INFLUENCE OF AGE FACTORS ON IMMUNIZABILITY OF MICE TO RABIES VIRUS

1. W-Swiss mice 60 or more days old are more readily immunizable against rabies virus infection than 20 day old or younger mice; this difference in immunizability with increasing age is most conspicuous when vaccination with virulent virus is followed by intracerebral test infection and least apparent when vaccination with avirulent virus is followed by intramuscular test infection. 2. The titre of circulating neutralizing antibodies does not parallel the titre of immunity.     

INFLUENCE OF AGE FACTORS ON SUSCEPTIBILITY OF MICE TO RABIES VIRUS

1. 7 to 9 day old mice are more susceptible than older mice to injections of fixed or street virus by any route. 2. 20 day old mice are more susceptible than 60 day old mice to peripheral but not intracerebral injection of fixed virus. 3. 20 day and 60 day old mice are equally susceptible to street virus.     

A NON-VIRULENT,SINGLE-DOSE RABIES VACCINE FOR PROPHYLACTIC IMMUNIZATION OF DOGS

Our studies on rabies vaccines thus far have led us to the view that in order to develop and test vaccines, quantitative methods are necessary, and that such quantitative methods may be exploited to greatest advantage by using mice, preferably W-Swiss, as the test animal. Dogs, due to their variability and susceptibility to intercurrent infections when kept under experimental conditions, are useful chiefly to check whether or not a vaccine produces a high grade of immunity; they remain of limited value in testing the comparative potencies of weak vaccines. A second point is that the Pasteur strain of virus has proved as potent as any tested for the preparation of vaccines. Another point is that virus material for preparing vaccines must titre at least 330,000 mouse doses per cc. to be effective. This requirement has eliminated all culture vaccines thus far reported, with the possible exception of Plotz''s (7) and leaves virus-containing brain tissue as the sole potent source of vaccine. In summary, we believe that a single injection of non-virulent irradiated vaccine, prepared as herein described, immunizes mice and dogs effectively against a subsequent test inoculation of virulent rabies virus and does so to a greater degree than do other vaccines now obtainable. It is easily and quickly prepared, keeps well, and has a low nitrogen content.     

ULTRA-VIOLET LIGHT AND VACCINE VIRUS : I. THE REACTION OF IRRADIATED SKIN TO VACCINE VIRUS.

Rabbit skin treated for a few minutes with ultra-violet light and then inoculated at once with vaccine virus is less susceptible to the action of the virus than is untreated skin. If 24, 48, or 72 hours elapse between the time of irradiation and inoculation, the treated skin appears to be more susceptible than is untreated skin. Skin repeatedly exposed to ultra-violet light is less susceptible to the action of vaccine virus than is non-irradiated skin.     

STUDIES ON THE MECHANISM OF ADAPTATION OF INFLUENZA VIRUS TO MICE

1. When strains of influenza A virus which have been isolated in chick embryos are introduced into the mouse lung, the virus multiplies readily and achieves initially a titer which is as high as is even obtained, even after repeated passage. The high initial titer of virus may be unaccompanied by any lethal or visible pathogenic effects; but with four or five mouse passages the agent becomes lethal in high titer and causes extensive pulmonary consolidation, though its capacity to multiply in the lung has not increased. In one example the adaptation to mouse lung was accompanied by increasing capacity to agglutinate guinea pig red cells without a corresponding increase in agglutinating power for chicken cells. Influenza B virus, in preliminary tests, did not behave in a similar fashion. 2. The adaptation of influenza A virus to mice is accompanied by changes in antigenic pattern, as detected by cross-tests with the agglutination inhibition method. Two strains, initially similar, with passage, changed in pattern along divergent paths so that they became not only unlike the parent strains but unlike each other. This finding has important implications for the interpretation of the strain difference problem in human influenza.     

THE THYMUS AND RECOVERY OF THE SHEEP ERYTHROCYTE RESPONSE IN IRRADIATED MICE

The role of the thymus in the recovery of the sheep erythrocyte response after lethal irradiation has been studied in adult CBA mice with the hemolytic plaque technique of Jerne. This immunological parameter is markedly thymus-dependent. 10 wk after irradiation and after antigenic challenge the thymectomized animal has only one-twentieth to one-fortieth the number of plaque-forming cells as does the irradiated animal with intact thymus. The thymus continues to function into the 7th and 8th month of life in this strain. Unlike the drug-tolerant animal, the incompetent irradiated thymectomized mouse retains base line plaques (plaques without antigenic stimulation). Thymectomy 18 days after irradiation is as effective as prior thymectomy in preventing recovery of the sheep cell response. Thymectomized animals receiving grafts of isogenic neonatal thymus (placed beneath the kidney capsule) 1 day, 1 wk, or 2 wk after irradiation are somewhat more responsive at 10 wk than intact animals. Grafts in place for 1 or 2 wk after irradiation and then removed result in one-fifth the recovery of grafts in place the entire time, while only slight restoration is obtained from grafts in place for the final 3 wk of the experiment. The results indicate that the thymus is not required for the 18 days after irradiation, that a period of at least 3 wk residence is required for complete restoration, and that the thymus itself is somewhat radiation-sensitive. Allogeneic thymus grafts failed to restore the hemolysin response of irradiated thymectomized animals.     

