ENCEPHALOMYELITIS OF MICE : II. A METHOD FOR THE MEASUREMENT OF VIRUS ACTIVITY

In a study of mouse encephalomyelitis the following observations were made:— 1. A definite relationship exists between the amount of virus inoculated intracerebrally and the length of the incubation period. 2. The reciprocal of the incubation period was found to be approximately proportionate to the logarithm of the amount of virus inoculated. The relationship can, therefore, be given a simple mathematical form. 3. This fact provides a basis for a new method for measurement of the activity of mouse encephalomyelitis virus.     

WILD-TYPE GROSS LEUKEMIA VIRUS AND THE PATHOGENESIS OF THE GLOMERULONEPHRITIS OF NEW ZEALAND MICE

The pathogenesis of the spontaneous glomerulonephritis of NZB and (NZB x NZW) F₁ hybrid mice is related at least in part to the formation of natural antibody against antigens of the G (Gross) system, and apparently to the deposition in the glomeruli of immune complexes of G natural antibody with G soluble antigen (GSA), type-specific antigen specified by wild-type Gross leukemia virus. G natural antibody and GSA are detectable in the acid-buffer eluate of the kidneys of NZB mice during the course of the glomerulonephritis. (NZB x NZW) F₁ hybrid mice develop glomerulonephritis and produce GSA and free G natural antibody earlier in life than do NZB mice. The proteinuria manifestation of the gomerulonephritis of (NZB x NZW) F₁ hybrid mice becomes increasingly prevalent as GSA undergoes immune elimination from the circulation. Gross leukemia virus-specified antigens together with bound immunoglobulins are located in the glomerular lesions of (NZB x NZW) F₁ hybrid mice, both in the mesangium as observed in NZB mice and also in the wall of the peripheral capillary loops of the glomeruli.     

THE PATHOGENESIS OF HERPES VIRUS ENCEPHALITIS : I. VIRUS PATHWAYS TO THE NERVOUS SYSTEM OF SUCKLING MICE DEMONSTRATED BY FLUORESCENT ANTIBODY STAINING

The pathogenesis of herpes simplex virus encephalitis and myelitis was studied in suckling mice using routine titration procedures and fluorescent antibody staining for the identification of infected cells. After intracerebral inoculation virus was shown to disperse rapidly in the cerebrospinal fluid (CSF), multiply in meninges and ependyma, and then invade the underlying parenchyma infecting both neurons and glia. Following extraneural inoculation virus gained access to the central nervous system (CNS) by both hematogenous and neural pathways. After intraperitoneal and intranasal inoculation virus was found to multiply in viscera and produce viremia; foci of CNS infection then developed around small cerebral vessels. After subcutaneous and intranasal inoculation neural spread of virus was demonstrated along corresponding peripheral and cranial nerves. This spread resulted from the centripetal infection of endoneural cells (Schwann cells and fibroblasts). Antigen was not found in axons even after infection of the corresponding ganglion cell perikaryon. Subsequent spread within the CNS was unrelated to neural tracts, and there was no evidence of axonal spread of virus in the host-virus system studied. These findings are discussed in relation to previous and current theories of the viral "blood-brain barrier" and neural pathways of infection.     

