QUALITATIVE DIFFERENCES IN THE ANTIGENIC COMPOSITION OF INFLUENZA A VIRUS STRAINS

A study of the PR8, Christie, Talmey, W.S., and swine strains of influenza A virus by means of antibody absorption tests revealed the following findings: 1. Serum antibody could be specifically absorbed with allantoic fluid containing influenza virus or, more effectively, with concentrated suspensions of virus obtained from allantoic fluid by high-speed centrifugation or by the red cell adsorption and elution technique. Normal allantoic fluid, or the centrifugalized sediment therefrom, failed to absorb antibodies. Influenza B virus (Lee) caused no detectable absorption of antibody from antisera directed against influenza A virus strains, but it specifically absorbed antibody from Lee antisera. 2. The neutralizing, agglutination-inhibiting, and complement-fixing anti-bodies in ferret antisera were completely absorbed only by the homologous virus strain, even though 2 absorptions were carried out with large amounts of heterologous virus strains. 3. PR8 virus appeared to have the broadest range of specific antigenic components for it completely absorbed the heterologous antibodies in Christie and W.S. antisera and left only those antibodies which reacted with the respective homologous strains. The other virus strains (Christie, Talmey, W.S., swine) were more specific in the absorption of heterologous antibodies and completely removed only those antibodies which reacted with the absorbing virus. 4. The absorption tests revealed a higher degree of specificity and individuality of the virus strains than the various cross reactions previously reported. The strain specificity of PR8 virus was equally manifest in absorption tests with ferret sera and with human sera following vaccination. 5. The amount of homologous antibody remaining in a PR8 ferret serum after absorption with PR8 virus, obtained by the red cell adsorption and elution method, varied inversely as the concentration of virus used for absorption. A given concentration of virus, however, absorbed a greater percentage of neutralizing antibodies than either agglutination-inhibiting or complement-fixing antibodies.     

A STUDY OF THE NEUROTROPIC TENDENCY IN STRAINS OF THE VIRUS OF EPIDEMIC INFLUENZA

The demonstration by Stuart-Harris that the W.S. strain of epidemic influenza virus can induce a fatal nervous disease in mice has been confirmed. In contrast, however, no previous period of adaptation to chick embryonic brain was required. By serial brain to brain passages in mice originally inoculated with the virus cultivated in the usual chick embryo culture medium a fatal disease, essentially meningeal in character, is produced. The Melbourne strain has been similarly enhanced while other strains have failed to reveal any neurotropic tendencies. The evidence indicates that the neurotropic characteristic is present in the two strains as an inherent quality which is quantitatively heightened and does not represent the acquisition of a property not previously present.     

QUANTITATIVE ASPECTS OF HOMOLOGOUS AND HETEROLOGOUS ACTIVE IMMUNITY TO STRAINS OF THE VIRUS OF EPIDEMIC INFLUENZA

When mice are immunized by one intraperitoneal inoculation with active or inactive influenza virus (strain PR8, W.S., and Melbourne) the quantity required for protection against heterologous strains is about 10 times the homologous minimal immunizing dose. Three injections increase the immunity to all strains, but the ratio between the homologous and heterologous minimal immunizing dose is not altered. Swine influenza virus given intraperitoneally fails to immunize against human strains unless the quantity injected is 1,000 times the minimal amount required for homologous immunity. Intranasal immunization of mice with 1/100 M.L.D. of attenuated ferret passage strains PR8 and Philadelphia, or the tissue culture strain of swine influenza, gives a solid resistance to infection with heterologous strains. When smaller amounts of virus are given intranasally, strain specificity becomes more apparent, and with minimal doses the immunity may be effective only against the homologous and closely related strains.     

