STUDIES IN RODENT POLIOMYELITIS : I. FURTHER EXPERIMENTS WITH THE MURINE STRAIN OF SK POLIOMYELITIS VIRUS

1. SK murine virus maintained over more than 200 serial mouse passages increased in virulence for mice from an initial intracerebral titer of about 1:1 million to a maximum titer of not less than 1:1 billion dilution activity. 2. Following intracerebral injection with murine virus of remote mouse passages, 5 of 13 rhesus monkeys developed a characteristic encephalitic syndrome. Repeated intravenous injection of massive doses of virus caused localized flaccid paralysis in 2 of 14 monkeys. 3. Intracerebral injection of graded doses of murine virus into mice of different age groups caused fatal paralysis in young and old animals alike. Infection with small doses of virus by peripheral routes, while uniformly fatal to young mice, was followed by survival of almost half of the old mice. 4. The incubation period of the disease in young mice infected intracerebrally with a standard dose of murine virus, when studied throughout the period of 1 year, was found considerably lengthened during the summer months. 5. Cross neutralization tests furnished no evidence for any serological relationship between SK murine virus and lymphocytic choriomeningitis virus. Theiler''s virus was found to be neutralizable by antimurine horse serum and, to a lesser extent, by concentrated antipoliomyelitis horse serum; however, such inactivation, in both cases, was distinctly inferior to that occurring with SK murine virus. On the other hand, no neutralization whatsoever was obtained between SK murine virus and normal adult mouse serum, whereas the same serum completely neutralized Theiler''s virus. Mice surviving infection with Theiler''s virus, though acquiring immunity to this virus, remained fully susceptible to reinfection with SK murine virus. 6. Neutralization tests with SK murine virus against poliomyelitis-convalescent monkey sera gave irregular results, but neutralization of murine virus occurred regularly with a hyperimmune antipoliomyelitis horse serum. Hyperimmune antimurine horse and rabbit sera, on the other hand, failed to inactivate three strains of monkey poliomyelitis virus (SK, RMV, Aycock) by intracerebral tests in monkeys. The same sera inactivated murine virus in mice by intraperitoneal, but not by intracerebral injection of virus-serum mixtures. 7. The identity of SK murine virus and its relation to other rodent strains of poliomyelitis virus is discussed on the basis of the available data.     

STUDIES IN RODENT POLIOMYELITIS : V. INTERFERENCE BETWEEN MURINE AND MONKEY POLIOMYELITIS VIRUS

1. The murine strain of SK poliomyelitis virus interferes with the propagation in rhesus monkeys of SK, Aycock, and RMV poliomyelitis monkey virus. 2. This interference is demonstrable by intracerebral injection of mixtures of murine and monkey virus prepared in vitro as well as by separate injection of the two viruses by diverse routes. 3. Mixture tests carried out with graded doses of murine and monkey virus show that 0.5 cc. of a 10 per cent suspension prepared from the brains of paralyzed mice is capable of counteracting at least 100 minimal paralyzing doses of two strains of monkey virus. 4. No interference was demonstrable with suspensions of brains infected with murine virus which had been inactivated by heating for ½ hour at 75°C., or with suspensions prepared from normal mice, or with brain suspensions prepared from mice infected with herpes virus. 5. When murine virus is introduced into monkeys by the intravenous route, before or after intracerebral infection with monkey virus, distinct prophylactic or therapeutic results may be obtained. 6. Analysis of the figures shows that the success of interference depends upon (a) the size of the infecting dose of monkey virus, (b) the amount of murine virus injected, and (c) the choice of proper intervals between the injection of monkey and murine virus. 7. The mechanism of the interference phenomenon here described is discussed in the light of the available data.     