SPONTANEOUS ENCEPHALOMYELITIS OF MICE,A NEW VIRUS DISEASE

1. The characteristics of a filterable virus obtained from mice found spontaneously paralyzed and showing lesions of encephalomyelitis are described. 2. The course of the disease in mice, following intracerebral inoculation, is briefly as follows: After an incubation period varying from 7 to over 30 days a flaccid paralysis of one of the limbs appears. This paralysis usually spreads rapidly until all four limbs are affected. Young mice are more susceptible than older ones, and very young mice, less than 4 weeks of age, usually die without showing signs of paralysis. 3. Adult mice often show no signs of infection after an intracerebral inoculation of virus. A number of these mice, although showing no signs of paralysis, nevertheless have become infected, a fact which is demonstrated by recovery of the virus from the mice as well as by histopathological studies. 4. Intranasal instillation of the virus is the only other method of producing the infection. This method, however, produces paralysis in only a small percentage of the mice. Following intranasal instillation of the virus, there often develops a slight immunity to a subsequent intracerebral injection of virus. 5. The paralysis in the surviving mice recedes gradually, but a permanent residual paralysis, usually of the hind legs, is almost invariable. Such mice, however, are virus carriers, as the virus can be recovered from the spinal cord for 1 year after infection. 6. Paralyzed mice are immune to a subsequent intracerebral injection of the virus. There is evidence that neutralizing substances are present in the immune mice. A considerable proportion of the mice which have remained well after an intracerebral injection of virus are immune to a second injection. 7. The virus resists the action of 50 per cent glycerine at from 2–4°C. for at least 150 days. It passes all grades of Berkefeld filters with ease. By the use of graded collodion filters, the size of the virus particle has been determined to be probably about 13 to 19 mµ. 8. The virus of mouse encephalomyelitis is not pathogenic for rhesus monkeys. No evidence of immunological relationship with the virus of human poliomyelitis has been obtained. 9. The anatomical basis of the paralysis is an acute necrosis of the ganglion cells of the anterior horn of the spinal cord. Isolated ganglion cells of the cerebrum also undergo necrosis. Following the acute necrosis of the ganglion cells, there is a marked neuronophagia. A perivascular infiltration is observed in the brain and spinal cord.     

THE VIRAL ENVELOPE GLYCOPROTEIN OF MURINE LEUKEMIA VIRUS AND THE PATHOGENESIS OF IMMUNE COMPLEX GLOMERULONEPHRITIS OF NEW ZEALAND MICE

The use of monospecific antisera for the analysis by radioimmunoassay and immunofluorescence study of two major viral proteins, gp69/71 and p30 of murine leukemia virus, that could be of significance in the pathogenesis of immune complex glomerulonephritis of mice, particularly NZB and B/WF₁ hybrid mice, yielded the following conclusions. A remarkably high concentration of viral envelope glycoprotein, gp69/71, was detected in the spleen and serum of New Zealand mice (NZB, NZW, B/WF₁, and W/BF₁); the concentration in the spleen was 10-fold greater than that found in AKR mice and 30-fold greater than that present in C57BL/6 mice. The gp69/71 was deposited along with bound immunoglobulins, apparently as an immune complex, in the diseased kidneys of mice, and the glomerular site and extent of deposition of gp69/71 was related to the severity of the glomerulonephritis. This study suggests that the pathogenesis of immune complex glomerulonephritis (and vasculitis) in mice is related to the expression of this specific viral envelope glycoprotein and to the host immune response to this protein.     

LOCALIZATION OF THE VIRUS AND PATHOGENESIS OF EPIDEMIC POLIOMYELITIS

The virus of poliomyelitis is capable of penetrating the retina without producing apparent injury, to reach the central nervous organs. The virus injected into the blood is deposited promptly in the spleen and bone marrow, but not in the kidneys, spinal cord, or brain. Notwithstanding the affinity which the nervous tissues possess for the virus, it is not removed from the blood by the spinal cord and brain until the choroid plexus and blood vessels have suffered injury. The intervertebral ganglia remove the virus from the blood earlier than do the spinal cord and brain. An aseptic inflammation produced by an intraspinous injection of horse serum facilitates and insures the passage of the virus to the central nervous organs, and the production of paralysis. The unaided virus, even when present in large amounts, passes inconstantly from the blood to the substance of the spinal cord and brain. When the virus within the blood fails to gain access to the central nervous organs, and to set up paralysis, it is destroyed by the body, in course of which destruction it undergoes, as a result of the action of the spleen and, perhaps, other organs, diminution of virulence. The histological lesions that follow the intravenous injections of the virus in some but not in all cases differ from those which result from intraneural modes of infection. In escaping from the blood into the spinal cord and brain, the virus causes a lymphatic invasion of the choroid plexus and widespread perivascular infiltration, and from the latter cellular invasions enter the nervous tissues. A similar lymphoid infiltration of the choroid plexus may arise also from an intracerebral injection of the virus. The histological lesions present in the central nervous organs in human cases of poliomyelitis correspond to those that arise from the intraneural method of infection in the monkey. The virus in transit from the blood through the cerebrospinal fluid to the substance of the spinal cord and brain is capable of being neutralized by intraspinous injection of immune serum, whereby the production of paralysis is averted. Carmin in a sterile and finely divided state introduced into the meninges and ventricles sets up an aseptic inflammation, but is quickly taken up by cells, including ependymal cells. When an aseptic inflammation has been previously established by means of horse serum, or when the nervous tissues are already injured by the poliomyelitic virus, the pigment appears to enter the ependymal cells more freely. The experiments described support the view that infection in epidemic poliomyelitis in man is local and neural, and by way of the lymphatics, and not general and by way of the blood. Hence they uphold the belief that the infection atrium is the upper respiratory mucous membrane.     

