EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : I. THE PERIOD OF TRANSMISSIBILITY

An experimental model has been developed for the reproducible transmission of influenza virus infection from experimentally infected mice to uninfected cage mates. Infector mice transmit influenza virus infection most readily during the period 24 to 48 hours after initiation of their infection. This restricted period of transmission is not due to declining titers of infective virus in the nose, trachea, or lungs of infector mice after 48 hours of infection, since peak titers in these tissues are maintained for another 48 hours. A mouse-adapted strain of A2 virus was found to be more readily transmitted than the mouse-adapted CAM strain of influenza A1 virus, although the CAM strain induced higher pulmonary virus titers and more extensive lung lesions.     

EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : III. DIFFERING EFFECTS OF IMMUNITY INDUCED BY INFECTION AND BY INACTIVATED INFLUENZA VIRUS VACCINE ON TRANSMISSION ON INFECTION

Immunization of mice by infection or intraperitoneal injection with homotypic A₂, heterotypic A₀, or recombinant A₀A₂ virus have differing effects on transmission of influenza A₂ virus infection. Immunization by infection with A₂ virus resulted in refractoriness to reinfection either by artificial aerosols or by exposure to infected cage-mates. Immunization by inoculation with inactivated A₂ virus vaccine resulted in a decreased susceptibility to transmitted infection in immunized contacts, but following A₂ virus challenge, transmission of infection by immunized infectors was not altered. Immunization by infection with influenza A₀ virus or recombinant A₀A₂ virus resulted in a decreased susceptibility to transmitted A₂ virus infection in immunized contacts, and to decreased transmission after A₂ virus infection in immunized infector mice. These differing effects on transmission of infection are attributed to differences in specific local immunologic responses following the various immunization procedures.     

EXPERIMENTAL TRANSMISSION OF INFLUENZA VIRUS INFECTION IN MICE : II. SOME FACTORS AFFECTING THE INCIDENCE OF TRANSMITTED INFECTION

Evidence has been presented that with the experimental model described, infected mice vary in their ability to transmit influenza virus infection. This variation is not explained by differences in titers of influenza virus in the nose, throat, trachea, or lungs of good transmitters. Older mice acquire transmitted influenza virus infection more readily than younger mice. Seasonal variations in the incidence of transmitted influenza virus infection occur.     

EXPERIMENTAL INFECTION WITH INFLUENZA A VIRUS IN MICE : THE INCREASE IN INTRAPULMONARY VIRUS AFTER INOCULATION AND THE INFLUENCE OF VARIOUS FACTORS THEREON

Following intranasal inoculation of influenza A virus (strain PR8) there is a rapid increase of the virus in the lungs which with large doses reaches a maximum within 24 hours. With smaller doses, although the proportional increase is greater, the maximum concentration is not reached until 48 hours following inoculation. If a lethal dose is administered, the ultimate concentration of the virus in the lungs is the same, irrespective of the size of the dose. If a sublethal dose is given, the titer of the virus in the lungs does not achieve the titer reached in mice receiving a lethal dose. Within 48 hours following inoculation of a sublethal dose the lungs of a mouse may contain at least 76,000 M.L.D., yet the mouse survives. The intranasal instillation of sterile fluid (distilled water, varying concentrations of NaCl, broth, or 10 per cent normal serum) into a mouse sublethally infected produces a sharp rise in the virus content of the lung usually followed by death within 3 to 8 days. If, however, the instillate consists of 10 per cent immune serum, there is no rise in the virus titer, and no apparent harm results from the instillation. The implications of these phenomena are discussed and an hypothesis presented to explain their occurrence.     

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.     

