STRUCTURE OF TYPE 5 ADENOVIRUS : II. FINE STRUCTURE OF VIRUS SUBUNITS. MORPHOLOGIC RELATIONSHIP OF STRUCTURAL SUBUNITS TO VIRUS-SPECIFIC SOLUBLE ANTIGENS FROM INFECTED CELLS

Purified type 5 adenovirus was disrupted at pH 10.5 and the capsid shown to be comprised of two characteristic morphological subunits: (a) Hollow, polygonal structures corresponding to the virus capsomeres seen in preparation of purified virus and (b) thread-like strands also identifiable in preparations of purified virus. These structures were compared morphologically with purified preparations of the group- and type-specific soluble antigens characteristically produced in mammalian cells infected with adenoviruses. The group-specific soluble antigen was a homogeneous preparation of hollow, polygonal rods identical with the virus capsomeres. The type-specific soluble antigen corresponded to the thread- or fiber-like components of the purified virus particle. Inspection of disrupted virus preparations confirmed earlier immunological data which indicated that the major virus antigen was the group-specific soluble antigen. These data provide convincing evidence for the hypothesis that the adenovirus-induced soluble antigens represent virus subunits produced in excess.     

Cyclic appearance of defective interfering particles of herpes simplex virus and the concomitant accumulation of early polypeptide VP175.

Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.     

PRECIPITIN REACTIONS OF HIGHLY PURIFIED INFLUENZA VIRUSES AND RELATED MATERIALS

Antisera to purified PR8 virus, to purified protein from normal allantoic fluid, and to purified normal mouse lung particles were obtained from hyper-immunized rabbits and used in quantitative precipitin tests employing various purified preparations of influenza virus and related materials as antigens. The results of those tests indicated that the most highly purified preparations of PR8 or of Lee influenza virus obtained from infectious allantoic fluid contain an antigen characteristic of normal allantoic fluid and likewise that highly purified mouse lung PR8 virus contains an antigen characteristic of normal mouse lungs. Since the infectivity of virus preparations which were ultracentrifugally and electrochemically homogeneous was precipitated by the appropriate antisera to normal antigens, it was concluded that the normal antigens constitute a part of the 100 mµ particles with which influenza virus activity is at present deemed to be associated. It was estimated from quantitative precipitin data that the most highly purified preparations of PR8 and of Lee influenza viruses obtained from infectious allantoic fluid contain at least about 20 and 30 per cent, respectively, of an antigenic structure characteristic of the sedimentable protein of normal allantoic fluid.     

Isolation and characterization of C-type viral gene products of virus-negative mouse cells

Antigens which immunologically cross-react with two mouse C-type viral polypeptides, p30 and p12, are present at very low levels in normal virus-negative mouse cells. These two antigens have been purified by 50–300-fold from cell extracts and shown to cochromatograph with the corresponding labeled viral polypeptides in several systems. Their type-specific antigenicities are shown to be distinct from those of previously tested MuLV isolates suggesting that they may be components of a new class of endogenous C-type virus. The methods utilized in the present studies for concentration of virus-specific antigens of normal mouse cells provide an approach for detection of C-type viral antigens in cells of other species.     

Antigens of leukemias induced by naturally occurring murine leukemia virus: their relation to the antigens of gross virus and other murine leukemia viruses

Leukemias can be induced in W/Fu inbred rats by neonatal inoculation of normal thymus cells of C58 mice. These leukemias are not transplantable to C58 mice or to adult W/Fu rats, but they can be kept in passage in W/Fu rats aged 0 to 7 days. Adult W/Fu rats inoculated repeatedly with these isogenic leukemias produce cytotoxic and precipitating antibodies. These antisera are of particular value in the analysis of the antigens of leukemia cells and of leukemia viruses because their mode of preparation precludes the formation of antibody against any normal constituents of the cell. Analysis based on the cytotoxic test indicates the presence of 2 distinct cell surface antigens in leukemias induced by Passage A Gross virus or occurring spontaneously in mice of high-incidence strains. All leukemias and other tissues known to contain G (Gross) leukemia antigen have both determinants, but certain leukemias of low-incidence strains have only 1 of them and so were previously classified G-. Immunoprecipitation with these antisera reveals the presence of a cellular antigen common to G+ cells and absent from G- cells; the same antigen can be demonstrated in ether-treated Gross virus, but not in intact virus. This antigen is present also in ether-treated preparations of the Friend, Moloney, and Rauscher leukemia viruses, but not in Bittner (mammary tumor) virus. Thus it may be regarded as a group-specific antigen of murine leukemia viruses, in contrast to the type-specific cellular antigens demonstrable by the cytotoxic test. Four additional antigens associated with leukemias induced by wild-type Gross virus have been demonstrated by immunoprecipitation, but their relation to viral and cellular antigens has not been determined.     