STUDIES IN RODENT POLIOMYELITIS : VI. FURTHER OBSERVATIONS ON INTERFERENCE BETWEEN MURINE AND SIMIAN STRAINS OF POLIOMYELITIS VIRUS

1. Attempts to separate by processes of physical segregation, i.e. ultrafiltration, ultracentrifugation, or dialysis, from live SK murine poliomyelitis virus a non-pathogenic agent capable of interfering with simian poliomyelitis virus were unsuccessful. Neither was it possible to convert live SK murine virus into a non-pathogenic interfering agent by processes of chemical inactivation, i.e., phenolization or formalinization. 2. Preparations of SK murine virus, which had been markedly attenuated by ultraviolet irradiation, gave evidence of having retained some interfering power in rhesus monkeys. 3. MM murine poliomyelitis virus interfered, both in mixture tests and by peripheral administration, with two simian strains of poliomyelitis virus. With adequate amounts, distinct protective effects could be obtained in rhesus monkeys which had received murine virus (animal passage or tissue culture virus) up to 48 hours after intracerebral infection with simian poliomyelitis virus. 4. Theiler''s virus of spontaneous mouse encephalomyelitis, when tested in mixture with simian poliomyelitis virus, gave some evidence of irregular and low grade interference. Interference could not be shown conclusively in experiments to prevent poliomyelitic infection or to modify its effects. 5. The nature of the interfering agent present in murine virus is discussed.     

STUDIES OF A MURINE STRAIN OF POLIOMYELITIS VIRUS IN COTTON RATS AND WHITE MICE

1. A neurotropic murine virus was isolated by passing poliomyelitis virus (SK strain) from the monkey to cotton rats and white mice. 2. The murine virus has been grown in tissue culture consisting of embryonic mouse brain in ox serum ultrafiltrate. 3. The symptoms and lesions produced by the murine infection compare in all respects with those of poliomyelitis in monkey and man. 4. The murine virus, while highly pathogenic for mice and cotton rats, is non-pathogenic for albino rats, guinea pigs, and rabbits. It possesses limited pathogenicity for rhesus monkeys. 5. Although producing no paralysis in the above mentioned refractory animals, the murine virus may be recovered in active form from neural and extraneural sites of infected albino rats, guinea pigs, and monkeys, but not from rabbits. 6. The identity of the murine and monkey virus is further suggested by cross-neutralization between the murine virus and homologous (SK) and related (Aycock) antipoliomyelitis sera, as well as between homologous and related monkey poliomyelitis virus and antimurine virus sera. 7. Immunization of monkeys with live murine virus, in the form of mouse brain or tissue culture, seems to confer some degree of resistance against subsequent infection with the homologous poliomyelitis monkey virus. 8. The presence of the murine virus in the central nervous system of infected monkeys appears to interfere with the propagation of SK and Aycock poliomyelitis monkey virus in the same animal.     

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS : III. QUANTITATIVE STUDIES OF SERUM ANTIVIRAL BODIES IN ANIMALS IMMUNIZED WITH ACTIVE AND INACTIVE VIRUS

An analysis of the preceding experiments discloses that antiviral bodies are demonstrable not at all or in small amounts in the sera of guinea pigs injected with a quantity of active virus not sufficient to induce immunity against the described intracerebral test for induced resistance. However, neutralizing bodies are found in immune animals, although in low concentration, and are regularly manifested when serum is added to low multiples of infective doses of virus under optimal conditions of time and temperature. Hyperimmune serum, on the other hand, reveals a distinct increase in the amount of antiviral bodies present. Irrespective of the mode of procedure for revealing neutralizing bodies, there does not appear to be any notable difference in the content of such bodies in the serum of animals immunized with active virus or with formolized vaccine in which active virus could not be demonstrated. In other words, the antigenic complexes in active as well as in inactive virus produce similar degrees of antibody reaction. The formolization of virus tissue suspensions, therefore, can be considered as a process whereby the virus is inactivated but the antigenicity of the suspensions is preserved, as is also shown in the preceding paper of this series in tests on tissue immunity. In that article is described the remarkably high degree of tissue immunity which results from injections of inactive virus; now we demonstrate that this resistance is associated with a minimal degree of serum antibody. Finally, the question may well be asked, if practically no antiviral bodies are demonstrable immediately or soon after mixing immune serum and virus, and are recognizable in a tenfold increase when functions of time and temperature are brought into play, whether the bodies are "neutralizing" or the phenomenon is due merely to aggregation of virus particles by the serum. From the recent work on the same virus and immune serum (9) by Merrill, there appears to be warrant for the belief in aggregation of virus particles which in turn diminishes the virus activity to the indicated degree.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : V. SURVIVAL OF THE VIRUS IN COLLODION SACS IMPLANTED INTRAABDOMINALLY IN GUINEA PIGS.