THE INFLUENCE OF INFLUENZA VIRUS INFECTION ON EXOGENOUS STAPHYLOCOCCAL AND ENDOGENOUS MURINE BACTERIAL INFECTION OF THE BRONCHOPULMONARY TISSUES OF MICE

Mice infected with a non-mouse-adapted Asian strain of influenza A virus suffered an impaired capacity to destroy or remove staphylococci introduced by the respiratory route. This temporary inhibition of local defense mechanisms was of 7 to 10 days' duration. The persistence of staphylococci in the lung following influenza did not appear to alter the nature of the pathologic reaction to influenza virus. The presence of influenza virus infection in the respiratory tract of the mouse did not alter the fate of intravenous staphylococci in the lung or other organs. In 40 to 50 per cent of mice with influenza, purulent bronchopneumonia and infection with Pasteurella and Hemophilus of murine origin were noted. A minority of control animals evidenced such infection. The administration of antimicrobials to which the murine bacteria were susceptible prevented both the appearance of the endogenous infection with Pasteurella or Hemophilus and the purulent sequelae to influenza virus infection. The true picture of uncomplicated bronchopulmonary influenza virus infection was thus separated from the combined virus-bacteria effect otherwise encountered.     

CONTRIBUTIONS TO THE PATHOLOGY OF EXPERIMENTAL VIRUS ENCEPHALITIS : IV. RECURRING STRAINS OF HERPES VIRUS.

In this paper, three strains of the herpes virus have been dealt with. The H.F. II strain was obtained from the subject H.F. 4 years after the H.F. I strain was secured. H.F. is a victim of recurrent herpes. If the subject is also a chronic carrier of the herpes virus, then it is not one, but two or more strains which are persistently carried. The H.F. II strain is of mitigated pathogenic action for the rabbit, as compared with the H.F. I strain; it is to be classed as dermatotropic rather than neurotropic. And yet, in the subject there was no indication that the attack of herpes provoked was different from the other attacks associated with the H.F. I virus. The other two herpes strains derive their interest from the fact that they came also from persons who suffer from repeated attacks of labial herpes. One strain proved highly neurotropic, resembling in this respect the H.F. I strain; the other was hardly neurotropic at all, but was none the less definitely dermatotropic. It may be possible at a later date to secure other samples of virus from these individuals for comparison. The dermatotropic F. strain penetrates to the central nervous system far more readily and certainly from the skin than from corneal surfaces. The recovered inoculated rabbits showed only relative protection to reinoculation of the herpes virus. A notable difference appeared in the degree of protection acquired, on the one hand by the cornea and on the other by the brain. While the one was partial, the other was complete. The complete resistance of the brain was shown (a) by the complete failure of the intracerebral inoculation, and (b) by the absence of circling movements following corneal inoculation.     

THE DEMONSTRATION OF LESIONS AND VIRUS IN THE LUNGS OF MICE RECEIVING LARGE INTRA-PERITONEAL INOCULATIONS OF EPIDEMIC INFLUENZA VIRUS

Following the intraperitoneal inoculation of mice with large doses of epidemic influenza virus (50,000 to 1 million intranasal M.L.D.) it can be recovered from the lungs in high concentration, and pulmonary lesions of moderate extent may be observed. The virus reaches its highest titer in the lungs 48 to 72 hours after intraperitoneal injection and may persist for 10 days. Virus may be recovered from the blood in the first 24 hours, but is readily detected in the omentum and peritoneum for 5 to 6 days. Mice which as a result of the intraperitoneal injection of virus show a high concentration of virus in the lungs do not die but become solidly immune to intranasal infection. Moreover, as early as 24 to 48 hours after intraperitoneal inoculation of large amounts of virus the animals may exhibit resistance to infection with fatal doses of virus given intranasally. Influenza virus given intravenously to mice is rapidly removed from the blood but persists in the lungs and induces pulmonary lesions. Virus can also be recovered from the liver for several days. With subcutaneous inoculation of influenza virus, however, the virus does not reach the blood or lungs in detectable amounts although the regional lymph nodes may yield considerable quantities of the agent. A brief consideration is presented of the mechanisms of infection and resistance which may be involved.     

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.     

