Single-Dose Assay Technique for Variola

RILEY, JEAN M. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.), AND MICHAEL D. ORLANDO. Singledose assay technique for variola virus. Appl. Microbiol. 12:7-9. 1964.-A biological assay for variola virus was needed that would (i) require a minimum of time and (ii) have acceptable precision. Preliminary titrations made in 11-day-old embryonated eggs and in suckling mice (6 to 24 hr of age) demonstrated a linear relationship between the concentration of variola virus injected and the mean reciprocal time to death (MTD) of both hosts. This linear response indicated that the injection of a single dose of virus suspension and the measurement of the MTD should result in an acceptable assay method. Seven replicate samples of liquid preparations (20% chorioallantoic membrane in Heart Infusion Broth) and the freeze-dried material obtained with these suspensions were assayed in triplicate for pock infectious units and for MTD. The variance of the pock counts was far greater than was expected from a Poisson distribution, and coefficients of variation ranged from 25 to 61 %O. Variances obtained with the single-dilution assay were all far below that expected from a Poisson distribution, and coefficients of variations ranged from 5.2 to 13.6%. The use of the MTD assay resulted in a saving of time, a saving in the number of hosts necessary per assay, and increased precision. The standard measurement for assaying pox viruses is titration of pock infectious units (PIU) on the chorioallantoic membrane (CAMI) of embryonated eggs. Theoretically, these pock counts should follow a Poisson distribution. However, a literature survey indicated that, regardless of the pox virus that was being investigated, the variance obtained in pock counting was far greater than expected for a Poisson distribution. Kaplan and Belyavin (1957), who made no adjustments in their assays of vaccinia virus, found that the variance of their pock counts was so wide that no estimate of the coefficient of variation was possible. In other cases, adjustments were made in the counts to reduce the coefficient of variation to about 25 %. These adjustments were made by assigning double value to counts the investigator considered to be in the "normal" range (Burnet and Faris, 1942), or by counting the pocks on only those membranes having no nonspecific lesions or encroachments of the albumin sac l Presented in part before the American Society of Microbiology, 63rd Annual Meeting, Cleveland, Ohio, 5-9 May, 1963. 2 In conducting the research reported herein, the investigators adhered to "Principles of Laboratory Animal Care" as established by the National Society for Medical Research. (Westwood, Phipps, and Boulter, 1957). These adjustments resulted in considerable improvement in variance, but not enough to approach the variance expected with a Poisson distribution. We needed a biological assay for variola virus that would require a minimum of time and have acceptable precision. Cabasso and Moore (1957) found that an LD5o assay in eggs was feasible and of acceptable precision. These investigators also noted that there seemed to be a linear relation between the log-dilution injected and the reciprocal survival time of the embryo. In addition, several investigators (Sarkar, Neogy, and Lahiri, 1959; Mayr and Herrlick, 1960; Marshall and Gerone, 1961) found that suckling mice less than 24 hr of age were susceptible to variola virus. A close examination of their data indicated that there was also a linear relation of dose and time-to-death of the mice. These observations offered the possibility of (i) an LD5o titration and (ii) an assay based on the meani time-to-death (MTD) of a group of embryonated eggs or of suckling mice given a single dilution of virus. The purpose of this report is to show that a dose-time response relationship exists for variola infections in both embryonated eggs and suckling mice and that the precision of a single-dilution assay in eggs or mice is far greater than that of the pock-counting assay. MATERIALS AND METHODS Virus. The seventh egg passage of the Yamada strain of variola virus was used throughout these studies. The liquid preparations were 20 % suspensions of CAM in Heart Infusion Broth (HIB). Some of this suspension was freeze-dried to provide dried material for assay. Hosts. Swiss-Webster albino mice ranging in ages from 6 to 24 hr were used. Litter sizes varied from 4 to 15 mice, so the litters were pooled and the mice were redistributed to give uninoculated mothers 8 young for the duration of the observation. In addition, 11-day-old embryonated eggs were used. The CAM was dropped as described by Hahon, Louie, and Ratner (1957). Assay procedures. Liquid slurries were diluted in HIB containing 500 units of penicillin and 100 ,ug of streptomycin per ml. When dry preparations were assayed, a 0.1-g sample was added to 99.9 ml of HIB plus antibiotics and blended for 1 min in a Waring Blendor. Serial tenfold dilutions were made from this. Pock counts were made in the usual manner by inocu7 on Jne 1, 2017 by gest ht://aem .sm .rg/ D ow nladed fom