The typhus virus contained in the blood of guinea pigs at the height of the experimental disease remains infective for 31 days in collodion sacs placed within the abdominal cavity of guinea pigs.     

FURTHER STUDIES CONCERNING THE FILTRABLE VIRUS PRESENT IN THE SUBMAXILLARY GLANDS OF GUINEA PIGS

1. It has been shown that the guinea pig virus localizes in the submaxillary glands of young guinea pigs following subcutaneous, intraperitoneal, or intravenous injection of active material, and that the specific lesion is demonstrable in the glands in 12 to 15 days. When an active infection of the gland has been produced in this way, the guinea pigs are refractory to intracerebral inoculation of the virus. 2. No lesion develops in the submaxillary glands of young guinea pigs injected subcutaneously with guinea pig virus which has been inactivated by heat. Young guinea pigs which have received injections of heat-killed virus do not become refractory to intracerebral inoculation of the virus. 3. When young guinea pigs from which both submaxillary glands have been removed are injected subcutaneously with active virus, the virus localizes in the parotid gland, and the animals become refractory to intracerebral inoculation. 4. It has been impossible to demonstrate virucidal properties in the sera of adult guinea pigs which have become spontaneously infected with the virus, or in the sera of young guinea pigs which have been artificially rendered refractory to intracerebral inoculation. 5. It has been possible to transmit the virus from guinea pig to guinea pig continuously in series through seven animals by direct inoculation from submaxillary gland to submaxillary gland. 6. The fact that the virus regularly localizes in the submaxillary glands following subcutaneous inoculation has been utilized in passing the virus from guinea pig to guinea pig. 2 weeks after the subcutaneous inoculation of the virus into young guinea pigs, the active agent was present in the submaxillary glands. Emulsions of the submaxillary glands of these animals were then used for the subcutaneous injection of another group of young guinea pigs. In this way the virus was transmitted continuously from skin to submaxillary gland through a series of seven animals.     

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS : II. IMMUNIZATION WITH FORMOLIZED VIRUS

From a study by quantitative methods, the conclusion is reached that a resistance of high degree may be induced in guinea pigs and mice against experimental equine encephalomyelitis by means of formolized vaccines in which no active virus can be demonstrated. The induced resistance is not due to residual traces of active virus which might possibly have escaped detection in the formolized tissue preparations.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : VI. SKIN LESIONS IN EXPERIMENTAL TYPHUS FEVER OF GUINEA PIGS.

The skin of guinea pigs in which the virus of typhus fever is propagated, when mildly irritated in advance of the febrile reaction, shows a characteristic exanthem during the height of the experimental disease. More drastic methods of irritation, however, cause a dermatitis which obscures the rash but produce in the corium more marked specific histopathological changes. The exanthem may aid the study of the specific incitant of typhus fever in the lesions.     

STUDIES OF THE DISTRIBUTION OF POLIOMYELITIS VIRUS : V. THE VIRUS IN FAMILIAL ASSOCIATES OF CASES

The occurrence and duration of the carrier state in familial associates of recognized cases of poliomyelitis was studied by the examination for virus of stool specimens collected from the members of four families at regular intervals for a period of over 2 months. The results indicate that: (1) virus may persist in their stools continuously for 4 to 5 weeks; (2) virus may be encountered intermittently in the stools; (3) in some instances virus may be present for brief periods only; (4) children are more likely to maintain virus than are adults in the same family; (5) infection of a family takes place rapidly, suggesting again simultaneous infection from a common source.     

AN IMMUNIZING STRAIN OF THE VIRUS OF POLIOMYELITIS

In this communication we have described a strain of the virus of poliomyelitis derived from a rapidly fatal human case, which exhibits mild degrees of infective power and marked degrees of protective effect for the monkey. While Macacus rhesus displays perceptible differences in suceptibility to the attenuated virus, nearly all individuals respond to the inoculation and none succumbs to the infection induced. As compared with previously described examples of experimental poliomyelitis, the modified disease described in this paper is distinguished by its relatively benign nature and its tendency to end in recovery rather than, as with the earlier observed instances, in death.     

