John F. Enders

Propagation in tissue cultures of cytopathogenic agents from patients with measles.

Conclusion and Summary The findings just summarized support the presumption that this group of agents is composed of representatives of the viral species responsible for measles. Eight agents exhibiting the properties of viruses have been isolated in cultures of human or simian renal cells from the blood or throat washings of five cases of typical measles. Multiplication of the agents in vitro is accompanied by characteristic changes in the cells. Primarily these changes consist in the formation of syncytial giant cells wherein the chromatin assumes a marginal position and is replaced centrally by an acidophilic substance of unknown nature. The cytopathogenic effect of at least one of the agents is inhibited by convalescent phase measles sera from other patients with measles. Antigen appears during cultivation in vitro of the measles agents that reacts specifically in complement fixation tests with convalescent phase measles sera.

Further studies on an inhibitor of viral activity appearing in infected cell cultures and its role in chronic viral infections

Abstract Fluids from primary human kidney and amnion cell cultures infected with a chick embryo-adapted strain of type 2 poliovirus inhibit the activity of several different viruses. The factor (or factors) responsible for this inhibition is inactivated by treatment with trypsin and is not dialyzable. It cannot be neutralized with type 2 poliovirus antiserum nor sedimented with infectious virus. Attempts to identify it either with ribonucleic acid or ribonuclease have failed. Evidence is presented indicating that the factor interferes with the spread of virus and with viral reproduction. The initiation and maintenance of a chronic infection with type 2 poliovirus in primary human amnion cell cultures depends at least in part upon the presence of this factor. It has been demonstrated in a chronic infection of HeLa cells, but here its role in maintaining the infection has not been finally established.

Multiplication and Cytopathogenicity of Simian Vacuolating Virus 40 in Cultures of Human Tissues.

Summary Multiplication of SV40 virus has been demonstrated in primary cultures of several human tissues. The virus multiplied without cytopathic effect during the first passage in these systems. In subsequent passages in kidney cell cultures, CPE was noted which included nuclear changes, increased cell proliferation and necrosis.

Complement fixation with Brunhilde and Lansing poliomyelitis viruses propagated in tissue culture.

Summary Antigens which gave specific complement fixation were concentrated from the supernatant fluids of tissue cultures infected with poliomyelitis viruses (Lansing and Brunhilde strains) by a method of ultrafiltration. Their anticomplementary activity was abolished by heating. The drop method of Fulton and Dumbell was used and offered a practical way of performing the tests with minimal amounts of antigen. Specific antibodies were demonstrated in the sera of hyperimmunized monkeys as well as in sera of poliomyelitis patients.

Propagation of Measles Virus in Cultures of Chick Embryo Cells.

Summary An egg-adapted strain of measles virus has been propagated in cultures of chick embryo cells throughout 24 serial passages. Beginning with the 6th passage an abrupt decrease occurred in the time required to attain maximal concentration of virus in the fluid. The concentration of virus was comparable to that found in cultures of infected primate cells. In the 5th chick cell passage the virus began to produce cytopathic changes closely resembling those occurring in human epithelial cells infected with measles virus. The identity of the agent was confirmed in neutralization tests with measles antisera. In chick cells maintained with a medium of known composition virus production approached that in cultures nourished with serum and tissue extracts. Inoculation of rabbits with the virus propagated in chick cells was followed by development of neutralizing antibodies specific for the agent of measles.

Cultivation of Measles Virus in Human Amnion Cells and in Developing Chick Embryo.

Summary 1. A strain of measles virus originally isolated in cultures of human renal cells has been propagated throughout 28 serial passages in cultures of human amnion cells. In the latter system it induces 2 types of cytopathic change: (1) formation of “syncytia” or “multinuclear giant cells” in which intranuclear and intracytoplasmic inclusions are prominent features; (2) only recently recognized, the assumption by individual epithelial cells of a characteristic fusiform or stellate configuration. In certain of these affected cells eosinophilic intranuclear inclusions are present that resemble those found in the nuclei of the syncytia. Eventually both types of change terminate in cellular necrosis and disintegration. In contrast to the dual response of amnion cells only the formation of syncytia has been observed in cultures of renal cells infected with the virus. 2. The Edmonston strain of measles virus from the 28th passage in human amnion cells was inoculated into chick embryos. In this host it has been maintained throughout 12 successive passages. Multiplication of the agent was demonstrated by addition of chick embryonic materials to cultures of human amnion cells. This procedure was necessary since no definite indication of viral activity has as yet been distinguished within the egg. 3. The virus present in the 9th chick embryo passage was identified as the measles agent in complement fixation and virus-neutralization tests with acute and convalescent phase measles sera.

