THE PREPARATION OF HIGHLY PURIFIED PR8 INFLUENZA VIRUS FROM INFECTED MOUSE LUNGS

Highly purified preparations of PR8 influenza virus were obtained from perfused, infected mouse lungs by a combination of methods involving adsorption of the virus on and elution from chicken red cells and differential centrifugation. Such preparations were found to possess 50 per cent infectivity end-points at 10^–11 to 10^–11.8, and 10^–13 to 10^–14.3 gm. of nitrogen in mice and in chick embryos, respectively. A sedimentation constant of 683 S was obtained for the material and electron micrographs revealed essentially spherical particles about 100 mµ in diameter. The material was isoelectric at pH 5.4 and chemical analyses on several of the preparations indicated the presence of 10.1 per cent of nitrogen, 1.06 per cent of phosphorus, 6.2 per cent of carbohydrate and about 33 per cent of lipid. Positive tests were obtained for both ribonucleic and desoxyribonucleic acids. The virus was precipitated strongly by antisenim to purified PR8 virus obtained from the allantoic fluid of infected chick embryos and this serum inhibited the agglutination of red cells by the mouse virus to a dilution of about 40,000. In general, the properties of the virus isolated from infected mouse lungs were found to coincide with those of the virus obtained from the allantoic fluid of infected chick embryos.     

INHIBITION OF PHOSPHORYLATION OF GLUCOSE IN MOUSE BRAINS BY VIRUSES AND ITS PREVENTION BY PREPARATIONS OF DIPHOSPHOPYRIDINE NUCLEOTIDE

1. Inhibition of glucose utilization in homogenates of brains of mice infected with poliomyelitis virus (Lansing strain) is reported. The inhibition occurs with glucose, or fructose-6-phosphate as substrate. No inhibition occurs in the presence of hexosediphosphate. 2. Brain homogenates of mice infected with the Theiler FA strain of mouse encephalomyelitis virus show inhibition ranging between 45 and 85 per cent (average 73 per cent). 3. Purified preparations of the Lansing and the Theiler FA strain invariably inhibit glycolysis when added to homogenates of normal mouse brain. A similar, but much less consistent inhibition is provoked by adding high concentrations of non-neurotropic viruses (influenza A and tobacco mosaic virus) to normal mouse brains. 4. The magnitude of inhibition caused by the purified virus is a function of the virus concentration and depends on temperature and time of incubation of the virus-brain mixture. 5. The inhibition of glycolysis in the brains of mice infected with Theiler FA virus and in normal brain-Theiler FA virus mixtures is prevented by the addition of preparations of DPN and glucose.     

Morphological and quantitative comparison between infectious and non-infectious forms of influenza virus

Electron microscopic study has revealed the morphological entity responsible for the rise in viral hemagglutinin observed in brains of mice after intracerebral inoculation of non-neurotropic strains of influenza virus. This rise in hemagglutinin, although dependent on inoculation of fully infectious virus, is not associated with an increase in infectious titer. The hemagglutinating principle is functionally similar to the "incomplete" influenza virus which can be obtained from chick embryos by serial egg-to-egg transfer of undiluted, infected allantoic fluid according to the method of von Magnus. A method has been described which facilitates selective adsorption of viral particles recovered from organ extracts on saponine-lysed ghosts of fowl erythrocytes. This procedure has been utilized in studying the morphology of non-infectious, hemagglutinating virus from chorio-allantoic membranes or mouse brains and in comparing these two forms with each other and with ordinary, infectious (standard) influenza virus. Standard virus isolated from allantoic fluids or membranes of infected eggs was found to contain uniform particles of predominantly spherical shape with smooth surface and even density, resembling those described by others. The appearance of such particles was not affected by the procedure of extraction and concentration used. In contrast, non-infectious, hemagglutinating virus obtained either from allantoic sacs ("undiluted passages") or from mouse brain was pleomorphic and seemed to consist of disintegrating particles. The majority appeared flattened and bag-like and had a rough, granular surface and reduced, uneven density. 37 per cent of the non-infectious particles isolated from mouse brain infected with the non-neurotropic strain WS had diameters in excess of 170 mµ, as compared with only 2 per cent of the particles of the parent strain itself. Regardless of whether or not the contrast in appearance of standard and of non-infectious particles was due to differing resistance to the preparatory treatment, it indicated the existence of basic structural differences between the two types of virus. Correlation of particle counts with hemagglutinin titers has shown that the non-infectious virus obtained from mouse brain is, unit for unit, an equivalent counterpart of standard virus derived from infected eggs. The end-point of hemagglutination in a pattern test corresponds for both forms to that dilution at which the ratio virus particles/red cells approaches one. The quantitative data based on particle counts support the assumption that non-infectious virus arises in mouse brain as a product of viral multiplication.     

