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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.

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 mmicro 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 mmicro in diameter, probably more nearly 10 mmicro, while the remainder is reversibly aggregated. Reversal to the more disperse state may be effected by dilution, sonic vibration, or moderate heat treatment.

This report describes the results of histopathologic and virologic studies in six patients with undifferentiated carcinoma (malignant lymphoepithelial lesions) of the salivary glands. Epstein-Barr virus (EBV) was detected in tumors from all six patients by DNA hybridization, while adjacent non-tumorous salivary gland tissue was negative for EBV in two patients tested for DNA and in three patients tested for Epstein-Barr nuclear antigen (EBNA). These findings add more evidence that these unusual salivary gland tumors are EBV-associated, and that EBV is specific to the tumor.

A parametric study of multi-megawatt mm-wave transmission and launching antenna systems for various electron cyclotron wave (ECW) applications in next generation thermonuclear fusion devices such as NET or ITER has been carried out for the frequency range between 120 and 260 GHz. The paper presents a conceptual design of a 140 GHz, 20 MW, CW reference ECW system to assist gas breakdown, plasma formation and pre-heating, and for local current density profile control by “upshifted” noninductive current drive (ECCD) near the q = 2 magnetic surface in order to prevent major disruptions. The ECW system will preferably be made up from 1 MW, CW mm-wave generator and transmission units (comprised of gyrotron, transmission line and torus barrier window) and one common quasi-optical antenna module. The evacuated transmission lines combine the advantages of HE11 - mode waveguide and TEMOO - mode quasi - optical components. Tracking of the q = 2 surface is achieved by sweeping of the mm-wave beam in a horizontal plane (15°–25°) using one rotatable reflector in the 4-mirror antenna system. The expected total mm-wave transmission efficiency from the gyrotrons to the plasma is approximately 85%.

Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera.

A total of 172 sera from patients with multiple sclerosis (MS), theri relatives and nursing personnel, patients with other neurological and nonneurological diseases, and healthy donors living in the United State or East Africa under vastly divergent hygienic conditions were examined for their capacity to neutralize the MS-associated agent (MSAA), which induces in experimental animals a transitory depression of circulating polymorphonuclear leukocytes (PMN). A considerable proportion of sera from MS patients and their relatives or nursing personnel and East African donors revealed neutralizing activity, but only one of 59 sera from American donors without known contacts with MS patients revealed neutralizing activity. Some of the sera could be diluted 100- or 1,000- fold and still prevent, or substantially reduce, PMN depressions in mice. The neutralizing activity was shown to be associated with the immunoglobulin fractions of sera and therefore appears to be due to an antibody. Cerebrospinal fluids from MS, but not other, patients also strongly neutralized MSAA. Evidence has been presented that sera from MS patients may contain both MSAA and MSAA neutralizing antibodies. Antigen-antibody complexes were separated from such sera by high-speed centrifugation, and neutralizing antibodies were dissociated from them at a low pH. Whereas the data are as yet limited due to the vagaries and complexities of the test procedures, they provide further evidence that MSAA is not an indigenous virus of experimental animals, causes infections in man, and is indeed closely associated with MS. If it were the cause of MS, which remains to be ascertained, the data imply that not all infections by MSAA lead to the development of MS.

The post-perfusion syndrome is rarely due to Epstein-Barr virus (EBV), because most adult patients already carry protective neutralizing antibodies in their sera. Data are presented from two patients in whom heterophil-antibody-positive infectious mononucleosis developed 35 and 48 days after open-heart surgery. The diagnoses of their predominantly febrile illnesses were delayed because of late appearance of atypical lymphocytes and heterophil antibodies. EBV-specific antibody responses were also delayed, with peak titers appearing several weeks or months after onset. The differences in evolution of the present cases, compared to those of classic infectious mononucleosis, were presumably due to different portals of entry (blood stream versus oropharynx).