ENCEPHALOMYELITIS OF MICE : III. EPIDEMIOLOGY

1. In the feces of approximately two-thirds of normal mice 6 weeks of age an agent in all respects similar to the virus of mouse encephalomyelitis can be recovered. 2. In isolated mice, fed on sterile food and water, excretion of virus has been shown to persist up to 53 days after isolation. 3. In normal mice known to be virus carriers virus has been demonstrated in the gastro-intestinal tract but not in the central nervous system, thoracic or abdominal viscera, or any organs of the head. 4. The source of the virus excreted in the feces has been shown to be located in all probability in the intestinal wall. 5. Evidence is presented that the virus can invade the animal organism, as virus has been demonstrated in the mesenteric lymph glands.     

INFLUENCE OF AGE ON SUSCEPTIBILITY AND ON IMMUNE RESPONSE OF MICE TO EASTERN EQUINE ENCEPHALOMYELITIS VIRUS

The experiments described in this paper were carried out with the Rockefeller Institute strain of albino mice and with the Eastern strain of the virus of equine encephalomyelitis. 1. The observation was confirmed that with increasing age of mice there occurred a decrease in susceptibility to intraperitoneal injection of active virus; also, the length of incubation period of those which succumbed increased with age. 2. The mice of various age groups which survived an intraperitoneal injection of active virus were indistinguishable in their antibody response. 3. Young mice, vaccinated with formalin-inactivated. virus when 2, 5, and 7 days old, gave an immune response to such a degree that they showed (a) measurable peritoneal immunity which increased with small increments of age, (b) no cerebral resistance, and (c) detectable amounts of neutralizing antibody in their sera which paralleled, though at a considerably lower level, their peritoneal resistance. 4. The peritoneal resistance induced as a result of vaccination was shown to be not local, but a general, systemic immunity, specific for the Eastern strain. Such a peritoneal resistance was demonstrable by the 4th day after beginning of vaccination of 10-days-old mice. 5. After intraperitoneal injection of active virus, large amounts of virus were recoverable from the blood of non-vaccinated young mice; none was found in the blood of vaccinated young mice; a minimal amount was detectable in the blood of non-vaccinated adult mice. 6. The bearing of age on the degree of immune response of which mice are capable and on their susceptibility to the virus has been discussed.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) : I. THE PRECISION OF MEASUREMENTS IN VIVO OF THE VIRUS AND ANTIBODIES AGAINST IT

This study on pneumonia virus of mice (PVM) was carried out in order to obtain as accurate data as possible on the degree of variation which may be expected in titrations of the virus or of antibodies against it in vivo. It is believed that the knowledge gained will facilitate further investigations on this latent pneumotropic virus and make possible a more exact assessment of the significance of experimental results obtained with the agent. The reproducibility of 50 per cent maximum score titration end points with PVM in mice is such that the chances are 19 out of 20 that a difference of 1.084 log units (i.e. a twelvefold difference) in the end points obtained in two separate titrations is significant. The reproducibility of 50 per cent maximum score serum dilution end points in neutralization tests with PVM in mice is such that the chances are 19 out of 20 that a difference of 0.626 log units (i.e. a fourfold difference) in the end points obtained with two sera against similar amounts of virus is significant. It was found that there is a linear exponential relationship between the serum dilution end point and the quantity of PVM used in a neutralization test. This relationship appears to be identical with immune serum obtained from different animal species, and appears also to be identical to the linear relationship described previously in similar studies with influenza A virus.     

