Abstract: In the preceding experiments observations have been reported upon the nature of herpes virus which confirm the suspicion that the virus is intracellularly located in the infected nervous system. In regard to the immunological conditions existing in this disease, our experiments have reaffirmed that herpes virus can be neutralized with the serum of actively immunized animals and have offered an explanation for the irregularity of the results of others, as well as our own. It has been found that brain extracts possess some virus-neutralizing power, but considerably less than the serum of the corresponding animals. Attempts at passive immunization with neutralizing serum were uniformly negative, even when the serum was introduced into the cisterna magna 12 to 24 hours before infection with the virus. It has been shown that active immunity can be attained only when some degree of reaction to the living virus has occurred. Rabbits which survived neutralized serum-virus mixtures did not acquire immunity nor did those treated with virus phenolized to the extent of actual destruction. This point suggests a reinvestigation of the Semple method of rabies immunization. In so far as our studies touched upon the herpes-encephalitis problem we have uniformly failed in attempts to transfer herpes virus directly from man to rabbits. These results are in contradiction to those of most of the earlier workers, but in keeping with the recent reports of Flexner and Amoss. Attempts to overcome the difficulty of transfer by the recently published technique of Perdrau were unsuccessful. Furthermore, animals repeatedly treated with human encephalitis material, either fresh or glycerolated, as practised in the Perdrau method, failed to acquire the slightest degree of immunity to subsequent herpes inoculation. By the inoculation of very small doses or by infection of partially immunized rabbits, as described above, we have succeeded in modifying the characteristic herpetic syndrome in rabbits in a manner which simulates many of the clinical features of human encephalitis. Our own experience forces the conclusion that no valid proof exists upon which can be based an assertion concerning the identity of the virus of herpes with that of encephalitis lethargica. Either the two viruses are entirely unrelated, or else prolonged sojourn in the central nervous system of man attenuates the virus for rabbits to an extent analogous to that in which rabies virus is attenuated for man by passage through rabbits. The isolated successes of Levaditi and of Doerr and their assistants might thus be regarded as fortunate exceptions in which material incompletely attenuated had been at their disposal. We suggest this point of view as an alternative working hypothesis largely because the results we are reporting, as well as those of Flexner and Amoss, are in flat contradiction to the reported successes of earlier workers and the more recent experiments of Perdrau. The experiments of the latter, as described, cannot be explained by the occasional existence of spontaneous encephalitis in his rabbits, nor by the assumption that a herpes virus fortuitously coexisted with that of lethargic encephalitis in his material, inasmuch as this material alone at first injection or in the unglycerolated state failed to infect. It is also possible to conceive that human beings may, by repeated skin infections, attain a not inconsiderable partial immunity to herpes virus, which would explain the nature of the clinical course (as in our partial immunity rabbits) as well as the innocuousness of direct injections of herpetic virus into man, as reported by Bastai and Busacca, and the finding of herpes virus in human beings not suffering from lethargic encephalitis. These suggestions are discussed in order to give this important problem the broadest possible consideration. For the time being, however, such reasoning cannot be taken as more than a logical possibility impressed upon us by our partial immunization experiments. All other experimental evidence obtained by direct inoculations with the limited material at our disposal tends to render identity of the two varieties of viruses unlikely.

