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.     

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.     

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 QUANTITY OF IRRADIATED NON-VIRULENT RABIES VIRUS REQUIRED TO IMMUNIZE MICE AND DOGS

In the experiments described above, we found with respect to tissue culture rabies virus that 1 cc., which contains approximately 50,000 mouse intracerebral lethal doses, properly irradiated, was required to immunize a mouse; 500 cc., which contain 25,000,000 doses, were required to immunize a 20 pound beagle dog. Tissue culture virus concentrated ten times proved capable of immunizing mice in a dose one-tenth as large as that required for unconcentrated culture virus. Brain virus suspensions were centrifuged so as to remove a large part of the tissue particles without striking loss in the virulence of the supernatant. The centrifuged supernatants of 1 to 5 per cent brain virus suspensions were irradiated so as to destroy virulence and yet retain immunizing potency. Irradiated supernatants of mouse brain virus proved capable of immunizing mice as well as or better than similar supernatants treated with chloroform. 0.1 cc. of a 1 per cent irradiated dog brain virus containing approximately 50,000 mouse intracerebral lethal doses immunized mice effectively.     

EPIDEMIC KERATOCONJUNCTIVITIS : I. ISOLATION AND IDENTIFICATION OF A FILTERABLE VIRUS

1. A virus has been isolated from two patients suffering with epidemic keratoconjunctivitis. 2. At first the virus could be maintained only by the inoculation of tissue cultures (serum ultrafiltrate and embryonic mouse brain) with conjunctival scrapings or with emulsified mouse brains from early passage animals. Later it caused a fatal disease in every mouse into which it was inoculated, and could, then be readily maintained in mice. 3. The virus proved pathogenic for unweaned white Swiss mice by the intranasal, intraperitoneal, and intracerebral routes; for adult mice by the intranasal and intracerebral routes, and for rabbits by only the intracerebral route. 4. Although the titer of tissue cultures rarely exceeded 10^–2 or 10^–3, the virus, once established in mice, increased in potency until titers of 10^–5 and 10^–6 were attained. Mice injected with either the emulsion of mouse brain tissue or with the tissue culture material in these dilutions developed symptoms within a definite incubation period; once the disease was initiated, it followed a characteristic course for a period of a few to 24 hours, and consistently terminated in death. 5. The pathological changes in mice were limited to the central nervous system, and were not particularly distinctive. The neurotropic character of the virus is further shown by the fact that only the brain tissue was consistently pathogenic for mice. 6. Serial tissue cultures could be maintained only at room temperature, and when the inoculum from virus-infected cultures into fresh tissue cultures contained ground-up cells. 7. The highest level of potency in cultures occurred on about the 6th day at room temperature. 8. The virus passed without difficulty through an E-K Seitz filter (double pads) and through all grades of Berkefeld filters. 9. The virus passed consistently through graded collodion membranes with an A.P.D. of 75 to 100 millimicrons and to a lesser extent through those with an A.P.D. of 50 to 75 millimicrons. Membranes with an A.P.D. of less than 50 millimicrons retained the virus. 10. The mouse virus was not neutralized by anti-lymphocytic choriomeningitis serum, antiherpes serum, normal human serum, or serum from cases of non-specific conjunctivitis or keratitis. 11. Mice hyperimmunized to Theiler''s virus were susceptible to the mouse keratoconjunctivitis virus. The latter virus was also not neutralized by hyperimmune Theiler rabbit serum. 12. The mouse virus could be neutralized by serum from the two patients from whom the virus had been isolated, and also by that from the three patients convalescing from epidemic keratoconjunctivitis in California and the serum of a convalescent in New York. The neutralization data were confirmed by tests on 15 additional convalescent serums (unpublished data). 13. A mild but characteristic picture of epidemic keratoconjunctivitis was reproduced in a human volunteer following inoculation with the mouse virus. 14. The serum of the human volunteer, while not neutralizing the mouse virus before infection, contained neutralizing antibodies 1 month after infection. 15. Development of antibodies was demonstrated in one patient (R.H.) in the present series, and in six other patients of another series (unpublished data).     

