value as a medium for the in vivo cultivation of different

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1 THE BEHAVIOR OF THE VIRUS OF EQUINE ENCEPH- ALOMYELITIS ON THE CHORIOALLANTOIC MEMBRANE OF THE DEVELOPING CHICK' ELIZABETH HIGBIE AND BEATRICE HOWITT George Williams Hooper Foundation, University of California, San Francisco, California Received for publication, October 24, 1934 The use of the developing hen's egg has recently proven of value as a medium for the in vivo cultivation of different filtrable viruses. It has been shown by Rivers and Schwentker (1932) and Mackenzie (1933) that the undifferentiated cells of the developing embryo are more susceptible to infection than the differentiated tissues of the hatched chick. This embryonic tissue has also been used for the in vitro methods of cultivation. Although it was observed by Syverton, Cox and Olitsky (1933) that the virus of equine encephalomyelitis described by Meyer, Haring and Howitt (1931) can be cultivated in a medium of minced chick embryo suspended in Tyrode solution, yet chicks 1 and 2 days after hatching are refractory to the virus. It was therefore thought of interest to study the behavior of this virus upon the membranes and embryo of the developing egg. Two strains of the virus were used, the original one isolated in 1930 by Meyer, Haring and Howitt (1931) and an eastern strain kindly sent by Dr. C. Ten Broeck from New Jersey (1933). The technique employed for the inoculation of the developing eggs was essentially that of Woodruff and Goodpasture (1931) with only slight modifications. The fertile egg was candied after ten days' incubation, the position of the embryo marked 1 Read before the Ninety-fourth Meeting of the American Association for the Advancement of Science held at Berkeley, California, June 18-23,

2 400 ELIZABETH HIGBIE AND BEATRICE HOWITT and the surface of the shell cleansed with alcohol. A small window '-inch square was cut through the shell over the embryonic area with a dental drill fitted with a small abrasive disk. The opening was again cleansed with alcohol and sterile cotton and the shell membrane removed with aseptic technique, exposing the chorio-allantoic membrane. The opening was ringed with sterile vaseline and sealed with a sterile coverglass. All eggs so treated were returned to the incubator until inoculated. A 20 per cent suspension in saline of ground guinea-pig brain containing virus was used as the base for making varying dilutions. The inoculations of the eggs were made by inserting a needle through the vaseline ring under the cover-glass and dropping 0.1 cc. of the fluid on the membrane, using a 1 cc. syringe. Inoculated eggs were incubated at 380C. for periods ranging from three hours to ten days. Infected membranes and tissues of the embryo were tested for presence of the virus by injecting guinea pigs intracerebrally with 0.3 cc. of a ground saline suspension. The first inoculations were made with varying dilutions of the 2 viruses, ranging from 1:1,000 to 1:100,000 for the New Jersey strain and 1:50 to 1:1,000 for the Californian. The eastern strain was transferred for 3 generations from the membrane of the egg given the 1:10,000 dilution of original inoculum. A 1: 1,000,000 dilution of the ground chorio-allantoic membrane was infective for a guinea pig. The western strain passed through 4 generations from the egg given the 1: 1,000 dilution of original material and was found to be infective for a guinea pig in a dilution of the membrane of 1:1,000,000. Further continuous passages of the ground membranes were made with both viruses. The eastern strain was carried through at least 8 and the western through 7 generations. It was found practical to transmit the virus from egg to egg by inoculating the ground embryonic brain. Such transmission succeeded through 4 successive passages. Control inoculations into guinea pigs have been positive for both membranes and brain material, respectively, after each instance.

