Studies were made on the oncogenic response of 3086 young chicks to i.v. inoculation of MC29 avian leukosis virus from blood plasma of previous-passage birds or the supernatant fluid of cultures of chick embryo cells infected with strain MC29. Among the large variety of neoplasms of other tissues previously described, there occurred a high incidence of primary growths of the liver. Pathomorphology of the growths frequently differed greatly in both different hosts and the same bird, but some uniformity of the types of neoplasms was evident in many animals. Despite much variation in histopathology, the large proportion of growths could be grouped in several distinctive categories. Examinations by light and electron microscopy provided evidence of derivation of the tumors by alteration of hepatocytes originating principally in the portal regions as indicated by forms transitional from the parenchymal cells to the cells of the different types of growths. Neoplastic aspects of the growths were evident by infiltration and invasion of adjacent tissues, penetration of blood vessels, transplantability to other avian hosts (described in another report), and metastasis to distant organs including the lung, kidney, and spleen. There was no evidence of tumors arising from the biliary system, and growths of cells resembling the biliary type could be traced to altered hepatocytes. None of the findings suggested conversion of biliary-type cells to hepatocytes. Continued growth resulted in anaplastic and metaplastic changes in cell morphology and structural organization and in the formation of cartilage, osteoid, and sarcoma-like spindle-cell tumors of probable epithelial origin. Development of the growths wasnot associated with cirrhosis, and necrosis was limited to infrequent disseminated, essentially unicellular changes or necrobiosis of small groups of cells. The marked variations in the type of virus-induced growths demonstrated the remarkable capacity of cells morphologically inidistinguishable from the hepatocytes for the most diverse alterations in cell structure and tissue organization. This neoplastic response of hepatocytes to the MC29 strain constitutes the only demonstration thus far of the specific hepatocarcinogenic activity of an avian tumor virus.

Infection of avian bone marrow with strain MC29 leukosis virus in vitro resulted in infection, elaboration of virus, and growth of characteristic cells. Newly liberated virus with the infectious and neutralization properties of strain MC29 agent and growth of the cells were demonstrable 48 hr and 5–6 days respectively after infection. Studies by light and electron microscopy revealed similarities between the bone marrow cells and the myeloid elements (myelocytes) of myelocytomatosis notable in the relatively low nucleus/cytoplasm ratio; very large nucleolus; and ribosome-rich cytoplasm with a dense gray (protein) matrix and a grainy or “ground glass” appearance. Such cells differed markedly in these morphologic aspects from the myeloid cells (myeloblasts) of myeloblastic leukemia induced by BAI strain A. They also differed in their short-lived growth of 3–4 weeks when compared with myeloblast proliferation indefinitely in culture. The cells were similar to chick embryo cells morphologically altered by strain MC29 because of the characteristically large nucleolus, the high concentration of cytoplasmic ribosomes, and the slight rough endoplasmic reticulum. A notable difference was the high content of diffuse chromatin in the chick embryo cells and the reverse proportion of condensed chromatin in the myelocytes. The various aspects of strain MC29 specificity of influence on cell response are discussed.

Occurrence of aberrant cytoplasmic masses in avian virus tumor cells and their possible relation to virus synthesis by the cells were described in a previous report. As seen earlier, such masses consist of spheres somewhat resembling the nucleoids of the avian tumor viruses with interspersed dense bodies of the size and appearance of ribosomes. The composition of these structures was further investigated in the present work by ultracytochemical study of neoplastic cells of tumors induced by strain MC29 avian leukosis virus. Treatment with ribonuclease of thin sections of cells from growths arising in the kidney completely removed the dense, ribosome-like bodies, leaving a moderately densely stained amorphous mass. Structures exposed to pepsin showed essentially no staining except that of the dense granules removed by ribonuclease. This was in contrast to the relatively deeply stained residual material in the cytoplasm which indicated either a high proportion of protein in the structures or major differences between the constitution of protein in the structures and that of the cytoplasm. The possible origin of the collections of spheres and associated ribosomes and their significance were discussed.

