STUDIES ON THE ETIOLOGY OF PRIMARY ATYPICAL PNEUMONIA : II. PROPERTIES OF THE VIRUS ISOLATED AND PROPAGATED IN CHICK EMBRYOS

Experiments to determine the optimum conditions for propagation of the virus of atypical pneumonia in chick embryos are described. Variations in the activity of infected chick embryo material were investigated. The highest dilution of chick embryo suspension producing pulmonary lesions in hamsters and cotton rats is not over 10^–3. Dilutions of 10^–4 infect chick embryos. The virus is unstable at room temperature and also loses activity when stored in a dry-ice refrigerator unless the suspensions are kept in sealed glass tubes. Filtration experiments indicate a maximum particle size of 180 to 250 mµ. The virus propagated in chick embryos produces pulmonary lesions in hamsters and cotton rats which have been immunized to their own non-bacterial agents inducing pulmonary lesions. Of these, the pneumonia virus of hamsters most frequently causes intercurrent respiratory infections, and methods of controlling epizootics due to this agent are described.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) : II. IMMUNOLOGICAL EVIDENCE OF LATENT INFECTION WITH THE VIRUS IN NUMEROUS MAMMALIAN SPECIES

The results of neutralization tests with PVM and serum obtained from numerous animal species indicate that antibodies agaiust this virus were present in the blood of all mammalian species tested, as not in that of fowls, and that their incidence in various species was widely different. They indicate, also, that in certain species, particularly the cotton rat, there were marked seasonal variations in the incidence of such antibodies; in the late winter and spring the incidence was much higher than during the summer and fall seasons. Cotton rats and hamsters which did not possess neutralizing antibodies against PVM were susceptible to manifest pulmonary infection with this virus, irrespective of the effects of previous experiments upon them, whereas those which possessed such antibodies were immune. It is suggested that circulating antibodies against PVM were present as a result of preceding infection with a latent virus; either PVM or an agent closely related to it in antigenic composition. Appropriate non-specific stimuli, e.g. the intranasal injection of suspensions of normal chick embryos, induced the development of neutralizing antibodies against PVM with significantly greater frequency in each of three species than occurred in control animals. Materials derived from patients with primary atypical pneumonia yielded results almost identical to those obtained with normal chick embryo suspensions. It is suggested that such materials, like the other non-specific stimuli employed, were effective in evoking a specific antibody response, because they unbalanced an equilibrium which previously existed between animal host and latent pneumotropic virus.     

STUDIES ON THE ETIOLOGY OF PRIMARY ATYPICAL PNEUMONIA : III. SPECIFIC NEUTRALIZATION OF THE VIRUS BY HUMAN SERUM

Significant increases in neutralizing antibodies were demonstrated in 42 of a total of 69 persons with a clinical diagnosis of primary atypical pneumonia. Detailed titrations of virus-neutralizing antibodies in a representative group of 28 patients are presented. Increases of four- to 64-fold were demonstrated. Acute-phase titers were 4 or less in 83 per cent and convalescent titers were 16 or over in 86 per cent of these cases. Only about half of the number of patients having increases in neutralizing antibodies also developed cold agglutinins and agglutinins for the indifferent streptococcus No. 344. Patients from the Eastern United States as well as those from the Pacific Coast were shown to develop virus-neutralizing antibodies. Patients with pneumococcal pneumonia and pneumonias caused by influenza virus type A or viruses of the psittacosis group did not have significant increases in neutralizing antibodies for the virus of atypical pneumonia. Cold agglutinins appeared in 3 cases of type A influenzal pneumonia. Sera from persons with atypical pneumonia, when tested against the 3 most prevalent respiratory viruses isolated from cotton rats and hamsters, failed to neutralize these agents or showed no significant change in neutralization titer.     

Isolation of B virus (herpes group) from the central nervous system of a rhesus monkey

While passaging a recently isolated strain of poliomyelitis virus through a rhesus monkey, another virus was procured from its central nervous system. After intracerebral inoculation, the virus produced meningoencephalitis in monkeys, cotton rats, hamsters, guinea pigs, and rabbits; after intracutaneous inoculation a necrotic skin lesion was produced in the monkey and rabbit and this was often followed by myelitis. The virus could also be passed in newborn mice less than 48 hours old and in chick embryos by inoculation of the chorioallantoic membrane. Immunological and host range studies revealed this virus to be related to the B virus originally described by Sabin and Wright in 1934 (1). To our knowledge this is the first record of B virus having been isolated from a monkey, and lends support to the inclusion of this agent as the simian member of the herpes group. The infection is not uncommon in monkey stocks, as revealed by the finding of antibodies to the virus in their sera. In the present series 9 of 44 monkeys gave positive antibody tests. Gamma globulin prepared in the United States in 1945, 1951, and 1953, as well as a certain proportion of sera from normal individuals in Bombay, India, and elsewhere, showed neutralizing activity against the new strain of B virus, and also to herpes simplex virus. This may be the result of the partial crossing which exists between the two viruses.     

