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.     

A VIRUS-INDUCED EPIZOOTIC HEMORRHAGIC DISEASE OF THE VIRGINIA WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS)

A circumscribed natural outbreak of a highly fatal disease of deer, which we have designated epizootic hemorrhagic disease (EHD), has been studied. The disease has proven readily transmissible in deer but not in other experimental or domestic animals tested, nor in embryonating eggs or deer kidney cell cultures. The causative agent is a virus which is readily filterable and is capable of storage, either frozen or in glycerol, for relatively long periods of time. It produces a solid immunity in the few animals that survive and the blood sera of such convalescent animals contain virus-neutralizing antibodies. The disease is one in which large and small hemorrhages occur in both the viscera and skeletal structures of the body, as well as in the subcutaneous tissues. It is probably the same as one known popularly in the southeastern United States as "black tongue" of deer. It is unrelated to epidemic hemorrhagic fever of man or to the disease caused in horses by the equine arteritis virus. At least two serologically different types of EHD virus exist. The New Jersey strain is of greater lethality for experimental deer than the serologically different one obtained from an outbreak that occurred in South Dakota a year after the New Jersey epizootic.     

BIOCHEMICAL BASIS FOR ALTERATIONS IN STRUCTURE AND FUNCTION OF HELA CELLS INFECTED WITH NEWCASTLE DISEASE VIRUS

The ability of NDV-infected HeLa cells to synthesize DNA, protein, and RNA was investigated by measuring the incorporation of tritiated precursors into these substances at intervals after infection of cells with a virus/cell multiplicity of 500:1. A significant decrease in incorporation of precursors into DNA and protein was first observed at 3¼ hours after infection. By 4½ hours, an 80 to 90 per cent decrease had occurred, and by 5¼ hours, incorporation of precursors into DNA and protein was almost completely inhibited. Incorporation of precursor into RNA decreased gradually following infection; by the 10th hour, a 40 per cent decrease had occurred. These results, integrated with earlier observations on biological aspects of infection, suggest the following causal relationships among events in NDV-infected cells: (a) The cessation of virus production is probably caused by inhibition of protein or RNA synthesis, and is not due to inhibition of DNA synthesis or to interferon. (b) The production of infective virus does not per se interfere with the ability of an infected cell to divide, nor is inhibition of mitosis caused by either inhibition of DNA synthesis or development of marked degenerative changes in infected cells. Inhibition of mitosis may be the result of inhibition of protein or RNA synthesis, (c) Marked cell damage could have been caused by inhibition of protein, DNA, or RNA synthesis, (d) Interference by NDV with the multiplication of influenza virus was probably due to the inhibitory effects of NDV on cellular biosynthetic activities.     

CHARACTERIZATION OF SPLENIC LYMPHOID CELLS IN FETAL AND NEWBORN MICE

In order to clarify the cellular events that precede the onset of immunological competence in the mouse, we have characterized and quantitated the lymphoid cells of the spleen as a function of age. Our results show that T cells and B cells both appeared in the spleens of Swiss-L mice as early as the 15th-16th day of gestation. Antigen-binding cells specific for each of three different antigens were also first detected during this same 24 h interval. The B cells and three varieties of antigen-binding cells increased in number rapidly and in parallel until about 1 wk after birth. The T cells, which were more numerous than B cells at first, increased in number somewhat more slowly. Coincident with the onset of response to antigen, there was a further increase in B cell numbers and a decrease in the T cell to B cell ratio. The capacity to respond to antigen by cellular proliferation and synthesis of antibody did not arise until about 2 wk after birth although there were no quantitative changes in the total numbers of T cells, B cells, and antigen-binding cells between 1 and 2 wk of age. Some qualitative change, such as the functional maturation of an antigen-reactive cell, may be required during this interval for the onset of this immunological response. Although the numbers of antigen-binding cells present in fetuses and young animals were smaller than in adults, we have as yet been unable to detect any restriction in the variety of specificities that can be expressed in fetuses, either in the kinds of antigens bound or in the range of avidities with which a single antigen is bound.     