Burkitt's lymphoma (BL) has been widely investigated and has attracted attention because of the possible etiologic role of the Epstein-Barr virus (EBV). To further determine the role of EBV in the causation of this tumor, we measured EBV-specific nuclear antigen (EBNA) and EBV DNA using immunofluorescence and nucleic acid hybridization techniques, respectively. Of 34 BL biopsies, 27 tissues (79%) were EBNA-positive, whereas none of the 25 non-BL biopsy tissues were EBNA-positive. Of 15 BL tumors tested, 14 (93%) were EBV DNA-positive with a mean of 39 (range, 8-86) EBV genome equivalents per cell. Each of the 15 non-BL biopsy specimens subjected to nucleic acid hybridization had less than two virus genome equivalents per cell, although all had serologic evidence of past EBV infection. The findings further supported the possible etiologic role of EBV in African BL and negated the passenger hypothesis. The EBV genome could, therefore, be used as a separating marker between African BL and non-BL lymphomas.

Fifty-six patients with a clinical and laboratory diagnosis of infectious mononucleosis who had not been ill for more than seven days, were randomised for peroral treatment with acyclovir (800 mg five times daily) or placebo for seven days in a double blind trial. Clinical, virological and immunological parameters were monitored in each patient for six months. During treatment, shedding of Epstein-Barr virus' as assessed in 36 patients, was significantly reduced (p less than 0.001). However, virus production in the oropharynx returned to pre-treatment levels one week after the cessation of therapy. Virus was detected in 35 patients at enrollment and in 28 of 36 patients at the six-month control. No effect on the clinical course of the disease was noticed. The virus-specific antibody response was also unaffected. A significant reduction in spontaneous outgrowth of in vivo Epstein-Barr virus-infected B-lymphocytes was found at 180 days after treatment in four acyclovir-treated patients compared to six controls (p less than 0.001). In another three patients with over-whelming clinical symptoms causing airway obstruction and/or disseminated intravascular coagulopathy, treatment with intravenous acyclovir (10 mg/kg three times daily) was combined with prednisolone (0.7 mg/kg daily) for ten days. Virus shedding ceased transiently during treatment, but returned to initial levels within one week. A dramatic clinical effect on the pharyngeal oedema and general health of the two patients with airway obstruction was noticed, but was much less evident in a patient with intravascular coagulopathy.

6 of 20 cotton-top tamarins (Saguinus oedipus) inoculated with Epstein-Barr virus (EBV) developed diffuse malignant lymphoma resembling reticulum cell or immunoblastic sarcoma of man. Hyperplastic lymphoreticular lesions were induced in three additional animals; in two instances the hyperplastic lesions regressed. Inapparent infection with development of antibody occured in eight animals. In two animals there was no evidence of EBV infection. One animal died in the first week after inoculation of parasitic infection. 10 animals uninoculated or mock-inoculated developed neither lymphoproliferative disease nor antibody. The malignant lymphoma appeared to arise from a cell with an uncleaved vesicular nucleus found in the center of the germinal follicle. The prominent cytologic features of this cell were extensive formation or rough endoplasmic reticulum and elaboration of the cytoplasmic membrane with microvilli. Cell lines derived from these tumors did not have receptors for complement. IgFc, or sheep erythrocytes, and the cell lines adhered to glass and plastic. EB nuclear antigen was found in imprints of two lymph nodes, one with lymphoma and one with hyperplasia. EB virus DNA was detected directly in the tumors of three animals and in cell lines from two lymphomas. Typical herpes virus particles were found in supernatant fluids from cell lines obtained from lymph nodes with tumors and hyperplasia, as well as in lines derived from blood leukocytes of marmosets with inapparent infection. These virus preparations had the biologic property characteristic of EBV, namely, stimulation of cellular DNA synthesis and immortalization of human lymphocytes. The virus derived from two cell lines was neutralized by reference human sera with EBV antibody and not by antibody-negative human sera. The virus derived from the experimental lesions is thus indistinghishable from human EBV. The marmoset has enhanced susceptibility to oncogenesis by EB virus. Among identified factors which may play a role in the heightened tumorigenicity of EB virus in this species are the increased production of virus by transformed cells and the absence of membrane receptors for complement or IgFc on transformed cells.

