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 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.     

NEUTRALIZATION OF INFLUENZA A VIRUS BY HUMAN SERUM

A linear relationship exists between the quantity of human serum used and the quantity of influenza A virus neutralized. By means of this relationship it is possible to determine the maximum quantity of virus which a given human serum can neutralize. This quantity, the neutralizing capacity, is a fixed value and, unlike the serum dilution end point, is independent of the amount of virus used in the neutralization test.     

STUDIES ON THE ETIOLOGY OF RABBIT POX : IV. TESTS ON THE RELATION OF RABBIT POX VIRUS TO OTHER VIRUSES BY SERUM NEUTRALIZATION EXPERIMENTS

The results of serum neutralizing tests with the viruses of pox, vaccinia, and virus III disease herewith reported generally agree and supplement the results of reinoculation experiments on immune rabbits reported in the previous paper (1). The finding that pox virus is neutralized by pox immune serum indicates that the refractory state of recovered pox rabbits to reinoculation with pox virus and the failure of recovered pox rabbits to contract a second pox infection after adequate exposure is to be explained upon the basis of the development of an active immunity. The failure of virus III immune serum to neutralize pox virus is in agreement with the previous conclusion drawn from the positive results of reinoculation and exposure experiments that there is no specific relationship between pox virus and virus III. Rabbits which had recovered from a pox infection were completely refractory to inoculation with dermo-(culture) vaccine, while rabbits which had recovered from vaccinia were partially refractory to inoculation with pox virus. Vaccine immune adult rabbits did not show any clinical evidence of pox upon exposure to florid cases, but young recently weaned vaccine immunes developed definite although comparatively mild pox infections. The serum neutralization tests showed that pox immune serum neutralized vaccine virus although the action was not complete as shown by the positive results obtained with high dilutions of serum; vaccine immune serum possessed some but comparatively slight neutralizing properties against pox virus. The results of the crossed inoculation and serum neutralizing experiments with pox and neurovaccine viruses resembled those obtained with pox and dermo-(culture) vaccine but the differences were less pronounced. The differences in virus neutralizing ability on the part of the three immune sera paralleled the differences in virus potency as indicated by the character of the local lesions at the site of injection and by the general character of the clinical manifestations of the infection. The potency or virulence of pox virus was much greater than that of neurovaccine and vastly greater than that of dermo-(culture) vaccine. The complete identity of pox and vaccine virus could not be established, but a definite relationship between them was demonstrated and this was shown to have an immunological basis. From a practical standpoint vaccination with vaccine virus as a prophylactic measure against rabbit pox was clearly indicated.     

STUDIES IN AGGLUTINATION : III. ON THE MECHANISM OF THE AGGLUTINATION OF BACTERIA BY SPECIFIC AGGLUTINATING SERUM.

From the foregoing evidence, specific bacterial agglutination may be conceived of as follows: When bacteria are mixed with their homologous agglutinative sera, specific union between organism and agglutinin occurs. This interaction consists of specific coating of the bacteria by globulin. By virtue of the fact, noted by Loeb and illustrated in Fig. 6, that protein film formation gives the coated particle the characteristics of denatured protein, the bacteria now take on the character of particles of denatured globulin. Particles of denatured protein flocculate whenever their charge is reduced by electrolyte to a critical level lying somewhere between 12 and 14 millivolts. This is true even when the salt is strong, as, unlike bacterial particles, their cohesive force is not readily depressed by salt. The sensitized bacteria, now being essentially particles of denatured protein, likewise agglutinate as soon as their charge is reduced by electrolyte to this potential level.     

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.     

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.     

STUDIES ON THE ETIOLOGY OF JAGZIEKTE : I. THE PRIMARY LESIONS.

