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.     

THE ROUTE OF ENTERIC INFECTION IN NORMAL MICE

This study followed the early pathogenesis of orally induced murine typhoid fever. Intragastrically administered Salmonella enteritidis moves quickly through the normal undisturbed gut so that only a small residuum remains in the cecum and large intestine after the first few hours. Dye injection of the gut wall was used to show that lymph from discrete portions of the gastrointestinal tract drains to separate lymph nodes, probably via the regional Peyer's patches. Plating techniques capable of detecting a single colony-forming unit of S. enteritidis within the different Peyer's patches and draining lymph nodes indicate that, although the cecum and large intestine are exposed to large numbers of Salmonella for longer time periods than the small intestine, the primary site of bacterial penetration involves the distal ileum. This area of the small intestine as well as the cecum are both drained by the distal mesenteric lymph nodes, and were the only nodes which contained detectable numbers of viable Salmonella over the first 24 h of infection. Neither the pyloric nor the proximal mesenteric lymph nodes (which drain the stomach and duodenum) nor the pancreatic and caudal lymph nodes (which drain the transverse and descending colon) contained viable Salmonella. Salmonella were observed to infect the ileal mucosa and its Peyer's patches. With time, this infection progresses to the draining lymph node and ultimately reaches the liver and spleen. Some of the implications of these findings relative to the development of acquired resistance to enteric disease are discussed.     

MULTIPLE ENZYME CHANGES IN THE PLASMA OF NORMAL AND TUMOR-BEARING MICE FOLLOWING INFECTION WITH THE LACTIC DEHYDROGENASE AGENT

Within 72 hours after injection of the LDH agent into normal mice, five (LDH, ICDH, MDH, PHI, and GOT) out of the seven plasma enzymes studied were elevated. This elevation persisted for the duration of the experiment. Alkaline phosphatase and aldolase were not elevated. Plasma from mice bearing tumor SS-70429 and infected with the LDH agent showed 7 times more LDH, 8 times more ICDH, and 4 times more MDH activity than the plasma from mice with the same tumor but uninfected. The plasma aldolase activity from the infected tumor-bearing animal was approximately the same as that from the uninfected tumor-bearing animal. Somewhat similar results, but lower in magnitude, were found with mice bearing mammary carcinoma C₃HBA. The early rise in plasma enzyme activity (LDH, MDH, ICDH) prior to the actual appearance of the tumor was shown to be due not to the tumor, but to the LDH agent. Uninfected tumor-bearing mice showed a late increase in plasma enzyme activity which appeared to be related to tumor growth. The findings reported above suggest that contamination with the LDH agent may have been responsible for much of the increased plasma enzyme activity previously attributed to the tumor.     

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.     

THE APPEARANCE OF A HEPATOTROPHIC VIRUS IN MICE THYMECTOMIZED AT BIRTH

Inbred (C57BL; C3H/Bi), hybrid (C57BL x C3H/Bi), and outbred (TO) mice thymectomized within 24 hours of birth develop wasting symptoms and die prematurely and a proportion of these animals have pathological changes in the liver. The incidence of the liver lesions varies according to the strain of mice used and the lesions tend to occur in animals dying comparatively late. These lesions were shown, by passage of tissue suspensions and of cell-free liver extracts, to be due to a hepatotrophic virus probably mouse hepatitis virus-1 (MHV-1). The part played by the hepatotrophic virus in the premature death of thymectomized mice is discussed but, although neonatal thymectomy apparently alters a normally stable host-virus relationship, it is not thought that the virus is primarily responsible for the death of its host. The role of this virus in the production of the physical wasting is also considered to be problematic.     