3 BEHAVIOR OF VIRUS OF EQUINE ENCEPHALOMYELITIS 401 The outstanding observation noted from these egg inoculations was the marked lethal effect of the virus upon the developing embryo. Whenever the virus was active, death of the chick occurred within fifteen to twenty-four hours. Control injection of guinea pigs confirmed the presence of virus in the membranes. For this reason the lethal factor was found to be useful in determining the approximate minimum lethal dose of the virus. Varying dilutions of the 20 per cent suspension of guinea pig brain were made and the end-point determined by death of the embryo. Control inoculations into guinea pigs usually ran a parallel course, although the chick embryo was more sensitive to the higher dilutions than the rodent. In one instance the New Jersey strain killed the embryo in a dilution of 1: 1,000,000 but was only lethal for the guinea pig in a 1: 50,000 dilution. As a rule, the eastern strain was more virulent than the western, killing both chick and guinea pig in higher dilutions. It was noted that the incubation time of the eastern virus was often shortened from the usual three- or four-day period to two days. The activity of the western strain was increased by repeated passage. Marked changes were noticeable in the appearance of the chorio-allantoic membrane after inoculation of active virus. The gross and microscopic pathology of the infected membranes has been reported by Covell (1934) so that it is of interest here only to note certain changes observed on the membranes of all infected eggs whether or not there was sufficient virus present to infect guinea pigs. These changes were not obtained in the membranes inoculated with sterile saline solution or with broth. The membrane appeared thickened and edematous as early as three to six hours after inoculation. As the lesion progressed, the thickening and gelatinous edema increased, the membrane was opaque and the blood-vessels were practically obliterated. Patchy grey areas often appeared in the older lesions, while in the most severe infections these grey areas practically covered the entire surface of the membrane. After repeated inoculation of eggs with varying dilutions of

4 402 ELIZABETH HIGBIE AND BEATRICE HOWITT the viruses it was found that the eastern strain in a dilution of 1: 10,000 of guinea pig brain and the western in a dilution of 1:1,000 had the most consistent lethal effect on the developing embryo. These dilutions were used routinely for all further experiments. In order to determine the time interval involved in the multiplication of the virus, its lethal effect, and its migration to the tissues and fluids of the embryo, 2 series of eggs were inoculated, 1 with each strain of virus. A dilution of 1:10,000 was used for the eastern and one of 1:1,000 for the western strain. Two eggs inoculated with each strain, respectively, were sacrificed from each series every three hours for a twenty-four-hour period. The chorio-allantoic membranes, the amniotic fluid, the brain and the cord of the embryo were removed at each interval of time for intracerebral inoculation of guinea pigs, while tissues were also prepared for histological study. A report on the latter has been made elsewhere by Covell (1934). From the results of these 2 series of inoculations it was possible to plot growth curves for both of the strains as shown in figures 1 and 2. The abscissae represent the number of hours after inoculation, while the ordinates show the highest dilution of the ground membrane sufficient to cause the death of the guinea pig with characteristic symptoms. In the first series (fig. 1) with the eastern strain sufficient virus was apparently recovered from the original inoculum after three hours' incubation to cause the death of the guinea pig. Upon repetition, however, no virus was recovered from the membrane after three hours' incubation. All other tissues were negative and the embryo was active. After six hours' incubation, the chicks were alive and no virus was obtained from any tissue in any dilution. After nine hours a heavy suspension of the chorioallantoic membrane was infective for guinea pigs but not the higher dilutions, while virus was recovered from the brain, cord and amniotic fluid of the embryo. From then on the virus was present in high dilutions of the membrane and in all of the other tissues examined. After the fifteenth hour the embryos were no longer viable. Between the ninth and the twelfth hours the

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6 404 ELIZABETH HIGBIE AND BEATRICE HOWITT virus apparently multiplied extensively and was recovered in a 1:100,000 dilution of the ground membrane. At this interval the amniotic fluid became positive and continued so for the remainder of the period of observation. By the fifteenth hour the dilution of ground membranes sufficient to infect guinea pigs dropped to 1:10,000. Throughout the remainder of the twenty-four-hour period the chicks were dead when the eggs were opened, and the virus content of the membranes remained at a dilution of 1:10,000. The growth curve for the western strain of virus (fig. 2) ran a similar course, no virus being recovered for the first six hours from any of the tissues examined. It was first recovered from the chorioallantoic membrane after the ninth hour in a dilution of 1:1,000. The brain, cord and amniotic fluid were also positive and continued to contain virus thereafter, except for the disappearance from the amniotic fluid during the twelfth hour. At the latter period the virus had increased in the membrane so that a dilution of 1: 30,000 killed a guinea pig. Multiplication continued until a dilution of 1:100,000 proved lethal during the thirteenth hour after inoculation. Although the virus was recovered from all of the tissues examined during these later intervals, yet the embryos remained alive until the twenty-first hour, after which death occurred. The lethal effect with this strain was therefore delayed longer than with the more active New Jersey virus. Otherwise the behavior of the 2 viruses upon the membranes and upon the developing embryo was approximately the same. It was interesting to note that the virus could be recovered from the heart's blood of the developing chick. The pooled serum obtained after twenty-four hours from 6 embryos infected with the eastern virus produced typical symptoms upon intracerebral injection of a guinea pig. Upon repetition of the experiment the virus was recovered from the serum of embryonic chicks twelve, fifteen and twenty-one hours, respectively, after inoculation of the membranes but not after three or nine hours. This would suggest the absorption of the virus first through the chorioallantoic membrane into the blood stream and then a localization

