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In agreement with earlier observations the infectivity titer in the allantoic fluids of chick embryos injected with influenza A virus remains constant for 5 to 6 hours before an increase in this activity can be noted. In contrast, the titers of hemagglutinin and complement-fixing antigen (virus antigen) have already begun to rise after 3 hours. The origin of the hemagglutinating and complement-fixing but non-infectious material is still obscure. In the allantoic membrane development of both the soluble and virus antigens can be demonstrated after the 2nd hour of incubation and 1 hour prior to an increase in hemagglutinins and 2 hours prior to a rise in infectivity. Thus there remain the first 2 hours during which no record of virus activity in the tissues can be obtained. Similar relationships are noted on dilution of the seed both in the allantoic fluids and membranes as long as the various properties reach measurable levels during the experimental period of one infectious cycle. Injection of high titered immune serum following infection with about 10(9) ID(50) reduces the amount of demonstrable seed virus in the allantoic fluid and membrane without significantly affecting propagation of the agent in the tissues as measured by infectivity titrations. The production of hemagglutinins appears markedly reduced under these conditions whereas formation of complement-fixing antigen is only slightly delayed and decreased. No definite explanations for the various discrepancies between the infectivity and hemagglutination can be given at present.

Cultured Burkitt cells were examined by immunofluorescence, autoradiography, and electron microscopy in an effort to identify the stainable cells with those harboring herpes-type virus particles. Immediately after a 2-hr pulse of (3)H-thymidine, from 30 to 60% of the cells revealed heavy nuclear labeling. In most cases the grains were evenly dispersed, but in about 3 to 5% the grains showed a focal distribution and occasionally they extended into the cytoplasm. Such nuclear foci were rarely seen at 8 hr after the pulse. When the analysis was restricted to preselected immunofluorescent cells, up to 80% showed label at 8 hr and cytoplasmic grains were prominent. To reduce cellular deoxyribonucleic acid (DNA) synthesis, cells were X-irradiated with 3,000 to 6,000 R, and the isotope pulse was applied 1, 4, or 7 days later. Whereas the total number of labeled cells decreased in roughly twofold steps at the respective intervals (from 40 to 10%), the incorporation of (3)H-thymidine into fluorescent cells was not affected by X irradiation. In each series, about 70% of the fluorescent cells contained label when they were examined at 24 and 48 hr after the pulse, whereas at 8 and 72 hr fewer were positive. At the earlier intervals, unlabeled fluorescent cells most likely represented cells which had completed viral DNA synthesis prior to the pulse; at the later intervals, unlabeled fluorescent cells were probably cells which commenced viral replication after the pulse. These data support the conclusion that the immunofluorescent cells are the ones which harbor virus, and also confirm the expectation that the virus is a DNA virus from a member of the herpes group. This conclusion was firmly established by sectioning and electron microscopic examination of individual fluorescent cells, all of which contained numerous virus particles, whereas the nonstained cells prepared in a similar manner were free of them.

Henle, Gertrude (Children's Hospital of Philadelphia, Philadelphia, Pa.), and Werner Henle. Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91:1248-1256. 1966.-Indirect immunofluorescence tests led to the brilliant staining of a small proportion of the cells in five different cultures derived from Burkitt's (African) lymphomas. The reaction was not restricted to the 17 sera from cases of this disease but extended to many sera from American individuals, whether healthy donors or patients suffering from a variety of illnesses. The incidence of positive sera increased with age from about 30% in childhood to > 90% in adults. Fluorescein-isothiocyanate-conjugated human gamma-globulins were suitable for direct staining of the same proportion of cells. The stained cells appeared to be in varying stages of degeneration, but cultural conditions leading to an increase in the cellular death rates failed to result in a rise in fluorescent cells. Several observations suggest that the stainable cells might be those which are seen to harbor virus particles under the electron microscope. Two cell lines derived from leukemic patients in this country also contained a small fraction of stainable cells but two others, and numerous primary human leukocyte cultures, gave consistently negative results. Attempts to relate the staining to known viral antigens have failed to implicate herpes simplex, varicella, cytomegalo, and reo viruses types 1, 2, and 3. The nature of the virus carried by the lymphoma cells as well as of the staining reactions remains to be determined.