Journal Article Nucleic Acid of the Rabbit Papilloma Virus Protein Get access A. R. Taylor, A. R. Taylor From the Department of Surgery, Duke University School of Medicine, Durham, N. C. Search for other works by this author on: Oxford Academic PubMed Google Scholar Dorothy Beard, Dorothy Beard From the Department of Surgery, Duke University School of Medicine, Durham, N. C. Search for other works by this author on: Oxford Academic PubMed Google Scholar D. G. Sharp, D. G. Sharp From the Department of Surgery, Duke University School of Medicine, Durham, N. C. Search for other works by this author on: Oxford Academic PubMed Google Scholar J. W. Beard J. W. Beard From the Department of Surgery, Duke University School of Medicine, Durham, N. C. Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 71, Issue 2, September 1942, Pages 110–114, https://doi.org/10.1093/infdis/71.2.110 Published: 01 September 1942 Article history Received: 26 January 1942 Published: 01 September 1942

Chickens have been hyper immunized with formolized concentrates of the virus of avian erythromyeloblastic leukosis together with the untreated plasma of diseased birds containing the agent in high concentration. The resulting immune serums strongly neutralized the infectious properties of the virus and precipitated the characteristic virus particles as observed macroscopically and corroborated by electron micrographs of the precipitates. Precipitation of the virus particles was associated with proportional precipitation of the enzyme activity of the virus to dephosphorylate adenosine triphosphate. The findings constitute the critical specific criterion needed to establish the particles as the virus and the enzyme as a component inseparable, by all methods yet tried, from these virus particles.

A particulate component, which may be the etiological virus, was isolated by ultracentrifugation of the blood plasma of chickens with erythroblastosis. The particles were of spheroidal shape and variable size, averaging approximately 102 mμ diameter. Micrographs of unshadowed preparations showed circular images of an appearance suggesting that the particles consisted predominantly of a watery, low-electron contrast, gellike material surrounding a small relatively dense internal structure. The findings were compared with those obtained in the analogous studies of particles representing the virus of myeloblastosis and that of one form of lymphomatosis.

Changes have been made in the conditions of the micro test of plasmas from chicks with erythromyeloblastic leukosis for their capacity to dephosphorylate adenosine triphosphate. The principal modification has involved use of a balanced saline solution in the reaction mixture resulting in a very large increase in the precision, sensitivity and resolution of the procedure. With 3-λ of plasma, instead of the 5-λ volumes employed earlier, the expected error of a single determination in 2 of 3 cases is ±4 gamma phosphorus on the basis of measurements by electrometric titration. The greater sensitivity has extended the range of plasma activity possible for accurate measurement and has shortened the time necessary for the estimation.

Studies were made on the levels of Ca+ +, Na+, Mg++ and K+ in the plasma of birds with erythromyeloblastic leukosis. No changes from the normal were observed with the extracellular elements Ca++ and Na+. In contrast, the ordinarily intracellular elements Mg+ + and K+ were elevated in close relationship to the capacity of the plasmas to dephos-phorylate adenosine triphosphate and, consequently, to the virus content of the plasmas. The source of the abnormal amounts of Mg+ + and K+ have not been determined. The possible origin of the elements was discussed.

A micro procedure has been designed for the rapid testing, in 5 δ volumes, of the plasmas of chickens with erythromyelo-blastic leukosis for their capacity to dephos-phorylate adenosine triphosphate. Since this enzymatic activity is related both to the number of virus particles and to the infectivity of the plasmas, the test provides the means either for the measurement of those qualities or for the selection of chicks possessing the qualities in the desired degree. There is a wide variation in this enzymatic activity among the individuals of the diseased chick population. For practical application to the study of erythromyeloblastic leukosis, the test is comparable with the hemagglutinative reaction with the influenza virus. The test in its present form would not be useful in the diagnosis of erythromyeloblastic leukosis which can be accomplished much more easily by blood smear. No experiments have been made to learn whether the reaction might occur with the plasma of birds with visceral lymphomatosis which is much more difficult to diagnose in the intact bird.

A highly potent capacity for the dephosphorylation of adenosine triphosphate has been observed with preparations containing the virus of avian erythromyeloblastic leucosis. This enzymatic reaction occurred with the filtered plasma of birds diseased with the virus and with virus concentrates obtained from the plasma by ultracentrifugal procedures. The partition of enzymatic activity in these preparations has closely paralleled the distribution of virus infectivity of analogous materials measured in other work by titration of the virus in susceptible host chicks. No evidence of the reaction was obtained with the plasma from normal chickens. These experiments demonstrate the specific relationship of the dephosphorylation of adenosine triphosphate with avian erythromyeloblastic leucosis and indicate that the activity is a property of the etiological virus.