THE SIZE OF INFLUENZA VIRUS

The sedimentation behavior of influenza virus in dilute solutions of electrolyte was found to be quite variable. At times the virus activity appeared to sediment at a rate comparable with that of particles about 80 to 120 mµ in diameter, at other times at a rate comparable with that of particles about 10 mµ in diameter, and at still other times the bulk of the activity appeared to sediment at a rate comparable with that of the larger particles and the residual activity at a rate comparable with that of the smaller particles. However, in the presence of a sucrose density gradient, the virus activity was always found to sediment with a rate comparable to that of particles about 80 to 120 mµ in diameter; hence it appeared that the variable sedimentation behavior in dilute electrolyte solution was due to convection or mechanical disturbances during centrifugation. About 30 per cent of the high molecular weight protein present in the allantoic fluid of chick embryos infected with the F 12 strain of influenza virus was found to consist of a component having a sedimentation constant of about 30 S, and hence a probable particle diameter of about 10 mµ. The residual protein of high molecular weight was present in the form of a component having a sedimentation constant of about 600 S, and hence a probable particle diameter of about 70 mµ. The proportion of the 30 S component in allantoic fluid of chick embryos infected with the PR8 strain of influenza virus was found to be considerably less. The 600 S and 30 S components of F 12 allantoic fluid were purified and separated by differential centrifugation. The purified preparations of the 600 S component were found to possess a specific virus activity from 100 to over 10,000 times that of the purified preparations of the 30 S component, the difference in activity apparently depending only on the degree of fractionation of the two components. The purified 30 S component was found to sediment normally in the presence of 12 per cent sucrose, whereas the small residual virus activity of such preparations was found to sediment in the presence of a sucrose density gradient with a rate comparable to that of much heavier particles. It is concluded that influenza virus activity is not associated with material having a particle diameter of about 10 mµ, but is associated solely with material having a sedimentation constant of about 600 S and hence a probable particle diameter of about 70 mµ.     

STUDIES OF TWO KINDS OF VIRUS PARTICLES WHICH COMPRISE INFLUENZA A2 VIRUS STRAINS : II. REACTIVITY WITH VIRUS INHIBITORS IN NORMAL SERA

Inhibitors present in normal human and animal sera prevented hemagglutination by and neutralized infectivity of inhibitor-sensitive influenza A2 virus. Starch zone electrophoresis of sera indicated that the same serum components possess both hemagglutination-inhibiting and neutralizing activities. The greatest amount of inhibitory activity was found in normal horse serum, and the inhibitory activity increased with heating or treatment with concentrated solutions of urea. The inhibitory activities on human, ferret, and rabbit sera were markedly reduced but not completely eliminated by V. cholerae filtrate and purified neuraminidase. The inhibitory activity of horse serum was only moderately reduced by these agents. The nature of the horse serum inhibitor and the differences in the interactions of inhibitor-sensitive and insensitive influenza A2 virus particles and pre-1957 influenza viruses with receptors have been discussed.     

STUDIES ON THE VIRUS OF INFLUENZA

1. Evidence is presented indicating the presence of a filtrable virus in the nasopharyngeal secretions of individuals suffering from influenza. 2. An attempt to transfer influenza from one human being to another by means of filtered nasopharyngeal washings resulted in the production in the inoculated volunteer of a common cold. 3. A filtrable agent has been cultivated in tissue medium from the filtered nasopharyngeal washings of patients with influenza. 4. Inoculation of the cultivated virus into human volunteers results for the most part in the production of a severe common cold with a tendency to pronounced constitutional reaction. 5. In one instance following inoculation of culture virus an infection clinically resembling influenza has been produced. 6. The more closely the source of the virus approached the type of epidemic influenza, the more likely the virus was to provoke constitutional symptoms. 7. The presence of certain pathogenic bacteria in the upper respiratory tract of inoculated individuals was not observed to modify the course or character of the experimental infection. 8. On prolonged cultivation the virus loses the capacity to infect human volunteers.     

EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : IV. RELATIONSHIP OF TRANSMISSIBILITY OF DIFFERENT STRAINS OF VIRUS AND RECOVERY OF AIRBORNE VIRUS IN THE ENVIRONMENT OF INFECTOR MICE

A mouse-adapted Jap. 305 strain of influenza A₂ virus was found to be much more readily transmitted from one mouse to another than the NWS strain of influenza A₀ virus although the two viruses were equally pathogenic for mice as judged by pulmonary virus titers and lung lesions. The survival of artificially created aerosols of virus and the quantity of airborne virus required to initiate infection in mice were identical for the two viruses. The difference in transmissibility was associated with the recovery of infectious airborne virus in the environment of mice infected with the Jap. 305 strain during the period of their maximum infectiousness, but not in the environment of mice infected with the NWS strain.     

THE 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.     