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.     

STUDIES IN RODENT POLIOMYELITIS : I. FURTHER EXPERIMENTS WITH THE MURINE STRAIN OF SK POLIOMYELITIS VIRUS

1. SK murine virus maintained over more than 200 serial mouse passages increased in virulence for mice from an initial intracerebral titer of about 1:1 million to a maximum titer of not less than 1:1 billion dilution activity. 2. Following intracerebral injection with murine virus of remote mouse passages, 5 of 13 rhesus monkeys developed a characteristic encephalitic syndrome. Repeated intravenous injection of massive doses of virus caused localized flaccid paralysis in 2 of 14 monkeys. 3. Intracerebral injection of graded doses of murine virus into mice of different age groups caused fatal paralysis in young and old animals alike. Infection with small doses of virus by peripheral routes, while uniformly fatal to young mice, was followed by survival of almost half of the old mice. 4. The incubation period of the disease in young mice infected intracerebrally with a standard dose of murine virus, when studied throughout the period of 1 year, was found considerably lengthened during the summer months. 5. Cross neutralization tests furnished no evidence for any serological relationship between SK murine virus and lymphocytic choriomeningitis virus. Theiler''s virus was found to be neutralizable by antimurine horse serum and, to a lesser extent, by concentrated antipoliomyelitis horse serum; however, such inactivation, in both cases, was distinctly inferior to that occurring with SK murine virus. On the other hand, no neutralization whatsoever was obtained between SK murine virus and normal adult mouse serum, whereas the same serum completely neutralized Theiler''s virus. Mice surviving infection with Theiler''s virus, though acquiring immunity to this virus, remained fully susceptible to reinfection with SK murine virus. 6. Neutralization tests with SK murine virus against poliomyelitis-convalescent monkey sera gave irregular results, but neutralization of murine virus occurred regularly with a hyperimmune antipoliomyelitis horse serum. Hyperimmune antimurine horse and rabbit sera, on the other hand, failed to inactivate three strains of monkey poliomyelitis virus (SK, RMV, Aycock) by intracerebral tests in monkeys. The same sera inactivated murine virus in mice by intraperitoneal, but not by intracerebral injection of virus-serum mixtures. 7. The identity of SK murine virus and its relation to other rodent strains of poliomyelitis virus is discussed on the basis of the available data.     

STUDIES OF A MURINE STRAIN OF POLIOMYELITIS VIRUS IN COTTON RATS AND WHITE MICE

1. A neurotropic murine virus was isolated by passing poliomyelitis virus (SK strain) from the monkey to cotton rats and white mice. 2. The murine virus has been grown in tissue culture consisting of embryonic mouse brain in ox serum ultrafiltrate. 3. The symptoms and lesions produced by the murine infection compare in all respects with those of poliomyelitis in monkey and man. 4. The murine virus, while highly pathogenic for mice and cotton rats, is non-pathogenic for albino rats, guinea pigs, and rabbits. It possesses limited pathogenicity for rhesus monkeys. 5. Although producing no paralysis in the above mentioned refractory animals, the murine virus may be recovered in active form from neural and extraneural sites of infected albino rats, guinea pigs, and monkeys, but not from rabbits. 6. The identity of the murine and monkey virus is further suggested by cross-neutralization between the murine virus and homologous (SK) and related (Aycock) antipoliomyelitis sera, as well as between homologous and related monkey poliomyelitis virus and antimurine virus sera. 7. Immunization of monkeys with live murine virus, in the form of mouse brain or tissue culture, seems to confer some degree of resistance against subsequent infection with the homologous poliomyelitis monkey virus. 8. The presence of the murine virus in the central nervous system of infected monkeys appears to interfere with the propagation of SK and Aycock poliomyelitis monkey virus in the same animal.     