Chemical analyses have been made of several bacteriophages (l-9) in more or less purified preparations.However, only a few, the T:, (G-8) and, more recently, the Te (9) bacteriophages of Escherichia coli, have been studied in sufficient detail for comparison with analogous studies on plant and animal viruses.The results obtained on the bacteriophages thus far examined have shown that, while these agents are somewhat similar to the animal viruses in the constitution of their major components, the bacterial viruses differ, with few exceptions, from other viruses in the quantitative distribution of components, the difference being manifested principally in an exceedingly high content of nucleic acid in the bacterial viruses.Judging from phosphorus content and direct estimation, the content of nucleic acid has been uniformly high in the various bacteriophages (l-9), and it would appear that this property and certain others may be characteristic of these agents.Recently, studies (10) were undertaken on another bacteriophage, Tr of E. wli, which was chosen because of its apparently great difference from the Tz agent in size, morphology, and immunological properties (11).The Tr virus is spherical, has about 12 per cent of the mass of the T, (and TB) phage, has no tail, and is immunologically unrelated to either the Tz or Ta agents.The purification and some of the properties of the Tr bacteriophage were reported in a recent publication (10).The results of chemical analyses of the agent and a comparison with analogous findings with 'l's, Ts, and the host organism, E. coli (strain B), are described in the present paper. Materials and MethodsPurifiation-The T, bacteriophage and the host organism E. coli (strain B) were obtained from Dr. M. Delbtick.Cultivation of the agent for purification was carried out by the procedures already described (10).Most of the analyses were made on two different batches, A and B, prepared in the present work.Some estimations were made with the phage prepared in the previous experiments.

A disease easily characterizable and measurable in severity by clinical observations on the height and duration of fever, loss of weight and anorexia was produced in normal, unvaccinated swine by exposure to active swine influenza virus contained in chick-embryo chorio-allantoic fluid. The animals were uniformly highly susceptible to infection, giving a morbidity score of 94.1 per cent, but no deaths occurred. There was no evidence of synergistic bacterial infection. Vaccination with formalin-inactivated, purified swine influenza virus given subcutaneously produced an overall reduction in the incidence and severity of the clinical disease, resulting in a morbidity score of 59 per cent. No significant difference could be seen between the effects of 1, 2 or 3 injections of 0.5-mg amounts of the inactive virus, though the respective morbidity scores were numerically slightly smaller with repeated vaccinations. The results were not related to the age or size of the animals nor to the time of year of the tests. The antibody titers of the vaccinated animals were consistently greatly increased following exposure to active virus, whether symptoms developed or not. The levels of antibody titer 2 weeks after exposure to active virus were related directly to the number of vaccinations preceding the exposure. The degree of immunity or resistance conferred by vaccination, 37 per cent, was far less than that induced by exposure to active virus, 82 per cent, regardless of the severity or mildness of the disease, and the same was true for animals tested with active virus a second time after previous vaccination and exposure to active virus. The levels of antibody titer of animals in these categories rose little, or not ar all, on repeated exposure to active virus, indicating a very low degree, if any, of asymptomatic infection under these conditions. The findings are discussed in relation to their bearing on the control of influenza in man by vaccination.

Influenza virus B (Lee strain) increases to small degree in the chorio-allantoic fluid of 11-day-old chick embryos incubated for 60 hours at 39°C after inoculation with the virus. Increase was great at 37°C and still greater at 35°C. The pH of the chorio-allantoic fluid of the same embryos descended into the acid region at 39°C; at 37° C the pH remained essentially above 7.0; and at 35°C the reaction was maintained well in the alkaline region. These relations suggest the possibility that the extent of increase of the virus in the chorio-allantoic fluid may be influenced more by the pH of the fluid at the various temperatures than by the temperature per se.

