| Summary: | Thesis (M.A.)--Boston University === Studies on the virus of herpes simplex have been done in t he past by using t he embryonated egg and the newborn mouse. Only comparatively recently has attention been directed to the use of the tissue culture method for this purpose. Howe ver, relatively little has been accomplished up to the present time by this method in the quantitative studies of this virus. This can probably be attributed, for the most part, to the lack of adequate and accurate tissue culture techniques.
An interesting approach to the development of a more accurate tissue culture technique was made by Dulbecco in 1952, who devised a new tissue culture technique by which he succeeded in obtaining focal necrotic areas (plaques) in chick embryo monolayer tissue cultures by the viruses of Western equine encephalomyelitis and Newcastle Disease. Thus the possibility arose for the study of certain animal viruses along lines similar to the ones used in the related field of bacterial viruses. Using this new technique, Dulbecco and Vogt have recently proved that, indeed, it can be successfully used for as saying poliomyelitis virus.
The purpose of this work was to investigate the possibility of applying; this "plaque-production" method to the assay of herpes simplex virus and to simultaneously compare its plaque producing capacity in tissue culture with that on the chorioallantoic membrane of the developing chick embryo. The currently employed techniques were modified for the sake of simplicity and convenience.
The tissue cultures were set up as follows, in principle following the techniques devised by Dulbecco and Noyes:
Five to six 11-day-old chick embryos were washed in Hanks' balanced salt solution (HBSS) and pressed through a 30-mesh wire screen into 15 ml of HBSS by means of a 50-ml syringe, and this material centrifuged at 800 r.p.m. for 30 seconds. The sediment was resuspended with 15 ml of BBSS and after vigorous pipetting centrifuged at 800 r.p.m. for 20 seconds. The supernatant was removed and allowed to stand for 20 minutes. The sediment of this was discarded and the cell concentration of the suspension determined in a hemocytometer. An appropriate amount of this cell suspension was added to the nutrient medium, which consisted of 40% HBSS (with indicator), 40% unheated horse serum and 20% chick embryo extract in order to give a final concentration of 8 x 10^6 cells per ml, the pH of the suspension being 7.6. 3 ml of this were then introduced into each 50-mm Petri dish, coated with chicken plasma, and the dishes sealed with Parafilm. The cultures were incubated at 37 C for 48 hours and at that time the nutrient medium removed, the cultures washed with HBSS, and 0.1 ml or more of the virus inoculated with a hypodermic syringe, gauge 27 needle, and well distributed all over the cell layer. The virus used was the Z strain of herpes simplex which had undergone 3 tissue culture and subsequently 5 egg passages since isolation from primary sources.
Following inoculation the cultures were incubated at 37 C for 45 minutes and then covered with a thin layer of plasma, or agar incorporated in the nutrient medium (baiting a final concentration of 0.75%). If plasma overlay was used, 3 ml of nutrient medium were added per culture. The petri dishes were sealed then and incubated at 37 C. At the time of examination of the cultures they were stained with neutral red in order to facilitate the visualization of the plaques, the nutrient fluid or agar overlay removed, and the plaques counted and examined microscopically.
The observations and results were as follows:
Soon after incubation the cells settled out and began to grow, forming a continuous monolayer of cells covering the entire bottom of the Petri dish. They also exhibited good metabolic activity. At 48 hours the pH of the nutrient medium was down to 7.0-7.2 and a uniform cell layer had formed consisting mainly of healthy fibroblast-type cells. No satisfactory cell layer could be obtained without use of the plasma undercoating. Following inoculation of the virus, plaques could be already detected in the cell layer after a 48-hour incubation period in all the experiments carried out. They appeared as round to slightly elongated, colorless, necrotic areas standing out in the red background of stained living cells, and had a diameter of 1-2 mm on the third day. Microscopically they appeared as bounded areas of cellular debris and if examined earlier the gradual disintegration of the cells was apparent. No plaques were produced in control cultures without virus or virus inactivated by heat and their number increased with the concentration of the virus. The endpoint for plaque production of the 10-fold dilutions of this virus was at 10^-4. In cultures with undiluted virus all the cells were completely destroyed, while with 10^-1 inoculum, semi-confluent plaques were produced. At dilutions of 10^-2 , 10^-3, and 10^-4 discrete and separated plaques were formed. The discreteness of the plaques was dependent on the presence of plasma or agar overlay. Either one could be efficiently used and there was no difference between them in regard to plaque formation. A comparative study with the developing chick embryo, inoculated on the 11th day on the C A membrane, showed that the number of foci produced on the membrane correlates with the number of foci produced on the cell layer in tissue culture although the titer was approximately 10-fold lower in vitro with this particular herpes simplex specimen.
The results of these experiments have shown that the Z strain of herpes simplex virus produces plaques in monolayer chick embryonic tissue cultures and this assumption was based on the facts that - (1) No plaques were formed in cultures where the virus was absent or inactivated by heat, (2) the number of plaques increased in proportion to the concentration of the virus inoculum, and (3) there is a correlation between the number of plaques formed in tissue culture and number of pox produced on the CA membrane of the developing chick embryo.
The technique employed throughout the experiments described in this paper includes some modifications of the ones already practiced, which simplify and economize, the procedure and makes it easily adaptable for use in any bacteriological laboratory.
Further more detailed work is needed to evaluate the aspects and adequacy of this method for quantitative study of the virus of herpes simplex. If satisfactory, it would seem to offer some advantages over certain others currently used.
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