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.     

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.     

A NEW VIRUS DISEASE OF PIGEONS : I. RECOVERY OF THE VIRUS

A virus has been isolated from the tissues of a pigeon with visceral lesions that were characterized by focal necrosis of parenchymatous tissue, by the presence in affected cells of intranuclear inclusions of the herpetic type, and by secondary inflammatory reaction. This newly recognized virus, which has been tentatively called the I.N.I. agent is pathogenic for pigeons and embryonated eggs but is avirulent for rabbits, guinea pigs, and mice. The virus is smaller than the agent of psittacosis and is immunologically different from it. The I.N.I. agent and psittacosis virus were both of etiological importance in an epizootic among pigeons. Some birds were infected simultaneously with the two agents while others were infected with only one.     

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.     

A NEW MOUSE VIRUS CAUSING NECROSIS OF THE THYMUS IN NEWBORN MICE

A new mouse virus has been recovered from laboratory and wild mice. The agent induces a non-fatal disease in infant mice, characterized by acute massive necrosis of the thymic medulla and cortex, granulomatous reaction, and subsequent restoration of essentially normal architecture with scarring. Intranuclear inclusion bodies are produced. Virus may persist in tissues of convalescent mice for many months. Electron micrographs of acutely infected thymuses showed nuclear and cytoplasmic karyoannular particles and masses of parallel fibrils in nuclei.     

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.     

AUTOIMMUNE DISEASE IN NZB/BL MICE : I. PATHOLOGY AND PATHOGENESIS OF A MODEL SYSTEM OF SPONTANEOUS GLOMERULONEPHRITIS

This study, based upon 528 laboratory examinations and 16 complete autopsies of NZB/Bl mice, deals with autoimmune manifestations (as shown by hypergammaglobulinemia, Coombs positive hemolytic anemia, and the occasional presence of lupus- and rheumatoid-like factors) and mainly with the pathology and the pathogenesis of glomerulonephritis in these mice, a model system of membranous glomerulonephritis with spontaneous and insidious onset, progression through chronic stages, and almost certainly induced by immunological, and autoimmune, mechanisms. The earliest and lasting histological change was hyaline thickening of the capillary walls and adjacent intercapillary regions of the glomerular tufts, corresponding in location to polysaccharide-rich capillary basement membrane and mesangial materials. Distributed focally and diffusely in the glomerular tuft and eventually sparing no glomerulus, hyaline, granular, and fibrillar ("spongy fiber") materials produced narrowing of capillary lumens by concentric or eccentric encroachment upon them. In the later stages hyaline lobulation and sclerosis of the glomerular tufts occurred. Thus the lesions corresponded to those seen in human focal and diffuse membranous, chronic lobular, and lastly (intracapillary) sclerosing glomerulonephritis. In all instances of glomerulonephritis the glomerular tufts contained selective localizations of mouse immunoglobulins corresponding in distribution to that of the hyaline and (PAS-positive) polysaccharide-rich materials in the focal and diffuse membranous and lobular lesions and in amounts increasing with the severity of glomerular disease. The mouse immunoglobulins were extracted from frozen sections of glomerulonephritic kidneys and were then capable of recombination with glomerular tufts in sections of autologous or isologous glomerulonephritic kidneys from which in vivo localized immunoglobulins had been extracted. The pattern of recombination with glomerular tufts was similar to that of in invo localized immunoglobulins. The extracted immunoglobulins did not show affinity for mouse red cells (in the indirect Coombs test) nor for autologous or isologous cell nuclei (in the immunofluorescence test). The serum of mice with severe glomerulonephritis contained immunoglobulins with in vitro affinity for extracted autologous or isologous glomerular tufts. Thus circulating as well as localized antibodies were demonstrated. The immunogenic materials (autoantigens) may have been formed in the glomerular tufts or accumulated in them from some other source, such as the circulating plasma; however they corresponded in location to polysaccharide-rich capillary basement membrane and mesangial materials. The spleen was identified at the cellular level as the main site of formation of autoantibodies to red cells, as well as the main site of red cell destruction. Some evidence was brought forth suggesting that these autoantibodies were "heavy" or γM-globulins. More studies are in progress.     

