Frederick C. Robbins

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.†

Isolation of Attenuated Rubella-Vaccine Virus from Human Products of Conception and Uterine Cervix

Abstract To evaluate the fetal hazard of accidental administration of live rubella vaccine during pregnancy, the vaccine was given to 35 women certified for legal abortion. Twenty-four of these wer...

DIFFERENCES IN INTERFERON CONTENT IN TISSUES OF MICE OF VARIOUS AGES INFECTED WITH COXSACKIE B1 VIRUS.

Summary Mice 24 hours to 6 weeks old were infected with Coxsackie B1 virus. Their tissues were assayed for virus and interferon content. It was demonstrated that adult mice produced interferon in all the tissues which became infected, whereas suckling mice produced small amounts of interferon in their livers only. There is a direct relationship between interferon titers and rapidity with which virus disappears from the tissues. A hypothesis is offered that the difference in outcome of Coxsackie B1 infection in the suckling and older mouse is in large part to be explained by inability of the cells of the immature animal to elaborate interferon.

Effect of Actinomycin D on Growth of Rubella Virus in Tissue Cultures

Summary Multiplication of rubella virus in primary and continuous-line African green-monkey kidney cells was delayed by addition of low doses of actinomycin D to the culture medium before or at the time of infection. Addition of the drug 2 hr after infection was less effective. However, virus replication became normal after 3 to 8 days, although cellular RNA synthesis in drug-treated control cultures continued to be inhibited.

Comparison of Neutralization Rate and Agar Diffusion Methods for Intratypic Sero-Differentiation of Polioviruses.

Discussion and summary Although these data are derived from examination of only 5 strains of type 1 poliovirus they are representative of the results of a larger number of similar observations(6). It is evident that when antigenic differences were detected by the agar diffusion method they were confirmed by the neutralization rate test. Thus, the results obtained with these 2 methods of determining intratypic antigenic differences would appear to be highly comparable. When these findings are considered with those of Nakano and Gelfand(4), who showed that the neutralization rate test and their modification of the method of Wecker gave similar results, it can be concluded that in spite of the potential differences, the 3 technics are in remarkable agreement. Our results are at variance with those of McBride(2) in that a clear difference could be demonstrated between the Mahoney and LSc-2ab strains, the latter being derived from the former, whereas he found no such distinction, but employed only antiserum prepared against Mahoney. Nakano and Gelfand(4) did find the 2 viruses to be antigenically distinct, but this was most evident in tests with the antiLSc serum. The discrepancy in results in the 3 laboratories might be explained by differences in the viruses tested or more likely by differences in specificity of the antisera employed. That the latter is the probable explanation is demonstrated by the results of tests in our laboratory with anti-Mahoney serum kindly supplied by Dr. Gelfand, which were in agreement with those of Nakano and Gelfand(4) and failed to differentiate clearly LSc and Mahoney viruses. However, with suitable technics not only can the related Mahoney and LSc strains be distinguished one from the other, but alterations which occur during multiplication in the human intestine can be detected. Changes during passage in the human intestine are shown by the few data given here and further documented by more extensive observations in our laboratory(6).† Nakano and Gelfand(4) indicate that they too have demonstrated antigenic differences between the viruses excreted by vaccinated persons and the vaccine virus. Similar observations have been made by Wassermann and Fox(7). Thus, it is apparent that the antigenicity of polioviruses is not such a stable trait as had been hoped.

Experimental Rubella in Rhesus Monkeys.

Summary In an attempt to transmit rubella to rhesus monkeys, 6 yearling male animals from an isolated, and therefore presumably susceptible population, were inoculated by several routes with throat washings and acute phase sera from selected rubella patients. During 21 days of observation following inoculation, none of the animals showed any physical signs of rubella. Serial daily total and differential leukocyte counts showed leukopenia and lymphocytosis of slight degree. In none of the animals tested was neutralizing antibody to rubella virus demonstrable prior to inoculation, but neutralization tests performed on pre-inoculation and post-inoculation sera showed a significant rise in titer in 4 of the 6 animals inoculated. It is concluded, therefore, that rhesus monkeys are probably susceptible to infection with rubella virus, but that the infection is usually of the inapparent type.

Toxic Effect on the Chick Embryo of Homologous Tissue Suspensions Following Intravenous Inoculation

Conclusion The toxic effect in vivo of certain tissue derivatives exhibits biologic specificity. This specificity appears to parallel that of thromboplastin on the clotting of blood plasma in vitro.