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 TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : I. THE PERIOD OF TRANSMISSIBILITY

An experimental model has been developed for the reproducible transmission of influenza virus infection from experimentally infected mice to uninfected cage mates. Infector mice transmit influenza virus infection most readily during the period 24 to 48 hours after initiation of their infection. This restricted period of transmission is not due to declining titers of infective virus in the nose, trachea, or lungs of infector mice after 48 hours of infection, since peak titers in these tissues are maintained for another 48 hours. A mouse-adapted strain of A2 virus was found to be more readily transmitted than the mouse-adapted CAM strain of influenza A1 virus, although the CAM strain induced higher pulmonary virus titers and more extensive lung lesions.     

STUDIES ON EASTERN EQUINE ENCEPHALOMYELITIS : II. PATHOGENESIS OF THE DISEASE IN THE GUINEA PIG

After inoculation with equine encephalomyelitis virus by various routes, guinea pigs were sacrificed at early stages, before symptoms were apparent. The brains were studied histologically, with serial sections; all lesions were noted, and subjected to topographical analysis. Nine cases are presented in detail. With any given mode of inoculation the distribution of lesions varied very widely from one instance to another. In some cases, affected regions bore a striking and definite anatomical relationship to each other. These distributions can be explained only by the assumption that the anatomical pathways played some rôle in the spread of the virus. In other instances lesions were present in areas, the anatomical connections of which were entirely normal. Attention is called to the frequency of lesions in the neocortex, with intact subcortical centers. Such distribution is held to render nerve spread extremely improbable. The only satisfactory explanation of such random distributions is by direct passage of virus from the blood stream into the brain tissue. There is no histological difference between lesions which result from blood spread and those resulting from nerve spread.     

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 TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : IV. RELATIONSHIP OF TRANSMISSIBILITY OF DIFFERENT STRAINS OF VIRUS AND RECOVERY OF AIRBORNE VIRUS IN THE ENVIRONMENT OF INFECTOR MICE

A mouse-adapted Jap. 305 strain of influenza A₂ virus was found to be much more readily transmitted from one mouse to another than the NWS strain of influenza A₀ virus although the two viruses were equally pathogenic for mice as judged by pulmonary virus titers and lung lesions. The survival of artificially created aerosols of virus and the quantity of airborne virus required to initiate infection in mice were identical for the two viruses. The difference in transmissibility was associated with the recovery of infectious airborne virus in the environment of mice infected with the Jap. 305 strain during the period of their maximum infectiousness, but not in the environment of mice infected with the NWS strain.     

IN VITRO INTERACTION OF MOUSE HEPATITIS VIRUS AND MACROPHAGES FROM GENETICALLY RESISTANT MICE : I. ADSORPTION OF VIRUS AND GROWTH CURVES

Peritoneal macrophages from genetically resistant C₃H mice and genetically susceptible Princeton (PRI) mice adsorbed the MHV (PRI) strain of mouse hepatitis virus equally well. The difference between the permissive cells and the nonpermissive ones seems to reside in the ability of the former to "eclipse" the virus and, subsequently, support virus replication. C₃H cells exposed to low multiplicities of the virus remained intact with no demonstrable viral replication. Virus, taken up by the resistant cells, was protected from heat and underwent slow inactivation while few or no virus particles were released into the medium.     

THE VACUOLATING VIRUS OF MONKEYS : I. ISOLATION, GROWTH CHARACTERISTICS, AND INCLUSION BODY FORMATION

A vacuolating virus isolated from uninoculated patas monkey kidney cultures was found to be serologically identical with SV₄₀, a virus previously found in association with rhesus and cynomolgus monkeys. Detection of the patas virus was facilitated when patas cells were exposed to x-ray treatment. Rhesus monkey and human cells were relatively insusceptible to the virus, although it persisted in these cells for a long period of time. Distinct intranuclear inclusions were detected in infected patas cultures 2 to 3 days before cytoplasmic vacuoles were noticeable. Cultures previously infected with PA-57 virus did not affect yields of poliovirus, and doubly infected cells were easily distinguished in stained preparations.     