INFLUENCE OF pH AND OF CERTAIN OTHER CONDITIONS ON THE STABILITY OF THE INFECTIVITY AND RED CELL AGGLUTINATING ACTIVITY OF INFLUENZA VIRUS

A study has been made of the pH stability of centrifugally purified strains of influenza virus with respect to the biological properties of mouse infectivity and chicken red blood cell agglutinating activity. Observations also were made on the importance of composition of buffer, temperature of storage, and concentration of virus protein to the stability of the virus. When tested for stability at a protein concentration of 0.1 mg. per cc. in phosphate buffer, the infectivity of PR8 virus was found to be most stable at pH 6.5–7; the swine virus, at pH 7–7.9; and the Lee strain, at a pH of 7.9 or higher. The CCA activity of the PR8 virus in phosphate buffer was most stable at pH 7, that of the swine virus at pH 7–8, and that of the Lee virus at a pH greater than 9. Furthermore, the Lee virus was much less stable in dilute solution in phosphate buffer, even under optimum conditions of pH, than either the PR8 or swine strains. The different strains of influenza virus were found to possess certain characteristics in common. They lost infectivity and CCA activity on the acid side of optimum pH conditions much more rapidly than on the alkaline side. Under suitable conditions of buffer and pH, the infectivity decreased while the CCA activity remained unchanged. In general, the rate of loss in infectivity was greater than the rate of loss in CCA activity. When tests of stability were carried out at a protein concentration of 0.1 mg. per cc. in a composite phosphate-glycine-NaCl buffer, the virus strains showed less marked differences and possessed much higher stabilities of CCA activity and infectivity than when stored at the same concentration in phosphate buffer alone. Under the modified conditions, all three viruses possessed maximum stabilities of CCA activity and infectivity at pH 7–8 with the exception of the PR8 virus whose infectivity appeared more stable at pH 7 than at pH 8. In detailed experiments with the Lee virus, it was found that the infectivity and CCA activity of this strain at pH 7 and at a protein concentration of 0.1 mg. per cc. were maintained best in the composite phosphate-glycine-NaCl buffer, less well in a buffer containing glycine and NaCl, and least well in phosphate buffer alone. In tests with PR8 virus, the activity was found to be much more stable at 4° C. than at 23° C. When stored at a concentration of 2 mg. per cc. at 4° C. in phosphate buffer at pH 7, the PR8 and Lee strains were found to be much more stable than when stored at the concentration of 0.1 mg. per cc. At the higher concentration, no significant losses in either infectivity or CCA activity were observed over a period of 2 months.     

COMPLEMENT FIXATION WITH THE NEUROTROPIC VIRUSES

Antigens capable of fixing complement specifically with the appropriate antibodies have been prepared from brain tissue of hamsters and mice infected with the viruses of St. Louis, Japanese, Western, and Eastern encephalitis, and with the West Nile virus. The antigens were freed of the material which reacts with normal serum by means of centrifugation at relatively high speed. In addition, the infectivity of the preparation was destroyed by irradiation with ultraviolet light. Cross reactions were demonstrated by means of the complement-fixation technique with materials from animals infected with the viruses of Eastern and Western equine encephalitis. No relationship was detectable by this procedure between St. Louis, Japanese, and West Nile viruses. These findings emphasize the need for further investigation and correlation of the immunological reactions of the groups of neurotropic viruses, since the equine agents are apparently unrelated when studied by neutralization and cross-immunity tests while these methods provide evidence of the presence of common antigenic structures in the St. Louis, Japanese, and West Nile agents.     

