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Purification, pH Stability and Culture of the Mumps Virus^*

From the Department of Surgery, and the Department of Pediatrics, Duke University School of Medicine, Durham, N. C.

Abstract

Experiments on the multiplication of mumps virus in embryonated eggs showed that the greatest and most rapid increase of the agent in the chorio-allantoic fluid occurred in eggs inoculated after incubation for 7 days at 37.5 C. The highest levels of infectious capacity were reached on the 4th to 6th days after inoculation with seed virus and further incubation at 35 C. The increase in hemagglutinative capacity continued until the 8th post-inoculation day or the 15th day of total incubation. The discrepancy between the increases in the two properties of the virus was interpreted as due to inactivation of the infectious capacity of the virus in the chorio-allantoic sac without proportional destruction of the hemagglutinative property. The infectious titer of the chorio-allantoic fluid for embryos under optimum conditions was in the region of 10⁹ 50 per cent endpoint units per ml, and the average yield of chorio-allantoic fluid was from about 6 to 8 ml per egg, depending on the time of harvest. The pH of the chorio-allantoic fluid ranged from about 7.5 to 8.5 on the 4th and 5th days after inoculation, decreasing to a level of about pH 6.2 on the 7th or 8th day after inoculation. No differences could be seen in the virus yield resulting from the use of various dilutions, from 10⁰ to 10^–3, of previous passage chorio-allantoic fluid as seed virus. Penicillin and streptomycin or both together had no appreciable inactivating effect on the virus in 14 to 28 days and were useful in preventing bacterial growth in the titration in eggs of contaminated materials.

By means of ultracentrifugal and related procedures, the mumps virus has been concentrated and, judging from electron micrographs, obtained in preparations of considerable homogeneity with respect to particle kind. The particles vary greatly in size and shape, and in micrographs of untreated preparations, the average diameter of the images was 233 ± 35 mµ. In formolized preparations the average diameter was 171 ± 31.3 mµ. The particles were irregular in shape, some having an ameboid appearance. Many of the particles treated with formalin were flattened spheres in shadowed micrographs.

Sedimentation diagrams showed two boundaries broadly spreading, a principal one with a sedimentation constant of S = 1311 x 10^–13 and a secondary peak of S = 1940 x 10^–13.

The yield of nitrogen varied from 0.5 to 0.8 mg per 100 ml of chorio-allantoic fluid. The 50 per cent point infectious unit was about 10^–13.5 gms of nitrogen, a value judged to be very high due to much virus inactivated during the purification procedure. The hemagglutinative unit was about 10^–7.0 gms of nitrogen. The optimal range of pH stability was from 5.8 to 8.0 but about 99 per cent of the virus was inactivated in this range in 4 weeks.

Footnotes

^* This work was aided by a grant to Duke University from Lederle Laboratories Inc., Pearl River, N. Y., and by the Dorothy Beard Research Fund.