EXPERIMENTAL STUDIES ON ENCEPHALITIS : III. SURVIVAL OF ENCEPHALITIS VIRUS (ST. LOUIS TYPE) IN ANOPHELES QUADRIMACULATUS

Anopheles quadrimaculatus mosquitoes, fed on mice in which encephalitis virus (St. Louis type) is present in the blood stream, take up and retain the virus for the duration of their lives. The titre of the virus in mosquitoes 4 hours after engorging on mice with a maximum blood stream infection represents about 10,000 lethal mouse intracerebral doses per mosquito. This titre drops during the following 2 weeks to about 100 lethal doses per mosquito, but from the 3rd week to the death of the mosquito, usually increases to approximately the original level and remains there. The titre of virus in mosquitoes which have engorged on mouse blood containing smaller quantities of virus exhibits the same drop and subsequent rise to the original level. The virus-containing mosquitoes did not infect mice or monkeys by biting.     

STUDIES IN RODENT POLIOMYELITIS : III. EXPERIMENTAL POLIOMYELITIS IN GUINEA PIGS PRODUCED WITH THE MURINE STRAIN OF SK POLIOMYELITIS VIRUS

1. Murine SK poliomyelitis virus has been transferred from mouse to guinea pig with the establishment of a fixed strain of cavian passage virus. 2. The disease thus produced in guinea pigs is characterized by the occurrence of flaccid paralysis. Typical poliomyelitic lesions are found in the anterior horn of the spinal cord. 3. Guinea pigs are susceptible to infection with murine virus by the intracerebral, intravenous, intraperitoneal, and subcutaneous route; cavian passage virus produces paralysis only upon intracerebral or intravenous injection. Neither virus paralyzes guinea pigs by feeding or nasal instillation. 4. The potency of the virus (murine or cavian) in guinea pigs is considerably lower than in mice and compares with the titer of the original SK strain in monkeys. In paralyzed guinea pigs the virus is found only in the central nervous system and not in extraneural sites, such as blood or abdominal viscera. 5. Attempts to cultivate cavian passage virus in tissue culture have yielded evidence of some in vitro propagation but no passage virus has as yet been obtained by this method. 6. Cross neutralization tests with cavian passage virus in guinea pigs and with murine virus in mice have established the serological identity of the two viruses. Inactivation of cavian passage virus in guinea pigs by poliomyelitis-convalescent monkey sera is irregular. Complete neutralization has been obtained with a concentrated poliomyelitis horse serum. 7. Resistance to reinfection with potent virus can be demonstrated in convalescent guinea pigs as well as in guinea pigs which have survived a symptomless infection with either murine or cavian virus. This immunity is demonstrable by the power of the serum of such animals to neutralize the virus in vitro and by the ability of nerve tissue to dispose in vivo of the infectious agent. 8. Cavian passage virus has a limited pathogenicity for rhesus monkeys. Of a total of 35 monkeys injected intracerebrally with guinea pig passage virus 26 failed to respond with any manifest symptoms of disease; 8 monkeys showed various signs of definite involvement of the central nervous system consisting of tremor, convulsions, facial palsy, and localized pareses; 1 monkey developed typical flaccid paralysis. 9. Following injection with cavian virus the virus may be recovered from the tissues of normal monkeys but not from the tissues of convalescent monkeys shortly after a paralyzing attack of poliomyelitis due to SK or Aycock virus. 10. Immunization of monkeys with early cavian passage virus by the subcutaneous route has given no clear-cut evidence of protection against intracerebral reinfection with SK poliomyelitis virus. Neither has there been any evidence of effective interference in monkeys injected intravenously with early cavian passage virus and intracerebrally with RMV poliomyelitis virus. 11. The bearing of the experimental data upon the epidemiology of the human disease is discussed.     