STREPTOCOCCAL M ANTIGEN LOCATION AND SYNTHESIS, STUDIED BY IMMUNOFLUORESCENCE

Streptococcal M protein has been studied directly in the intact streptococcal cell by specific immunofluorescence. By this method, it can be seen to be concentrated in or on the cell wall, but cannot be detected in the capsule. The lack of type-specific (but not group-specific) immunofluorescence after trypsinization; and the inhibition of group-specific immunofluorescence by unlabeled type-specific antibody, are observations most compatible with a location of the M antigen determinants on the cell surface superficial to the group antigen. M antigen is not "resynthesized" after trypsinization of living cells, but appears anew only at sites of new cell wall growth. A limited amount of such growth, leading sometimes to detectable amounts of M in the gross, can take place in deficient media without detectable increases in optical density of the cell population.     

FURTHER IMPLICATION OF MURINE LEUKEMIA-LIKE VIRUS IN THE DISORDERS OF NZB MICE

Further evidence implicating murine leukemia-like virus in the disorders of NZB mice was afforded by a study of antigens associated with murine leukemia virus (MuLV). MuLV group antigens were prevalent in extracts of spleen, kidney, and, to a lesser extent, thymus throughout a substantial portion of the life span of NZB mice as well as in extracts of lymphomas and sarcomas indigenous to the strain. G (Gross) soluble antigen, type-specific antigen, was first detected in plasma of untreated NZB mice at 3 months of age. G soluble antigen production increased thereafter in line with age, with 50% of reactions becoming positive at 5.3 months and 100% at 7 to 9 months. From months 3 to 9, the time-response curve for positive conversion of direct antiglobulin (Coombs) tests in untreated NZB mice corresponded closely to that for G soluble antigen production. Beyond the 9th month, G soluble antigen underwent elimination from the plasma of NZB mice, with positive reactions reduced to 50% at 13.3 months and to 0% at 18 months. G natural antibody was first detected in the serum of NZB mice at about 10 months of age and increased thereafter in line with age. The curves for G antibody production and G soluble antigen elimination bore a reciprocal relation to each other with crossover at 50% response occurring at 13.3 months. Significant proteinuria, a functional manifestation of membranous glomerulonephritis, became increasingly prevalent in female NZB mice as G soluble antigen was eliminated from plasma. Cumulative mortality of female NZB mice, mainly attributable to renal glomerular disease, increased in phase with G antibody production. MuLV group antigens were identified in the glomerular lesions by the immunofluorescence method. Positive conversion of direct antiglobulin tests was significantly delayed by vaccinating baby NZB mice with formaldehyde-inactivated cell-free filtrates of older NZB mouse spleens. The plasmas of vaccinated NZB mice with negative direct antiglobulin reactions at 4 to 7 months were likewise negative when tested for G soluble antigen. The 50% response time for G antibody production in the vaccinated NZB mice occurred at 7.3 months, that is, 6 months earlier than in untreated NZB mice. The collective findings implicate murine leukemia-like virus in the etiology of autoimmune hemolytic disease and membranous glomerulonephritis, as well as malignant lymphoma, of NZB mice and suggest that virus-specified cell-surface and soluble antigen is a factor in the immunopathogenesis of the renal disease and possibly also the autoimmune hemolytic disease.     