ISOLATION FROM NORMAL MICE OF A PNEUMOTROPIC VIRUS WHICH FORMS ELEMENTARY BODIES

1. A pneumotropic virus which forms elementary bodies has been isolated from apparently normal albino Swiss mice. 2. The antigenic relationship of this virus to those of meningopneumonitis, lymphogranuloma venereum, hamster pneumonia (7), and human pneumonitis (8) was established either by cross-immunity or complement fixation or both. 3. In spite of a relationship to other viruses, the virus could be differentiated from all the others studied by certain of its properties.     

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.     

THE LIMITED NEUROTROPIC CHARACTER OF THE ENCEPHALITIS VIRUS (ST. LOUIS TYPE) IN SUSCEPTIBLE MICE

1. St. Louis encephalitis virus injected intracerebrally into susceptible mice multiplies there to reach a titre of 10⁹ intracerebral lethal doses. It is found also in the blood in small amounts immediately following injection and preceding death. 2. Injected intraperitoneally or subcutaneously the virus circulates in the blood for several hours and survives in the spleen for days. It does not multiply in the brain and cause encephalitis, however, unless overwhelming doses are injected or the brain is traumatized. 3. Virus dropped into the nares is demonstrable in the olfactory bulbs at 24 hours, in the piriform lobes at 24 to 48 hours, in the remainder of the brain at 3 days, and in the spinal cord at 4 days. In the brain it reaches a titre of 10⁹ in 6 days. Virus is not readily demonstrable in the blood but is present in the spleen after 48 hours. 4. Virus survives and is capable of multiplying in the spleen. 5. Lesions following nasal instillation of virus appear first in the olfactory bulbs on the 3rd day, in the piriform lobes on the 4th, and in Ammon''s horn on the 5th day. The character of the lesions in order of their appearance is exudation of mononuclear cells about superficial blood vessels and in the pia, hyperplasia of the endothelium of the pia, and necrosis of nerve cells of the olfactory tract.     

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.     

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.     

THE EFFECT OF SULFHYDRYL GROUPS ON PNEUMONIA VIRUS OF MICE (PVM)

Evidence is presented which indicates that PVM is affected adversely in concentrated lung tissue suspensions or in the presence of glutathione. Because iodoacetamide inhibits or eliminates these effects in a similar manner, it is concluded that sulfhydryl groups are essential to their development.     

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.     

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.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE : II. STRUCTURE AND MORPHOGENESIS OF THE VIRUS PARTICLE

Pneumonia virus of mice (PVM) particles are spheres 80–120 mµ in diameter, or filaments of similar diameter with lengths up to 3 µ. The particles possess an outer spike-covered envelope and helical internal component 120–150 A in diameter. Virus particles acquire their envelope by a budding process at the cell membrane; mature particles are seen only extracellularly. Dense inclusions are prominent in the cytoplasm of PVM-infected BHK21 cells by 48 hr after inoculation. The inclusions appear to consist of aggregates of the internal component of PVM, and the helical component has been isolated in a cesium chloride gradient from extracts of osmotically shocked cells. Murine erythrocytes, which are agglutinated by PVM, adsorb to the surface of infected cells and to budding and extracellular PVM particles. On the basis of its structure and morphogenesis, PVM appears to be a myxovirus; however, it does not fit into either of the established subgroups of myxoviruses. The 120–150 A diameter of the PVM internal component differs from the diameters of the internal components of the two established subgroups of myxoviruses, and suggests that a third subgroup of these viruses may exist.     