THE INFECTION OF MICE WITH SWINE INFLUENZA VIRUS

The experiments confirm the earlier observation of Andrewes, Laidlaw and Smith that the swine influenza virus is pathogenic for white mice when administered intranasally. Two field strains of the swine influenza virus were found to differ in their initial pathogenicity for mice. One strain was apparently fully pathogenic even in its 1st mouse passage while the other required 2 or 3 mouse passages to acquire full virulence for this species. Both strains, however, were initially infectious for mice, without the necessity of intervening ferret passages. There is no evidence that bacteria play any significant rôle in the mouse disease though essential in that of swine, and fatal pneumonias can be produced in mice by pure virus infections. Mice surviving the virus disease are immune to reinfection for at least a month. In mice the disease is not contagious though it is notably so in swine. The virus, while regularly producing fatal pneumonias when administered intranasally to mice, appears to be completely innocuous when given subcutaneously or intraperitoneally. Prolonged serial passage of the virus in mice does not influence its infectivity or virulence for swine or ferrets. It is a stable virus so far as its infectivity is concerned, and can be transferred at will from any one of its three known susceptible hosts to any other. In discussing these facts the stability of the swine influenza virus has been contrasted with the apparent instability of freshly isolated strains of the human influenza virus. Though the mouse is an un-natural host for the virus it is, nevertheless, useful for the study of those aspects of swine influenza which have to do with the virus only.     

EXPERIMENTAL INFECTION WITH INFLUENZA A VIRUS IN MICE : THE INCREASE IN INTRAPULMONARY VIRUS AFTER INOCULATION AND THE INFLUENCE OF VARIOUS FACTORS THEREON

Following intranasal inoculation of influenza A virus (strain PR8) there is a rapid increase of the virus in the lungs which with large doses reaches a maximum within 24 hours. With smaller doses, although the proportional increase is greater, the maximum concentration is not reached until 48 hours following inoculation. If a lethal dose is administered, the ultimate concentration of the virus in the lungs is the same, irrespective of the size of the dose. If a sublethal dose is given, the titer of the virus in the lungs does not achieve the titer reached in mice receiving a lethal dose. Within 48 hours following inoculation of a sublethal dose the lungs of a mouse may contain at least 76,000 M.L.D., yet the mouse survives. The intranasal instillation of sterile fluid (distilled water, varying concentrations of NaCl, broth, or 10 per cent normal serum) into a mouse sublethally infected produces a sharp rise in the virus content of the lung usually followed by death within 3 to 8 days. If, however, the instillate consists of 10 per cent immune serum, there is no rise in the virus titer, and no apparent harm results from the instillation. The implications of these phenomena are discussed and an hypothesis presented to explain their occurrence.     

A TRANSMISSIBLE AGENT (THEILER'S VIRUS) IN THE INTESTINES OF NORMAL MICE

Every experiment with the contents of one, or with those pooled from two to five of the normal stock of Rockefeller Institute strain of albino mice, 1 to 2 months of age, revealed the presence of a virus which, after intracerebral inoculation into normal mice, induced characteristic paralytic encephalomyelitis, indistinguishable from Theilerapos;s disease. No difference was seen in this effect of intestinal contents deriving from animals paralyzed during the course of spontaneous encephalomyelitis and from normal mice. The influence of age on carriage of virus, as well as on the persistence of the carrier state, is discussed. The present, as well as previous work has shown that the virus found in normal (or paralyzed) mice is similar to that of Theiler''s disease in all of its properties thus far investigated; among the strains of the latter now at hand it can be classified with those having a low degree of invasiveness after peripheral inoculation. The virus has thus far been recovered from intestinal contents, intestinal walls, and mesenteric glands but not from the central nervous system of normal mice; from these sites, as well as from the central nervous system, in paralyzed mice. In order of concentration of virus, the contents have more, the walls less, and the glands least.     

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.     

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.     