Discussion and Summary The results of the present work show clearly that the degree of antibody response of swine is related to the dose per unit weight of formalin- or ultraviolet light-inactivated swine-influenza virus. Within the limits of the range of doses employed, 0.1 to 5.0 mg per 100 1b, the relation of log antibody titer to log dose of vaccine per unit weight appeared linear. With a single vaccination with formolized virus, a 10-fold difference in dose resulted in a 2.5-fold difference in antibody titer; a similar difference in the dose of ultraviolet vaccine was associated with a 1.6-fold difference in titer. The analogous relations for the second vaccination were a 1.6-fold difference in titer with a 10-fold difference in the dose of formalin vaccine and a 1.7-fold difference in titer with a 10-fold difference in the dose of ultraviolet light vaccine. The results likewise show clearly that the influence of dose on the peak level of antibody titer within the range of doses studied is relatively small in comparison with factors associated with the immunological status of the host with respect to experience with the influenza virus prior to the time of vaccination. Whereas at the first vaccination the difference between the log titers of the smallest dose of formalin vaccine, 0.1 mg per 100 lb, and the largest, 5.0 mg per 100 lb (a 50-fold range in dose) was log 0.67 (4.6-fold range of titer), the difference between the log titer associated with the dose of 0.1 mg per 100 lb at the first vaccination, log 1.96, and that following a like dose at the second vaccination, log 2.84, was log 0.88 (7.6-fold range in titer). From inspection of the results of a single vaccination (figure 8) it might appear that doses of vaccine in the range studied called forth responses which were near the limit of reaction possible for the host. That this is not the case is seen from the results with like doses of vaccine at the second vaccination (figure 13), and the results obtained with the animal, No. 65 (figure 22), which possessed-demonstrable antibodies before any vaccination. It is thus evident that while the magnitude of dose is a factor of relative influence on the antibody level under given conditions, the ultimate limits of the capacity of the host to respond are determined to a greater extent by immunological mechanisms inherent in the host itself. These findings are by no means unique; instead, the results conform closely to the generality of experience relative to host response to other antigenic materials. In so far as the character and the scope of the experiments are comparable, the findings of Hirst, Rickard, Whitman and Horsfall (2) in studies in man are similar to those with swine. These authors found that a 100-fold range in dose of influenza virus A vaccine was associated with a 2.5-fold range of antibody response (table 2 of (2)). In the instance of influenza virus B vaccine, there was a 2.4-fold range of response corresponding with a 40-fold range of dose. These results with man are quantitatively comparable with the present findings in swine. It should be noted, however, that the interpretation by Hirst, Rickard, Whitman and Horsfall “that the average antibody response of human beings is directly related, though not strictly proportional, to the amount of virus given” is somewhat of an overstatement in view of the quantitative relations actually found. Further parallelisms between the behavior of swine and man are the rapid loss of antibodies after vaccination and the more rapid decline in titer in those individuals in which the peak of titer was high than in those exhibiting the lower titers. The significance of the results of the studies on dose-response in swine is of chief interest in the possible bearings of the findings on the problem of vaccination of man for protection against infection with the influenza virus. With access to preparations of highly concentrated and partially purified virus, it is obviously desirable to consider the use of such material for preparing vaccines. Especially attractive in this respect is the possibility of grading the dose of vaccine to optimal proportions relative to 1) response; 2) the toxicity of the vaccine; and 3) the cost per dose of producing the vaccine. The range of dosage used in the experiments with swine, 0.125 to 2.0 mg per individual, was based on practical consideration. The quantity of 2.0 mg of virus per ml was attained by a concentration of the virus to a degree approximately 100 times that present in the chorio-allantoic fluid. A volume of 100 ml of chorio-allantoic fluid is approximately that contained in 10 to 11 eggs at the time of harvest, 42 hours after inoculation with the swine virus. With consideration of the technical procedures and materials, including eggs, necessary for producing large quantities of the vaccine, this amount, 2.0 mg per ml, seemed about the maximum desirable. The smallest dose, 0.125 mg of virus (vaccine), represented the quantity that could be recovered from about 6.2 ml of chorioallantoic fluid and thus lay within the range attainable by other methods which have already been extensively used for concentration, as, for example, the adsorption on and elution from chicken red blood cells. The yield of influenza virus B (Lee strain) is about 5 mg per 100 ml of chorio-allantoic fluid infected with this strain (13) and of influenza virus A (PR8 strain) 