THE SWINE LUNGWORM AS A RESERVOIR AND INTERMEDIATE HOST FOR SWINE INFLUENZA VIRUS : III. FACTORS INFLUENCING TRANSMISSION OF THE VIRUS AND THE PROVOCATION OF INFLUENZA

1. During a 3 year study of the lungworm as intermediate host for the swine influenza virus 98 transmission experiments, using 216 swine, have been conducted. Of these, 50 gave negative results. In the remaining 48, transmission of swine influenza virus by way of the lungworm was demonstrated in one or more animals of each experiment. Irregularities in the results would appear to be due not so much to lack of transmission of masked virus by the lungworms as to failure to evoke its pathogenic capabilities. 2. The stimulus of choice that was most successful in the provocation of swine influenza consisted of multiple intramuscular injections of H. influenzae suis. In several experiments pigs developed swine influenza virus infections 9 to 17 days after infestation with infected lungworms in the absence of any known provocative stress. In these instances an immune response of the swine to the lungworms themselves is suspected of having furnished the provocation. 3. During May, June, July, and August, swine prepared by the ingestion of lungworms carrying virus were absolutely refractory to the provocation of influenza, and they were relatively refractory in September and October. The masked virus was activated most readily during the first 4 months of the year. 4. In a single experiment we succeeded in demonstrating by direct means the presence of swine influenza virus in the neighborhood of lungworms at the base of the lung at a time when the virus was not demonstrable anywhere else in the respiratory tract. 5. Masked swine influenza virus was found to be present in lungworm ova obtained either from the respiratory tracts or the feces of infected swine. 6. In a number of instances, masked swine influenza virus has been found to persist for over a year in lungworm larvae within the earthworm intermediate hosts, and in one case its presence was demonstrated after 32 months. 7. Two varieties of a single species of earthworm, namely, Allolobophora caliginosa f. typica (Savigny) and A. caliginosa f. trapezoides (Dugès), have been found separately capable of serving as intermediate hosts for virus-infected lungworms. 8. Lungworm ova, obtained from convalescent swine which are no longer carrying swine influenza virus in infectious form in their respiratory tracts, contain masked virus.     

STUDIES ON HERPETIC INFECTION IN MICE : IV. THE EFFECT OF SPECIFIC ANTIBODIES ON THE PROGRESSION OF THE VIRUS WITHIN THE NERVOUS SYSTEM OF YOUNG MICE

Two-week-old mice inoculated with herpes virus on the pad of a hind foot regularly developed paralysis of the infected limb followed by paraplegia and encephalitis terminating fatally 5 or 6 days after inoculation. Hyperimmune rabbit serum given intraperitoneally at the time virus was inoculated on the foot pad prevented the formation of an herpetic lesion of the foot pad. When the antiserum was given 12 hours after inoculation of the virus, a typical infection of the epithelium of the foot pad developed, but the virus was prevented from causing obvious signs of infection of the nervous system in many of the animals. Amputation of the foot 2 hours after the inoculation of the virus prevented the paralysis of the hind leg. Some of the mice died of a delayed encephalitis. Amputation of the foot at 24 hours neither prevented nor delayed the sequence of paralysis of the hind leg, encephalitis, and death. In order to study immune serum therapy of an infection of the nervous system uncomplicated by a peripheral focus of infection or by traumatic disturbance of the central nervous system, 2-week-old mice were inoculated on the foot pad, the infected feet were amputated 24 hours later, and the immune serum was administered at varying intervals thereafter. Using litter mate controls and statistically significant numbers of mice, it was shown that hyperimmune rabbit serum, administered during the first one-third of the incubation period, retards and, in some cases, arrests the progress of herpetic infection within the nervous system.     

A STUDY IN VITRO OF COMPONENTS IN THE TRANSMISSION CYCLE OF SWINE INFLUENZA VIRUS

Swine lungworm extracts and suspensions of swine lungworms contain receptor-like substances capable of adsorbing influenza virus, a result consonant with the hypothesis (5–8) that the lungworm may be involved in the swine influenza cycle. Yet no evidence for multiplication of virus or even persistence of infectious virus in lungworms at undimished titer was found. Clearly much more information is needed, and it is hoped that the present demonstration of the practicality of studying the components of the transmission cycle proposed by Shope, will provide important tools requisite for further investigation of this problem. Studies on the role of the earthworm in the transmission of swine influenza suggest that, at best, that role would be a passive one.     