STUDIES ON EASTERN EQUINE ENCEPHALOMYELITIS : III. INTRAOCULAR INFECTION WITH FIXED VIRUS IN THE GUINEA PIG

The behavior of a fixed strain of Eastern equine encephalomyelitis virus was studied in guinea pigs after intraocular inoculation. Such inoculation concerns the central and not the peripheral nervous system. The susceptibility to intraocular injection lies midway between the highly virulent intracerebral and the quite avirulent peripheral routes. The virus must act for 10 to 13 hours in order to induce a fatal infection. Removal of the inoculated eyeball before this interval almost always prevents fatality although it may allow immunity to develop. The virus, at suitable intervals after injection into the eye, may be recovered from successive and appropriate optic centers before it is demonstrable in non-optic portions. Approximately 24 hours are required for the virus to reach a significant concentration in the contralateral geniculate body, 36 hours in the contralateral visual cortex. Significant amounts of virus may be present in the optic chiasm and tract prior to involvement of the higher centers. Virus placed in contact with the retina produces an insignificant, essentially non-specific reaction comparable to that produced at the site of direct intracerebral inoculation. In the retina there is no ganglion cell necrosis unless there is a complicating intraocular infection. In the cerebral visual centers the first reaction is inflammatory and interstitial, and may appear in the lateral geniculate body as early as 24 hours after injection. Neuronal necrosis is not the primary action of the virus on the nervous system in these experiments. The distribution of lesions in the brain is in excellent agreement with the method of direct testing for virus content, and is far more accurate than the latter. The virus in its primary distribution through the nervous system follows the nerve pathways of the optic system. This occurs within the central nervous system, where presumably there is first an involvement of the nerve cell body and then a spread along the cell process or axone.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : I. CONCURRENT INFECTIONS DURING THE COURSE OF EXPERIMENTAL TYPHUS FEVER IN GUINEA PIGS.

The work reported in this paper relates to the bacteria which can be cultivated from the blood and spleen of guinea pigs at different stages of infection with the virus of typhus fever. The studies show that during the period of incubation and before the onset of fever no ordinary bacteria appear in the cultures, while on the 1st day of the febrile reaction different bacteria were found in 6 of 26 guinea pigs cultured; on the 2nd day, in 10 of 16; on the 3rd day, in 3 of 4; and on the 4th day in cultures of all of the 4 guinea pigs observed. The findings indicate that the virus of typhus fever is distinct from ordinary cultivable bacteria, and, as the disease set up by the virus progresses, the infected guinea pigs become subject to invasion by secondary or concurrent bacteria which thus induce a mixed infection. The bacteria which under the influence of the virus of typhus fever thus invade the body of the guinea pig are of several kinds, and vary not only among themselves, but also with the day of the fever on which the examination is made. Thus, on the 1st day of the fever Plotz'' bacilli were recovered twice and anaerobic streptococci, proteus bacilli, aerobic diphtheroids, Gärtner type bacilli, and Staphylococcus aureus each once. On the 2nd day Plotz'' bacilli were found four times, anaerobic streptococci three times, Gärtner type bacilli, aerobic diphtheroids, Bacillus welchii, aerobic Gram-positive diplobacilli, and Staphylococcus aureus each once. On the 3rd day Plotz'' bacilli were recovered once, as were anaerobic streptococci and Grtner type bacilli. On the 4th day Staphylococcus aureus was found twice and Plotz'' bacilli and Bacillus proteus each once. This variation in the kind of bacteria as well as the lack of predominance of one kind over another during the different stages of the febrile reaction in guinea pigs leads us to infer that they occur concurrently with the typhus virus. And since the more unusual of these organisms, the Plotz bacillus, the anaerobic streptococcus, the aerobic diphtheroid, and the diplobacillus are non-pathogenic for guinea pigs, while the more common bacteria such as the Gärtner type bacillus, Welch''s bacillus, the proteus bacillus, and the staphylococci induce distinctive effects, and since all the bacteria could be suppressed without their reappearance in guinea pig passages of the virus containing them, we believe that they are independent and unrelated to the true virus of typhus fever.     