THE ENTRY AND DISTRIBUTION OF HERPES VIRUS AND COLLOIDAL GOLD IN HELA CELLS AFTER CONTACT IN SUSPENSION

The way in which herpes virus of a well adapted strain penetrates susceptible HeLa cells has been investigated using thin sectioning techniques for electron microscopy. Mature virus particles and cells were mixed together in suspension cultures for 15, 30, 60, or 120 minutes so that the stages in virus uptake could be followed in sequence. The ingestion of particles of colloidal gold by HeLa cells under similar conditions was studied for comparison in parallel experiments. After 15 minutes' contact, the mature virus was found adsorbed on the surface of the cells but separated from them by a narrow gap in which phosphotungstic acid staining was sometimes able to reveal an extraneous coat which appeared as an amorphous layer on the outer aspect of the plasma membrane. When mixing continued for longer the particles were present in deep invaginations or actual cytoplasmic vacuoles, with their outer layers in various stages of stripping and digestion. The stripped, naked, central portion of the virus was occasionally found in these vacuoles but was more commonly free in the cytoplasmic matrix; the mode of transition between these sites could not be determined. Where contact continued for 2 hours these phenomena were much less frequently observed. The larger particles of colloidal gold were ingested in the same way as the virus, but smaller ones were taken up in micropinocytosis vesicles. The gold passed through membrane-bounded cytoplasmic spaces to accumulate in vacuoles from which, in contrast to herpes particles, it did not escape. These findings are discussed, and considered with particular reference to their bearing on the initiation of infection, the uptake and disposal of particles by cells, and the influence on the latter of virus morphology.     

BACTERIOLOGICAL STUDIES ON AN EPIZOOTIC OF INTESTINAL DISEASE IN SUCKLING AND NEWLY WEANED MICE

It is now quite generally conceded that the presence in animals of bacteria of Salmonella type is indicative of an active, or potentially active, pathogenic agent (26). In the present instance bacteriological studies of the organs of acutely ill infant mice revealed the presence of one of the Salmonella group. It showed itself to be such by an absence of fermentation of lactose and saccharose, by other cultural reactions, and by cross-agglutination with certain members of the Salmonella group in very low dilutions of their antisera.⁶ However, the bacterium cannot be included in any of the known species of the group because of its indol-forming properties, differences in carbohydrate reactions, and specific serological reactions. Its presence in affected mice, growth on artificial media, ability to cause the disease following enteral and parenteral inoculations, and the fact that it can be recovered from artificially infected mice fulfill the postulates of Koch. Furthermore, agglutinins are present in the blood of recovered mice and not in that of normal animals, and recovery of mice from natural infection evokes an apparent resistance against the special recovered Salmonella bacterium. The organism would appear to fall in the Asiaticusdivision of the genus, as designated by Castellani and Chalmers (27).     

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.     

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.     

THE ENDURING PARTNERSHIP OF A NEOPLASTIC VIRUS AND CARCINOMA CELLS : CONTINUED INCREASE OF VIRUS IN THE V2 CARCINOMA DURING PROPAGATION IN VIRUS-IMMUNE HOSTS

The V2 carcinoma—a transplanted rabbit cancer derived originally from a virus-induced papilloma and carrying in masked or altered form the virus primarily responsible for it—was propagated in five successive groups of animals all previously hyperimmunized against the papilloma virus. The cancer grew as well in the hyperimmunized hosts as in normal animals implanted during the same months; and serological tests, made when the tumor was eventually returned to ordinary hosts, proved that the virus was still associated with the carcinoma cells: it had increased to the usual extent as the tumor grew in the hyperimmune animals. The continued increase of the neoplastic virus during propagation of the V2 carcinoma in hyperimmunized hosts contrasts sharply with the elimination of certain extraneous passenger viruses when the tumors they ride upon are grown in hosts previously immunized against them. The facts as a whole would seem to warrant a distinction between the enduring partnership of a neoplastic virus and carcinoma cells on the one hand and the casual association of passenger viruses with tumor cells on the other.     

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.     

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.     

PRODUCTION OF RUNT DISEASE IN TOLERANT MICE BY THE INJECTION OF SYNGENEIC LYMPHOID CELLS

When chimeric A strain mice tolerant of (A x C57BL/1)F₁ hybrid skin grafts are injected with spleen cells from normal A donors the recipients develop weight loss, clinical evidence of runting, and death in some animals. Similar recipients injected with spleen cells from A strain donors immunized against C57BL/1 tissue show a more rapid onset of the runting process and increased mortality. Runting in. these experiments therefore results from an immune attack by the injected A strain lymphoid cells against the (A x C57BL/1)F₁ hybrid tissue harbored by the chimeric recipients. Since the hybrid tissues of the chimeric recipients were derived from spleen cell populations we conclude that the immunologic rejection of lymphoid and hematopoietic tissue is sufficient to cause the runting syndrome. C3H mice tolerant of A strain skin grafts because of the prior injection of viable or disrupted A strain spleen material were given 400 r of x-irradiation and an injection of C3H spleen cells. Only the chimeric C3H mice harboring viable A strain cells developed weight loss and clinical evidence of disease, showing again that runting occurs only when an attack can be made against viable lymphoid and hematopoietic tissue. Normal A strain mice injected intravenously with 850 million (A x C57BL/1)F₁ hybrid spleen cells reject hybrid skin grafts and do not develop runting, whereas the rejection of similar hybrid tissue present in chimeric A strain mice results in runting. It is concluded that runting will occur only when the immunologic attack is directed against lymphoid and hematopoietic tissue which has become established within host tissues. The possibility that runting may result from hypersensitivity reactions occurring in the lymphoid tissues is discussed.     