THE PATHOGENESIS OF HERPES VIRUS ENCEPHALITIS : II. A CELLULAR BASIS FOR THE DEVELOPMENT OF RESISTANCE WITH AGE

The resistance to herpes virus encephalitis which develops with age was studied in mice using fluorescent antibody staining. Adult mice remained susceptible to intracerebral inoculation, and the infection of the central nervous system was identical with that found in immature mice. A "barrier" to the spread of virus inoculated extraneurally developed with maturation, and the limitation of spread appeared to coincide with the infection of peritoneal and tissue macrophages. In vitro, suckling and adult mouse macrophages were infected with equal ease. However, suckling mouse macrophages infected other cells in contact with them, while infected adult mouse macrophages did not. Studies failed to reveal the nature of this change in macrophages which developed with age. The role of macrophages in the pathogenesis of herpes virus encephalitis is discussed. The hypothesis is made that an alteration in the macrophages of the maturing mouse plays an important role in its development of resistance to herpes virus encephalitis.     

LABORATORY TRANSMISSION OF JAPANESE B ENCEPHALITIS VIRUS BY SEVEN SPECIES (THREE GENERA) OF NORTH AMERICAN MOSQUITOES

In the present studies ten common species of Western North American mosquitoes have been tested for their ability to act as vectors of Japanese B encephalitis virus (see summary Table XII). The strain of Japanese B encephalitis virus which was used was adapted to direct mouse brain passage, probably a disadvantage, but no freshly isolated strain was available. Of the ten species of mosquitoes tested, seven were demonstrated to be laboratory vectors. These seven species represent three genera (Culex, Aedes, and Culiseta). In previously reported work Japanese and Russians had only incriminated five species of two genera (Aedes and Culex) (1–3). Transmission was made to mice 21 times and to a chicken once. Two attempts to infect mosquitoes from an infected chicken were unsuccessfui, but no significance is attached to so few experiments. Repeated tests for virus in the eggs, or in imagines reared from eggs of infected female mosquitoes have been negative. In this we failed to confirm results claimed by Japanese investigators (5, 6). These data, in addition to the published accounts by Japanese and Russian workers of the natural epidemiology of this disease lead us to believe that this virus might well establish itself in North America, especially if introduced in those areas where our native encephalitides are now endemic. These studies also indicate that species of mosquitoes (Culex tarsalis, Culex pipiens, See PDF for Structure Aedes dorsalis, and Culiseta inornata) now known to be fully incriminated vectors of the Western equine or St. Louis encephalitis viruses can also serve as laboratory vectors of the Japanese B virus. Methods for the effective abatement of these species should be further developed and put into practice if future epidemics of encephalitis of the Western equine, St. Louis, or Japanese B types in Western North America are to be prevented or brought under control.     