TITRATION OF INFLUENZA VIRUS IN CHICK EMBRYOS

A study was made to establish the reproducibility of end points obtained in the titration of influenza viruses in chick embryos. Six tests were performed, each composed of five replicate titrations of a purified preparation of the PR8 strain of influenza virus. The data from these titrations were subjected to statistical analysis which revealed that the chances are 19 out of 20 that differences in end points of 0.37 and 0.62 logarithmic units are significant in titrations employing ten embryos and five embryos per dilution, respectively. Additional simultaneous titrations in embryos and in mice showed that chick embryos are sensitive to considerably smaller amounts of virus than are mice, and that the mouse titration is adversely affected by inactive virus under conditions which are without apparent influence on the embryo titration.     

IMMUNOCYTOLOGICAL EFFECTS OF VARIED INOCULA OF INFLUENZA VIRUS

Variation in the amount and quality of influenza virus injected into the amniotic sac of the chick embryo led to differences not only in the yield of virus but also in the immunofluorescent cytology of the infection. The production of infectious virus was associated with a predominance of cells in which the virus antigens were first detected along the cell surface in contact with the amniotic fluid, later deeper in the cytoplasm, and finally, though to a lesser extent, in the nucleus. When the virus yield was primarily non-infectious hemagglutinin the virus antigens appeared in reverse sequence; i.e., nucleus first, then adjacent cytoplasm, and ultimately throughout the entire cell. Under conditions of "autointerference," the immunofluorescence seen in some of the cells failed to progress beyond the nuclear or early cytoplasmic stage, while many other cells remained unreactive throughout the experimental period. With the largest dose used a pale intranuclear reaction was localized 1 hour after injection but the embryo died shortly thereafter.     

IMMUNOLOGICAL STUDIES WITH THE VIRUS OF INFLUENZA

Following infection with the virus of influenza, both ferrets and mice develop a state of active immunity to reinfection. The serum of these animals contains neutralizing antibodies, as evidenced by the capacity of the serum to confer passive protection to mice against infection with the P.R.8 and Phila. strains of the virus of human influenza. Rabbits which are apparently insusceptible to infection with the virus of influenza produce specific antibodies in response to repeated injection of virus-containing material. The serum of immunized rabbits affords passive protection to mice against mouse-virulent virus. Although the subcutaneous or intraperitoneal injection of the living virus does not produce infection in mice, animals so treated acquire active immunity against subsequent infection by the intranasal route. Neutralization tests with the serum of patients before and after recovery from influenza, pneumonia and the common cold indicate that neutralizing antibodies arise as a specific response to infection with the virus of influenza. The immunological identity of strains of influenza virus recovered from human sources has been established, and the possible existence of strains of related, but not identical, antigenic structure is discussed.     

STUDIES OF TWO KINDS OF VIRUS PARTICLES WHICH COMPRISE INFLUENZA A2 VIRUS STRAINS : III. MORPHOLOGICAL CHARACTERISTICS: INDEPENDENCE OF MORPHOLOGICAL AND FUNCTIONAL TRAITS

Seven strains of influenza A2 virus were serially passed in the chick embryo, and morphological characteristics of the passages were examined in the electron microscope. With serial passage there was a change from a prominently filamentous appearance in early passages to an almost entirely spherical appearance in later passages. The number of passages required for the conversion to spherical morphology varied with different strains. The filament-sphere variation was found to be independent of the properties which differentiate "+" and "-" influenza A2 virus particles, and both highly filamentous and spherical populations of "+" and of "-" virus particles were obtained. The usefulness of these pairs of independent markers for genetic studies is discussed.     

THE MOUSE INFECTIVITY TITRATION OF INFLUENZA VIRUS

A study has been made to establish the statistical significance of results obtained in mouse infectivity titrations of influenza virus. Five titrations, each composed of five replicas, were carried out and 50 per cent end points were calculated for each titration. Three criteria for evaluating the end points were employed, namely, the presence or absence of pulmonary lesions, the occurrence of death, and a weighted composite taking into account both the extent of lung consolidation and the occurrence of death. Standard deviations of the distribution of end points obtained by each method were computed, and from these data levels of probabilities for significance in the differences between end points were determined. It was found that the chances are 19 out of 20 that differences of 0.99, 0.77, and 0.73 logarithmic units, respectively, for the lesion, the death, and the weighted end points are significant.     