THE YIELD OF RABIES VIRUS IN THE CHICK EMBRYO

Rabies virus was inoculated intracerebrally in 8 day old chick embryos and the virus activity of pools of embryos titered after incubation at 35–36°C. for different lengths of time. The virus reached a titer of 10^–5.5 to 10^–6.5 in 5 to to 6 days and remained at a rather constant level until the 9th day of the infection. The report by Kligler and Bernkopf that rabies virus will invade the very young embryo after inoculation on the chorioallantoic membrane was confirmed.     

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.     

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.     

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.     

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.     

THE DEMONSTRATION OF LESIONS AND VIRUS IN THE LUNGS OF MICE RECEIVING LARGE INTRA-PERITONEAL INOCULATIONS OF EPIDEMIC INFLUENZA VIRUS

Following the intraperitoneal inoculation of mice with large doses of epidemic influenza virus (50,000 to 1 million intranasal M.L.D.) it can be recovered from the lungs in high concentration, and pulmonary lesions of moderate extent may be observed. The virus reaches its highest titer in the lungs 48 to 72 hours after intraperitoneal injection and may persist for 10 days. Virus may be recovered from the blood in the first 24 hours, but is readily detected in the omentum and peritoneum for 5 to 6 days. Mice which as a result of the intraperitoneal injection of virus show a high concentration of virus in the lungs do not die but become solidly immune to intranasal infection. Moreover, as early as 24 to 48 hours after intraperitoneal inoculation of large amounts of virus the animals may exhibit resistance to infection with fatal doses of virus given intranasally. Influenza virus given intravenously to mice is rapidly removed from the blood but persists in the lungs and induces pulmonary lesions. Virus can also be recovered from the liver for several days. With subcutaneous inoculation of influenza virus, however, the virus does not reach the blood or lungs in detectable amounts although the regional lymph nodes may yield considerable quantities of the agent. A brief consideration is presented of the mechanisms of infection and resistance which may be involved.     

ALLOGENEIC THYMUS GRAFTS AND THE RESTORATION OF IMMUNE FUNCTION IN IRRADIATED THYMECTOMIZED MICE

Irradiated and thymectomized CBA mice are markedly depressed in several immunological parameters (skin homograft rejection, graft-vs.-host activity and hemolytic plaque-forming cells of the spleen, hemolysin and hemagglutinin formation, and peripheral lymphocyte counts). In the present experiments the ability of homografts of neonatal thymus placed beneath the kidney capsule to restore immunological capacity of such animals was studied. Thymus homografts which share the same H-2 locus with the CBA mouse were permanently tolerated and immunological restoration was complete. Skin from the thymus donor was specifically retained, but third party skin with even minor (non-H-2) incompatibility was normally rejected and hemolytic plaque-forming cells of the spleen were restored. Thymus homografts which differ at the H-2 locus were promptly rejected and led to accelerated rejection of skin subsequently grafted from the thymus donor. With such H-2 incompatible thymus grafts, third party skin with minor histo-incompatibility was retained while there was slight to moderate restoration of rejection of skin with major (H-2) incompatibility. Graft-vs.-host activity was restored, but there was no return of plaque-forming spleen cells, hemolysins, hemagglutinins, or peripheral lymphocyte counts. In view of the cross-reactivity at the H-2 locus in CBA mice between thymus and third party skin donors, it was felt that restoration of skin rejection and graft-vs.-host activity could be adequately explained on the basis of immunization by the thymus graft and did not require the postulation of true immune restoration or a thymus hormone.     

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 EFFECT OF VITAMIN B1 DEFICIENCY AND OF RESTRICTED FOOD INTAKE ON THE RESPONSE OF MICE TO THE LANSING STRAIN OF POLIOMYELITIS VIRUS