STUDIES ON LEUKEMIA IN MICE : I. THE EXPERIMENTAL TRANSMISSION OF LEUKEMIA

Lymphatic leukemia has occurred with great frequency in a particular strain of mice which have been inbred by brother-sister matings since 1921. In addition to typical cases of leukemia are others which, because of the absence of leukemic changes in the blood, correspond to "pseudoleukemia" and others which, by the presence of unusually great enlargement of certain lymph node groups resemble the "leukosarcomatoses" as observed in man. Examinations of the blood of leukemic mice have shown that leukemic blood pictures are not necessarily early in their appearance, nor are they constant. The blood picture may not, therefore, be used as a criterion for the separation of the two diseases (leukemia and pseudoleukemia) but merely indicates different phases of the same condition. Likewise, cases with lesions intermediate between the local growths of "leukosarcomatosis" and the more general lymphatic enlargements of leukemia suggest that these conditions differ only in the distribution of lesions but not in their nature. Lymphatic leukemia occurring spontaneously in this strain may be transmitted to other mice of the same strain, and carried, apparently, for an unlimited number of transfers in animals at an earlier age than that at which leukemia occurs spontaneously. In each of 10 such experiments transmissions were obtained. The lesions produced by inoculation correspond to those of spontaneous cases, in that they consist of growths of abnormal lymphoid cells which infiltrate tissues and organs and often appear in the circulating blood. Only minor differences have occurred, some of which are characteristic of certain experimental lines. After repeated transfers, the disease tends to run a more acute course. Among the cases in which transmissions occurred, are some without leukemic changes in the blood, and many with local growths at the site of inoculation or in certain node groups. The differences in the blood pictures and distribution of lesions (which latter may be influenced to some extent by the method of inoculation) correspond to similar differences which are sometimes observed in the spontaneous cases.     

THE SWINE LUNGWORM AS A RESERVOIR AND INTERMEDIATE HOST FOR SWINE INFLUENZA VIRUS : II. THE TRANSMISSION OF SWINE INFLUENZA VIRUS BY THE SWINE LUNGWORM

1. The swine lungworm can serve as intermediate host in transmitting swine influenza virus to swine. The virus is present in a masked non-infective form in the lungworm, however, and, to induce infection, must be rendered active by the application of a provocative stimulus to the swine it infests. Multiple intramuscular injections of H. influenzae suis furnish a means of provoking infection. Swine influenza infections can be provoked in properly prepared swine during the autumn, winter, and spring, but not during the summer. The phenomenon, while not regularly reproducible, occurs in well over half the experiments conducted outside the refractory period of summer. No explanation for the failures is apparent. 2. The virus can persist in its lungworm intermediate host for at least 2 years. 3. Swine infected with swine influenza virus by way of the lungworm intermediate host exhibit a more pronounced pneumonia of the posterior lobes of the lung than do animals infected intranasally with virus. The situation of the worms providing the virus will account for this. 4. Occasional swine infested with lungworms carrying influenza virus fail to become clinically ill after provocation but instead become immune. In these it is believed that lungworms containing the virus are localized outside the respiratory tract at the time of provocation. 5. It is believed that the experiments described furnish an explanation for the findings recorded in the preceding paper, in which swine influenza virus infections were induced in apparently normal swine by multiple injections of H. influenzae suis. 6. In a single experiment swine lungworms failed to transmit hog cholera virus.     

SWINE INFLUENZA : I. EXPERIMENTAL TRANSMISSION AND PATHOLOGY

Swine influenza has been induced in pigs by the intranasal instillation of material from spontaneous cases of the disease as occurring epizootically in eastern Iowa. The experimental disease has the same features as the epizootic. It has been maintained for study by serial passages accomplished either by intranasal instillation or by pen contact. Eight strains of the virus have been established experimentally during three epizootic periods. The clinical disease induced by these eight strains has been in general the same although its severity and mortality have varied. The principal features of the pathology of swine influenza are an exudative bronchitis accompanied by marked damage of the bronchial epithelium and its cilia, a peribronchial round cell infiltration, and massive pulmonary atelectasis. The latter is modified somewhat by a round cell infiltration of the alveolar walls. The lymph nodes, especially the cervical and mediastinal ones, are hyperplastic and edematous. There is usually a mild to moderate, acute splenic tumor. The mucosa of the stomach and colon is congested. The pneumonia following swine influenza is, characteristically, lobular in type and of the same general distribution as the atelectasis. The non-pneumonic areas of lung are extremely edematous and congested.     

