Modification of the virulence of yellow fever virus by cultivation in tissues in vitro

Abstract 1. 1. A pantropic or natural strain of yellow fever virus has been cultivated during a period of 21 months and through more than 150 subcultures in vitro without intercurrent animal passage. 2. 2. A simple and practical technique for the cultivation of yellow fever virus is described. 3. 3. Pantropic yellow fever virus has been cultivated for more than 130 subcultures in media consisting of serum-Tyrode solution and minced tissue of mouse embryo, chicken embryo or adult mouse testis. The same strain of virus has been grown for more than 55 passages in a medium of serum-Tyrode solution and minced adult guineapig testicular tissue, and for more than 20 transfers in the same fluid medium and chicken embryo dermis. 4. 4. Neurotropic yellow fever virus of both relatively early and late mouse passage has been cultivated for more than 120 subcultures in a medium of serum-Tyrode solution and minced chicken embryo tissue. Neurotropic yellow fever virus of late mouse passage has been grown for 55 transfers in a medium of serum-Tyrode solution and minced mouse embryonic tissue. Neurotropic yellow fever virus of late mouse passage has been cultivated for 86 subcultures in a medium of Tyrode solution and minced, chicken embryo tissue. 5. 5. The cultivated strains of pantropic yellow fever virus have exhibited consistently in Macacus rhesus a progressive loss of the power to provoke yellow fever following inoculation. Evidence is advanced to show that the degree of neurotropism for mice and monkeys of pantropic strains of yellow fever virus does not increase during cultivation in various tissue media. Experimental data upon the virulence of a pantropic strain from the time of its isolation from man through its passage in monkeys, its cultivation in mouse embryonic tissue and its passage again through monkeys is presented. 6. 6. A strain of pantropic yellow fever virus grown for 92 subcultures in a medium of serum-Tyrode solution and mouse embryonic tissue when passaged for 30 transfers in Macacus rhesus only slowly regained its virulence for monkeys. The increase of virulence thus provoked was manifested by the death of 6 of 30 monkeys in the series of yellow fever. None died of yellow fever virus encephalitis. 7. 7. Monkeys become highly immunized against yellow fever if inoculated with the pantropic virus cultivated in mouse embryonic tissue together with human immune serum. Demonstrable serum immunity is produced even if the inoculum of virus contains no more than 34 minimal lethal doses for a mouse and the amount of immune serum (titre 128) equals 5·0 c.c. per kilogram of body weight. The concomitant injection of human immune serum (titre 128) in a ratio of 0·5 c.c. per kilogram of body weight completely or nearly completely protects monkeys against the demonstration of circulating virus from the day of inoculation to the appearance of serum immunity. 8. 8. The antigenic power for immune man of the neurotropic virus of mouse brain origin and the cultivated pantropic virus of mouse embryonic tissue origin is approximately the same. For previously immunized man the serum antibody titre rises rapidly following inoculation of virus, to reach a peak usually at 2 weeks, and falls rapidly thereafter to approach its initial level at 4 weeks. 9. 9. The results of the immunization of 26 persons with pantropic yellow fever virus cultivated in mouse embryonic tissue, in the presence of an existing passive immunity produced by the concomitant injection of a titrated quantity of human immune serum are recorded. The reactions following inoculation in this short series were minimal or absent. The sera of thirteen individuals in this group which were titrated for protecting antibodies during the period from 14 to 28 days following inoculation showed titres ranging from 8 to —256.