The lungs of South African sheep, in a district in which jagziekte is appearing sporadically, differ from those of normal American sheep. Their structure is in general variable. They are more subject to the action of bacteria, demonstrable histologically, and to the effect of inhaled foreign material. The most significant difference, however, centers in the interalveolar tissue, which, in about 33 per cent of the animals, is definitely thickened beyond the range of variation in this direction observed in American sheep. The thickenings occur in localized areas several millimeters in diameter. They begin with engorgement of the alveolar capillaries and accumulation of macrophages and of lymphocytes. Many of the macrophages pass into the alveolar lumina and assume the appearance of typical epithelioid cells. These infiltrative and exudative changes are primary to the epithelial proliferations which always arise in tissues modified in this way and which it is proposed to describe in a second paper.     

OBSERVATIONS ON THE PHAGOCYTOSIS OF THE PNEUMOCOCCUS BY HUMAN WHOLE BLOOD : II. THE NEUTRALIZATION OF THE ANTI-PHAGOCYTIC ACTION OF THE SPECIFIC SOLUBLE SUBSTANCE BY ANTISERUM

1. In vitro phagocytic experiments with human blood, antipneumococcus serum, pneumococcus specific soluble substance, and living virulent pneumococci show that there is a definite phagocytic inhibition zone when strong antiserum is used. 2. If the antiserum is further diluted, there is a zone where phagocytosis is effective. If the serum is diluted still more, phagocytosis gradually falls off, as the very dilute antiserum fails to neutralize the specific carbohydrate, which has a specific antiphagocytic action. 3. The inhibition zone is apparently caused by the specific precipitate (formed by the antiserum and the specific carbohydrate) interfering, perhaps mechanically, with the ingestion of the pneumococci by the leucocytes. 4. The inhibition zone is better marked with Type III than with Type I pneumococcus. 5. As the concentration of antiserum in the zone of effective phagocytosis in vitro does not correspond with the concentration of antiserum generally used in vivo in the serum therapy of pneumonia, this question is discussed.     

STUDIES ON THE ETIOLOGY OF RABBIT POX : III. TESTS OF THE RELATION OF RABBIT POX VIRUS TO OTHER VIRUSES BY CROSSED INOCULATION AND EXPOSURE EXPERIMENTS

Experiments are reported in which it was shown that rabbits which had recovered from experimental or spontaneous rabbit pox were refractory to inoculation of pox virus injected by various routes, and in addition did not develop clinical manifestations of the disease under conditions of exposure to florid cases of pox. It was found that pox recovered rabbits were susceptible to inoculation with the virus of virus III disease of rabbits and that virus III recovered rabbits could be successfully inoculated with pox virus. Furthermore, virus III recovered rabbits developed pox when subjected to room exposure in the same manner as did normal rabbits. It thus appears that there is no specific relationship between the two viruses. Rabbits which had recovered from experimental or spontaneous pox were found to be just as susceptible to inoculation with the virus of infectious myxoma of rabbits as were normal rabbits, a result which demonstrates that there is no specific relationship between these viruses. Rabbits which had recovered from experimental or spontaneous pox were refractory to inoculation with culture dermovaccine virus, but vaccine recovered rabbits were not completely refractory to inoculation with pox virus. Under conditions of exposure to clinical cases of pox, adult vaccine immune rabbits did not develop clinical manifestations of pox, but young, recently weaned vaccinated rabbits did contract mild but definite clinical pox. Experimental pox recovered rabbits were partially refractory to inoculation with neurovaccine virus and neurovaccine recovered rabbits were partially refractory to inoculation with pox virus. The refractory condition of the pox immune rabbits appeared to be more pronounced than that of the neurovaccine immunes. The cutaneous lesions which developed from the intradermal injection of pox, neurovaccine, and culture vaccine viruses showed definite differences with respect to the rate and persistence of active growth, amount of edema, hemorrhage, and necrosis, and the degree of tissue destructiveness. These features were most pronounced in the lesions of pox virus and were least marked in the lesions of culture vaccine virus. The differences were particularly apparent in normal rabbits, but they were also present in the lesions which developed in immune animals. It was found that the calf was susceptible to inoculation with pox virus applied to a scarified skin area. There were many similarities in the appearance and course of the pox lesions to those resulting from culture vaccine virus, the New York Board of Health vaccine, and neurovaccine virus similarly inoculated. But the pox lesions were most numerous, much the largest and most destructive, and by far the most persistent while next in order were those of the Board of Health dermovaccine. The results of these various experiments showed that a close relationship obtains between pox virus, on the one hand, and vaccine virus and neurovaccine virus, on the other, but it cannot be said that pox virus is identical in all respects with either one of these viruses. The findings indicated that the relationship between pox and neurovaccine viruses is closer than that between pox and culture vaccine viruses. Upon the basis of the results observed in culture (dermo) vaccine immune rabbits inoculated with or exposed to pox, it appeared that vaccination with vaccine virus offered a method of protection against rabbit pox.     