5 to 10 mg for a like volume (13). Thus the smallest dose of swine-virus vaccine, 0.125 mg, likewise corresponds to an amount of the human types of virus which can be obtained from a single egg and concentrated into a volume of 1 ml. From the point of view of yield and cost of virus and the difficulty associated with the technical procedures of virus concentration, the range of swine-virus vaccine, 0.125 to 2.0 mg, corresponded to a range of influenza-virus B (Lee strain) vaccine of 0.3 to 5.0 mg and of influenza-virus A (PR8 strain) vaccine of 0.3–0.6 to 5.0–10 mg. The range of swine-virus vaccine dose employed in the present work, therefore, parallels a range of doses of human vaccine varying from quantities technically simple and relatively inexpensive to obtain to amounts bordering on the limits of accessibility from a practical point of view. A factor of considerable importance in the choice of dose is the toxicity of the vaccine, that is, the general reaction of the host to administered vaccine aside from that concerned with the infective properties of the agent. In swine the largest dose of formolized vaccine per individual tolerated without evident reaction was 0.5 mg. This is likewise approximately, or perhaps slightly more than, the amount which has been administered on a large scale to man (5, 6). This statement is based on the consideration that if influenza A or B virus-infected chorioallantoic fluid contains 0.05 mg per ml of the respective viruses, a quantitatively 10-fold concentration by red blood cell adsorption and elution would yield 0.5 mg of virus per 1.0 ml of the concentrate. In the final choice of dose, it would appear desirable to employ the largest quantity of vaccine compatible with the various factors. From the point of view of toxicity, the dose 0.5 mg is within the limits of host tolerance. This quantity can be obtained simply and relatively inexpensively in a volume of 1.0 ml and is of a size which would permit the inclusion in a single inoculum of a mixture of the two types of virus (A and B) in quantities of 0.25 mg each. It would be possible also to employ mixtures of various strains of the respective types currently responsible for infection in the general population. The above discussion has been concerned for the most part with dosage, that is, with a consideration of the amount of vaccine that would be introduced at a single injection under any given set of conditions. Another problem, which is actually of greater importance from the point of view of practical protective vaccination, is concerned with the manner or sequence of applying the optimal dose. The experiments with swine show that, irrespective of the dose, a single vaccination is relatively ineffective and inefficient in the induction of antibodies in comparison with the effects of repeated vaccination. Furthermore, as shown both in swine and in man, the effects of either 1 or 2 vaccinations are shortlived. In man, inasmuch as antibodies are nearly universally present, it can be considered that a single vaccination would be equivalent, essentially, to a second vaccination in a host that had not had previous infection or contact with the virus. Such a concept, while adaptable to the conditions at the moment, fails to take into account the possibilities of future years. The hopes of success of a practice of a single vaccination under this concept would be largely dependent on the necessity for previous infection or contact of the host with the infectious virus in nature. Moreover, it would be necessary either to assume that the time relations between previous infection or contact and the moment of vaccination were uniform within the population or discount to a considerable extent the possible influence exerted by this relation. It must be recognized, of course, that the degree of protection against subsequent infection afforded either by previous infection or by vaccination under any conditions yet employed is relatively not great. Yet this knowledge cannot be permitted to interfere with the application of optimum immunological principles in the attempt to lessen the frequency of infection with influenza-virus. From this point of view it is evident that, if resistance to infection is related or proportional to, though not necessarily dependent on, the degree of antibody response and to the longevity of the antibodies induced, then multiple administrations of vaccine, even in minute quantities, would be expected to be of far greater importance than the magnitude of the dose to be given at one time. The question of the optimal frequency of vaccination is still to be investigated. The experiments with swine show that the antibodies arising as the result of a single vaccination decline to a low level by the end of the 3-weeks' post vaccination period. In contrast, the level of antibodies attained after a second vaccination was still high a month later and no lower after 6 weeks than the antibody level reached in the decline in 3 weeks following the first vaccination. Regardless of whether resort will be had finally to multiple injections of vaccine throughout the influenza season, it is clear from the results already at hand that repetition of vaccination provides a far greater promise of maintaining a high antibody level through the influenza season than the application of a single injection of the largest dose of vaccine thus far investigated.