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS : IV. EFFECT OF IMMUNE SERUM ON ANTIGENICITY OF ACTIVE AND INACTIVE VIRUS

A study was undertaken on the effect in vivo, in the guinea pig, of equine encephalomyelitis virus antiserum upon the antigenic response to active, as compared with that to formolized, inactive virus. It was found that when animals were given subcutaneously a proper amount of hyperimmune serum 1 hour before inoculation, in the subcutis, of either active or of inactive virus, no immunity was induced against an intracerebral test of more than 1,000 and less than 10,000 M.L.D. of virus. This preventive power of the serum was lost by its dilution, the loss being proportional to the dilution, and, on the other hand, more serum was needed to obtain the blocking effect as the quantity of virus was increased. When an insufficient amount of serum was introduced into the animals along with the same quantities of active virus or formolized vaccine, a certain number of those receiving the untreated virus succumbed to virus infection in the course of the inoculations, but the survivors were rendered resistant to the intracerebral test; all the guinea pigs treated with higher dilutions of serum and with formolized material were brought safely to an immune state. The point to be stressed then is that antigenic stimuli present in untreated active virus and in formolized virus tissue suspensions in which no active virus is demonstrable by drastic tests (1) and which are wholly noninfective in animals (1), are completely inhibited from acting by the use of proper amounts of immune serum. The mechanism underlying this preventive power of adequate amounts of serum may be explained on the basis of facts deduced in preceding papers of this series (1, 3) and in the present article. We have shown that 3 x 10⁷ m.i.u. of active virus contains a sufficient amount of antigen to induce immunity without the necessity of its multiplication in the animal body. This has been fully established by the similar degree of resistance brought about by 3 x 10⁷ m.i.u. of virus formolized to a degree in which no active virus could be revealed (1). The assumption that the blocking effect of serum in the quantity employed prevents multiplication of the virus which is reflected in the production of inadequate amounts of antigen, is therefore untenable, since this effect was obtained when a sufficient amount of antigen was present in "living" as well as in "killed" virus. On the other hand, with insufficient amounts of immune serum (to be noted in higher dilutions shown in Table II), only the active virus could multiply—the formolized vaccine was not affected in respect to its antigenicity by these quantities of serum—and so produce more antigenic substance. This substance, in turn, brought about greater resistance in the host. The precise action of proper amounts of serum in preventing development of immunity by both active and inactive virus is not definitely known. However, two hypotheses are offered for consideration: the first implies that the action of the serum is direct, that is, by entering into combination with the antigens to bar antigenic capacity; the second ascribes to the serum an indirect action, on the cells of the body, in such a way as to make them unable to react to the antigenic stimuli present in the inoculated materials. The identity of these antigenic stimuli in virus suspensions containing the active, infective agent or this agent inactivated by formalin is at the present time undetermined. If virus were obtainable in pure state, free from extraneous material, the answer to this question might be readily given, but it is quite a different matter when the substance called virus is a mixture of the infective agent, of inflammatory tissue products, of tissue, etc. We have, however, shown that induced immunity is not due to the presence of "living" virus, but whether the antigenic action originates from "killed" virus or from another constituent of the suspension is not clear. On the other hand, Sabin (7) suggests the possibility that the virus may not be the direct antigenic stimulus but that some substance on which it acts and which becomes liberated from infected cells may be the factor responsible. While this subject awaits the results of further study, we believe that formalin inactivates the infective agent in virus suspensions and preserves the antigenic component therein, whatever its nature may be. It would be of interest if this phenomenon of prevention of antigenic capacity by proper amounts of immune serum might apply to such materials which by their very nature do not multiply in the body of the host, e.g., toxin and antitoxin. Theobald Smith (8) and later Park (9) demonstrated that in mixtures of diphtheria toxin-antitoxin, when smaller amounts of immune serum (antitoxin) are used, the toxicity of the mixture is retained and immunity results; if the serum is increased, toxicity is reduced and immunity occurs irregularly, and if more serum is added, no toxicity nor immunity results. This is supported by the experiments of Hartley (10) on washed precipitates from underneutralized, neutral, and overneutralized mixtures of antitoxin and toxin: those derived from underneutralized material are toxic and powerfully antigenic; those from neutralized, atoxic and of good antigenic action, and from overneutralized, atoxic and of low antigenicity. Hartley states, moreover, that the precipitate reactions of toxicity and antigenicity bear a close relationship to the nature of the mixture from which they are produced. There is, therefore, a connection between the preventive reactions of the serum on the two forms of virus and of antitoxin on toxin in respect to toxicity and antigenicity. Furthermore, the toxin is rendered atoxic with retention of immunizing capacity by formalin: the production of toxoid or anatoxin (Glenny and Hopkins (11), Ramon (12))—again a condition related to the effects following formolization of the virus. It has, however, been stated that "in an immunizing mixture prepared with modified [formolized, but partially detoxified] toxin the antitoxin present does not within wide limits affect the antigenic power" (Glenny, Hopkins, and Pope (13)). It is not known whether a preliminary injection of antitoxic serum could have prevented the antigenic power of fully detoxified toxin, that is, after the passive immunity induced by the serum disappears. If a preventive action of antitoxic serum could be shown under these circumstances, a remarkable correlation of the reactions of proper amounts of antitoxin to toxoid and of proper amounts of immune serum on the virus would be evident. Finally, the inhibition of antigenic power of both active and inactive virus by immune serum has been demonstrated to apply to the virus of equine encephalomyelitis in guinea pigs and no generalizations of the application of the phenomenon to other viruses are intended.     

