STUDIES ON THE ETIOLOGY OF PRIMARY ATYPICAL PNEUMONIA : A FILTERABLE AGENT TRANSMISSIBLE TO COTTON RATS,HAMSTERS, AND CHICK EMBRYOS

1. A filterable virus from certain cases of primary atypical pneumonia was transmitted to chick embryos by inoculation into the amnion of suspensions of bacteriologically sterile lung tissue or filtered sputum, and three strains were adapted by passage. 2. After intranasal inoculation into cotton rats or hamsters, suspensions of the infected chick embryo tissues produced pulmonary lesions which were similar to those seen after instillation of infective human material. 3. The agent propagated in chick embryos was specifically neutralizable by serum from patients recovered from primary atypical pneumonia and was not neutralized by the acute-phase specimens. 4. Passages of the virus in cotton rats and hamsters gave confusing results because of contamination with latent respiratory agents already present in the animals.     

THE YIELD OF RABIES VIRUS IN THE CHICK EMBRYO

Rabies virus was inoculated intracerebrally in 8 day old chick embryos and the virus activity of pools of embryos titered after incubation at 35–36°C. for different lengths of time. The virus reached a titer of 10^–5.5 to 10^–6.5 in 5 to to 6 days and remained at a rather constant level until the 9th day of the infection. The report by Kligler and Bernkopf that rabies virus will invade the very young embryo after inoculation on the chorioallantoic membrane was confirmed.     

THE PREPARATION OF HIGHLY PURIFIED PR8 INFLUENZA VIRUS FROM INFECTED MOUSE LUNGS

Highly purified preparations of PR8 influenza virus were obtained from perfused, infected mouse lungs by a combination of methods involving adsorption of the virus on and elution from chicken red cells and differential centrifugation. Such preparations were found to possess 50 per cent infectivity end-points at 10^–11 to 10^–11.8, and 10^–13 to 10^–14.3 gm. of nitrogen in mice and in chick embryos, respectively. A sedimentation constant of 683 S was obtained for the material and electron micrographs revealed essentially spherical particles about 100 mµ in diameter. The material was isoelectric at pH 5.4 and chemical analyses on several of the preparations indicated the presence of 10.1 per cent of nitrogen, 1.06 per cent of phosphorus, 6.2 per cent of carbohydrate and about 33 per cent of lipid. Positive tests were obtained for both ribonucleic and desoxyribonucleic acids. The virus was precipitated strongly by antisenim to purified PR8 virus obtained from the allantoic fluid of infected chick embryos and this serum inhibited the agglutination of red cells by the mouse virus to a dilution of about 40,000. In general, the properties of the virus isolated from infected mouse lungs were found to coincide with those of the virus obtained from the allantoic fluid of infected chick embryos.     

A STUDY OF CONDITIONS FOR THE OPTIMUM PRODUCTION OF PR8 INFLUENZA VIRUS IN CHICK EMBRYOS

In order to determine the conditions for the optimum production of PR8 influenza virus in chick embryos, a study has been made of the róles of concentration of virus in the inoculum, temperature of incubation of infected embryos, length of time of incubation of infected embryos, and age of embryos at the time of inoculation. Relative amounts of virus in different preparations were measured indirectly by means of determinations of chicken red blood cell agglutination titers. Frozen infectious allantoic fluid which produced infection in chick embryos at a maximum dilution of 10^–7 was employed as a stock inoculum. Best results were obtained with an amount of stock inoculum of 0.1 cc. of a 10^–5 dilution, a temperature of incubation of 35°C., a length of time of incubation of 36 to 48 hours, and with embryos brought to 10 or 11 days of age at 37°C. or 9 or 10 days of age at 39°. An uncontrolled factor arising from inherent variations in the properties of different embryos and different batches of embryos was discussed.     

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.     

PROPAGATION OF RABIES VIRUS IN TISSUE CULTURE

Rabies virus has been propagated in serum-Tyrode solution containing either embryo mouse brain or embryo chick brain. The culture virus reached a titre of 3 x 10^–5 cc. after 4 days'' incubation at 37°C., and survived at least 2 months at 5°C. in the liquid or dry state.     