7 BEHAVIOR OF VIRUS OF EQUINE ENCEPHALOMYELITIS 405 in the nerve tissues of the developing embryo. This would further supplement the observations recently reported by one of us (Howitt, 1934) for the guinea pig, that after intranasal instillation the virus gained entrance first into the blood stream with a subsequent invasion of the nerve tissues. It has previously been reported (Howitt, 1932) that the virus of equine encephalomyelitis was capable of being neutralized by serums of hyperimmunized animals (rabbit, guinea pig, monkey and horse). Since the present observations have shown the marked lethal effect of the virus upon the embryo of the developing chick, it was thought of interest to determine the value of the egg for use in this test. Preliminary titrations for potency of both the western and eastern strains of virus were made by inoculating varying dilutions into both eggs and guinea pigs, respectively. The serums to be used were found capable of neutralizing the homologous virus when injected intracerebrally into guinea pigs. Equal parts of diluted virus and of immune serum were then mixed, incubated one hour at 370C. and allowed to stand four hours at room temperature. Of each mixture 0.1 cc. was inoculated upon the membranes of duplicate eggs. Sterile saline, serum with saline alone and virus with saline were inoculated, respectively, into control eggs. Observations were made at regular intervals during the incubation, covering a period of several days. It was found that the egg could easily and successfully be utilized as a medium for determining the neutralizing value of the immune serum. In both series of experiments performed, the eggs given homologous virus and serum remained alive, while the control embryos inoculated with virus alone died within the time limits for the dilution used. Sterile saline and the serums themselves had no lethal effect upon the developing chick. In confirmation of neutralization tests previously noted for guinea pigs by Giltner and Shahan (1933), Ten Broeck and Merrill (1933) and Howitt (1935), it was observed that the western strain of virus was not neutralized by the eastern immune serum and vice versa. All the results, therefore, ran parallel with those reported when using the guinea pig. Utilization of the egg as a

8 406 ELIZABETH HIGBIE AND BEATRICE HOWITf quick and inexpensive medium for performing the in vitro neutralization test has recently been reported by Burnet and Galloway (1934) for the virus of vesicular stomatitis. It may, therefore, prove of value for those viruses which give a pronounced lethal effect upon the developing embryo. SUMMARY 1. The virus of equine encephalomyelitis, both eastern and western strains, may be cultivated upon the chorio-allantoic membranes of the developing chick. 2. Inoculated upon the membranes of the egg, the virus may be recovered from the nerve tissues and the amniotic fluid of the embryo after a definite time interval. This progressive invasion is comparable with a growth curve ultimately leading to the death of the embryo. The virus could be recovered from the vitelline vein and from the pooled heart blood of several chick embryos after a definite incubation period. 3. Because of the rapidly lethal effect of the virus upon the embryo, the inoculation of the developing egg offers a simple and inexpensive method for titrating the potency of the virus and for carrying out the in vitro neutralization test. REFERENCES BURNET, F. M. AND GALLOWAY, I. A Brit. Jour. Exper. Path., 15, 105. COVELL, W. P Proc. Soc. Exper. Biol. and Med., 32, 51. GILTNER, L. T. AND SHAHAN, M. S Science, 78, 63. HowITT, B. F Proc. Soc. Exper. Biol. and Med., 32, 58. HOWITT, B. F Jour. Infect. Die., 51, 493. HOWITT, B. F Jour. Bacteriol. 29, 61. MACKENZIE, R. P Jour. Path. and Bacteriol., 37, 75. MEYER, K. F., HARING, C. M. AND HOWITT, B. F Science, 74, 227. RIVERS, T. M. AND SCHWENTKER, F. T Jour. Exper. Med., 55, 911. SYVERTON, J. T., Cox, H. R. AND OLITSKY, P. K Science, 78, 216. TEN BROECK, C. AND MERRILL, M. H Proc. Soc. Exper. Biol. and Med., 31, 217. WOODRUFF, A. M. AND GOODPASTURE, E. W Amer. Jour. Path., 7, 209.