ULTRACENTRIFUGATION STUDIES OF YELLOW FEVER VIRUS

1. It was possible to study in the ultracentrifuge by optical methods the behavior of yellow fever virus particles directly in the unaltered serum from infected monkeys. 2. The virus showed an extremely high light absorption in the spectral range of 320 to 440 mµ, which seemed to be its intrinsic property. In a 1 cm. thickness of fluid, the small amount of virus present in unaltered infective serum absorbed about as much light (approximately 25 per cent) in the middle of this range as did all the normal serum proteins present in a combined concentration some 1000 times as great. 3. The concentration of virus in the unaltered serum was found to be of the order of 0.00005 gm. per cc. 1 cc. of a 10^–9 dilution, which, as has been shown, may constitute a minimal infective dose for monkeys, would contain approximately 10,000 virus particles. The probability that most of the virus particles were in the inactive form is discussed. 4. In infective serum having a viscosity of 14 millipoises, the particles sediment with a blurred boundary at rates lying between approximately 18 and 30 x 10^–13 cm./sec./dyne. Evidence indicates that this spread is the result of an aggregation or association phenomenon. 5. Computations of size are in approximate agreement with those made from ultrafiltration studies. On the assumption that the density of the virus particle is near that of protein, its volume is computed to be at least that of a spherical particle having a diameter of 12 mµ. An assumed density of 1.15 gm. per cc. yields a diameter of 19 mµ, considering the shape as spherical.     

Surface-activated microtiter-plate microarray for simultaneous CRP quantification and viral antibody detection

Microarrays are widely used in high-throughput DNA and RNA hybridization tests and recently adopted to protein and small molecule interaction studies in basic research and diagnostics. Parallel detection of serum antibodies and antigens has several potential applications in epidemiologic research, vaccine development, and in the diagnosis of allergies, autoimmunity, and infectious diseases. This study demonstrates an immobilization method for immunoassay-based microarray in conventional 96-well polystyrene plates for a serologic diagnostic method combined with quantitative C-reactive protein (CRP) assay. A synthetic peptide (HIV-1), a recombinant protein (Puumala hantavirus nucleocapsid), and purified virus preparations (Sindbis and adenoviruses) were used as antigens for virus-specific antibody detection and monoclonal anti-CRP antibody for antigen detection. The microarray was based on conventional enzyme immunoassays and densitometry from photographed results. Peptide and recombinant antigens functioned well, while whole virus antigens gave discrepant results in 1 out of 23 samples from the reference method, tested with human sera with various antibody responses. The CRP results were in concordance in the concentration range 0.5–150 mg/L with 2 commercially available CRP assays: ReaScan rapid test (R² = 0.9975) and Cobas 6000 analyzer (R² =0.9595). The results indicate that microtiter plates provide a promising platform for further development of microarrays for parallel antibody and antigen detection.     

SYNTHESIS OF VIRUS-SPECIFIC POLYMERS IN ADENOVIRUS-INFECTED CELLS: EFFECT OF 5-FLUORODEOXYURIDINE

Biochemical synthesis in adenovirus-infected HeLa cells was studied utilizing 5-fluorodeoxyuridine (5-FUDR), a potent inhibitor of deoxyribonucleic acid production. Synthesis of saline-soluble DNA and infectious virus was completely suppressed by addition of the analogue to cells as late as 10 hours after infection. The inhibitory effect of this compound was totally reversed by addition of 10^–6 M thymidine to the culture medium. Synthesis of DNA essential for virus production began 10 hours after infection and was completed by 16 hours after infection. These data support the hypothesis that the saline-soluble DNA is a precursor of infectious virus particles. Studies of antigen production indicated that formation of virus-specific proteins was directly dependent upon synthesis of DNA.     

CHARACTERIZATION OF A HUMAN ACUTE PHASE PROTEIN FOUND IN ASSOCIATION WITH RUBELLA VIRUS INFECTION

A precipitating antigen, rho, was first detected in the blood of persons with rubella and in rubella virus-infected cell culture fluids (1). Partially purified antigens from both sources were examined and shown to have similar properties, although antigen from serum sedimented more heterogeneously, with estimated coefficients from 15 to 21 S, while that from culture fluids sedimented in the 11–14 S region. In each case, antigen was located in the β-1 zone after electrophoresis in agarose, and at a density of 1.305 g/ml after centrifugation in CsCl. Stability characteristics were typical of protein antigens. Immunofluorescent microscopy revealed that rubella virus induced the appearance of rho antigen scattered throughout the cytoplasm of infected cells. When cells containing antigen were exposed for 24 h to 5 µg/ml actinomycin D rho was no longer detectable, indicating the probable cellular origin of the antigen. Also, titers in medium of infected cultures showed a reduction after actinomycin treatment, but levels of the virus-specified antigen, iota, were relatively unaffected. Rho appears to be a protein common to man and many animals. In vitro, it was induced by rubella virus and by adenovirus. In vivo, rho titers were shown to be elevated after rubella virus infection and, to a lesser extent, after infection with certain other viruses. High titers were also demonstrated in women late in pregnancy and in patients with rheumatoid arthritis. In man and the chimpanzee, the appearance and decline of rho in the blood after rubella virus infection were temporally similar to the patterns of CRP, although rho seemed to be a more sensitive indicator of infection. The data presented indicate that rho is a newly recognized acute phase protein inducible by certain virus infections and by other unidentified stimuli present prominently in pregnancy and rheumatoid arthritis.     