GENETIC INFLUENCES AFFECTING THE OCCURRENCE OF A DIABETES MELLITUS-LIKE DISEASE IN MICE INFECTED WITH THE ENCEPHALOMYOCARDITIS VIRUS

The M variant of encephalomyocarditis virus produces a diabetes mellitus-like disease in DBA/2 mice but not in animals of the C3H strain. Fewer than one-third of infected F₁ (DBA/2 x C3H) progeny exhibit the disease, whereas the prevalence in backcrosses (F₁ x DBA/2, F₁ x C3H) is comparable to the parental inbred strain. Thus, the mode of inheritance of the diabetic predisposition appears to be polygenic. DBA/2 animals develop striking inflammatory and necrotizing lesions of the islets of Langerhans; in contrast, alterations of the insular tissue in the C3H mice are minimal. Although metabolic abnormalities appear to be consequent to lesions of beta cells, the factors influencing the severity of these insular changes are incompletely understood.     

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 PROPAGATION OF THE VIRUS OF EPIZOOTIC HEMORRHAGIC DISEASE OF DEER IN NEWBORN MICE AND HELA CELLS

The New Jersey strain of EHD virus has been propagated in newborn Swiss mice by the intracerebral route and is regularly lethal beyond the first serial mouse passage. A complement-fixing antigen prepared from the brains of infected mice reacts positively with the sera of deer recovered from infection with either the New Jersey or South Dakota strain of virus, but not with the serum of normal deer. The mouse-passaged virus induced an inapparent infection in an experimental deer. The virus can also be grown serially in HeLa cell culture and induces a characteristic cytopathic effect. It is neutralizable in such cultures to high titer by the sera of deer recovered from EHD (New Jersey strain) and to lower titer by the serum of a deer recovered from EHD (South Dakota strain). Normal deer serum does not neutralize the virus in tissue culture. The HeLa cell-passaged virus induced typical lethal EHD in an experimental deer and virus could be recovered from most of the tissues of this animal in HeLa cell culture. An unexplained prozone of inhibition of cytopathogenicity at low dilutions was observed in cultures of some of the organs. The fact that EHD virus exhibited a limited sensitivity to sodium desoxycholate suggests that it may belong in the arbor virus group.     

ORAL PAPILLOMATOSIS OF RABBITS: A VIRUS DISEASE

Papillomas occur frequently on the oral mucosa of domestic rabbits procured in the metropolitan area of New York. They are small and benign, and are situated mostly on the under side of the tongue. A filtrable virus can be extracted from them with which growths can be reproduced in the oral mucosa of several species of rabbits and hares but which fails to cause lesions when inoculated into other rabbit tissues and into the oral mucosa of other species. The virus differs notably from the Shope virus, which causes cutaneous papillomas in rabbits but proves innocuous to oral mucosa: rabbits solidly immune to the oral papilloma virus are fully susceptible to the Shope virus and vice versa. The oral papillomas are not highly contagious, for susceptible animals kept in individual cages in the same rooms with others carrying the growths, fed the same kind of food, and cared for by the same attendants, do not "catch" them. They are found much more frequently in the offspring of dams that carry the growths than in those of mothers free from them, and the causative virus can be recovered from the mouth washings of rabbits having no growths. The observations indicate that the virus may be spread by transfer from the mother to the young during the period of suckling, and that it may lie latent in the mouth, doing no harm unless the mucous membrane is injured. The slight trauma occurring now and then when coarse foods are chewed may furnish the required tissue nidus under natural conditions, for papillomas occasionally appear after virus has been dropped into the mouths of uninoculated rabbits; but the more extensive injury and healing resulting from experimental tattoo inoculations proves regularly effective in this respect. Tar can also act as an efficient adjuvant to the virus, the incidence of "spontaneous" oral papillomas being much higher in domestic rabbits that had had the opportunity to lick tar from their ears and paws during long periods than in normal control animals. The virus is recoverable in quantity from the oral papillomas of tarred domestic rabbits, and the findings indicate that it is their essential cause, the tar acting merely to prepare the tissue for the virus'' action. For the same tar does not elicit oral papillomas in wild cottontail rabbits, which do not carry the causative virus though fully susceptible to it. The implications of the findings are discussed.     

EFFECTS OF SPONTANEOUS DISEASE ON ORGAN WEIGHTS OF RABBITS

A group of 127 rabbits presenting clinical manifestations of disease of spontaneous origin was studied with a view to determining whether any relation could be detected between physical constitution as represented by organ weights and functional activity as measured by the efficiency of the reaction to disease. The results of the investigation are presented in tabular form and the values obtained are compared, by means of graphs, with corresponding values for normal rabbits. It was found that animals that were the subjects of disease showed decided changes in the weights of nearly all organs, changes which appeared to be of functional origin in that the values obtained for organ weights and coefficients of variation tended to conform, in general, with the efficiency of the reaction displayed by the various groups of animals into which the entire series was divided.     