ACTIVE IMMUNICATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS : I. QUANTITATIVE EXPERIMENTS WITH VARIOUS PREPARATIONS OF ACTIVE VIRUS

Active Eastern or Western equine encephalomyelitis virus in three forms,—chemically untreated but simply passaged through series of mice; adsorbed on alumina Gel C, and precipitated by tannin,—yielded practically the same results when employed for the immunization of guinea pigs. The virus is not inactivated by the process of adsorption or precipitation : guinea pigs and mice inoculated in the brain with these materials develop lethal encephalomyelitis in the same manner as when chemically untreated mouse passage virus has been used. Moreover, there is no difference in the rate of absorption in vivoof the chemically treated and untreated virus preparations. After storage of the three immunizing preparations—the longest periods thus far studied being 2 to 3 months for mouse passage and for precipitated suspensions, and 6 months for adsorbed material—each was found to contain an amount of virus sufficient to produce immunity in animals against the usual intracerebral test inoculation. Finally, the protection afforded by the three preparations is apparently durable, as is true of many active viruses utilized in preventive treatments. The amount of the virus necessary to confer protection may be defined as that which immunizes (a) with the least number of antigenic units and (b) with the minimum of febrile reaction and blood infection. In proportion as this amount is exceeded, the incidence of fever and of circulating virus increases and, on the other hand, as this amount is decreased, the degree of induced immunity is diminished. We have thus shown that for this particular virus and in the guinea pig, one or two subcutaneous doses of I cc. of any of the different virus preparations, each containing 3 x 10³ to 3 x 10⁴ mouse infective units, bring about protection regularly against experimental infection by way of the nose or subcutis. The results are irregular when the test is made by way of the brain. By three injections, resistance is invariably obtained against as many as 10³ to 10⁴ lethal doses, given intracerebrally. No matter in what form the virus is given, as mouse passage, or adsorbed, or precipitated material, in certain instances fever occurs and virus circulates. With the amount of virus adequate for immunization (3,000 to 30,000 m.i.u.) a mild or subclinical infection may occur in the guinea pig without other manifestation of disease. Lesser quantities of virus apparently fail to gain a foothold in the animal and thus fail to bring about resistance. To conclude, a quantitative basis has been established for the comparison of the immunizing capacities of preparations employed in experimental equine encephalomyelitis in guinea pigs.     

EXPERIMENTAL PNEUMONIA IN GUINEA PIGS : II. EFFECT OF ANTI-AUTOLYSATE SERA ON PNEUMOCOCCUS PNEUMONIA IN GUINEA PIGS

1. Anti-pneumotoxic sera prepared in rabbits or horses by immunization with sterile filtrates of the pneumotoxin, under certain conditions protect against the pneumonia caused by the intratracheal injections of mixtures of living pneumococci and toxic autolysates. 2. The protection against the development of pneumonia is heterologous, at least as regards Type I, Type II, viz.: an anti-autolysate serum prepared by the immunization with a pneumotoxin from one type of pneumococcus will prevent the development of pneumonia caused by the injection of pneumococci and autolysate from another type. 3. Certain anti-pneumococcus horse sera used in the treatment of pneumonia in man, either contain no heterologous pneumonia-preventing antibodies or slight amounts only. These sera, however, protect against the pneumonia produced by injections of pneumococci and pneumotoxin of the homologous strain, the degree of protection depending on the amount of specific protective substances such sera contain. 4. Anti-pneumotoxic sera produced in rabbits or horses by the injection of sterile Berkefeld filtrates of the toxic autolysates contain no pneumococcus specific protective substances.     