STUDIES ON THE NATURE OF THE VIRUS OF INFLUENZA : II. THE SIZE OF THE INFECTIOUS UNIT IN INFLUENZA A

The pathogenic agent of influenza A has been sedimented from infected extra-embryonic fluids of the developing chick, embryo by ultracentrifugation. Material so obtained contains two fractions resolvable in the analytical centrifuge cell. The first, a homogeneous fraction, showed a sedimentation constant S₂₀ = 20 to 31 x 10^–13. The second showed a sedimentation constant S₂₀ = about 800 ± 100 x 10^–13, was much less homogeneous than the first, and was shown to consist principally of aggregated particles of the more disperse fraction. Both fractions contained the virus in essentially equal amounts per unit of protein weight as calculated from nitrogen determinations. Electron micrographs of the isolated virus protein indicated that the predominating unit is roughly spherical in shape and has a modal particle diameter of about 11 mµ, in good agreement with the sedimentation data in indicating a molecular weight of about 650,000. Approximately 300 of the particles having molecular weight of 650,000 were present in the minimal dose producing infection in mice after nasal instillation. The influenza A virus may now be regarded as one of the smallest pathogenic agents thus far isolated. Preliminary analyses indicate that it is also one of the least complex, being composed principally of nucleoprotein.     

COMPARISONS OF INFLUENZA VIRUS STRAINS FROM THREE EPIDEMICS

Some of the peculiarities of strains of influenza A and B virus from two epidemics have been described. The influenza B virus of 1945–46, when compared with influenza A virus, proved to be much more difficult to isolate from human sources by any known means. Its adaptation to the chick embryo (by any route) or to mice was much slower than that of A virus. It did not keep nearly as well on storage at –72°C. either in throat garglings or as passage material. Its adaptation to amniotic growth was usually much better than to allantoic growth even after repeated allantoic passages. It failed to show primary evidence of occurring in the O form, although many of the secondary O characteristics were present and persisted. Its titer in throat washings was not demonstrably high as compared with certain strains of A virus, which were demonstrated in garglings at dilutions of 10^–5 and 10^–6. The antigenic patterns of influenza A strains from two epidemics were compared. No antigenic differences of significant degree were found among the strains of either epidemic and the difference between the strains of the two epidemics was very slight. A similar study was made of the influenza B strains of the epidemic of 1945–46. This also showed complete lack of significant strain differences. The implications of these findings for influenza prophylaxis are discussed.     

THE INFLUENCE OF AGE OF HOST AND TEMPERATURE OF INCUBATION ON INFECTION OF THE CHICK EMBRYO WITH VESICULAR STOMATITIS VIRUS

Chick embryos after 7 days of incubation were found to be much more susceptible to infection with vesicular stomatitis virus than were 10 day embryos. They had a 100 per cent mortality and were very suitable for titrations of the virus. The rate of increase of virus in 7 and 10 day embryos was studied. Two different temperatures of incubation were employed, 35–36°C. and 39–40°C., and the growth curves for the virus under the different conditions are presented. 10 day embryos were highly resistant and at 39–40°C. more than half of them survived. At the lower temperature of incubation, 35–36°C., all 10 day embryos died, but they survived much longer than did 7 day embryos. In the 7 day embryos death occurred after about 12 hours at 39–40°C. and after about 16 hours at 35–36°C., or earlier at the higher temperature. In embryos of both ages the virus titer reached at the higher temperature was only about 1 per cent of that reached at 35–36°C., even in those that died.     

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.     

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.     

THE COURSE OF EXPERIMENTAL INFECTION OF THE CHICK EMBRYO WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS

The titration curve for the virus of Eastern equine encephalomyelitis inoculated into the 10 day old chick embryo shows that the maximum increase in virus content continues until shortly before the generalized destruction of the embryo is apparent. This is followed by a stationary phase. Histological studies of infected embryos fail to demonstrate selective tissue destruction, and titrations show the virus to be distributed throughout the egg, although concentrated in the embryo. The chorioallantoic membrane gradually becomes increasingly resistant with age to both the Eastern and Western viruses. Increased resistance with age is also apparent in the hatched chick. These findings are based on the use of the chick embryo itself as the test animal to determine the 50 per cent mortality end-point. The limits of accuracy of this method are defined.     