SYNERGISTIC ACTION OF HEMOPHILUS INFLUENZAE SUIS AND THE SWINE INFLUENZA VIRUS ON THE CHICK EMBRYO

The synergistic effect of Hemophilus influenzae suis and swine influenza virus in the pig can be reproduced by the inoculation of these agents on the chorioallantoic membrane of 9 to 10 day old chick embryos. Two strains of human influenza virus that were studied failed to substitute for the swine virus in the synergistic reaction. No loss of synergistic effect was noted when the swine influenza virus was put through 11 chick embryo passages. Recently isolated and old stock strains of Hemophilus were equally able to enhance the effect of the virus. Heat-killed cultures of H. influenzae suis can be substituted for the bacterial component of the reaction. Infection of the embryo with swine influenza virus predisposes to infection with H. influenzae suis. The combination of H. influenzae suis and swine influenza virus causes a selective destruction of the embryo lungs, not produced by the individual components. This pneumonia exhibits the essential features of the natural disease.     

Studies on primary atypical pneumonia. I. Localization,isolation, and cultivation of a virus in chick embryos

By means of fluorescein-labelled antibody, the primary atypical pneumonia virus was found to multiply exclusively in the cytoplasm of the epithelial cells lining the bronchioles and air sacs of developing chick embryos. When 13-day old embryos were inoculated intra-amniotically and incubated at 35°C. for 5 days or longer, over 90 per cent of the inoculated embryos became infected. Between 1954 and 1956, seven strains of PAP virus were isolated from sputums or nasopharyngeal washings in patients during the acute stage of the PAP infection. One strain of virus was isolated from the frozen lung of a patient who died at Boston in 1943. All eight recently isolated strains and the Mac strain isolated by Eaton et al. in California in 1944 were antigenically closely related if not identical. PAP virus is not related antigenically to agents of psittacosis, Q fever, adenovirus (Types 1 to 6), influenza A or B, or PVM.     

Studies of mouse polyoma virus infection. 1. Procedures for quantitation and detection of virus

Three procedures have been compared for usefulness in titration and detection of polyoma virus: production of cytopathic effect (CPE) in mouse embryo tissue culture, production of HI antibody after inoculation into weanling mice (MAP test), and production of tumors in suckling hamsters during a 3 to 5 week observation period. The tissue culture and mouse antibody production tests were generally comparable in sensitivity, reproducibility, and time required to obtain results. Titration by tumor production in suckling hamsters was not suitable for quantitation because of marked variation in susceptibility among animals. Virus was detected in tissues of normal mice from spontaneously infected colonies by either production of CPE in mouse embryo tissue culture or by the MAP test; virus was found in organs of 15 (58 per cent) of 26 mice with antibody, and 2 (8 per cent) of 24 mice without antibody.     

EXPERIMENTAL MENINGOCOCCUS INFECTION OF THE CHICK EMBRYO

1. A strain of meningococci obtained directly from the spinal fluid of a patient has been propagated in serial passage in 10 to 12 day old chick embryos without change in its essential characteristics. 2. The chick embryo is susceptible to infection with the meningococcus, and, depending on its stage of development, reacts to the infection with more or less specific lesions. 3. In chick embryos of 15 days incubation, following the utilization of definite portals of entry, such as the nasopharynx, or by inoculation of the amniotic fluid or by inoculation of the body wall, the meningococcus is localized in specific areas, namely in the cranial sinuses, the lungs or meninges, or in all of these areas. 4. The lesions of the meningococcus infection in man, a septicemia, sinusitis, pneumonia and meningitis can be reproduced in the chick embryo by choosing embryos at the proper state of development and utilizing the various portals of entry experimentally available.     