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.     

PROPAGATION OF VACCINIA VIRUS IN THE RABBIT FETUS

1. A method is described for propagation of bacteria-free vaccinia virus in the rabbit fetus. 2. For the production of severe generalized infection, affording a high yield of virus, the following conditions were found to be favorable: the use of 24 day fetuses, a virus dosage of 10,000 adult rabbit skin units, injection into multiple sites, and an incubation period of 4 days in utero. 3. Under these conditions the virus was found to disseminate widely in the infected fetus, being recoverable particularly from liver, lungs, brain, skin, placenta, and kidney. 4. Under varied conditions of virus dosage, fetal age, and incubation period, all grades of reaction were observed in the fetus, ranging from the mildest infection, with only an occasional small lesion demonstrable, to generalized vaccinia with pocks in nearly every organ of the body, and often death. 5. It was shown that the virus could be carried in serial passage through fetuses. A series of 27 such passages was made by using fetal skin for subinoculation at each transfer. Similarly, two other strains of the virus were evolved in shorter series of transfers by using respectively fetal brain and fetal liver as the subinocula. 6. The passage strains maintained their identity and titer, as judged by the intradermal inoculation of adult rabbits. However, these strains underwent a significant reduction in their capacity to infect by the scarification method, and the lesions induced by intradermal inoculation were much milder than those produced by the parent strain. None of the passage strains manifested properties characteristic of the so called neurovaccine strain, despite the frequent use of intracerebral inoculation in the fetuses. 7. In explanation of the reduction of virulence of the passage strains, it is suggested that a given suspension of virus may be considered as consisting of particles of varying degrees of virulence, and that when cultivation is carried out in highly susceptible tissues a relative overgrowth of the less virulent elements is permitted, so that apparent attenuation results.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE : I. REPLICATION IN BABY HAMSTER KIDNEY CELLS AND PROPERTIES OF THE VIRUS

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 10⁶ TCID₅₀ per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm.     

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.     

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.     

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.     

THE VISCERAL LESIONS PRODUCED IN MICE BY THE SALIVARY GLAND VIRUS OF MICE

Extensive visceral lesions containing intranuclear inclusions have been produced in mice by intraperitoneal and intracerebral inoculations of the homologous salivary gland virus. Rarely small pancreatic lesions containing inclusions have been encountered 2 weeks after subcutaneous inoculation. Many of the animals injected intraperitoneally died between the 4th and 7th day after inoculation. In spite of the extensive lesions produced in the liver and spleen, the virus could not be transferred with an emulsion of these organs.     

FACTORS DETERMINING PATHOGENICITY OF VARIANTS OF ECHO 9 VIRUS FOR NEWBORN MICE

While some strains of ECHO 9 virus were found to be completely incapable of multiplying in newborn mice or even of being adsorbed by their tissues (e.g., the prototype Hill strain), other naturally occurring strains readily multiplied even after inoculation of as little as 3 TCD₅₀ of virus. With the multiplying strains, the infection remained clinically inapparent except after inoculation of very large doses, usually in the range of 10⁵ to 10^7.5 TCD₅₀. Investigation of the question why such large doses were required to produce paralysis indicated that for paralysis to occur virus multiplication had to reach a level of 10⁸ TCD₅₀ or more within 4 days after inoculation of mice less than 1 day old. The reason for this was found in the fact that at 5 to 6 days of age the mice lost their susceptibility to paralysis even when multiplication was capable of progressing to the indicated high level. Thus, speed of multiplication and extent of muscle involvement before the 5th day of life were the determining factors. Passage in tissue culture had no effect except to yield a larger dose for inoculation, while serial propagation in mice resulted in a gradual enrichment of virus particles capable of more rapid multiplication in mice and in a concurrent greater paralytogenic activity of smaller doses.