A SEDIMENTABLE COMPONENT OF ALLANTOIC FLUID AND ITS RELATIONSHIP TO INFLUENZA VIRUSES

Macromolecular material was isolated from normal allantoic fluid by a centrifugation procedure comparable to that currently employed for the concentration and purification of influenza viruses. The yield of material was found to vary with the age of the embryo, reaching a maximum average value after 14 days of incubation at 39°C. of about 0.02 mg. per ml. of allantoic fluid. The purified material was found to contain protein, carbohydrate, and lipid and to have a general composition similar to purified preparations of PR8 influenza virus. A typical preparation of normal material had an isoelectric point at pH 2.3. Sedimentation studies indicated that the normal material can give a variety of sedimentation constants depending upon the concentration and viscosity of the preparations. The sedimentation constant, corrected for viscosity, of the major component of a fresh preparation was 170 S. The diameters of the predominant particles shown in electron micrographs of the normal material and of preparations of PR8 influenza virus were about 40 and 100 mµ, respectively. Serological tests indicated that the normal material is a good antigen and that preparations of both A and B types of influenza virus obtained from allantoic fluids by centrifugation show a strong serological relationship to the normal material. Freezing and thawing of allantoic fluid, and repeated adsorption of virus on red cells, failed to provide a practical basis for the separation of normal protein from the virus entity in the case of PR8 virus. In the cases of similar preparations of F12 and of Lee viruses, a partial separation of a small component was accomplished by fractional centrifugation and this component and the normal protein were shown to be identical or very closely related. Antiserum to the purified normal material inhibited red cell agglutination by A and B types of influenza virus at serum dilutions of 600 to 700, but failed to show significant neutralizing capacity in chick embryo and in mouse tests at a serum dilution of 100. Rabbit antiserum to purified preparations of PR8 virus gave a 50 per cent red cell agglutination inhibition endpoint at a serum dilution of 112,000. Some of the implications of the findings are discussed.     

CHEMICAL AND IMMUNOLOGICAL STUDIES ON THE AGENT PRODUCING LEUKOSIS AND SARCOMA OF FOWLS

The activity of the agent producing sarcoma or leukosis in material deposited by high speed centrifugation is the same as that of the original crude extracts. Material sedimentable at high speed containing approximately 10^–5 mg. N produces tumors at the site of injection. Small quantities of material sedimentable at high speed are present in normal chicken sera, and about twice as much in leukemic sera (strain 1). Normal chicken and mouse spleens and all other human and mouse tissues examined contain large amounts of material sedimentable at high speed. Extraction of leukemic blood cells with saline yields little additional virus. The washed cells readily produce leukosis even after irradiation with amounts of x-rays sufficient to destroy the leukemic cells but not the virus. Freezing at –60°C. preserves the activity of the high speed deposits for at least 6 months. Addition of 5 per cent of saturated Na₂SO₄ solution slightly delays deterioration of high speed deposits in the ice box. Most of the agent measured by inoculation of chickens and the fraction sedimentable at high speed measured by its nitrogen content is precipitated by one-third saturation with sodium sulfate.     

THE CONSTITUTION OF MITOCHONDRIA AND MICROSOMES,AND THE DISTRIBUTION OF NUCLEIC ACID IN THE CYTOPLASM OF A LEUKEMIC CELL

1. Rat tumor extracts, containing chiefly the cytoplasmic constituents of leukemic cells, were fractionated into three main portions, the different components separating in the centrifuge according to size. 2. Mitochondria were isolated by centrifugation at relatively low speed. Elementary composition of purified mitochondria was found to correspond to about 11.5 per cent nitrogen, 1.6 per cent phosphorus, and 27 per cent lipids. Phosphorus and nitrogen content of the lipid portion suggests that as much as 75 to 80 per cent of the lipids of mitochondria is represented by phospholipids. Tests for ribose nucleic acid were positive. 3. Microsomes were separated by means of centrifugation at 18,000 x g. A relation between the high phosphorus content of the microsomes and the marked basophilia of the cytoplasm of leukemic cells is suggested. 4. Phosphorus distribution in the tumor extract, and light absorption analysis of the third fraction, seem to demonstrate that nucleic acid was not present either in a free condition, or in the form of nucleoprotein of relatively low molecular weight. The nature of the results suggests that ribose nucleic acid occurs in the cytoplasm of leukemic cells only in association with formed elements of relatively large size, namely microsomes, and mitochondria.     