STUDIES ON THE ETIOLOGY OF RABBIT POX : V. STUDIES ON SPECIES SUSCEPTIBILITY TO RABBIT POX VIRUS

The white mouse, the guinea pig, the calf, and probably the rat, were found to be susceptible to infection with the virus of rabbit pox. Serial transmission of the virus in mice by brain to brain passage was characterized by a fatal outcome usually on the 5th or 6th day after inoculation. Infection of the guinea pig was accomplished by intratesticular injection and the virus was continued to the 2nd passage in this species. Guinea pigs developed a well marked cutaneous reaction from the intradermal injection of both rabbit and guinea pig tissue virus. Active virus was demonstrated in the testicles of rats 8 days after intratesticular injection by rabbit subinoculation. In the calf inoculation of the scarified skin was followed by the development of large papular lesions with marked hemorrhage and necrosis. The results of the investigations on the etiology of rabbit pox and of the experimentally induced infection reported in this and the four preceding papers (1–4) are discussed with special reference to the relation of pox virus to other viruses and of rabbit pox to other pock diseases.     

THE RECOVERY OF VACCINE VIRUS AFTER NEUTRALIZATION WITH IMMUNE SERUM

1. When vaccine virus and its specific antiserum are brought together, no evidence of stable union between them can be determined by the experimental methods employed. 2. A definite relationship exists between the degree of dilution of a neutral vaccine virus-antiserum mixture and the size of the lesion produced by endermic inoculation of rabbits of any given dilution.     

NEUTRALIZATION OF THE VIRUS OF POLIOMYELITIS BY NASAL WASHINGS

1. The results of 56 experiments have shown that washings of the nasal and pharyngeal mucosas possess definite power to inactivate or neutralize the active virus of poliomyelitis. 2. This power is not absolutely fixed, but is subject to fluctuation in a given person. Apparently inflammatory conditions of the upper air passages tend to remove or diminish the power of neutralization. But irregularities have been noted, even in the absence of these conditions. 3. Too few tests have been made thus far to ascertain whether adults and children differ with respect to the existence of this neutralizing property in the nasal secretions. While the inactivating property was absent from the secretions of one child during the first days of poliomyelitis, it was present in another to whom immune serum was administered, and in still another on the 15th day of illness when convalescence was established. 4. The neutralizing substance is water-soluble and appears not to be inorganic; it appears to be more or less thermolabile, and its action does not depend upon the presence of mucin as such. 5. It is suggested that the production of healthy carriers through contamination with the virus of poliomyelitis may be determined by the presence or absence of this inactivating or neutralizing property in the secretions. Whether this effect operates to prevent actual invasion of the virus and production of infection can only be conjected. Probably accessory and not the essential element on which defense against infection rests. It is more probable that other factors exist which help to determine the issue of the delicate adjustment between contamination and infection.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : III. FILTRATION EXPERIMENTS.

We have presented experiments to show that the typhus virus in the tissues of the guinea pig during the height of reaction to the experimental disease does not lose its infecting power when the cells of the brain or of the spleen are disintegrated by repeated freezing and thawing, or by freezing and desiccating, or by crushing by mechanical means, or by grinding into a homogeneous pulp with sand. The virus after such treatment is as actively infective as in the same tissue not subjected to the disintegrating influences. The possibility exists, therefore, of an extracellular condition of the typhus virus. Fourteen attempts to filter through Berkefeld V and N candles the virus contained in the disintegrated tissue have all resulted in failure.