Efforts were made to elucidate the nature of the resistance to vesicular stomatitis virus (VSV) observed in MCN cultures persistently infected with Newcastle disease, mumps, or 6-6 viruses (MCN(NDV), MCN(Mps) and MCN(6-6), respectively). Cells derived from persistently infected cultures adsorbed VSV to the same extent as their uninfected counterparts. Only a fraction of the adsorbed virus could be recovered from the cells indicating that it enters into an eclipse in all of the cell types. While propagation of VSV in MCN cells is largely inhibited at low pH levels, the resistance of persistently infected cultures could not be ascribed to their increased lactic acid formation. Resistance was not absolute in that a few cells in persistently infected cultures apparently supported VSV reproduction. Furthermore resistance of the cultures was found to be transitory in that the VSV infection gradually gained the upper hand after 2 to 4 weeks of incubation. Addition of ultraviolet-inactivated NDV to MCN cultures induced resistance to VSV as long as the equivalent of at least one ID(50) (for chick embryos) of inactivated virus was provided per cell. Establishment of resistance required some time and its duration depended upon whether or not the free inactivated NDV was removed or neutralized after given adsorption periods. The transitory nature of resistance in persistently infected cultures, or in MCN cells following adsorption of inactivated NDV, is most likely explained by the fact that the cells continue to divide and that the daughter cells are, at least in part, susceptible to VSV. The results are compatible with the conclusion that the resistance observed represents another example of interference between 2 viruses.

In previous reports of this series, it was shown that persistent infection of MCN cultures with certain myxoviruses rendered the cells insusceptible to superinfection by several cytopathogenic viruses. It was thought that production of an interferon might be the cause of this resistance and efforts to confirm this suggestion have been presented. Addition of ultraviolet-inactivated myxoviruses (mumps, Newcastle disease, influenza A, and Sendai) to MCN cultures for periods of 2 to 3 hours, followed by washing and refeeding of the cells, led to the subsequent release into the media of a substance which induced in fresh MCN cells a transitory resistance to infection by vesicular stomatitis virus, and prevented incomplete reproductive cycles of influenza A and Sendai viruses. Media containing this substance were free of detectable hemagglutinating activity and viral complement-fixing antigens. The substance was not neutralized by specific antiviral sera; it was not sedimentable by high speed centrifugation; it was not adsorbed onto red cells; but it was inactivated by trypsin. Thus, its properties matched those of the interferon described by Isaacs and his associates. A comparison of the extent of resistance induced in MCN cells by decreasing doses of ultraviolet-inactivated myxoviruses (interference test) and the protection afforded by the media removed from the cultures prior to challenge and transferred to fresh MCN tubes (interferon test) revealed that wherever interference became detectable in the cells, the media of the corresponding cultures contained some interferon. Interferon was obtained by inactivated myxoviruses also from primary cell cultures by the same techniques, but not from HeLa cells. Interferons derived from one type of culture may protect others equally well or show a certain degree of host specificity in that resistance in homologous cells may be somewhat more pronounced than in heterologous cultures. No resistance could be induced in HeLa cells by the interferon preparations employed. Interferon was detected also in MCN cultures, persistently infected with mumps virus. Its concentration was apparently too small in carrier cultures maintained as routine to be measurable. However, when the cells were grown in heavy sheets in roller bottles, and especially when the volume of medium was reduced for several days prior to harvest, interferon became readily detectable. These results strengthen the suggestion that interferon may play a decisive role in the establishment and maintenance of persistent infections in the system under study. Its nature, source, mode of action, and exact role in persistent infection remains to be elucidated.

Armstrong, Donald (Children's Hospital of Philadelphia, Philadelphia, Pa.), Gertrude Henle, and Werner Henle. Complement-fixation tests with cell lines derived from Burkitt's lymphoma and acute leukemias. J. Bacteriol. 91:1257-1262. 1966.-Cells of various lines isolated from Burkitt's lymphomas and acute leukemias and disintegrated by freezing and thawing or sonic treatment were found to react in complement-fixation tests with a considerable proportion of human sera. At least 10(7) cells per milliliter were required for antigenic activity. All but one of 13 sera from Burkitt lymphoma patients were positive, with titers ranging from 1:8 to 1:320. About 20% of sera from American children and 60% of sera from adults, regardless of diagnosis, showed titers in a similar range. Sera giving positive tests with one of the neoplastic white cell antigens usually reacted also with many if not all of the others, but rarely with antigens derived from normal peripheral leukocyte cultures and not at all with HeLa or other human nonleukocytic cells. Various observations indicate that the complement-fixation test measures mainly antigens which are different from those detected by immunofluorescence. The nature of the reactions described remains obscure.