STUDIES ON INFLUENZA VIRUS : THE COMPLEMENT-FIXING ANTIGEN OF INFLUENZA A AND SWINE INFLUENZA VIRUSES

Influenza complement fixation tests designed for use with ferret serum are described. Complement-fixing antigens derived from influenza ferret lungs were unsatisfactory due to their low content of soluble antigen; those prepared from mouse lungs or developing chick embryo membranes proved to be better antigenically and were reliable when the various reagents in the test were properly adjusted to eliminate non-specific fixation of complement. The results of cross complement fixation tests indicated that the soluble antigens of the PR8 and W.S. strains of influenza A virus were closely similar, if not identical. They indicated also that the soluble antigen of swine virus possessed components present in the antigens of the human strains of virus.     

ELECTROPHORESIS OF THE COMPLEMENT-FIXING ANTIGEN OF HUMAN INFLUENZA VIRUS

1. An electrophoretic study has been made of normal mouse serum, influenza mouse serum, and influenza mouse lung suspension. The mobilities of the protein fractions present have been determined at various pH''s by the optical method. 2. The pH-mobility curve of the soluble (complement-fixing) antigen present in the lung suspension has been determined analytically by sampling and application of the complement fixation test. The results show that the complement-fixing antigen has a mobility definitely smaller than that of serum albumin and close to that of a globulin, with an isoelectric point close to pH 5.     

THE INFECTION OF MICE WITH SWINE INFLUENZA VIRUS

The experiments confirm the earlier observation of Andrewes, Laidlaw and Smith that the swine influenza virus is pathogenic for white mice when administered intranasally. Two field strains of the swine influenza virus were found to differ in their initial pathogenicity for mice. One strain was apparently fully pathogenic even in its 1st mouse passage while the other required 2 or 3 mouse passages to acquire full virulence for this species. Both strains, however, were initially infectious for mice, without the necessity of intervening ferret passages. There is no evidence that bacteria play any significant rôle in the mouse disease though essential in that of swine, and fatal pneumonias can be produced in mice by pure virus infections. Mice surviving the virus disease are immune to reinfection for at least a month. In mice the disease is not contagious though it is notably so in swine. The virus, while regularly producing fatal pneumonias when administered intranasally to mice, appears to be completely innocuous when given subcutaneously or intraperitoneally. Prolonged serial passage of the virus in mice does not influence its infectivity or virulence for swine or ferrets. It is a stable virus so far as its infectivity is concerned, and can be transferred at will from any one of its three known susceptible hosts to any other. In discussing these facts the stability of the swine influenza virus has been contrasted with the apparent instability of freshly isolated strains of the human influenza virus. Though the mouse is an un-natural host for the virus it is, nevertheless, useful for the study of those aspects of swine influenza which have to do with the virus only.     

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.     

THE NUCLEIC ACID AND CARBOHYDRATE OF INFLUENZA VIRUS

Both ribonucleic and desoxyribonucleic acids have been obtained from purified particles of PR8 influenza virus. These particles were also found by extraction with formamide to contain carbohydrate in addition to that of the nucleic acids. Carbohydrate-rich fractions, essentially devoid of nucleic acid, were obtained not only from the particles representing PR8 virus but from those of Lee influenza virus as well. The carbohydrate in each case appeared to be a polysaccharide composed of mannose, galactose, and glucosamine units.     

DISSOCIATION OF HEMAGGLUTINATING AND ANTIBODY-MEASURING CAPACITIES OF INFLUENZA VIRUS

Preparations of Type B influenza virus, propagated in the embryonated egg and obtained in the form of allantoic fluid, were found after heating at 56°C. for 30 minutes to retain the capacity to agglutinate erythrocytes but no longer measured specific antibody when used as antigen in titrations of serum antibody. The dissociation of the two activities suggests the presence in such virus preparations of a complex virus antigen comprising, (1) a heat-stable component which agglutinates erythrocytes and reacts primarily with specific antibody; (2) a heat-labile component reacting with a factor of normal serum which ordinarily tends to inhibit the hemagglutinating activity of influenza virus. The relation of the reagents to other known serological activities of influenza virus is being studied.