IMMUNOLOGICAL DETERMINANTS OF POLYOMA VIRUS ONCOGENESIS

Adult mice and hamsters can be made resistant to an isologous transplantable polyoma tumor by an inapparent infection with polyoma virus. This resistance is cell-mediated and seems not to be related to anti-viral serum antibodies. The basis of the resistance appears to be a transplantation type of cellular immunity directed against a "foreign" antigen contained in the tumor cell. Evidence has been presented to demonstrate this tumor antigen. It is possible that this phenomenon may explain the lack of oncogenesis by polyoma virus infection of adult mice, and the rarity of naturally occurring polyoma tumors.     

ENDURING IMMUNITY FOLLOWING VACCINATION OF MICE WITH FORMALIN-INACTIVATED VIRUS OF RUSSIAN SPRING-SUMMER (FAR EASTERN,TICK-BORNE) ENCEPHALITIS : CORRELATION WITH SERUM-NEUTRALIZING AND COMPLEMENT-FIXING ANTIBODIES

A single course of two intraperitoneal injections of formalin-inactivated virus of Russian spring-summer encephalitis induced in albino mice a solidly immune state which endured almost throughout life. Active virus is therefore not essential for the production of a high degree of lasting immunity. The immune response to vaccination consists of resistance to peripherally introduced active virus and development of circulating antibody. A correlation has been found to exist throughout the long period of the immune state between the titer of neutralizing antibody, as determined by the intraperitoneal method described, and the degree of immunity to peripherally introduced active virus. Thus laboratory tests for the immunizing power of a vaccine suggest themselves, to be carried out by an estimation in vaccinated mice of (a) immunity to peripherally inoculated active virus, and (b) serum virus-neutralizing antibody determined by the intraperitoneal method. The rôles as indicators of immunity in vaccinated mice of complement-fixing antibody in the serum, of the intracerebral challenge dose of virus, and of the intracerebral method for testing neutralizing antibody are discussed. Finally, if the immune response of man to vaccination with formalin-inactivated virus of Russian spring-summer encephalitis follows the pattern of the response of mice as here described, and if the correlation of neutralizing antibody with immunity to peripherally introduced virus applies to man as to mice, then possibly the degree of immunity in human beings following vaccination can be appraised by a peripheral test for neutralizing antibody in the serum.     

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.     

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.     

A LATENT VIRUS IN NORMAL MICE CAPABLE OF PRODUCING PNEUMONIA IN ITS NATURAL HOST

1. A virus capable of producing fatal pneumonia in mice has been isolated repeatedly from the lungs of certain apparently healthy mice. Not all mice carry the virus. It was obtained only from mice supplied by three breeders although mice from eight different sources were studied. 2. The virus was avirulent as it occurred in normal mouse lungs and became virulent only after serial mouse lung passage. It was strictly pneumotropic for mice and produced pneumonia when given intranasally but showed no evidence of infection when given by other routes. The virus was non-infectious for ferrets and did not become pathogenic for this species after numerous serial passages. It was also non-pathogenic for rabbits, guinea pigs, rhesus monkeys, voles, deer mice, skunks, wood-chucks, opossums, and Syrian hamsters. 3. All strains of the virus which have been tested have been immunologically identical, as indicated both by cross immunity and cross neutralization tests in mice. 4. The virus was antigenic both in mice and in rabbits and was readily differentiated from viruses of human influenza and of swine influenza by means of either cross immunity or cross neutralization tests. 5. The virus was also neutralized by about 30 per cent of normal human sera tested. 6. The virus was extremely labile, and suspensions prepared in saline or broth became inactivated within a few hours at room temperature. The addition of normal horse serum to the virus suspensions, however, exerted a definite stabilizing effect. 7. Ultrafiltration results indicated that the virus particles have a diameter of about 100 to 150 millimicrons. 8. Evidence is presented which indicates that this virus is different from other viruses which various investigators have found in normal mouse lungs.