Virginia I. Babcock

Virus biographies. II. Growth of herpes simplex virus in tissue culture

Abstract Monolayer cultures of the human epidermoid cancer cell line HEp 2 were infected with the HF strain of herpes simplex virus (herpes virus hominis) by using an inoculum of approximately 1 infectious unit per cell. At 1, 2, 4, and 7 days thereafter the cultures were studied for number of remaining cells, infectivity of cells and culture medium, cytopathic changes, viral antigen (fluorescent antibody technique) and viral morphology and development (electron microscopy). At day 1 (16 hours) infectivity of the culture medium had decreased from the initial level and about 1% of the cells were infected. There was no obvious change in number of cells and the only evidence of cell damage was a probable reduction of cytoplasmic RNA (as judged by acridine orange staining) and margination of the chromatin in a few nuclei. A very few cells had viral antigen sharply concentrated at the nuclear membrane or distributed irregularly and diffusely throughout the nucleus. Rarely a cell contained scattered intranuclear single-membraned virus particles or dense particles resembling viral nucleoids with no membrane. At day 2 about 15% of cells contained infective virus and there was a concordant increase in antigen and virus particles and nuclear DNA, but there was still no major evidence of cell destruction. By day 4 cell destruction was apparent on routine microscopy and progression of the infectious process was also evident by the fluorescent antibody and electron microscopic studies, but the characteristic changes of the herpes virus infection were most frequent in the day 7 study when a majority of the cells which remained on the glass were infected. Intranuclear inclusions were not seen by either optical or electron microscopy. The most characteristic location of viral antigen was at the nuclear membrane as either a fine line or a thickened band encircling part or all of the nucleus. Antigen was often present within the nucleoplasm also. On days 1 and 2 this intranuclear antigen was usually distributed irregularly and diffusely but at 4 and 7 days it also occurred as distinct sharply outlined particles. The usual intranuclear virus particle consisted of a dense spherical nucleoid with a maximum diameter of 60 mμ, surrounded by a single membrane of about 100 mμ in diameter, but empty particles and naked nucleoids were numerous, and double-membraned particles of about 160 mμ in diameter were occasionally seen within the nucleus in advanced infections. Virus particles were never found in crystalline arrays. They were most numerous near or in contact with the inner lamella of the nuclear membrane or between the inner and outer lamellae. There was striking thickening and progressive evagination of the inner lamella at points of contact with virus particles, and it seems clear from these morphologic alterations that this altered inner lamella of the inner nuclear membrane becomes the second membrane of the virus particle. Extreme proliferation and plication of the nuclear membrane was common, such that the perinuclear region became a complex mixture of virus particles and membranous projections. This often produced structures resembling pods of peas, with the membrane enclosing a row of single- or double-membraned virus particles. Virus particles in the cytoplasm were almost always surrounded, either individually or as a group, with a thin third membrane, thus resulting in triple membrane particles with a total diameter of 185 to 200 mμ or a cluster of double membrane particles in a thin-walled sac. The results of this study are compared with previously published studies of herpes simplex and other viruses of the herpes group.

Obstructive Uropathy in Laboratory Mice.

Summary Proteinaceous bladder plugs are reported as a cause of acute urinary obstruction and death in laboratory mice.

Effect of Steroids on Human Cell Cultures; Sustaining Effect of Hydrocortisone.

Summary The effect of 8 steroid hormones on 12 lines of human cells was studied in tissue culture. The steroids included estrogens, androgens, progestins, and hydrocortisone. The cell lines included amnion, epidermoid carcinomas, adenocarcinomas and sarcomas. Cytotoxic effects were not acute, but developed after several days if at all. No consistent relationship was apparent between minimum cytotoxic dose and endocrinologic characteristics of the steroids or type of cell. Testosterone and androsterone were toxic to most cell lines at 10 γ/ml. The ovarian and cervix adenocarcinoma cells were sensitive to most of the steroids at 10 γ/ml. Toxicity at 1.0 γ/ml was rare. Hydrocortisone at 2.5 to 10 γ/ml greatly extended the duration of good growth in cell lines HEp 1 and RPMI-41.

Effect of cortisone, related hormones, and adrenalectomy on susceptibility of mice to virus infections.

Summary Large doses of cortisone greatly increased the susceptibility of mice to lethal infection by West Nile, Ilheus, and Bunyam-wera viruses. ACTH in massive dosage produced the same effect with West Nile virus. Desoxycorticosterone, testosterone, progesterone, and estradiol in massive doses had no significant effect on these virus infections in mice. Anopheles B virus was not infective by intraperitoneal route even in cortisone treated mice. Adrenalectomy did not influence susceptibility to virus infection. The possible mechanisms of the cortisone effect are discussed and it is concluded that the effect of cortisone upon virus infection is probably due to a direct effect of cortisone upon the host.