ELECTROPHORETIC STUDY OF ANTIVIRAL SERA

1. Sera of animals immunized against Japanese B encephalitis, Venezuelan equine encephalomyelitis, and Western equine encephalomyelitis viruses were fractionated by electrophoresis. 2. Electrophoretic patterns of rabbit sera before and after immunization against Japanese B virus showed no consistent change traceable to antibody formation. 3. To determine the antibody content, the electrophoretic fractions of the respective sera were mixed in varying dilutions with infected mouse brain suspensions, and the neutralizing titers of the fractions were compared. 4. In all instances serum fractions containing γ-globulin were protective, whereas in no case did serum albumin show any virus-neutralizing activity. The Japanese B encephalitis antibody appeared to be associated entirely with the γ-globulin. The Venezuelan and Western equine encephalomyelitis antibodies were associated with the β- and γ-globulins and probably possessed an average electrophoretic mobility between that of β- and γ-globulins. 5. Normal rabbit serum similarly separated electrophoretically showed no neutralizing properties. 6. Chickens, whose electrophoretic serum pattern is markedly different from that of rabbits, were also immunized against the Japanese B encephalitis virus. Their antisera were electrophoretically fractionated and similarly subjected to neutralization tests. The specific neutralizing capacity of chicken serum was considerably lower than that of rabbit serum and no neutralizing activity was found in the fractions containing the faster moving components. The antibody appeared to be associated with component 4 which had a mobility of approximately 2.3 x 10^–5 cm.²/volt/sec.     

Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis

Rosmarinic acid (RA) reduced the mortality of mice infected with Japanese encephalitis virus (JEV). Significant decreases in viral loads (P < 0.001) and proinflammatory cytokine levels (P < 0.001) were observed in JEV-infected animals treated with RA compared to levels in infected mice without treatment, at 8 to 9 days postinfection.     

IMMUNOLOGICAL RELATIONSHIPS AMONG CENTRAL NERVOUS SYSTEM VIRUSES

From observations carried out with the viruses of Russian spring-summer encephalitis, louping ill, W.E.E., and the Japanese B, St. Louis, and West Nile types of encephalitis, the following facts and inferences have been derived. 1. Russian encephalitis and louping ill viruses showed a close relationship by complement fixation, neutralization, and intraperitoneal cross-resistance tests. Intracerebral cross-resistance tests, on the other hand, failed to reveal any connection between them. Neither Russian nor louping ill virus appeared to be related to the remaining viruses tested. 2. Japanese B, St. Louis, and West Nile types of encephalitis, as a group, showed a certain degree of group relationship, but it was not so close as that between the Russian and louping ill viruses. In complement fixation tests, besides homologous reactions, Japanese serum brought about reactions with both St. Louis and West Nile antigens; St. Louis serum reacted with Japanese antigen, and West Nile serum with Japanese antigen. In neutralization tests with mouse sera, no relationship was found amongst these three viruses, while in similar tests with either hamster or guinea pig serum,—which gave higher homologous titers,—it was found that Japanese serum protected against West Nile and St. Louis viruses, St. Louis serum protected against West Nile virus, and West Nile serum against Japanese virus. In intracerebral and intraperitoneal cross-resistance tests, no relationship was found to exist between these three viruses. Moreover, the Japanese B, St. Louis, and West Nile viruses appeared to be unrelated to any one of the three other viruses tested. 3. W.E.E. virus stood apart in all tests as unrelated to any of the other viruses studied. 4. The homologous titers of complement-fixing antibodies in mouse sera showed a gradual decline with the passing of time after vaccination, and this loss of homologous titer was paralleled by a similar drop in the titer of the heterologous reactions. In the case of the Japanese B, St. Louis, and West Nile viruses, with which at the start the amount of crossing was not high, a point was reached beyond which heterologous reactions could no longer be detected. 5. A comparison of the specific levels of complement-fixing and neutralizing antibodies for the viruses in mouse hyperimmune sera showed their rate of persistence to differ. Complement-fixing antibodies which had highest titers on the 10th day, diminished gradually until, when tested on the 50th day, all titers had reached levels from one-fourth to one-eighth of their values on the 10th day. On the other hand, the levels of neutralizing antibodies for the same samples of serum were, on the 50th day, as high as or higher than those found on either the 25th or 10th days, save in the case of the Japanese B virus. 6. The state of immunity of animals following vaccination with the viruses discussed was found to be different at a given time, depending on the method employed to determine it. Thus, mice vaccinated with St. Louis virus had positive complement-fixing antibodies on the 10th day and no neutralizing antibodies. The state of immunity changed in the course of time. For this reason it is felt that in order to detect whether two viruses are related or not, multiple observations are necessary, over a considerable time and employing all available methods of immune comparison.     

