THE LIMITED NEUROTROPIC CHARACTER OF THE ENCEPHALITIS VIRUS (ST. LOUIS TYPE) IN SUSCEPTIBLE MICE

1. St. Louis encephalitis virus injected intracerebrally into susceptible mice multiplies there to reach a titre of 10⁹ intracerebral lethal doses. It is found also in the blood in small amounts immediately following injection and preceding death. 2. Injected intraperitoneally or subcutaneously the virus circulates in the blood for several hours and survives in the spleen for days. It does not multiply in the brain and cause encephalitis, however, unless overwhelming doses are injected or the brain is traumatized. 3. Virus dropped into the nares is demonstrable in the olfactory bulbs at 24 hours, in the piriform lobes at 24 to 48 hours, in the remainder of the brain at 3 days, and in the spinal cord at 4 days. In the brain it reaches a titre of 10⁹ in 6 days. Virus is not readily demonstrable in the blood but is present in the spleen after 48 hours. 4. Virus survives and is capable of multiplying in the spleen. 5. Lesions following nasal instillation of virus appear first in the olfactory bulbs on the 3rd day, in the piriform lobes on the 4th, and in Ammon''s horn on the 5th day. The character of the lesions in order of their appearance is exudation of mononuclear cells about superficial blood vessels and in the pia, hyperplasia of the endothelium of the pia, and necrosis of nerve cells of the olfactory tract.     

EXPERIMENTAL STUDIES ON ENCEPHALITIS : I. TRANSMISSION OF ST. LOUIS AND KANSAS CITY ENCEPHALITIS TO MICE

1. Mice of special strains injected intracerebrally with a 10 per cent emulsion of bacteria-free brain tissue from fatal cases of encephalitis in St. Louis and Kansas City develop a characteristic and fatal encephalitis. 2. Transmission of the disease can be continued indefinitely by injecting the bacteria-free brain tissue from the infected mice into healthy mice. 3. In the injected mice there is a 3 to 4 day incubation period, followed by hyperesthesia, coarse tremors, convulsions, prostration, and death in from 4 to 6 days. 4. The lesions in the mice with experimental encephalitis consist chiefly of perivascular accumulations of mononuclear leucocytes throughout the brain, stem, cord, and the pia, and destruction of pyramidal cells in the lobus piriformis and cornu Ammonis. 5. The human encephalitis brain tissue preserved in glycerine from the time of death of the patient apparently loses its infectivity for mice in about 32 days.     

EXPERIMENTAL STUDIES ON ENCEPHALITIS : II. THE SPECIFIC VIRUS CHARACTER OF THE INFECTIOUS AGENT FROM CASES OF ST. LOUIS AND KANSAS CITY ENCEPHALITIS, 1933

1. The infectious agent from fatal cases of St. Louis and Kansas City encephalitis passes Seitz pads in high dilution without appreciable loss of infectivity and traverses collodion membranes with an average pore size of 66 mµ or greater. 2. It is highly infectious for mice by the intracerebral and intranasal routes, but practically innocuous by the subcutaneous and intraperitoneal routes. 3. Certain strains of mice are more susceptible than others. 4. The agent administered to mice intranasally causes tremors and convulsions after a 6 to 7 day incubation period, followed by prostration and death in 8 to 10 days. Lesions are demonstrable in the olfactory bulbs 3 days after infection, in the piriform lobe after 4 days, and in Ammon''s horn after 5 days. 5. In Macacus rhesus monkeys, the agent provokes a mild, non-fatal reaction and the development of specific neutralizing bodies. On passage in monkeys, the virus becomes progressively weaker. 6. In rabbits, guinea pigs, rats, and sheep the agent is apparently without effect. 7. All available strains of the agent proved alike in effects in animals and in immunological response. 8. The available data enable one to conclude that the agent is a filtrable virus differing from those studied heretofore.     