Cultivation of poliomyelitis virus in cultures of human foreskin and embryonic tissues.

Recently, the propagation in vitro of the Lansing strain of poliomyelitis virus in human embryonic tissues was reported and evidence presented that this virus is capable of multiplying in cells other than those of nervous origin. 1 These experiments have been continued and this agent now has been carried for a total period of 224 days through 13 serial cultures in which the tissue consisted of mixed human embryonic skin and muscle. This strain has also been maintained for 173 days in two lines of 11 serial cultures each and composed respectively of human embryonic intestine and brain. Additional experiments described here in a preliminary manner are reported. Two objectives were in mind: (a) to determine whether the Lansing strain was capable of multiplying in completely differentiated non-nervous tissue as well as in embryonic tissue; (b) to determine whether the Brunhilde strain of poliomyelitis virus-a strain immunologically distinct from the Lansing group 2 and not adaptable to rodents-could, like the Lansing strain, be cultivated in non-nervous human embryonic tissues. Propagation of the Lansing strain in human foreskin tissue. As a source of completely differentiated non-nervous tissue fragments of human foreskin were employed. The use of this tissue was suggested by the report of Blank, Coriell, and Scott, 3 who explanted it on the chorioallantoic membrane according to the method of Goodpasture. The material was derived from patients between 4 and 11 years of age. Each prepuce was sufficient for the preparation of at least 8 cultures and before mincing was washed 2 or 3 times in nutrient fluid 4 containing 50 units each of streptomycin and penicillin per ml. The fluid phase of the cultures, which contained the same concentration of antibiotics was removed and replaced at intervals of 4 days.

Cytopathogenic Effect of Poliomyelitis Viruses In vitro on Human Embryonic Tissues.

Summary and discussion The experiments which have been described demonstrate the capacity of the Lansing and Brunhilde strains of poliomyelitis virus to cause cell injury and death. This cytopathogenic property is revealed (1) by degenerative changes produced in infected tissue fragments in flask cultures and apparent on histologic examination; (2) by failure of such tissue fragments to exhibit normal cell migration when explanted to plasma cultures; (3) by degeneration of newly emigrated cells in roller-tube cultures; (4) by decreased acid production by infected cells. The conclusion that certain of these manifestations of injury are induced as a result of infection by the virus is further supported by the fact that type specific immune serum prevents their development. These phenomena are of interest from two general points of view. First, they leave no doubt that poliomyelitis virus in vitro can multiply in cells other than those of the nervous system and cause profound injury of such cells. Secondly, they provide criteria by which the presence of the virus can be recognized in vitro and hence may afford a basis of technics for isolating virus from patients or animals, for the quantitative assay of virus, for serologic typing and possibly for the screening of chemotherapeutic and antibiotic substances. Further study will be required of the reliability and practicability of the application of these phenomena to such ends.†

Bovine Amniotic Fluid as Tissue Culture Medium in Cultivation of Poliomyelitis and Other Viruses.

Summary Bovine amniotic fluid induced active cell growth when substituted for balanced salt solution and bovine serum ultranltrate in a medium used for the cultivation of embryonic and mature human tissues in roller tube cultures. The growth was more luxuriant than that observed in comparable cultures maintained with the balanced salt mixture. Moreover, superior yields of poliomyelitis viruses have been obtained in cultures nourished with bovine amniotic fluid as a constituent of the medium and in such cultures the minimal infecting dose of these agents has also proved to be smaller. Bovine amniotic fluid does not interfere with the neutralization of poliomyelitis virus by specific antibody. Through the use of bovine amniotic fluid the labor and expense involved in the preparation and maintenance of tissue cultures applied to the propagation of poliomyelitis virus and certain other agents of this class have been reduced.

An interferon appearing in cell cultures infected with measles virus.

Summary An inhibitor of viral activity appears in cell cultures inoculated with live measles virus. This inhibitor has been separated from the infectious particles and complement fixing antigens produced in the same cultures. It is not neutralized by measles antiserum. It does not inactivate the virus when mixed with it, but reduces the extent of cytopathic changes as well as quantity of virus produced.