Stable cell line of rat nephroma in tissue culture.

Summary An embryonal nephroma, which occurred spontaneously in a rat and had been maintained by serial transplantation, has now been established in tissue culture as a stable cell line and has been maintained in continuous passagefor more than 5 years. It is readily transplantable to young rats, subcutan-eously or intraperitoneally, by inoculation of a million or more cells butsuch transplants often regress spontaneously.

SELECTIVE TISSUE LOCALIZATION OF HUMAN CANCER TRANSPLANTS IN NEWBORN RATS

SUMMARY Newborn rats were inoculated via the umbilical vein with suspensions of tissue-cultured cell lines of human cancer (HEp 2 and J-111). Fluorescent antibody studies demonstrated that many of these cells were deposited in the liver, but they seldom grew to produce tumor nodules in the liver. When they did grow in liver (1/11 rats with HEp 2 and 4/16 with J-111), implants also grew in the lungs. This is in contrast to results of transplantation of these cells into newborn rats via the tail vein, which produced progressively growing tumors in the lungs and adrenals of a great majority of the recipients, and never produced implants in the livers. The observations are interpreted as indicating a role of local tissue factors in determining receptivity to the growth of tumor cell implants. The observations provide experimental evidence in support of the concept that distribution of tumor metastases may be due in part to differences in the susceptibility of various tissues for the growth of randomly distributed cancer cells.

SPECIFIC ADOPTIVE AND PASSIVE IMMUNOTHERAPY BY PARABIOSIS FOR SYNGENEIC MOUSE AND RAT TUMORS

A laboratory model of cancer that is suitable for immunotherapy studies, and that simulates the conditions encountered in clinical oncology, requires experimental animals that either have progressively growing autochthonous or syngeneic primary tumors or have had such tumors treated by methods that leave a residuum of local or metastatic tumor cells. In the studies of adoptive and passive imrnunotherapy summarized here, we utilized both of these tumor situations. Of the numerous methods that can be used for immunotherapy, the various methods of transferring cellular immunity are of particular interest to oncologists because of the many indications that cell-mediated immunity plays a major role in specific immunologic resistance to tumor growth. In clinical medicine, the technique of plasmapheresis permits the frequent transfer of large numbers of lymphoid cells. This technique has not been scaled down for application to small laboratory animals, but a similar massive and sustained transfer of leukocytes (and serum) can be achieved by parabiosis. By using syngeneic animals as “donors,” the immunity that is transferred to the “patients” can be tumor specific. Of course, this technique provides a combination of adoptive and passive immunotherapy: it does not permit dissection of mechanisms; the result is the sum of the effects of all mechanisms that affect the tumor. Further details of these studies will be published elsewhere.’-:’

Propagation of Bunyamwera. West Nile, Ilheus, and Br I viruses in human cells in tissue culture.

Summary 1. Bunyamwera virus was grown in roller-slide tissue cultures of human lymph nodes (fibroblastic type cells). Propagation of virus was demonstrated by increases in virus titers in short-term virus growth studies, by long persistence of virus in tissue cultures as contrasted with rapid disappearance in cell-free preparations, and by persistence of virus after serial passages had diluted the original virus inoculum far beyond its minimal infective dilution. 2. Similar but less extensive studies demonstrated the propagation in human fibroblast tissue cultures of West Nile, Egypt 101, Ilheus, and Br I viruses. 3. Bunyamwera virus showed changes interpreted as an adaptation phenomenon during serial passages in tissue cultures. These changes were reversed by serial intracerebral passage in mice. 4. None of these viruses caused appreciable cytopathol-ogy under these experimental conditions, even after prolonged growth in tissue culture. 5. Bunyamwera, Egypt 101, and Ilheus viruses were also grown in tissue cultures from various human cancers. It was not possible to determine whether virus was propagating in the cancer cells, or in nonneoplastic stromal cells. 6. Egypt 101 virus propagated in cells from an immune patient, but not in the presence of immune serum.

Growth of Human Cancer Cells as Lung Metastases in Immunologically Tolerant Rats

Summary Newborn rats were made tolerant to human cell antigens by intravenous injection of Amnion B cells, a permanent human cell line of normal origin. At various intervals thereafter each animal, and non-tolerant litter mate controls were challenged by SC and IV injections of the malignant human cell line J-111. Tumor nodules of J-111 cells grew SC and in the lungs of most of the tolerant rats challenged at ages from 7 to 14 days, but not in their controls. Challenge at age 19 days produced SC tumors but there was no growth from the IV inoculum.