EXPERIMENTAL ENCEPHALITIS (ST. LOUIS TYPE) IN MICE WITH HIGH INBORN RESISTANCE : A CHRONIC SUBCLINICAL INFECTION

1. St. Louis encephalitis virus injected intracerebrally or intraperitoneally in maximum doses in resistant mice is distributed and is effective in a manner generally similar to that in susceptible mice. The minimum infecting dose is at least 1,000 times greater in resistant than susceptible mice and virus injected in the brain tends to remain at a relatively low titre, persist for a few days, and then disappear. 2. Virus dropped in the nares is demonstrable and progresses in the brains of resistant mice as in susceptible mice, but does not increase in titre beyond the 5th day, does not bring about fatal encephalitis, and persists for at least 4 weeks. 3. Lesions in the brains of resistant mice following nasal instillation of virus do not appear until the 8th day, reach a maximum at 40 days, and are still present, though resolving, at 3 months. The changes resemble those seen in the human disease and in other unnamed forms of encephalitis. 4. The quantity of virus drops 1,000-fold when recovered from resistant mice and becomes non-infective by the nasal route. Passage in susceptible mice promptly restores its full titre.     

EXPERIMENTAL STUDIES ON ENCEPHALITIS : IV. SPECIFIC INACTIVATION OF VIRUS BY SERA FROM PERSONS EXPOSED TO ENCEPHALITIS,ST. LOUIS TYPE, 1933 WITH A NOTE ON THE EVALUATION OF THE RESULTS OF MOUSE TESTS OF SERA

1. A protection test for measuring serological protective properties against the encephalitis (St. Louis type) virus is described. 2. Normal non-contact sera and sera from persons supposed to have had no exposure to the disease do not protect against the virus. 82.5 per cent of sera from tested St. Louis encephalitis convalescents and at least 66 per cent of sera from tested persons thought to have had the disease do show protective properties. 3. The protective activity of sera is maintained for at least 2½ years after onset of the disease. In vitro aging of serum decreases its activity. 4. Protection tests indicate that the virus was present as early as 1932 in Paris, Illinois, spread through the north central states and reached New York in 1933, and was again active in the north central states in 1934.     

ST. LOUIS ENCEPHALITIS VIRUS IN THE BLOOD OF EXPERIMENTALLY INOCULATED FOWLS AND MAMMALS

1. Of three species of mammals tested by peripheral inoculation (guinea pig, cat, and horse) none showed viremia under conditions which suggested that any of these species would serve as a frequent source of mosquito infection. 2. Of the birds tested (chicken, duck, and dove) all developed viremia and might readily serve as natural sources of mosquito infection. Chickens were shown to be very highly susceptible to infection by minute amounts of virus inoculated subcutaneously. 3. Virus may appear in the blood of chickens within 16 hours after inoculation and it has persisted till at least the 120th hour. No fowl showed any sign of illness as a result of the infection.     

LABORATORY TRANSMISSION OF ST. LOUIS ENCEPHALITIS VIRUS BY THREE GENERA OF MOSQUITOES

1. St. Louis virus has been successfully transmitted in the laboratory by the following 9 species of mosquitoes from 3 genera: Culex tarsalis, Culex pipiens, Culex coronator, Aedes lateralis, Aedes taeniorhynchus, Aedes vexans, Aedes nigromaculis, Theobaldia incidens, and Theobaldia inornata. 2. Though transmission has not been demonstrated, survival of the virus for more than a few days was shown to occur in Culex quinquefasciatus, Culex stigmatosoma, Psorophora ciliata, and Anopheles maculipennis freeborni. 3. In experiments with Culex tarsalis, infection occurred from feeding on chickens and ducks which had been previously inoculated by the subcutaneous route. After an incubation period these mosquitoes infected other chickens and virus was in turn demonstrated in the blood of these. This is interpreted as proof that fowl may serve as reservoirs of virus in nature. Since mosquitoes have been repeatedly found naturally infected with St. Louis virus and epidemiologic evidence supports their incrimination, their rôle as vectors is now established. The fully incriminated species is Culex tarsalis.     