INFLUENZA : I. THE HEMAGGLUTINATION AND INFECTIVITY TITRE CURVES OF PR8 INFLUENZA VIRUS CULTIVATED IN EMBRYONATED EGGS AT DIFFERENT TEMPERATURES

Groups of embryonated eggs infected with the PR8 strain of influenza virus A were incubated at 34°, 37.5°, and 40°C. At frequent intervals, for periods ranging up to 96 hours, pooled allantoic fluids were tested simultaneously for infectivity and hemagglutination. After about 12 hours of virus growth, fluids often showed infectivity titres greater than 10^–5, but were incapable of causing hemagglutination. At later time intervals, marked disagreement between the two tests for viral activity was noted at all temperatures, but most strikingly at 40°C. Hemagglutination titres were highest and best sustained in eggs incubated at 34°C., while incubation at 37.5°C. resulted in the highest and best sustained infectivity titres. Hemagglutination titre determinations do not reflect accurately the rate of influenza virus multiplication. Possible reasons for the lack of correspondence between hemagglutination and infectivity are discussed.     

STUDIES OF GENETIC TRANSMISSION OF MURINE LEUKEMIA VIRUS BY AKR MICE : II. CROSSES WITHFv-1b STRAINS OF MICE

The transmission of murine leukemia virus (MLV) to hybrids between AKR and Fv-1^b mice was studied in order to evaluate the effect of the Fv-1 gene on endogenous MLV infection and to attempt to determine if the genetic loci contributed by AKR carry viral genetic determinants. Fv-1 was shown to have a marked suppressive effect on time of appearance of detectable infectious virus and on the titers attained in vivo, but did not affect the ability of the cells to produce virus in vitro after induction with 5-iododeoxyuridine. The host range type of the virus detected in the hybrid mice was almost always of the type carried by AKR, although the low-virus Fv-1^b parents carry the genome of a different host range type. This finding provides strong, but not conclusive, evidence that the virus-inducing loci of AKR contain MLV genetic determinants.     

STUDIES ON THE NATURE OF THE VIRUS OF INFLUENZA : I. THE DISPERSION OF THE VIRUS OF INFLUENZA A IN TISSUE EMULSIONS AND IN EXTRA-EMBRYONIC FLUIDS OF THE CHICK

A considerable fraction of the influenza A virus contained in infected allantoic fluid of the developing chick is not sedimentable under conditions which remove virus activity almost completely from filtrates of emulsified mouse lung. The infectious unit from tissue suspensions is about 100 mµ in diameter and is of the same chemical composition as particles of the same size and abundance separated from normal tissues by an identical procedure. Evidence has been presented showing that the infectivity can be, and probably is, carried on such normal cell components as an adsorbate. Other non-infective particles such as erythrocytes may also become infectious units through adsorption of the virus. The virus occurs in allantoic fluid in two states of dispersion. A variable percentage is associated with particles considerably less than 100 mµ in diameter, probably more nearly 10 mµ, while the remainder is reversibly aggregated. Reversal to the more disperse state may be effected by dilution, sonic vibration, or moderate heat treatment.     

STUDIES OF GENETIC TRANSMISSION OF MURINE LEUKEMIA VIRUS BY AKR MICE : I. CROSSES WITHFv-1n STRAINS OF MICE

AKR mice, which regularly contain infectious murine leukemia virus, were mated with four Fv-1ⁿ strains of mice which show little or no expression of virus. F₁, F₂, and first and second backcross generation hybrids were tested for virus in tail tissue at 2 and 6 wk of age. The segregation data indicate that the AKR mouse contains two unlinked, autosomal, chromosomal loci, either of which suffices to induce detectable levels of infectious virus in Fv-1ⁿ progeny by 6 wk of age. One of the loci (tentatively referred to as V₁) is on linkage group I, 25–30 map units from the locus for albino; the gene order tentatively appears to be N₁-c-Hbb.