STUDIES ON THE TOXICITY OF INFLUENZA VIRUSES : II. THE EFFECT OF INTRA-ABDOMINAL AND INTRAVENOUS INJECTION OF INFLUENZA VIRUSES

Upon intra-abdominal or intravenous injection of allantoic fluids infected with influenza viruses, mice frequently died within 8 to 96 hours. Similar results were observed upon injection of rabbits, rats, and guinea pigs. Autopsy of the mice revealed widespread necrosis of liver and spleen, hemorrhages into the intestines, pleural exudation, and other occasional findings. Survivors frequently developed pulmonary consolidation or jaundice. The dominant type of lesion depended on the strain of virus used. All attempts to demonstrate propagation of the influenza viruses outside of the respiratory tract failed. It was concluded that the early lesions were the result of toxic activities of the virus and not of virus multiplication in the affected tissues. Injection into chick embryos of highly diluted inocula produced higher titers of virus, hemagglutmin, and toxicity in the allantoic fluids than the use of more concentrated seed culture. Serial passage of various strains in high dilution frequently increased the toxic activity. The infectivity often reached its peak in 24 hours when tests for toxicity were still negative. Maximal toxicity was usually not attained before 48 hours. The toxic activity could not be separated from the infective property by such means as differential centrifugation and adsorption onto and elution from chicken red cells. However, upon heating, formalinization, and irradiation with ultraviolet light, the ability of the agents to propagate was lost at a faster rate than the toxic property. The toxic property remained stable for 2 to 3 months at 4°C. This stability was comparable to that of the infectivity for chick embryos. Specific immune sera neutralized in high dilution the toxic activity of the homologous virus. Non-specific neutralization occurred in low dilutions of normal and heterologous immune sera. Strain differences were indicated by this method of testing. Vaccination of mice by the subcutaneous or intra-abdominal routes protected mice specifically against the toxic effects of intra-abdominally or intravenously injected preparations of virus.     

AN EVALUATION OF METHODS FOR THE CONCENTRATION AND PURIFICATION OF INFLUENZA VIRUS

The concentration and purification of influenza virus by means of differential centrifugation in a vacuum type centrifuge, by adsorption on and elution from adult chicken red cells, by elution of the precipitate formed on freezing and thawing of allantoic fluid, by adsorption on and elution from embryonic chick red cells, and by combinations of the first method with each of the three succeeding methods, have been studied. Over-all yields of virus of about 50 to 70 per cent were obtained by these methods and combinations of methods except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed. However, the purified products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 per cent of non-virus protein. The purified products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component having a sedimentation constant of about 600 S. Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10^–14 gm. of the materials gave 50 per cent infectivity end points in chick embryos. The Sharples centrifuge was found to be almost as efficient as the vacuum type centrifuge for the concentration and purification of influenza virus and, because of its larger capacity, is recommended for the preparation of purified virus on a large scale.     

STUDIES ON THE BACTERIOPHAGE OF D'HERELLE : IV. CONCERNING THE ONENESS OF THE BACTERIOPHAGE.

Lytic filtrates, active against Bacillus dysenteriœ Shiga, Bacillus coli, Bacillus pestis caviœ, and staphylococcus respectively, proved to be differently affected by changes in hydrogen ion concentration. Anti-staphylococcus lysin was the least resistant of the four, showing deterioration in 3 hours at 7°C. beyond the zone of hydrogen ion concentration limited by C_H = 6.3 x 10^–5 and C_H = 1.6 x 10^–9. Under the same conditions, the zone of resistance of anti-coli filtrate lay between C_H = 2.7 x 10^–3 and C_H = 2.5 x 10^–11, and that of anti-Shiga between C_H = 1-7 x 10^–4 and C_H = 1-3 x 10^–11. Anti-pestis caviœ filtrate was most resistant of the four, retaining its full activity in the zone from C_H = 1 x 10^–3 to C_H = 3.5 x 10^–12. The fact that these differences in individual resistance persisted, notwithstanding the repeated passage of lytic filtrates through cultures of bacteria other than those against which they were primarily active, seems to offer evidence in favor of a multiplicity of bacteriophages.     

THE PREPARATION AND PHYSICOCHEMICAL CHARACTERIZATION OF THE SERUM PROTEIN COMPONENTS OF COMPLEMENT

1. Methods for the separation from guinea pig serum in highly purified form of three of the components of complement are described. These substances are the so called mid-piece, end-piece, and 4th component. 2. Mid-piece has been separated as a euglobulin, with an electrophoretic mobility of 2.9 x 10^–5 in phosphate buffer of ionic strength 0.2 at pH 7.7, and with a sedimentation constant of 6.4 x 10^–13 in potassium chloride of ionic strength 0.2. 3. End-piece and 4th component were found together in a euglobulin fraction of serum which contained 10.3 per cent carbohydrate and had an electrophoretic mobility of 4.2 x 10^–5 in phosphate buffer of ionic strength 0.2 at pH 7.7.     