Serial sera from patients with infectious mononucleosis were examined for the emergence of antibodies reactive in antibody-dependent cellular cytotoxicity tests, using Epstein-Barr virus-superinfected Raji cells as targets. For this specific purpose, the antibody-dependent cellular cytotoxicity test proved to be of limited sensitivity because only relatively high serum dilutions can be tested dependably, due to prozone effects at low serum concentrations, and because antibody-dependent cellular cytotoxicity reactions at the 5% level are not always statistically significant. Under the conditions of the test, antibody-dependent cellular cytotoxicity-reactive antibodies were not measurable, or only barely measurable, in early-acute-phase sera, but they became detectable during convalescence and increased thereafter, gradually over many months to the range of titers seen in healthy persons after long-past-primary Epstein-Barr virus infections. The percentages of antibody-dependent cellular cytotoxicity ultimately attained were on the order of 20% in most patients and healthy individuals, but in others did not exceed 10%. The likely identity of the antibodies reactive in the test with antibodies to late Epstein-Barr virus-determined cell membrane antigens has been discussed.

Summary Partially purified preparations of the L and S components of Newcastle disease virus were obtained from infected allantoic fluid and membrane suspensions, respectively, by differential centrifugation. The L component could be separated also from the infected tissues but S could be detected in allantoic fluid only in relatively low concentrations. The S component was found to be partially sedimentable by centrifugal forces far in excess of those needed for removal of L. Suspension of the L component revealed ID50/HA ratios of the order of 106. In contrast, S preparations gave ratios as low as 100.6 and generally below 102. Whereas L was readily absorbed onto red cells in the cold, only relatively small amounts of S appeared to combine with these cells under the same conditions. No inhibitor of adsorption could be detected in S preparations. The data presented suggest that the S component either possesses greater eluting activity than L or that an elution-promoting factor is present in such preparations. The L component readily causes hemolysis whereas S according to all indications lacks this property. When red cells were exposed to S and subsequently washed they resisted hemolysis by the L component. Heated S or control material had no such effect. Only the L component showed the phenomenon of extension of titers in hemagglutinin assays upon repeated resuspension and settling of the red cells at 37°C. Only red cells previously exposed to L at 37°C until stabilized could be agglutinated by dilute NDV-immune serum or were capable of agglutinating normal red cells. These properties, according to Burnet, are closely linked with the hemolytic activity. The facts that S failed to reveal these reactions and was found to be non-hemolytic appear to be in agreement with this suggestion. Under carefully graded conditions S precedes the L component in the receptor gradient. Various problems concerning the mechanisms of interaction of these components with red cells have been discussed.

