Cultivation of Vaccinia Virus by Rivers' Method

There are at present two reliable methods of growing vaccinia virus in avian tissues under bacteriologically sterile conditions, namely, those of Rivers and his associates 1 and of Goodpasture and his coworkers. 2 Our experience, which was confined to the former technique, has convinced us of its extreme practicability for the cultivation of vaccinia virus. Previous reports of the successful application of this means of propagation of vaccinia virus, for use in Jennerian prophylaxis in man, have been made by Rivers, 1 Rivers and Ward, 3 Herzberg, 4 and Coffey. 5 With the exception of those of Rivers and his colleagues, none of these communications deal with prolonged cultivation of the virus.

Two strains of vaccinia virus have been grown during our cultivation experiments, one known as neurolapine (Reichgesundheitsamt) given to us by Haagen 6 after 24 mouse-brain passages; the other, Rivers' second revived cultivated strain of dermal vaccine virus after 88 subcultures in minced chicken embryo tissue and Tyrode solution. 3

The neurolapine virus was cultivated for 30 passages in a medium of whole chicken embryonic tissue and normal monkey serum—Tyrode solution. The first series of cultures were inoculated with the supernatant fluid after centrifugalization of a 2% suspension of virus infected mouse brain in 0.9% sodium chloride solution. Each subsequent transfer was made at 4- to 7-day intervals. The infectivity of the supernatant fluid from each series of subcultures was routinely tested by the intracerebral inoculation of a group of 6-12 mice, of which number nearly all died of vaccinal meningitis 4 to 6 days following inoculation.

Rivers' second revived strain of cultivated vaccinia virus was grown for 94 subcultures during a period of 15 months without intercurrent animal passage in a medium of minced whole chicken embryo tissue and Tyrode solution.