Experiments on the role of the chicken mite,Dermanyssus gallinae. and the mosquito in the epidemiology of St. Louis encephalitis

The present experimental results concern primarily the question, whether or not mosquitoes feeding on chickens having viremia, as a result of the bite of infected mites, can acquire the virus of St. Louis encephalitis and whether or not mosquitoes thus infected, can transmit the virus to chickens and hamsters. During the course of the investigation, 7 species of mosquitoes of 3 genera were infected with the virus in one or two or all of three ways: by feeding on a suspension of infected mouse brain tissue, by feeding on chickens in which viremia had been produced by subcutaneous inoculation of virus, and by feeding on chickens having viremia as a result of the bite of infected mites. These mosquitoes transmitted the virus to chickens at periods varying from 5 to 33 days after the infective meal. The virus of St. Louis encephalitis was transmitted to hamsters by Culex pipiens at periods varying from 4 to 27 days after feeding on chickens having viremia as a result of the bite of infected mites. While viremia was demonstrated readily in hamsters, signs of encephalitis did not develop. In all transmission experiments the method of chorioallantoic passage proved necessary for the demonstration of viremia. A concept of the epidemiology of St. Louis encephalitis is presented: two blood-sucking vectors may be involved, one an arachnid, the mite, maintaining the virus in nature by transovarial passage, and the other, an insect, the mosquito, which carries the infection from birds to other vertebrates including man.     

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.     

ENCEPHALOMYELITIS OF MICE : I. CHARACTERISTICS AND PATHOGENESIS OF THE VIRUS

1. The two strains of virus named GD VII and FA, respectively, accidentally discovered during experiments with yellow fever, have been shown to be immunologically related to each other, as well as to the virus of mouse encephalomyelitis. 2. Infection of the central nervous system can be produced with both strains by intracerebral, intranasal, or intraperitoneal inoculations. The cardinal symptom produced by the GD VII strain of virus by all three methods of inoculation is a flaccid paralysis of the limbs. The symptoms produced by the FA strain are referable to lesions of the brain when infection is produced by intracerebral and intranasal inoculation. Following intraperitoneal inoculation of the FA strain of virus, however, a flaccid paralysis is usually produced. 3. By the use of graded collodion membranes the particle size of the virus of mouse encephalomyelitis has been shown to be from 9 to 13 mµ 4. The stability of the virus at different hydrogen ion concentrations has been tested. It has been found that there are two optima of stability, one at about pH 8.0 and the other at pH 3.3. 5. The virus is readily inactivated at 37°C. by 1 per cent hydrogen peroxide. 6. Of organic solvents tested, ether had no action, whereas ethyl alcohol in 20 per cent concentration almost completely inactivated the virus after 45 minutes in the cold. 7. The virus can be precipitated by means of ammonium sulfate. 8. With increasing age mice acquire a relative resistance to the virus. 9. Immunity to a subsequent intracerebral inoculation can be produced by intraperitoneal, as well as intranasal, administrations of relatively large amounts of virus. 10. Mice infected by the intracerebral inoculation of a relatively avirulent virus acquire a high degree of immunity to a subsequent inoculation of a highly virulent strain. 11. The course of infection in mice following intracerebral, intranasal, and intraperitoneal inoculation of the FA strain of virus has been studied.     