JAPANESE B ENCEPHALITIS VIRUS: ITS DIFFERENTIATION FROM ST. LOUIS ENCEPHALITIS VIRUS AND RELATIONSHIP TO LOUPING ILL VIRUS

1. Japanese B encephalitis virus, obtained from Japanese investigators, has proved virulent for mice and monkeys, confirming the reports from Japan. It has also been found virulent for monkeys when instilled intranasally and for sheep when introduced intracerebrally or intranasally. 2. Japanese B encephalitis virus has been differentiated from St. Louis virus and found similar to louping ill virus according to its reactions in animal species. Serologically, however, it is distinct. 3. Japanese B encephalitis and its related group of primary virus encephalitides of man have been discussed with regard to their differentiation and mode of spread.     

INFLUENCE OF AGE ON SUSCEPTIBILITY OF MICE TO ST. LOUIS ENCEPHALITIS VIRUS AND ON THE DISTRIBUTION OF LESIONS

1. Young mice are more susceptible than older mice to the virus of St. Louis encephalitis inoculated intraperitoneally, but with virus inoculated intracerebrally or intranasally, there is no significant age difference in susceptibility. The greatest change in the resistance to the virus inoculated intraperitoneally occurs between the 2nd and 3rd weeks of life. 2. The distribution of the lesions of St. Louis encephalitis in the C.N.S. of young and of old animals following intraperitoneal inoculation indicates that the virus may reach the brain either by the ascending pathway from the spinal cord or by the olfactory pathway irrespective of the age of the animal. However the ascending pathway is most frequently concerned. 3. The distribution of lesions does not offer evidence that the virus enters the C.N.S. of young animals directly from the blood stream following intraperitoneal inoculation. 4. Although widespread lesions occur earlier in the C.N.S. of young mice than in that of older mice inoculated intraperitoneally with large doses of virus, this fact is not satisfactorily explained by assuming the more rapid increase of the virus in the C.N.S. of young animals, since the latter are not more susceptible to virus inoculated directly into the brain. 5. The observations can be explained by the hypothesis that a greater amount of virus survives and reaches the portals of the C.N.S. in young animals following intraperitoneal inoculation and that this is an important factor in the influence of age on susceptibility to the virus.     

INFLUENCE OF ANESTHESIA ON EXPERIMENTAL NEUROTROPIC VIRUS INFECTIONS : II. IN VITRO STUDIES WITH THE VIRUSES OF WESTERN AND EASTERN EQUINE ENCEPHALOMYELITIS,ST. LOUIS ENCEPHALITIS,POLIOMYELITIS (LANSING), AND RABIES

1. Experimental neurotropic virus infections previously shown to be altered by ether anesthesia are caused by viruses destroyed in vitro by anesthetic ether; this group includes the viruses of Eastern equine encephalomyelitis, Western equine encephalomyelitis, and St. Louis encephalitis. 2. Experimental neurotropic virus infections which were not altered by ether anesthesia are caused by viruses which are refractory to the in vitro virucidal activity of even large amounts of anesthetic ether; this group includes the viruses of poliomyelitis (Lansing) and rabies. 3. Quantitative studies of the in vitro virucidal activity of ether indicate that concentrations of this anesthetic within the range found in central nervous system tissues of anesthetized animals possess no virucidal activity. 4. The lowest concentration of ether possessing significant virucidal capacity is more than fifteen times the maximum concentration of the anesthetic tolerated by the experimental animal. 5. Concentrations of ether 50 to 100 times the maximum amount tolerated by the anesthetized animal are capable of destroying large amounts of susceptible viruses, the average lethal dose (LD₅₀) being reduced more than 5 log units. 6. On the basis of the studies presented in this report, it cannot be concluded that direct virucidal activity of ether is not the underlying mechanism of the inhibition by anesthesia of certain experimental neurotropic virus infections. Indirect inhibition of the virus by the anesthetic through an alteration in the metabolism of either the host cell or the host animal as a whole appears at this point to be a more likely possibility.     