THE USE OF YELLOW FEVER VIRUS MODIFIED BY IN VITRO CULTIVATION FOR HUMAN IMMUNIZATION

The response of rhesus monkeys to a subcutaneous inoculation with varying amounts of virus modified by prolonged cultivation in vitro has been studied. The tissue components of the medium consisted of chick embryo tissue containing minimal amounts of nervous tissue. The immunity produced in monkeys, as measured by the antibody titer developed, has no relation to the amount of virus inoculated. Monkeys inoculated subcutaneously with the tissue culture virus are rendered immune to a subsequent injection of a highly virulent yellow fever virus. This resistance is already present 7 days after vaccination. The subcutaneous inoculation of the culture virus into immune persons leads to a substantial increase of the serum antibody titer. The results of vaccinating eight normal persons with culture virus are presented. The reactions were minimal. The highest temperature recorded following vaccination was 37.4°C. The sera taken from the eight vaccinated persons 2 to 4 weeks after inoculation with the tissue culture virus showed the presence of yellow fever antibodies.     

SARCOMAS IN HAMSTERS AFTER INJECTION WITH ROUS CHICKEN TUMOR MATERIAL

Newborn hamsters were injected subcutaneously with a suspension of finely minced Rous chicken sarcoma (Schmidt-Ruppin strain). After an interval of about 2 weeks, progressively growing sarcomas developed at the site of injection in almost all animals. Also in adult hamsters inoculated intramuscularly with the same material sarcomas developed at the site of injection within 2 to 4 months. Secondary growths appeared on the peritoneal surface, in the retroperitoneal and mediastinal lymph nodes and in the lungs. The sarcomas usually had a pleomorphic appearance and showed a certain resemblance to rhabdomyosarcoma, but sometimes they had the character of spindle cell sarcomas of varying degree of maturity. Sarcomas were not only obtained in hamsters injected with cellular material from the Rous chicken sarcoma but were also seen in hamsters which were injected at birth or when 2 months' old with supernatant fluid obtained by repeated centrifugation of suspensions of homogenized chicken sarcoma, and presumed to be cell-free. The hamster sarcoma was transplanted to a newborn hamster and could then without difficulties be passed in series in hamsters. All attempts to transfer the sarcoma from hamster to hamster by means of cell-free material from the hamster sarcoma failed. On the other hand, material from the hamster sarcomas inoculated into chickens induced rapidly growing Rous sarcomas at the site of inoculation. This proved possible not only with material from the first but also from later passages of the tumor in hamsters. It is concluded that the strain of Rous virus used has the capacity to induce sarcomas not only in chickens but also in hamsters.     

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 INFLUENCE OF CORTISONE ON EXPERIMENTAL VIRAL INFECTION : VI. INHIBITION BY HYDROCORTISONE OF INTERFERON SYNTHESIS IN THE CHICK EMBRYO

The initial observations that cortisone may act as an inhibitor of viral interference (11, 4) are now explicable as an inhibitory effect on interferon synthesis. The suggestion that the action of interferon is also inhibited by cortisone or its analogues (6) has not been confirmed in a plaque reduction type of interferon assay system in which autointerference by the challenge inoculum is a lesser problem. In this respect, the present results are in accord with those obtained by DeMaeyer and DeMaeyer (8) with hydrocortisone in a system in which a low multiplicity (0.1) Sindbis virus infection in monolayer culture was employed with cytopathic effect (CPE) as an end-point. It has been shown that hydrocortisone is restrictive to the synthesis of interferon induced by inoculation of either infective or inactivated virus into the chick embryo, and that this inhibitory effect is temporary. However, in another study in the chick embryo, three spaced injections of hydrocortisone (0.25 mg/dose) prevented the appearance of detectable interferon during the entire 64 hr observation period following inoculation of 10^3.3 EID₅₀ of Lee virus (12). The importance of explicit definition of experimental conditions in assessing hormonal effects on infection is illustrated by the capacity of hydrocortisone either to inhibit or increase interferon synthesis in vitro, depending on the proportion of inactivated and infective virus in the inoculum employed, and the time at which interferon is measured. As suggested previously, it is not unlikely that similar shifts in hormone-virus-interferon balance may operate in vivo to influence the outcome of infection.     