A FILTERABLE VIRUS PRESENT IN THE SUBMAXILLARY GLANDS OF GUINEA PIGS

In the lesions of herpes simplex and similar conditions due to filterable viruses, cells are present which show characteristic alterations, particularly in the nucleus. The nucleus of these cells contains a mass which stains with acid dyes. Surrounding this mass is a clear space or halo, within which there are large granules staining with basic stains. These cells are little if at all enlarged. In a few human cases, especially in infants, enlarged cells have been found which contain nuclei showing changes similar to those seen in the abnormal cells of herpes simplex. In the ducts of the submaxillary glands of guinea pigs, Jackson observed structures which she considered to be protozoan parasites. Our own studies indicate, however, that these structures are greatly swollen epithelial cells with nuclei having the same characters as the nuclei of the atypical cells in the lesions of herpes simplex. These cells are usually surrounded by a mononuclear cellular reaction. They were found in 84 per cent of the full grown guinea pigs examined but they were present in only three of forty-three young guinea pigs less than 1 month old. The resemblance of these cells, except as regards size, to the atypical cells present in lesions due to filterable viruses suggested that they also may be the result of an infection with a similar agent. That they are usually not present in guinea pigs less than 1 month old indicates that natural infection usually occurs after this period. Experiments were therefore undertaken to determine whether or not an infective agent is concerned in this condition and if so to learn something of its nature. When an emulsion of the submaxillary glands of full grown guinea pigs is injected into the brains of young guinea pigs the animals have fever and exhibit symptoms of cerebral irritation. They usually die in 5 to 7 days and in sections of the brain a diffuse subacute meningitis is found. In the exudate there are large numbers of cells having all the characteristics of the abnormal cells of herpes simplex. Similar cells are present in the lesions resulting from the injection of the same emulsion into the testicle, lung, tongue, and submaxillary glands of young guinea pigs. In none of these lesions, however, are the cells greatly enlarged as they are in the lesions in old guinea pigs. These results support the view that the lesion in the submaxillary gland of old guinea pigs is due to an infective agent. Attempts were therefore made to transmit this agent through a series of young guinea pigs. When the injections were all made into the same organ all the experiments but one gave negative results, but when the site of injection was changed at each transfer it was possible in a number of instances to reproduce the lesions through two animals in series and in one experiment through three animals in series. By modifying the technique, efforts were made to transmit the infection indefinitely but these attempts were unsuccessful. No explanation can be offered for this failure. Studies made to determine some of the properties of the infective agent have shown that it is destroyed by heating at 54° for 1 hour, and that it is not injured by preservation in 50 per cent glycerol for as long as 11 days. After the material had remained in 50 per cent glycerol for 28 days, however, it was found to be no longer infective. The infective agent was not held back by a Berkefeld N filter which was impermeable to bacteria. It seems probable therefore that the infective agent belongs in the group of filterable viruses, though further work will be necessary to learn more of its exact nature. These observations present additional evidence that the presence of cells with nuclear inclusions in any lesion indicates that the injury is probably due to an infective agent belonging in the group of filterable viruses.     

EXPERIMENTS WITH THE VIRUS OF POLIOMYELITIS

1. Efforts to adapt the virus of poliomyelitis to the rabbit organism and to produce poliomyelitis in rabbits by testicular injection and by brain injection after testicular passage produced no evidence that the virus could be adapted in this manner. Suggestive symptoms produced in very young rabbits were duplicated in non-specifically treated and in uninoculated controls. The admixture of a vaccine virus, adapted to the rabbit organism, with the poliomyelitis virus in similar injections and passages did not aid the adaptation. The virus of poliomyelitis did not survive 24 hours in the rabbit testicle—whether alone or mixed with vaccine virus. Repeated intraperitoneal and intradermal injection of poliomyelitis virus and of poliomyelitis and vaccinia virus mixtures produced no disease in rabbits. Massive doses of concentrated virus by stomach tube in conjunction with meningeal irritation produced no symptoms in rabbits. 2. No neutralizing substances against poliomyelitis virus could be produced in rabbits by the repeated intraperitoneal and intradermal injection of poliomyelitis virus or of poliomyelitis-vaccinia virus mixtures. 3. Although attempts to infect monkeys by intrastomachic injections, after bile irritation of the mucosa, were entirely negative, evidence was obtained that repeated intrastomachic injection after bile irritation may produce an appreciable degree of immunity. 4. No evidence could be obtained that the cellular elements of the blood contain the virus in any greater proportion than the whole blood. 5. One attempt to immunize by neutral virus-serum mixtures was entirely negative.     