INFLUENCE OF ANESTHESIA ON EXPERIMENTAL NEUROTROPIC VIRUS INFECTIONS : I. IN VIVO STUDIES WITH THE VIRUSES OF WESTERN AND EASTERN EQUINE ENCEPHALOMYELITIS,ST. LOUIS ENCEPHALITIS,POLIOMYELITIS (LANSING), AND RABIES

Anesthesia with diethyl ether significantly alters the course and outcome of experimental infections with the equine encephalomyelitis virus (Eastern or Western type) or with the St. Louis encephalitis virus. No comparable effect is observed in experimental infections produced with rabies or poliomyelitis (Lansing) viruses. The neurotropic virus infections altered by ether anesthesia are those caused by viruses which are destroyed in vitro by this anesthetic, and those infections not affected by ether anesthesia are caused by viruses which apparently are not destroyed by ether in vitro. Another striking difference between these two groups of viruses is their pathogenesis in the animal host; those which are inhibited in vivo by ether anesthesia tend to infect cells of the cortex, basal ganglia, and only occasionally the cervical region of the cord. On the other hand, those which are not inhibited in vivo by ether anesthesia tend to involve cells of the lower central nervous system and in the case of rabies, peripheral nerves. This difference is of considerable importance in view of the fact that anesthetics affect cells of the lower central nervous system only in very high concentrations. It is obvious from the complexity of the problem that no clear-cut statement can be made at this point as to the mechanism of the observed effect of ether anesthesia in reducing the mortality rate in certain of the experimental neurotropic virus infections. Important possibilities include a direct specific effect of diethyl ether upon the virus and a less direct effect of the anesthetic upon the virus through its alteration of the metabolism of the host cell.     

EFFECT OF PREGNANCY UPON THE IMMUNITY OF MICE VACCINATED AGAINST ST. LOUIS ENCEPHALITIS VIRUS

1. Virgin and pregnant Swiss mice are equally susceptible to intracerebral inoculation of St. Louis encephalitis virus. 2. Following subcutaneous vaccination with the St. Louis virus, the great majority of virgin Swiss mice become immune to subsequent intracerebral injection of 10,000 M.L.D. of the virus. 3. The majority of mice vaccinated during pregnancy do not become immune to even as little as 500 intracerebral M.L.D. of the virus. The depression of the ability to acquire immunity against the virus is most marked when the vaccination is carried out late in pregnancy, but it is also demonstrable when the mice are vaccinated early in the gestation period and during the first 2 weeks postpartum. At 7 weeks postpartum the response to vaccination is more nearly like that of virgin mice. 4. Pregnancy not only interferes with the development of acquired immunity but it also diminishes a previously established immunity. 5. Offspring of the mice vaccinated during pregnancy are not immune to 100 M.L.D. of virus.     

MULTIPLICATION AND SPREAD OF THE VIRUS OF ST. LOUIS ENCEPHALITIS IN MICE WITH SPECIAL EMPHASIS ON ITS FATE IN THE ALIMENTARY TRACT

1. Beginning at 24 hours after intravenous injection of about 10 million intracerebral LD₅₀ of virus there was evidence of simultaneous, progressive multiplication in the brain and intestinal tract. 2. When the virus was introduced directly into the brain or the nasal cavities and mouth, none was found in the intestinal tract until there was general centrifugal spread from the central nervous system during the last stages of the infection at 96 or 120 hours after inoculation when the virus in the entire brain had reached a concentration of about 3 billion LD₅₀. 3. Centrifugal spread began when the virus in the brain reached a concentration of about 400 million LD₅₀ and virus appeared in the pharynx, tongue, and adrenals before it was demonstrable in the intestinal tract, blood, or viscera such as the spleen, liver, and kidneys. 4. Despite the high concentrations of virus which developed in the intestinal tract following intravenous inoculation, it was not demonstrable in the stools, differing in this respect from Theiler''s virus in mice and poliomyelitis virus in human beings and monkeys. 5. No antiviral agent was found in the stools, but the urine of normal mice having a pH of 5.6, inactivated large amounts of St. Louis encephalitis virus. 6. There was no evidence of multiplication in the nasal mucosa of mice which succumbed with encephalitis following nasal instillation of the virus, the course of events being comparable in this respect to the behavior of the M.V. poliomyelitis virus in rhesus monkeys. 7. At the terminal stage of infection the virus content per milligram of tissue was as great in the leg muscles as in the sciatic nerves. Since this was also true for the urinary bladder, heart, lungs, and tongue among other tissues, and since the amount in the blood was too negligible to account for it, it would appear that the virus either accumulated in these tissues by diffusion from the nerve fibers, along which it was spreading from the central nervous system, or that it multiplied in some constituent other than the nerve fibers.     