THE EFFECT OF PROLONGED CULTIVATION IN VITRO UPON THE PATHOGENICITY OF YELLOW FEVER VIRUS

1. Experimental evidence is presented to show that prolonged cultivation of yellow fever virus in vitro results in a change in its pathogenicity, and that this change varies with the type of tissues used for the cultivation. 2. In the tissue cultures used for the propagation of the virus, three different types of tissues were used. They included whole mouse embryo, chick embryo from which the head and spinal cord had been removed, and testicular tissues of mice and guinea pigs. 3. The changes in the pathogenicity of the virus cultivated for a period of over 3 years in a medium containing the tissues of whole mouse embryo were not striking. The viscerotropic virulence of the virus appeared somewhat diminished, in that when injected subcutaneously into rhesus monkeys or hedgehogs it failed to produce a fatal infection, although there is evidence to indicate that a generalized infection takes place as demonstrated by the appearance of virus in the circulating blood in relatively high concentration during infection. The neurotropic virulence of the virus remained unaltered during the cultivation in this medium. 4. The changes in the pathogenicity of the virus cultivated in medium containing tissues of chick embryo from which the head and spinal cord had been removed were very pronounced. The viscerotropic virulence of the virus was lost to a large extent. When injected subcutaneously into monkeys there was as a rule a very mild generalized infection, as demonstrated by the minimal quantities of virus found in the circulating blood. Its neurotropism was also much diminished. When injected into monkeys intracerebrally, it no longer produced a fatal encephalitis but only a moderate febrile reaction, followed by recovery and solid immunity to reinoculation with a highly virulent strain of virus. When injected intracerebrally into mice, the mortality ratio was not diminished but the incubation period was markedly prolonged. 5. The changes in the pathogenicity of the virus cultivated in medium containing testicular tissues were somewhat similar to those observed after cultivation in chick embryo medium which contained only a minimal amount of nervous tissue. Its viscerotropic affinity had been largely lost and only very small amounts of virus were found in the circulating blood of monkeys inoculated subcutaneously. Given intracerebrally, it produced death from encephalitis in monkeys. The incubation period in mice inoculated intracerebrally with this virus was also prolonged but somewhat less so than with the virus grown in chick embryo tissues without the central nervous system.     

STUDIES ON PRIMARY ATYPICAL PNEUMONIA : III. A FACTOR IN NORMAL SERUM WHICH ENHANCES THE REACTION BETWEEN PAP VIRUS AND CONVALESCENT SERUM

A factor present in fresh normal human or guinea pig serum enhances the primary atypical pneumonia (PAP) antibody titers in serological reaction with its homologous virus as tested by indirect fluorescent antibody staining. This factor is not properdin. It has many properties similar to complement but may not be identical with complement. The addition of this enhancement factor to human sera convalescent from primary atypical pneumonia has made the serological test more sensitive without affecting its specificity.     

EXPERIMENTAL INFECTION OF THE CHICK EMBRYO WITH THE VIRUS OF PSEUDORABIES

The chick embryo responds to experimental infection with the virus of pseudorabies with a disease pattern simulating the natural infection. Virus lesions of the membrane are followed by infection of all tissues of the central nervous system. Fixed strains produce a hemorrhagic destruction of the central nervous system of the embryo, which is referable to destruction of blood vessel endothelium. Field strains lack the hemorrhagic tendency, but infect the brain when inoculated on the membrane. Neutralization of the virus by specific hyperimmune serum can be demonstrated by inoculation on the membrane. The reaction of the embryo to the virus varies with the age of the embryo. This is reflected both in the membranal lesion and in the subsequent encephalitis.     

EXPERIMENTAL TYPHUS INFECTION IN THE EASTERN COTTON RAT (SIGMODON HISPIDUS HISPIDUS)

1. The course of typhus infection in cotton rats has been described, and a relationship between the dose administered and the time of death has been demonstrated. 2. Rather incomplete pathological studies indicated that typhus in the cotton rat is an acute infection primarily involving mesothelial tissue, and tissues of mesothelial origin. Depending somewhat upon the route of infection, rickettsiae were most easily demonstrated in smears from liver, brain, and pericardial and peritoneal exudates. By subinoculation into the yolk sacs of fertile eggs, rickettsiae were readily isolated from these sources and, with rather less facility, from blood. 3. Although relatively large numbers of rickettsiae were found necessary to produce lethal infections, the susceptibility of the cotton rat as measured by the development of an immunizing infection proved to be very high. The supporting data suggest that cotton rats might be suitable for the isolation of new strains. 4. Contrary to the earlier report (1), age was not found to influence markedly the susceptibility of the cotton rat to lethal infection with any of the strains studied. 5. Comparative studies indicated that cotton rats were just as susceptible to murine infection as white mice and more susceptible to Breinl infection than the latter animal. The susceptibility of cotton rats and of guinea pigs to Breinl infection was found to be about the same. Comparative studies in these animals were not made with other strains. 6. No toxic activity of rickettsial suspensions could be demonstrated for cotton rats, although suspensions of infected cotton rat livers were shown to be toxic for mice. 7. A method has been described by which three strains of Rickettsia prowazeki were carried in serial intracardial passage as fatal infections for cotton rats. No evidence was obtained that prolongation of such passage increased the virulence of the strains for the rats. 8. Cotton rats surviving typhus infection were shown to be solidly immune to reinfection with homologous virus and to possess nearly complete immunity to rickettsiae of heterologous immunologic character. Sera from recovered rats, furthermore, were shown to possess neutralizing, complement-fixing, and antitoxic antibodies but no agglutinins for proteus OX19. 9. The technique of a serum neutralization test in cotton rats has been described in which the test antigen consists of suspensions of infected cotton rat livers. By this technique neutralizing antibody was demonstrated in human, rabbit, guinea pig, and cotton rat sera. Although some degree of cross-neutralization could be shown, serum titers against homologous antigen were uniformly greater than against comparable doses of antigen of heterologous immune nature.     