FURTHER IMPLICATION OF MURINE LEUKEMIA-LIKE VIRUS IN THE DISORDERS OF NZB MICE

Further evidence implicating murine leukemia-like virus in the disorders of NZB mice was afforded by a study of antigens associated with murine leukemia virus (MuLV). MuLV group antigens were prevalent in extracts of spleen, kidney, and, to a lesser extent, thymus throughout a substantial portion of the life span of NZB mice as well as in extracts of lymphomas and sarcomas indigenous to the strain. G (Gross) soluble antigen, type-specific antigen, was first detected in plasma of untreated NZB mice at 3 months of age. G soluble antigen production increased thereafter in line with age, with 50% of reactions becoming positive at 5.3 months and 100% at 7 to 9 months. From months 3 to 9, the time-response curve for positive conversion of direct antiglobulin (Coombs) tests in untreated NZB mice corresponded closely to that for G soluble antigen production. Beyond the 9th month, G soluble antigen underwent elimination from the plasma of NZB mice, with positive reactions reduced to 50% at 13.3 months and to 0% at 18 months. G natural antibody was first detected in the serum of NZB mice at about 10 months of age and increased thereafter in line with age. The curves for G antibody production and G soluble antigen elimination bore a reciprocal relation to each other with crossover at 50% response occurring at 13.3 months. Significant proteinuria, a functional manifestation of membranous glomerulonephritis, became increasingly prevalent in female NZB mice as G soluble antigen was eliminated from plasma. Cumulative mortality of female NZB mice, mainly attributable to renal glomerular disease, increased in phase with G antibody production. MuLV group antigens were identified in the glomerular lesions by the immunofluorescence method. Positive conversion of direct antiglobulin tests was significantly delayed by vaccinating baby NZB mice with formaldehyde-inactivated cell-free filtrates of older NZB mouse spleens. The plasmas of vaccinated NZB mice with negative direct antiglobulin reactions at 4 to 7 months were likewise negative when tested for G soluble antigen. The 50% response time for G antibody production in the vaccinated NZB mice occurred at 7.3 months, that is, 6 months earlier than in untreated NZB mice. The collective findings implicate murine leukemia-like virus in the etiology of autoimmune hemolytic disease and membranous glomerulonephritis, as well as malignant lymphoma, of NZB mice and suggest that virus-specified cell-surface and soluble antigen is a factor in the immunopathogenesis of the renal disease and possibly also the autoimmune hemolytic disease.     

PROPERTIES OF PNEUMONIA VIRUS OF MICE (PVM) IN RELATION TO ITS STATE

Additional evidence is presented that PVM is capable of combining firmly with lung tissue particles or erythrocytes from certain susceptible species. Release of the virus from such combination can be effected by treatment with alkali as well as by heating. Free virus expressed from infected lungs without grinding the tissues is infectious and causes hemagglutination when tested directly. The concentration of virus can be estimated more accurately by means of the hemagglutination technique than from the results of infectivity tests in mice. Present evidence indicates that PVM may occur in 4 different states and that in each state it manifests distinctive properties.     

STUDIES ON TRANSMISSIBLE LYMPHOID LEUCEMIA OF MICE

Lymphoid leucemia of the mouse is readily transmitted by intravenous inoculations. The majority of the mice inoculated successfully develop leucemic, a smaller number of them, aleucemic lymphadenosis. The data presented favor the view that leucemic and aleucemic lymphadenosis are essentially the same condition. Leucemia produced by transmission is preceded by an aleucemic stage, in which the lymph nodes and the spleen are uniformly enlarged, and the white blood count and the percentage of lymphocytes are within the normal range but immature lymphocytes are numerous in the circulating blood. Young as well as old mice may develop leucemia if leucotic material enters their circulation. Studies of transmissible leucemia favor the view that leucemia of mammals is a neoplastic disease. The basic problem of leucemia would seem to be determination of the factors that bring about a malignant transformation of lymphoid cells.     

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.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) : III. HEMAGGLUTINATION BY THE VIRUS; THE OCCURRENCE OF COMBINATION BETWEEN THE VIRUS AND A TISSUE SUBSTANCE

1. The cause for the phenomenon of hemagglutination with heated PVM suspensions has been sought. 2. Evidence in wide variety indicates that the component responsible for hemagglutination is the virus particle itself. 3. The virus is capable of combining with a substance present in lung tissue of certain mammalian host species susceptible to infection by PVM. The occurrence of such combination accounts for a number of unusual properties manifested by this pneumotropic virus.