CONTRIBUTIONS TO THE PATHOLOGY OF EXPERIMENTAL VIRUS ENCEPHALITIS : V. HERPES VIRUS ENCEPHALITIS IN THE GUINEA PIG.

The guinea pig is subject to cerebral and corneal inoculation of the herpes virus. The effects of the inoculations vary with the strength or degree of virulence of the virus. Weak strains of the virus are implanted on the cerebrum with difficulty and strong strains with ease. Weak strains are quickly suppressed by the brain and strong strains may be passed indefinitely from brain to brain of the guinea pig. Strains of intermediate potency can be passed for a limited number of times only. Weak strains induce keratoconjunctivitis without brain involvement, while strong strains invade the brain from the eye and produce fatal encephalitis. In the latter case, the brain contains active virus inoculable upon the cornea and into the brain of rabbits and guinea pigs. Strains of intermediate potency produce keratoconjunctivitis accompanied by mild symptoms of encephalitis, from which recovery results. The guinea pig serves even more definitely than the rabbit to distinguish grades of virus according to strength or virulence. There is no difference of kind but only of degree of response to inoculation of herpes virus in the rabbit and the guinea pig. The etiology of epidemic encephalitis has not, therefore, been brought appreciably nearer solution by experiments with herpes virus carried out in guinea pigs.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : II. SURVIVAL OF THE VIRUS IN AEROBIC AND ANAEROBIC CULTURE MEDIA.

The results of the foregoing experiments show that the typhus virus, found in the blood of guinea pigs during the height of typical experimental typhus fever, does not survive at 37°C. in anaerobic media for as long a period as in the same media under aerobic conditions. In media from which oxygen is excluded, the viability period is 24 to 48 hours; in the same media having no barrier to atmospheric oxygen, the period is usually 5 days, in one instance, 3 days. The dead virus fails to induce not only the typical experimental disease but also an immunity to further injections of typhus virus. That the death of the virus is due to exclusion of oxygen from the medium, rather than to a change in the hydrogen ion concentration therein is inferred from the fact that media with varying hydrogen ion concentrations, such as broth (pH 7.4), horse serum (pH 7.8), and human ascitic fluid (pH 8.0) have the same comparative effect on the virus, when under aerobic and anaerobic conditions. That is, in all, the anaerobic state causes a shortening of the viability period of the typhus virus. In the Smith-Noguchi tissue, ascitic fluid, sealed medium in which bacteria resembling Plotz'' bacilli grow luxuriantly and remain viable for several weeks, the typhus virus does not increase in virulence, and even dies after 24 hours. This evidence supports the conclusion previously presented that the Bacillus typhi exanthematici of Plotz is not identical with the active agent of typhus virus.     

CONTRIBUTIONS TO THE PATHOLOGY OF EXPERIMENTAL VIRUS ENCEPHALITIS : III. VARIETIES AND PROPERTIES OF THE HERPES VIRUS.