Use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections

No practical animal models for the testing of chemotherapeutic or biologic agents identified in cell culture assays as being active against measles virus (MV) are currently available. Cotton rats may serve this purpose. To evaluate this possibility, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and poly(acrylamidomethyl propanesulfonate) (PAMPS), two compounds that have been reported to inhibit MV in vitro, and ribavirin, an established antiviral drug with MV-inhibitory activity, were evaluated for their antiviral activities against MV and respiratory syncytial virus (RSV) in tissue culture and in hispid cotton rats. A single administration of PAMPS markedly inhibited pulmonary RSV or MV replication (>3 log(10) reduction in pulmonary titer compared to that for controls), but only if this compound was administered intranasally at about the time of virus inoculation. Both EICAR and ribavirin exhibited therapeutic activity against RSV and MV in cotton rats when they were administered parenterally. However, both of these compounds were less effective against MV. On the basis of the pulmonary virus titers on day 4 after virus inoculation, the minimal efficacious dose of EICAR against MV (120 mg/kg of body weight/day when delivered intraperitoneally twice daily) appeared to be three times lower against this virus than that of ribavirin delivered at a similar dose (i.e., 360 mg/kg/day). These findings correlated with those obtained in vitro. The data obtained suggest that cotton rats may indeed be useful for the initial evaluation of the activities of antiviral agents against MV.     

THE RECOVERY FROM PATIENTS WITH ACUTE PNEUMONITIS OF A VIRUS CAUSING PNEUMONIA IN THE MONGOOSE

1. A virus capable of producing pulmonary consolidation in the wild mongoose (Herpestes griseus) has been isolated from throat washings obtained from four patients with a clinical syndrome termed acute pneumonitis. 2. The virus was not pathogenic for ferrets, mice, guinea pigs, rabbits, monkeys, voles, hamsters, deer mice, skunks, opossums, or woodchucks. 3. The virus was filterable through Berkefeld V and N candles, was not inactivated by glycerin or by freezing and drying in vacuum, and was propagated for at least 30 serial passages on the chorio-allantoic membrane of the developing chick embryo. 4. Normal mongooses placed in contact with infected mongooses developed pulmonary consolidation. 5. The virus was neutralized by the serum of mongooses convalescent from the infection but was not neutralized by normal mongoose serum. 6. Serum of human beings convalescent from acute pneumonitis also neutralized the virus, but serum obtained from the same individuals during the acute phase of the disease failed to do so. 7. The evidence so far obtained strongly suggests that this virus is the cause of acute pneumonitis in human beings. It differs from other viruses known to cause infections of the respiratory tract in man.     

TRANSPLANTABILITY OF TISSUES TO THE EMBRYO OF FOREIGN SPECIES : ITS BEARING ON QUESTIONS OF TISSUE SPECIFICITY AND TUMOR IMMUNITY.

Inoculation of the Jensen rat sarcoma into the developing chick embryo gives a rapidly growing tumor at the site of inoculation, whether in the membranes or in the body of the chick itself. These tumors by transfer from embryo to embryo can be kept going for as long as forty-six days, and perhaps indefinitely in the foreign species. The rat cells show no morphological change even after a very long dependence. Their biological characters are also retained, as is shown by the fact that the cells when replanted in the rat, after a prolonged sojourn in the chick, will produce a rapidly growing sarcoma of the Jensen type. These rat tissues grown for long periods in the chick show no adaptation to the new species, being destroyed even more rapidly when placed in the adult chicken than cells taken directly from the rat. Morphologically the cells retain a close resemblance to those in the original tumor. Other tissues grown in chick embryo are various embryonic cells from the chicken, mouse, and rat, the Ehrlich sarcoma and chondroma of the mouse, a mammary carcinoma of the mouse, the Flexner-Jobling adenocarcinoma of the rat, and a human sarcoma.