutinating activity appeared after Tween-ether treatment. These components are assumed to be split products of the viria. Exposure of “complete” virus to cobra venom resulted in the appearance of noninfectious and nonhemagglutinating complement-fixing components with buoyant densities higher (1.346-1.307) than those of hemagglutinin and “complete” virus. Evidence is presented that these “heavyrd” components represent the viral core and parts of it.

Preparation of Inactivated Vaccines against Alphaviruses Using Semliki Forest Virus-White Mouse as a Model. II. Evaluation of Formalin-Inactivated Vaccines Treated with Tri(n-Butyl)Phosphate and/or Saponin and Properties of the Inactivated Virus

Double inactivated vaccines were prepared with formalin-inactivated Semliki Forest virus subsequently treated with Tween 80-tri(n-butyl)phosphate, with saponin, or with a combination of both. These vaccines possessed a similar or even higher protective potency when compared with vaccines containing untreated formalin-inactivated virus. A considerable part of the formalin-inactivated virions are not disrupted by Tween 80-TNBP treatment in spite of the release of lipid components resulting in a higher buoyant density of the virions; in these particles, virus RNA is accessible to RNase.

Presence of splenic suppressor cells in mice bearing regressively growing moloney sarcomas and their absence in progressor mice.

Abstract Cultured spleen cells prepared from mice bearing Moloney sarcoma at the stage of peak tumor size did not show induction of blastogenesis by Concanavalin A and phytohemagglutinin as measured by 3H-thymidine uptake. Moreover, in such cultures the spontaneous 3H-thymidine uptake was markedly depressed in the presence of the mitogens, especially of phytohemagglutinin. However the concomitantly assessed activity of these spleen cells in a 3H-proline microcytotoxicity assay was strong. The depression of the mitogen response was restricted to the peak tumor size phase of the Moloney sarcoma and could not be observed during development and regression of the tumor. Spleen cells of mice bearing a non-regressing tumor with a development comparable with that of the Moloney sarcoma in the stage of progression did not show a depression of mitogen response and had no cytotoxic activity. Therefore the absence of detectable cytotoxic effector cells was not due to the activity of suppressor cells, and the growth rate of a tumor seems without influence on the development of suppressor cells.

The Bovine Leukosis Virus

Lymphocytic leukosis of cattle is characterized by neoplasia of lymphoid tissue; an increased lymphocyte count, i.e., a persistent lymphocytosis is common, but not a constant feature. There are four forms of bovine leukosis distinctly classified by epizootiologieal, clinical, and patho-anatomical criteria. Sporadic bovine leukosis comprising the calf form and the thymic form, as well as cutaneous leukosis, have a low incidence. In the enzootic form, mainly affecting cattle three or more years old, scattered lymph nodes are enlarged and often the heart, abomasum and uterus are affected. The disease occurs as multiple cases within a herd. Enzootic bovine leukosis is the most important neoplastic disease in cattle and its occurrence and economic losses have been described throughout the world. In the Federal Republic of Germany, enzootic bovine leukosis occurs predominantly in Frisian cattle and to give an impression about the incidences some figures from the State of Lower-Saxony are mentioned [10]. In 1975, the blood from cattle of 87,095 herds was examined for persistent lymphocytosis, i.e. 84.3% of all herds in the State. In 4,238 of these herds, i.e. 4.9%, animals with persistent lymphocytosis were detected, and 12,455 leukotic animals had to be slaughtered. With regard to the etiology of the disease, epizootiologic and transmission studies performed in the past gave suggestive evidence for an infectious agent likely to be transmitted both horizontally and vertically without entering the germ line. But, not before Miller et al. [8] described type C particles in short-term lymphocyte cultures derived from leukotic cows, was an association between type C particles and bovine leukosis firmly based on experimental data. However, these lymphocyte cultures were not well suited for the characterization of the bovine C type particles, and, therefore, it was a great breakthrough when Van der Maaten et al. [ 11 ] were successful in establishing long-term cultures which continued to release adequate numbers of bovine C type particles for which, by transmission experiments, suggestive evidence was given that they represent the bovine leukosis virus (BLV).