STUDIES UPON EXPERIMENTAL MEASLES : I. THE EFFECTS OF THE VIRUS OF MEASLES UPON THE GUINEA PIG.

In general it may be said that three striking alterations occurred constantly in the animals reacting to intracardiac injections of blood from cases of measles; namely, pyrexia, leucopenia, and nephritis. The elevation in temperature usually began about the 9th day following inoculation, the rise being fairly abrupt from the normal to 104°F. and above, at which height it remained with slight fluctuations for 3 or 4 days, finally subsiding by lysis. During the period of fever, particularly in the animals of first transmission, often no objective signs of illness were to be noted, the guinea pigs remaining lively and eating as usual. When the animals showed any outward signs these were manifested by a loss of appetite, lusterless and ruffled hair, and indisposition to move even when disturbed. The typical exanthematous signs of human measles were not observed in any of the animals. In some there occurred a slight coryza and watering at the eyes; however, no special account was attached to these signs as they were rather indefinite and inconstant, especially for animals of first transmission. Coincident with the rise of temperature there always appeared a fall in the total leucocytic count; in some animals the drop in white cells preceded the rise in temperature by 2 to 3 days. The leucopenia, though well defined, varied for animals of the same series. In some the count was as low as 3,600 cells per c. mm.; in no case did we fail to note at least a moderate fall in the leucocytes. The average time for the appearance of this cellular change was 9 to 10 days following inoculation; in other words, the leucocytic reaction seemed to indicate the end of the incubation period. So constant and striking was the leucopenia that we stress this as the most significant feature of the reaction of the guinea pig to the injection of measles blood, particularly since normal blood produced not the slightest change in these white elements. The greatest leucocytic depression was observed around the 12th to 14th days, which corresponded approximately to the stage of temperature peak. Following the leucopenia, the leucocytic rise was very gradual, taking on the average 8 days to attain the normal level. The animals killed at the height of the reaction, or those dying presumably from the effects of the blood containing virus, showed as the most constant lesion gross evidences of acute nephritis. The kidneys in these cases were swollen, cloudy, and congested. In certain guinea pigs there were in addition well defined petechiæ and larger blood extravasations scattered throughout the cortical substance. In the animals dying after inoculation, the kidney alterations were found so constant and characteristic that we regarded them as a special index of the experimental infection. In these animals the hemorrhagic areas were not infrequently 2 to 5 mm. in extent, and when occurring on the surface of the organ produced a separation of the capsule. Occasionally free blood was noted in the pelvis, and in the absence of hemorrhage within the pelvic lining, we assumed that it came from the uriniferous orifices of the calicos. Microscopically the kidney sections revealed evidence of acute hemorrhagic nephritis, the hemorrhages for the most part being related to the capillaries of the tufts and the tubules of the pyramids. In the most pronounced cases practically all glomeruli were affected. Every stage from marked dilatation of the capillary whirl to well defined intercapillary blood extravasations that often filled the capsular space, and in consequence partially or completely obliterated the glomeruli, were found. Whether a destruction of the endothelium occurred, could not be determined. The absence of a neutrophilic reaction was of special significance, these elements of acute inflammation being nowhere found associated with the kidney lesion. The blood in relation to the tubules commonly occurred in the lumen of the collecting tubules and in the form of red blood cell casts. The spleen usually was found enlarged, particularly in the guinea pigs presumably dying as a result of the virus injections. Aside from the usual parenchymatous changes common to toxemias, the other internal organs showed nothing of special note.     

MULTIPLICATION IN VITRO OF PSEUDORABIES VIRUS IN THE TESTICLE TISSUE OF IMMUNIZED GUINEA PIGS

Pseudorabies virus was cultivated in vitro in washed testicle tissue from immune guinea pigs, and evidence was thus procured which indicated that the testicle cells themselves had not become immune to pseudorabies. The rate of multiplication of the virus was considerably greater in control cultures with normal guinea pig testis than in cultures with immune testis. The reason for this fact may be that even by repeated washing the immune tissue could not be completely freed from fluid antibodies, and that such antibodies somewhat inhibited the multiplication of the virus.