Summary The question whether antibodies are truly incapable of entering cells was studied in HeLa cultures infected with the PR8 strain of influenza A virus and in uninfected populations treated with antiviral or normal rabbit sera. The results were evaluated by staining of cells vitally or after acetone fixation with fluorescein isothiocyanate-coupled antibodies to PR8 virus or rabbit γ-globulin, as well as by hemagglutination and complement fixation tests. Replication of influenza virus in HeLa cells is restricted to one cycle because solely noninfectious hemagglutinins (NIHA) are produced. The ultimate yields of NIHA and the maximal percentage of cells containing influenza antigens were strictly proportional to the dose of seed virus employed. Few, if any, cells reacted with fluorescent antibodies in the second passage, and none after further passages. Differential staining of acetone-fixed cells from infected, untreated cultures showed that S antigen developed first and remained restricted to the nucleus. V antigen appeared initially in the region of the Golgi apparatus, from where it spread throughout the cytoplasm and, in time, reached the cell surface, as evident from vital staining with labeled anti-PR8 γ-globulin. Exposure of infected or uninfected cultures to antiviral or normal rabbit serum provided the following data: Vital staining of infected antiserum-treated cells with fluorescent anti-PR8 yielded essentially negative results, whereas labeled anti-rabbit γ-globulin produced brilliant fluorescence, restricted to the cell surface. The reverse was noted with infected cells maintained in the presence of normal serum; i.e., surface fluorescence with labeled antibodies to PR8 virus but not to rabbit γ-globulin. Uninfected cells, treated or not, failed to stain with either of the fluorescent antibodies.Acetone-fixed cells from antiviral or normal serum-treated cultures revealed the same degrees of virus-specific nuclear and cytoplasmic fluorescence. With labeled anti-rabbit γ-globulin, only the former became stained, but again apparently only at the cell surface in that no characteristic nuclear or perinuclear fluorescence could be elicited. Uninfected, treated or untreated cultures gave negative results with both types of fluorescent antibodies.Disintegrated cell suspensions from infected, antiserum-treated cultures fixed specifically some plement per se, but not those derived from populations exposed to normal rabbit serum. After addition of excess anti-V or anti-S, both preparations fixed the same numbers of complement units, indicating that the presence of antiviral serum in the medium did not affect the production of the two antigens.Exposure of infected cultures to anti-PR8 serum prevented the demonstration of NIHA formation as long as antibodies were present throughout or were added late in the incubation period, but not when they were removed at the 4th hr after infection. This secondary effect of antiserum may be accounted for by combination of viral antigen at the cellular surface with antibodies which on disruption of the cells may interact with liberated NIHA by means of free determinant groups or after dissociation in the course of reestablishing antigen-antibody equilibrium. All attempts to remove the attached antibodies were unsuccessful. As the data stand, no evidence was obtained to indicate entry of antibodies into normal or infected cells in sufficient amounts to be detectable by the immunofluorescence techniques employed or by reduction in production of viral antigens.

Summary The recall of complement-fixing antibodies was studied in guinea pigs previously immunized with one or several representative agents belonging to the poliomyelitis, Coxsackie A and B, and Echo groups. Although the studies were limited in scope, it became evident that poliomyelitis and Coxsackie B viruses readily recalled antibodies to the other members of the respective groups. Recalls were induced, although less frequently, by agents belonging to heterologous enterovirus groups but not by mumps virus. Antibodies were recalled often in the absence of detectable responses to the injected virus. A single dose of a given virus led to homologous antibody responses at an increased frequency if the animals had previous experience with another member of the same group of viruses. Broadening of the antibody spectra to include reactions with viruses not previously injected were observed only within the poliomyelitis group but the data are insufficient to exclude its occurrence among or between other groups of enteroviruses. Neutralization tests carried out with a few selected sera revealed that recalls of complementfixing antibodies were not accompanied by rises in neutralizing activity. The results presented appear to parallel the experience gained in the serodiagnosis of enterovirus infections of man.

Two institutional outbreaks of mumps have been analyzed with the aid of the complement-fixation reaction. The data indicate that mumps occurred only in children who failed to react significantly with the V-antigen at the onset of the epidemic. About 30% to 40% of all infections remained inapparent according to the clinical data of the outbreaks studied, or the records of past mumps. A few children with low antibody levels to both V- and S-antigens developed an increase in anti-V but not in anti-S, a type of response noted not infrequently following skin tests or subcutaneous vaccination. In one of the schools (M) no children were left at the end of the epidemic period who failed to react with the V-antigen. At School D at least eight of the children showed no serologic evidence of recent or past infection. Possible explanations for these various data are discussed.

It is a well-known fact that infection of a bacterium, plant or animal with one virus frequently prevents or partially inhibits simultaneous propagation of another viral agent in the same host. Such interference phenomena, which have been called also “sparing effect”, “cell blockade”, or “Schienenimmunität”, have aroused great interest on account of their theoretical, and possibly practical implications. As a result, the literature on this subject has been increasing rapidly in recent years. It may be profitable, therefore, to take stock at this time and to attempt formulation of general principles which have evolved in studies of the various phenomena. It will be apparent that the present knowledge is still very fragmentary, so much so that the plural in the title of this review is being employed advisedly because of the fact that it is not known whether the various instances of interference described are all based upon related mechanisms, or whether several entirely different reactions are involved in the various observations.