Abstract: We are submitting this series of experiments as observed facts, realizing that there are so many uncertainties in this form of indirect observation that great caution must be exercised in drawing conclusions of any kind. The most serious of the possible errors involved is that the active substances which we have studied, the enzyme—the bacteriophage—and the several varieties of virus, may not be free in our suspensions, but are adsorbed to larger particles. The peculiar difficulties encountered in filtering herpes virus particularly suggest a source of error of this kind, and if we are right in assuming the intracellular position of this virus in the nervous tissue, it is more than likely that most of the virus obtained in suspension may be closely associated with protein particles derived from the cells. Keeping all this in mind, we may, nevertheless, derive a certain amount of information from our experiments as follows: 1. The order of magnitudes of the pure proteins with which we have worked,—namely, crystallized egg albumen, crystallized serum albumen and purified casein,—follows the order of molecular weights of these substances as determined by Cohn. As far as casein is concerned, the size indicated by filtration in comparison with collargol is far greater than it should be by calculations which take a molecular weight of 192,000 as the point of departure. While one cannot be sure of the reason for this, there are many possible explanations such as considerable swelling of the casein partides, aggregation of molecules and the fact that casein is not at its isoelectric point under the conditions of filtration and surely present as a salt. 2. Trypsin, even in the certainly very impure condition in which we employed it, is but very slightly larger than serum albumen and distinctly smaller than casein. In its pure form it may well be much smaller even than our filtrations indicate, but certainly not larger. This relatively small size of trypsin may have considerable bearing upon the question of whether or not the lytic agents spoken of as "bacteriophage" are substances of the nature of enzymes, or whether they are more comparable to the filtrable virus, as supposed by d'Hérelle. 3. Herpes virus, the Rous chicken sarcoma and a staphylococcus bacteriophage were all subjected to filtration at pH 7.2 and at hydrogen ion concentrations higher than 8, which is given by Olitsky and Boëz as the isoelectric point of foot-and-mouth disease, but failed to pass membranes which, at the same pressures, were permeable for casein and collargol. The bacteriophage and the Rous sarcoma with considerable regularity passed through membranes which held back colloidal arsenic trisulfide. We have cited only a few of the experiments which were actually done, every one of the tests tabulated being merely representative of a number of others that were omitted for economy of space. The herpes virus we have had greater difficulty in filtering. We cite one experiment with a 2 per cent acetic-collodion membrane and another with a 1.5 per cent membrane through which the herpes virus passed, the membrane being so controlled that gross leakage could be excluded. We believe that the difficulty here is very largely due to the fact that in preparing the herpes virus for filtration it cannot be separated from considerable amounts of brain material, from which, perhaps, it is not easily dissociated. This would be natural if the herpes virus were intracellularly located, as we believe it to be. This experiment and similar ones, however, incline us to believe that the herpes virus is not far different from the Rous sarcoma virus and the bacteriophage, as far as filtration through membranes is concerned. It certainly is not smaller than either of these substances and probably, as we judge from a few experiments carried out at higher pressures, is not much larger. It may be assumed, therefore, that in the form in which we were able to procure the bacteriophage and the two varieties of virus investigated by us, they were of a magnitude larger than casein and collargol and smaller than colloidal arsenic. The weak point in drawing our conclusions is the fact that we were not in a position to measure for ourselves with any accuracy the actual sizes of collargol and arsenic trisulfide particles. Accepting the general views of Bechhold and others, however, our experiments would define the sizes of the separticular substances as larger than 20 mµ and probably smaller than 100 mµ. The order of magnitudes of the substances measured by us would then be as follows: Crystallized egg albumen Crystallized serum albumen Trypsin Collargol Casein Bacteriophage, Rous sarcoma virus, herpes virus Arsenic trisulfide Our experiments show little agreement with the work of Levaditi and Nicolau and of Levaditi, Nicolau and Galloway. In their recent filtration tests of foot-and-mouth disease this virus is reported by them as passing through membranes that held back trypsin, indicating a size much smaller than any of the viruses measured by us. Our results, on the other hand, are in actual measurements comparable to those of Olitsky and Boëz, not only in the fact that the viruses with which we worked correspond approximately to the size determined by them for foot-and-mouth disease, but that the percentage of collodion in membranes permeable for virus and impermeable for colloidal arsenic corresponds almost exactly to our own. This gives us confidence that the technique developed may be more easily standardized than we at first believed and that the method of ultrafiltration, owing to the great ease with which membranes of relatively standard size may be made, may have valuable applications in the investigation of bacteriological and immunological problems.

Abstract: The virus of herpetic encephalitis and the virus of vaccinia can be demonstrated in the filtrate, if a broth emulsion of fresh tissue containing the virus, is passed through a Berkefeld V filter.

Abstract: In working on the neutralizing powers of the serum of rabbits immunized against the agent of herpes, the writers noticed some years ago (1) that salt solution suspensions of this material taken from the brains of rabbits dead of the disease rapidly lost virulence, upon incubation at 37.5°C. There was a distinct diminution of infectiousness within two or three hours, and often complete loss of virulence within five or six hours, the time period varying with the densities of the suspensions. Suspensions in which the amount to be inoculated into a rabbit (0.1 to 0.25 cc.) contained not more than three or four fatal doses—that is, dilutions of 1:16,000 according to the potency of the virus—were often completely inactivated within four or five hours, whereas heavier suspensions would last correspondingly longer, probably because of the presence of minute particles of brain material within which the herpetic agent was protected to some extent from the deteriorating influences active in the salt solution.

Abstract: In 1926 Takaki, Bonis and Koref (1) reported that they had been able to identify and differentiate the various forms of encephalitic viruses by means of the complement fixation reaction. Working on rabies, Kraus and Michalka (2) in the same year published very favorable results in the diagnosis of this disease by complement fixation. They used “cooked” and glycerin extract antigens. Their results were so definite that it was decided to repeat their experiments with rabies, and to use the same technic with herpes, in view of the fact that in herpes studies during the last two years we (3) have carried out a large number of complement fixation experiments with herpes virus, always with negative results. Technic 1. Immunization of the rabbits Rabbit 1 was immunized against rabies by a prolonged treatment with Semple's vaccine (50 cc. in all) followed by three injections with living fixed virus (3.8 cc. in all).