After allantoic injection of chick embryos with a known amount of influenza virus, the process of adsorption of the agent onto host cells and infection of them can be interrupted at a given time by the administration of large quantities of heterologous virus inactivated by irradiation. A sudden great increase in the amount of free virus in the allantoic fluid occurring after 6 hours in the case of the PR8 strain, and 9 hours in that of the Lee strain, indicates that the untreated virus associated with the host cells has multiplied. The length of the period preliminary to this increase remains the same even though the concentration of the original inoculum is varied over a wide range. Since administration of the irradiated virus leaves no susceptible host cells, because of the interference phenomenon, and further adsorption of active virus is minimized or entirely prevented, practically the entire new increment of virus can be found in the allantoic fluid and assayed; for every ID(50) adsorbed about 50 ID(50) are released. Homologous irradiated virus, on the other hand, when injected after infection of the allantoic sac, reduces the yield of virus to a more or less considerable extent. Some inhibitory effect can still be observed when the homologous irradiated virus is given several hours after infection. This effect is linked to the virus particle and destroyed by prolonged irradiation.

Various strains of influenza virus produce a cytopathogenic effect in cultures of HeLa cells. The virus could not be passed in series. Virus partially or even completely inactivated with respect to infectivity by exposure to 37 degrees C. or ultraviolet light retained some of its cytopathogenic effect. No evidence has been obtained of an increase in infectious virus in HeLa cultures, but an increase in hemagglutinins and in both viral and soluble complement-fixing antigens became detectable during incubation. These virus materials apparently were not released from these cells prior to their destruction. These results suggested that HeLa cells are capable of supporting an incomplete reproductive cycle of influenza virus. The fact that radioactive phosphorus was readily incorporated into the hemagglutinin supplies strong evidence for this interpretation.

Cultured cells derived from male patients with Burkitt's lymphoma and harboring herpes-type virus particles were lethally irradiated. These irradiated cells induced normal peripheral leukocytes of female infants to grow within 2 to 4 weeks after mixed cultivation. Cells of a line free of this agent failed to stimulate growth. If either type of cell was cultured separately, it did not survive under the experimental conditions. Herpes-type viral antigen and C-group chromosomal marker previously described in cultured Burkitt cells were found in all of the female cell cultures that were obtained.

Exposure of fifteen children to mumps virus of fifth amniotic passage in chick embryos led to involvement of the salivary glands in six, orchitis in the absence of other manifestations of mumps in one, and to no signs of illness in eight. Attempts to isolate virus from the saliva of these individuals gave the following results: 1. All patients with involvement of the salivary glands excreted virus beginning on the 11th to 15th day after exposure, 2 to 6 days prior to onset of clinical signs of disease and extending up to the 4th day of illness. 2. The patient with primary orchitis without any recognized involvement of the salivary glands excreted virus for 2 days, beginning on the 15th day after exposure and 10 days prior to his illness. 3. Six of the eight children classified as having inapparent infections because of their serologic response in the absence of clinical signs of illness, began to excrete virus on the 15th to 16th day after exposure for from 1 to 9 or more days. The epidemiologic significance of these data is discussed.

After incubation at 37 C for 2 or more days, Basal Medium Eagle (BME) supplemented with 25% fetal calf serum (FCS) was not able to support adequate growth of EB3 and other lines of Burkitt tumor cells. The medium did, however, increase, by a factor of about 10, the number of cells synthesizing herpes-type virus with which the cultures were persistently infected. Not every lot of FCS produced these effects, nor were these effects seen when BME and FCS were incubated separately for 7 days before the medium was completed. At 37 C, appropriate lots of FCS interacted with several of the amino acids present in BME; this interaction resulted in an inhibition of cellular growth, whereas interaction with arginine yielded the virus-enhancing effect. Arginine-free BME, supplemented with 25% FCS and used without prior incubation, prevented cellular replication and promoted viral infection to a similar extent as did preincubated complete medium. Replenishment of arginine reduced, but did not regularly abolish, the virus-enhancing activity of preincubated media. RPMI-1629 medium was less affected by preincubation with FCS because it contained twice the amount of arginine that BME contained. The FCS factors which act upon arginine and other amino acids are not dialyzable and are partially resistant to heating at 56 or 60 C for 30 to 60 min. Calf and horse sera appear to be devoid of these activities. The nature of these interactions, as well as the mechanism by which arginine deficiency enhanced the viral infection, remains to be ascertained.

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