STUDIES OF THE HEMOLYSIS OF RED BLOOD CELLS BY MUMPS VIRUS : I. THE DEVELOPMENT OF MUMPS VIRUS HEMOLYSIN AND ITS INACTIVATION BY CERTAIN PHYSICAL AND CHEMICAL AGENTS

The conditions for the production of extra-embryonic fluids with hemolytic activity from chick embryos infected with mumps virus have been investigated. Infected fluids with strong hemolytic activity can be obtained by harvesting the fluids of 6- to 8-day-old chick embryos inoculated by the amniotic route after 5 to 6 days' incubation at 35°C. Under such circumstances, the hemolytic capacity of amniotic fluids is usually much higher than that of the allantoic fluids. The hemolytic activity and infectivity of the virus have been found to be reduced or destroyed by heat, formaldehyde, and ultraviolet irradiation under conditions which leave the hemagglutinating capacity practically unchanged. Ultraviolet irradiation appeared to have a greater deleterious effect on the infectivity of the virus than on its hemolytic capacity. The marked reduction or destruction of hemolytic activity of the virus produced by certain treatments with these various agencies was not accompanied by loss of the ability of the virus to elute following its adsorption on red blood cells during the process of hemagglutination. This test for hemolytic activity, which measures a more labile property of the virus than do determinations of virus hemagglutination or virus elution, may be useful in detecting changes which occur early during degradation of the virus.     

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.     

STUDIES ON THE PATHOGENESIS OF FEVER : X. THE EFFECT OF CERTAIN ENZYME INHIBITORS ON THE PRODUCTION AND ACTIVITY OF LEUCOCYTIC PYROGEN

The production of endogenous pyrogen by intact granulocytes obtained from acute peritoneal exudates is blocked by arsenite, iodoacetate, p-chloromercuribenzoate, and N-ethylmaleimide in concentrations of 2 x 10^–4 M. When the concentration of these sulfhydryl-reactive enzyme inhibitors is increased to 2 x 10^–2 M, only the iodoacetate inactivates the pyrogen molecule, whereas the arsenite, the p-chloromercuribenzoate, and the N-ethylmaleimide have no gross effect upon its thermogenic activity. Both diisopropyl fluorophosphate and dinitrofluorobenzene are even more potent inactivators of the pyrogen molecule than iodoacetate, although the action of the DFP cannot be blocked or reversed by known antagonists such as 2-pyridine aldoxime methiodide and hydroxylamine. Proteolytic enzymes, potentially capable of degrading leucocytic pyrogen, are released from polymorphonuclear leucocytes, along with the pyrogen, when the cells are incubated in normal salt solution. These enzymes are readily activated by a sufficient concentration of glutathione (2 x 10^–2 M). They are not present in preparations of partially purified leucocytic pyrogen from which much of the non-pyrogenic protein has been removed. Glutathione by itself, even at concentrations as high as 2 x 10^–1 M, does not affect in the gross the thermogenic activity of the purified pyrogen. The implications of these findings in relation to both the production and the chemical characteristics of leucocytic pyrogen are discussed.     

FURTHER OBSERVATIONS ON THE CULTIVATION OF VACCINE VIRUS IN LIFELESS MEDIA

We have made ten attempts to cultivate vaccine virus in tissue extracts prepared according to the method described by Eagles and Kordi (4). Renal, testicular, and chick embryo extracts were employed with a dermal strain of vaccine virus and with the Levaditi strain of neuro-vaccine virus. In no instance were we able to show that the virus multiplied in the extract media. Both of these strains of virus, however, multiplied in media containing bits of minced viable tissue. Furthermore, treatment of rabbit testicular tissue and chick embryo tissue in the manner described by Eagles and Kordi for the preparation of the extracts leaves some cells not only alive but capable of proliferation. Although the results of our work are not in accord with those obtained by Eagles and Kordi, we offer no explanation for the discrepancy. Nevertheless, one cannot examine the results of our work recorded in the six tables without recognizing the fact that in the types of media used the presence of viable cells appears to be essential for the multiplication of vaccine virus. Rabbit testicular tissue and bits of chick embryos support the regeneration of the active agent more efficiently than does rabbit renal tissue.