In several experiments it was shown that a deficiency of vitamin B₁ in the diet increased the resistance of mice to the Lansing strain of poliomyelitis. The source of the virus was a suspension of infected mouse brain in saline, which was injected intracerebrally. Both the mortality rate and the incidence of paralysis were lower in the deficient animals than in the normally fed controls. The protection was more pronounced with respect to paralysis than with respect to the number of deaths. Some deaths in the deficient groups were undoubtedly due to the vitamin deficiency, as indicated by numerous deaths among groups of animals which were given the deficient diet but injected with a suspension of normal brain. An attempt was made to maintain a state of chronic vitamin deficiency by giving small amounts of the vitamin. The results also seem to indicate that the effect of the deficiency was more in delaying the action of the virus than in preventing it. The greatest difference between normally fed and deficient animals receiving the virus came at about the 12th day after inoculation. Comparable results were obtained by restricting the intake of the complete diet to 1 gm. per mouse per day, which is about 40 per cent of the intake of the normally fed mice. Restriction of the caloric intake alone gave similar results. Restriction of food intake was effective in experiments in which extra vitamin B₁ was given in the diet and also when a diluted saline solution was given by stomach tube to assure a sufficient intake of fluid. Other data are necessary before an explanation can be given for the manner in which these deficiencies increase the resistance of the mice to the virus of poliomyelitis.     

THE EFFECT OF HISTOCOMPATIBILITY-2 TYPE ON RESPONSE TO THE FRIEND LEUKEMIA VIRUS IN MICE

Two types of quantitative response to the F-B strain of Friend virus in segregating generations of a cross involving a susceptible (DBA/2 or BALB/c; H-2²) and a resistant (C57BL/6; H-2^b) mouse strain show a marked correlation with the H-2 type of the mice. Essential susceptibility, as determined by the splenomegalic response to high virus doses, is controlled by a single pair of alleles which segregates independently with respect to the H-2 locus. However, relative susceptibility, as determined by the incidence of the splenomegalic response at moderate or low levels of virus dosage, is significantly greater among mice homozygous or heterozygous for the H-2^d allele than among H-2^b homozygotes in these populations. In addition, the incidence of recovery from splenomegaly induced by a given level of virus dosage is significantly greater in H-2^b homozygotes than in segregants of other H-2 types among their littermates. Possible mechanisms responsible for these effects are discussed.     

THE EFFECT OF EPITHELIAL REMNANT AND WHOLE ORGAN GRAFTS OF THYMUS ON THE RECOVERY OF THYMECTOMIZED IRRADIATED MICE

Work has been presented which suggests that thymus epithelial reticular cells are not effective in restoring the microscopic morphology of lymphoid tissues and their immunologic capacities. They function in recruiting precursors of thymus lymphocytes from the host animals to produce an organ which, after it becomes architecturally normal, can reconstitute the defective host. Intact thymus grafts in situ from 10–14 days, but not for shorter periods of time, have been shown to result in a return toward normal of these two parameters. Evidence is offered to show that few dividing cellular components in the lymphoid tissue originate from the thymus remnant grafts, and that a minor cellular component is contributed by the intact grafts. These data support the concept that the structural and functional development of the lymphatic tissue in thymectomized animals is dependent on thymus lymphoid cells and/or their products, and that the epithelial-reticular cells do not have a direct action in peripheral lymphoid reconstitution.     

COMPARISON OF A VIRUS OBTAINED BY KOBAYASHI FROM CASES OF EPIDEMIC ENCEPHALITIS WITH THE VIRUS OF RABIES

Since the symptoms produced in experimental animals by the encephalitic virus of Kobayashi and by the virus of rabies are similar, and are accompanied by lesions which are indistinguishable, and since a cross-immunity is demonstrable between the two viruses, the conclusion is advanced that the specimen of so called encephalitis virus isolated by Kobayashi is in reality a specimen of rabic virus.     

STUDIES ON THE IN VIVO AND IN VITRO MULTIPLICATION OF THE LDH VIRUS OF MICE

In vivo analysis of the virus titer in various loci, 24 hr after infection, showed that a titer similar to that in the blood plasma was found in the ascitic fluid of Erlich ascites cancer-bearing mice, and in lymph nodes, spleen, and thymus, i.e. loci which contain macrophages as a common cell type. However, only in the lymph nodes and in the ascitic fluid did the increase in virus titer precede or parallel the increase in the plasma. The LDH virus titer in the plasma of X-irradiated mice was similar to that of control mice, eliminating radiation-sensitive cells but not macrophages as target cells of the virus. Electron microscopic observation of infected lymph node cells revealed the presence of two types of particles: one consisting of small densely stained annuli, about 25 mµ in diameter and one of similar dense annuli with a halo extending the diameter to about 50 mµ. Such particles were repeatedly observed within single or double membraned vesicles. In vitro, the LDH virus multiplied only in cultures of mouse peritoneal macrophages, maintained in medium 199 with 10% FBS. The virus titer could be maintained for at least 33 days, during eleven serial passages, involving an overall dilution factor of 10¹¹. These results corroborate the findings of Evans and Salaman, who used peritoneal macrophages maintained in Eagle's medium and 5 to 10% lamb serum. However, in the serial passage experiments reported here, the virus titer could only be maintained following trypsinization of each successive inoculum. The role of macrophages as the target cell for LDH virus multiplication in vivo is discussed.