Mild strains of the virus of herpes are described the action of which tends to be confined and local. Unless, therefore, these mild strains are injected intracranially they do not tend to produce virus encephalitis in the rabbit. Recovery from infection with the mild strains confers immunity to virulent strains of the herpes and allied viruses. Long glycerolation reduces the number of viable organisms. This loss among the mild strains may reduce the virus below the strength required for an effective extracranial although not below the strength needed for an intracranial inoculation. Herpes virus carriage in man, even under highly favorable conditions, is difficult of detection by means of rabbit inoculation. The detection may be achieved by intracranial when it cannot be accomplished by intracorneal inoculation. The virus producing encephalitis in the rabbit attaches itself chiefly to and multiplies in the substance of the central nervous system. Hence its detection in the cerebrospinal fluid is rarely accomplished. When the inoculation of the virus is made intracranially and especially when the inoculum is composed of active brain tissue, the virus is discoverable in the cerebrospinal fluid by rabbit inoculation much more frequently than when the virus encephalitis follows an extracranial variety of infection. The herpes virus is capable of excretion by the kidney of the rabbit and of being detected in the urine by rabbit inoculation. Among the rarer symptoms of virus encephalitis is excessive lacrimation. While salivation is frequent, lacrimation is exceptional. A comparison of the Levaditi, Doerr, and Goodpasture strains of virus indicates the first to be of medium, the second of mild, and the third of high degree of neurotropic activity. The Doerr strain resembles the mild herpes strains described in this paper. The Goodpasture virus, while exceeding the Levaditi strain in affinity for the central nervous system, falls below the H. F. strain in this regard. Neutralization of virus by the serum of infected and recovered rabbits takes place regularly within certain quantitative limits. Neutralization with human serum is inconstant and capricious and without demonstrable relation to previous attacks of epidemic encephalitis. Comparison of the clinical types of encephalitis as presented by the epidemic variety in man and the experimental virus variety in rabbits brings out certain correspondences and certain differences. It is only in partial and essentially superficial aspects that the two diseases can be identified one with the other.     

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.     

BLOOD-SUCKING VECTORS OF ENCEPHALITIS: EXPERIMENTAL TRANSMISSION OF ST. LOUIS ENCEPHALITIS (HUBBARD STRAIN) TO WHITE SWISS MICE BY THE AMERICAN DOG TICK,DERMACENTOR VARIABILIS SAY

(1) The common dog tick, Dermacentor variabilis, is capable of being infected with the virus of St. Louis encephalitis, Hubbard strain, by feeding on inoculated animals and, once infected, can transmit the virus to normal susceptible animals by bite. (2) A female can transmit the infection to her offspring, through all stages of metamorphosis of the 2nd generation into the 3rd generation. (3) Ticks infected under laboratory conditions and kept inactive at a temperature of 12.5°C., remained infective for at least a period of 10 months. Eggs laid by an infected female and stored in a refrigerator (12.5°C.) for 10 months retained infective virus, and larvae hatched from such eggs at the end of the 10 months of dormancy were also infective. (4) The present work, a preliminary account of which appeared in December, 1941 (1, 2), is of theoretical significance since in so far as we are aware, it represents the first successful transmission of St. Louis encephalitis to experimental animals by a blood-sucking vector.     

ST. LOUIS ENCEPHALITIS : TRANSMISSION OF VIRUS TO CHICKENS BY INFECTED MITES DERMANYSSUS GALLINAE AND RESULTING VIREMIA AS SOURCE OF VIRUS FOR INFECTION OF MITES

Transmission of the virus of St. Louis encephalitis to normal chickens by the bite of infected mites (Dermanyssus gallinae) has been demonstrated. Both experimentally infected and naturally infected mites were shown to be capable of transferring the virus of St. Louis encephalitis to chickens by bite. Virus is present in the blood of such chickens in small amounts, so that demonstration of viremia was possible only by utilizing chorioallantoic passage in hens'' eggs. However, there is sufficient virus present in the blood for uninfected chicken mites to acquire the virus by feeding on chickens in which viremia has resulted from previous bite of infected mites. Thus it has been shown that the arachnid vector Dermanyssus gallinae is capable of transmitting the virus of St. Louis encephalitis to normal chickens by bite and that such chickens can serve as a source of virus for uninfected mites.