INHERITANCE OF RESISTANCE OF MICE TO ENTERIC BACTERIAL AND NEUROTROPIC VIRUS INFECTIONS

Under the conditions specified, there may be selected promptly from a hybrid stock of mice, of which 40 to 50 per cent die following a standard dose of B. enteritidis or St. Louis encephalitis virus, lines in which as high as 95 per cent and as low as 15 per cent succumb. Three lines,—one bacteria-susceptible-virus-susceptible, one bacteria-susceptible-virus-resistant, and one bacteria-resistant-virus-susceptible,—are regarded as remaining relatively stable after approximately twelve generations of selection and brother to sister or line inbreeding. Crossing susceptible with resistant lines and testing F₁, F₂, F₃, and backcross progeny resulted in mortality percentages in the neighborhood of those expected on the basis that resistance to B. enteritidis and to encephalitis virus is each inherited independently on a single factor basis with resistance dominant over susceptibility. A bacteria-resistant-virus-resistant line is being developed from a cross between bacteria-susceptible-virus-resistant and bacteria-resistant-virus-susceptible lines. All selected lines proved uniformly susceptible to a strain of mouse passage rabies virus.     

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.     

Hemagglutination with arthropod-borne viruses

Through the use of acetone and ether extraction of brain tissue from newborn mice infected with certain arthropod-borne viruses, it has been possible to demonstrate hemagglutinins for chick erythrocytes associated with the following viruses: dengue Type 1, dengue Type 2, Eastern equine encephalitis, Ilhéus, Japanese B, Ntaya, St. Louis, Sindbis, Uganda S, Venezuelan equine encephalitis, West Nile (Egypt 101 strain), Western equine encephalitis, and yellow fever (viscerotropic and neurotropic strains). On the basis of the temperature and pH required for reaction, the viruses can be assembled in two groups: A—those that require 37°C. and a pH of about 6.4, comprising Eastern, Venezuelan, and Western equine encephalitis and Sindbis viruses; and B—those that require either 4° or 22°C. and a pH of about 7.0, comprising dengue Types 1 and 2, Ilhéus, Japanese B, Ntaya, St. Louis, Uganda S, West Nile, and yellow fever viruses. A method of eliminating non-specific inhibitory substances present in sera was developed. The method consists essentially of filtration through Seitz pads. Extensive serological crossings were found among viruses of each group, while antisera of one group failed consistently to cross-react with antigens of the other. Antisera deriving from animals immunized with certain viruses for which no hemagglutinins could be developed by the present method, reacted with members of either one or the other group. Thus Semliki Forest virus would appear to belong to Group A, and Russian Far Eastern and louping ill viruses to Group B.     

THE COMPLEMENT FIXATION TEST IN THE DIAGNOSIS OF VIRUS INFECTIONS OF THE CENTRAL NERVOUS SYSTEM

A specific complement fixation test can be obtained in various central nervous system virus infections by using as antigens emulsions of infected brain tissue, freezing and thawing the brain emulsion, and then centrifuging it in an angle head centrifuge at 3500 R.P.M. for 1 hour. The method has proved reliable in the case of rabies, St. Louis encephalitis, Japanese B encephalitis, lymphocytic choriomeningitis, Eastern equine encephalomyelitis, Western equine encephalomyelitis, louping ill, and spontaneous encephalomyelitis of mice (Theiler''s disease). The specificity of the reaction, regardless of the virus involved, requires different temperatures of inactivation of the sera according to animal species: 56°C. for guinea pig, 60°C. for mouse, and 65°C. for rabbit and dog sera, all heated for 20 minutes. For human sera a temperature of inactivation of 60°C. also for 20 minutes has been adopted; at this temperature the reaction is in general specific. Complement-fixing antibodies in high titre were found in the sera of rabbits, guinea pigs, mice, and dogs immunized with rabies virus. Complement-fixing antibodies were present in high titre in sera drawn from two persons 8 years after an attack of louping ill, from five persons 2½ years after an attack of Eastern equine encephalomyelitis, and from two persons 2½ years after Western equine encephalomyelitis. In cases of St. Louis encephalitis and lymphocytic choriomeningitis, complement-fixing antibodies have been found shortly following infection but not after long periods.