IMMUNOPATHOLOGY OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTION OF NEWBORN MICE : ANTITHYMOCYTE SERUM EFFECTS ON GLOMERULONEPHROTIS AND WASTING DISEASE

Antithymocyte serum, when administered neonatally to mice, delayed the maturation of the lymphoid system, permitting development of cellular tolerance to LCM virus at an older age than is ordinarily possible. Humoral antibody formation was not prevented and the animals exhibited the paradox of high titers of both circulating virus and antibody. This, in turn, was followed by a chronic immunopathologic glomerulonephritis in most animals. Some animals developed wasting disease between 1 and 2 months of age, characterized by reticular cell hyperplasia and widespread infiltration into tissues and organs.     

EFFECT OF IN VITRO CULTIVATION ON THE PATHOGENICITY OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS

Continued in vitro cultivation in a Maitland type medium resulted in a marked modification of the extraneural pathogenicity of the Venezuelan equine encephalomyelitis virus. The ability of the virus to induce lethal infections after peripheral inoculation was almost completely lost for mice 42 or more days of age, was somewhat reduced for mice 28 days of age, but was still retained for mice 21 or less days of age. The virulence of the virus by the cerebral route remained essentially unaffected for mice of any of the experimental age groups. Prolonged cultivation also resulted in almost complete attenuation of the virus for rabbits and guinea pigs by the intraperitoneal route.     

THE ATTENUATION OF THE VIRUS OF EPIZOOTIC HEMORRHAGIC DISEASE OF DEER BY ITS SERIAL PASSAGE IN THE BRAINS OF NEWBORN MICE

Serial passage through the brains of newborn mice markedly attenuates the New Jersey strain of EHD virus. Deer inoculated with this attenuated virus show no clinical evidence of illness, but do develop virus-neutralizing antibodies in their sera. They also become solidly immune to infection with the regularly fatal unattenuated virus.     

WESTERN EQUINE ENCEPHALOMYELITIS VIRUS IN THE BLOOD OF EXPERIMENTALLY INOCULATED CHICKENS

1. Chickens inoculated subcutaneously with 0.2 cc. of a 10^–2 to 10^–7 dilution of Western equine mouse brain virus had the virus in the blood serum between the 12th and the 48th hour in most instances. The fowls showed no signs of illness. 2. Viremia could be induced regularly in chickens by inoculating subcutaneously the least amount of virus which would produce encephalitis in the mouse when inoculated by the intracerebral route. 3. Even the minimal infecting dose for a chicken led to such multiplication of the virus that it was detectable in the serum in a 10^–4 dilution. Moreover, a minimal infecting dose appeared to result in a longer period of viremia than was produced by a larger dose. 4. Virus has not been found to persist for more than 3 days after inoculation in any organ of the chicken tested for it and usually it did not persist over 2 days. Antibodies were present in the blood within at least 15 days after inoculation. 5. It is concluded that chickens may serve as sources of infection for mosquitoes or other blood-sucking ectoparasites for short periods of time after the infecting bite of a similar invertebrate vector. There is no evidence that the chicken serves as a latent carrier of the virus. 6. No virus could be found in the blood of 2 inoculated calves, and virus has not been demonstrated regularly or with the same case in the blood of horses or of men, as it has in that of chickens. It seems unlikely therefore that large mammals serve frequently as sources for mosquito infection. 7. These experimental data on fowls and mammals correlate well with other epidemiological and laboratory findings, in particular with the feeding preference of the mosquitoes found infected in epidemic areas.     

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.     

A VIRUS DISEASE OF PARROTS AND PARRAKEETS DIFFERING FROM PSITTACOSIS

The virus of parrots and parrakeets discovered by Pacheco, Bier, and Meyer is unrelated to the agent causing psittacosis either in birds or in man. The virus is fairly species-specific and manifests itself chiefly by the production of areas of focal necrosis in the liver and acidophilic intranuclear inclusions in affected cells.     

AN EPIZOOTIC DISEASE OF FERRETS CAUSED BY A FILTERABLE VIRUS

1. An epizootic disease of ferrets with a very high case fatality rate is described. 2. By the use of suitable material the natural disease can be transmitted experimentally. 3. The primary causative agent of the disease is a filterable virus. 4. Secondary invasion by bacteria of the respiratory tract of infected animals frequently occurs. The most important secondary invader is hemolytic streptococcus. 5. There seems to be no immunological relationship between the virus of the ferret disease and the viruses of canine distemper and of human influenza. 6. Histologically the disease is characterized by cytoplasmic and intranuclear inclusion bodies in epithelial cells of many organs. 7. These inclusion bodies are indistinguishable from those occurring in canine distemper.     

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.