INTRAPERITONEAL AND INTRACEREBRAL ROUTES IN SERUM PROTECTION TESTS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS : I. A COMPARISON OF THE TWO ROUTES IN PROTECTION TESTS

Young (12 to 15 day old) mice are approximately as susceptible to the virus of equine encephalomyelitis, Eastern or Western strain, when it is given intraperitoneally as are adult mice when the virus is injected intracerebrally. With this susceptibility by the intraperitoneal route as a basis, the injection of immune serum-virus mixtures intraperitoneally was found to result in protection in dilutions which give rise to infection after intracerebral inoculation. The difference of protective power by the two indicated routes was shown not to depend on the amount of inoculum nor on the age of the intracerebrally injected mice. Incubation at 37°C. for 2½ hours neither increases nor diminishes the protective action of immune serum when the intraperitoneal method is employed. The phenomenon of selective protection in different tissues is elicited by the sera of hyperimmunized mice, guinea pigs, and rabbits and by sera derived from horses infected with the disease in nature or exposed to it by contact. Of four horses recovered from the malady, all showed antibody in their sera; of others exposed by contact, four of nine animals revealed antiviral bodies, when the intraperitoneal technique was employed. These tests on horse sera have pointed to the potential value of this procedure for epidemiological studies. Finally, the reaction itself has significance through its bearing on the mechanism of immunity.     

PATHOGENESIS OF THE GLOMERULONEPHRITIS OF NZB/W MICE

The development of glomerulonephritis in NZB/W mice is closely related to the formation of antinuclear, particularly anti-DNA, antibodies. The developing inflammatory glomerular lesions are characterized by the deposition of γG- and β_1C-globulins plus DNA and possibly other nuclear antigens, presumably as complexes, in a granular to lumpy pattern along the capillary walls and in the mesangia. Elution studies revealed the γG-globulin in the glomeruli to be largely γG_2A-type antibody to soluble nuclear antigens. Enhancement of the antinuclear antibody response by active immunization of young NZB/W mice with DNA-methylated BSA hastens the development and increases the severity of the glomerulonephritis. Similarly, injections of soluble DNA into NZB/W mice with circulating anti-DNA antibodies but with as yet little nephritis causes rapid progression of nephritis.     

VIRUS PARTICLES IN THE THYMUS OF CONVENTIONAL AND GERM-FREE MICE

Electron microscope study of thymuses of both conventional and germ-free mice has revealed the presence of typical virus particles associated with the thymic lymphocytes or with the thymic epithelial cells. The particles resemble those associated with several murine leukemias and their viral nature seems convincingly substantiated by morphological observation. Germ-free mice are therefore not virus-free. The biological significance of these particles is still unknown and we can only speculate as to the possible relationship of these particles to the incidence of "spontaneous" leukemia, to the lymphocytosis stimulating factor of Metcalf, and to the numerous latent viral infections of laboratory mice.