Summary Studies have been presented on the labelling of influenza virus with P32 during propagation in the allantoic cavity of chick embryos. The labelled virus has been purified routinely by 2 cycles of adsorption onto and elution from chicken red cells with intermittent dialysis against large volumes of phosphate buffered saline solution. The degree of radioactivity of the virus progeny could be improved by an increase in the dose of P32 used, by prolongation of the interval between injection of the isotope and infection, and by a reduction in the age of the embryos employed. The ratio between counts per minute and hemagglutinin units of the yields could be increased further by the use of deembryonated eggs instead of intact chick embryos. It has not been possible to separate the radioactivity from the virus particles by various biological, physical and chemical means. Chemical fractionation of labelled virus revealed most of the radioactivity in the lipid and nucleic acid fractions.

Burkitt's lymphoma (BL) has been widely investigated and has attracted attention because of the possible etiologic role of the Epstein-Barr virus (EBV). To further determine the role of EBV in the causation of this tumor, we measured EBV-specific nuclear antigen (EBNA) and EBV DNA using immunofluorescence and nucleic acid hybridization techniques, respectively. Of 34 BL biopsies, 27 tissues (79%) were EBNA-positive, whereas none of the 25 non-BL biopsy tissues were EBNA-positive. Of 15 BL tumors tested, 14 (93%) were EBV DNA-positive with a mean of 39 (range, 8-86) EBV genome equivalents per cell. Each of the 15 non-BL biopsy specimens subjected to nucleic acid hybridization had less than two virus genome equivalents per cell, although all had serologic evidence of past EBV infection. The findings further supported the possible etiologic role of EBV in African BL and negated the passenger hypothesis. The EBV genome could, therefore, be used as a separating marker between African BL and non-BL lymphomas.

Several viruses induce tumors in animals under experimental or natural conditions. It is likely therefore that some human malignancies are also caused by viruses. Proof of this hypothesis can be provided only by indirect evidence based on the following criteria: (1) detection of viral antigens or viral genetic information in a given tumor; (2) transformation of normal human cells by the virus in tissue culture; (3) induction of tumors in animals by the virus; and (4) demonstration of enhanced titers of antibodies to the virus in patients bearing the tumor. These criteria have been fulfilled to support a causal relationship of the Epstein-Barr virus (EBV) in Burkitt's lymphoma and nasopharyngeal carcinoma. It is clear, however, that factors of a genetic, immunologic or environmental nature must play an additional role because EBV, the cause of infectious mononucleosis, is widely disseminated yet development of the tumors is a rare event.

In vivo and in vitro humoral and cellular immune responses were studied in a 2 1/2-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus. During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells. In vitro mitogen transformation of lymphocytes was reduced. In vivo antibody responses to the T cell-dependent antigens bacteriophage phi X 174 and Keyhole limpet hemocyanin were markedly depressed. These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis.

Serial sera from nine volunteers, injected 10 years ago with sheep erythrocytes and showing heterophile antibody responses comparable to those seen in infectious mononucleosis, were retrieved from storage and examined for antibodies to Epstein–Barr virus (EBV) related antigens. Eight of the nine volunteers had pre-existing antibodies to EBV, as determined by neutralization tests and by indirect immunofluorescence reactions with fluorescein-conjugated antibodies to human IgG, but not with anti-human IgM conjugates. The EBV-specific reactivities remained unchanged after immunization. The results indicate that: (a) EBV- or EBV antigen-containing lymphoblasts from carrier or freshly infected cultures contain no detectable heterophile antigen; (b) immunization with sheep erythrocytes does not evoke antibodies which interact with EBV or EBV-infected cells. These points are supported especially by the results obtained with the heterophile antibody-positive, anti-EBV-negative serum of one of the volunteers who subsequently sustained a primary EBV infection. It is unlikely, therefore, that immunization with heterophile antigen will induce immunity to infectious mononucleosis, apart from the fact that heterophile antibody responses are largely restricted to the IgM class.