ON THE NATURE OF TRANSPLANTATION IMMUNITY IN THE ADENOVIRUS TUMOR SYSTEM

The existence of a virus-induced, virus-specific transplantation, antigen in adenovirus 12-induced CBA mouse tumors was demonstrated. The antigen is virus-specific, but not related to structural virion or T antigens. It is a weak antigen, and required immunization with whole, infectious adenovirus 12 to produce considerable immunity. Comparable immunity could not be achieved with homologous cellular or subcellular materials, but some indication of enhancement was produced with low tumor dose. Immunization required at least 2 wk and was mediated by immune lymphoid cells. Serum of immunized animals showed no demonstrable cytotoxicity or enhancement. Animals immunized with virus and Freund's adjuvant showed diminished transplantation immunity, although these animals were actively immunized against adenovirus type 12 structural virion antigens.     

AUSTRALIA ANTIGEN (A HEPATITIS-ASSOCIATED ANTIGEN) : PURIFICATION AND PHYSICAL PROPERTIES

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated ³²P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85°C but was stable at 56°C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.     

WILD-TYPE GROSS LEUKEMIA VIRUS AND THE PATHOGENESIS OF THE GLOMERULONEPHRITIS OF NEW ZEALAND MICE

The pathogenesis of the spontaneous glomerulonephritis of NZB and (NZB x NZW) F₁ hybrid mice is related at least in part to the formation of natural antibody against antigens of the G (Gross) system, and apparently to the deposition in the glomeruli of immune complexes of G natural antibody with G soluble antigen (GSA), type-specific antigen specified by wild-type Gross leukemia virus. G natural antibody and GSA are detectable in the acid-buffer eluate of the kidneys of NZB mice during the course of the glomerulonephritis. (NZB x NZW) F₁ hybrid mice develop glomerulonephritis and produce GSA and free G natural antibody earlier in life than do NZB mice. The proteinuria manifestation of the gomerulonephritis of (NZB x NZW) F₁ hybrid mice becomes increasingly prevalent as GSA undergoes immune elimination from the circulation. Gross leukemia virus-specified antigens together with bound immunoglobulins are located in the glomerular lesions of (NZB x NZW) F₁ hybrid mice, both in the mesangium as observed in NZB mice and also in the wall of the peripheral capillary loops of the glomeruli.     

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

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

PREPARATION AND ANTIGENICITY OF M PROTEIN RELEASED FROM GROUP A, TYPE 1 STREPTOCOCCAL CELL WALLS BY PHAGE-ASSOCIATED LYSIN

The lysis of Group A type 1 streptococcal cell walls by phage-associated lysin has been described. In the preparation of lysin, a new semisynthetic non-protein media to support growth of the propagating strain of Group C streptococci was employed. Following lysis of the cell walls, the resulting digest was partially purified by ion-exchange chromatography and ammonium sulfate precipitation. In addition to M protein, the resulting preparation (called lysin M protein) contained the group-specific carbohydrate and the T protein—but did not contain antigens, detectable by precipitin tests, which cross-reacted with absorbed heterologous group or type antisera. Capillary precipitin reactions between the lysin M protein and type-specific antiserum did not occur in the presence of high ionic strength buffers; these buffers did not similarly affect precipitin reactions of acid M protein. Type 1 lysin M protein is shown to be a good antigen. A total of 1.5 mg. injected intradermally in saline produced bactericidal antibody in eight of nine rabbits; when injected in adjuvant in one rabbit, protective serum antibodies developed. Streptococci grown in sera from seven of nine rabbits immunized with lysin M protein demonstrated significantly longer chains than when grown in normal rabbit serum. Antibody as demonstrated by each of these three tests was shown to be type-specific. No local or systemic toxicity was noted following intradermal injection in rabbits of lysin M protein.     

Empty capsids in column-purified recombinant adenovirus preparations

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.     

A STUDY OF THE ANTIGENS INVOLVED IN ADENOVIRUS 12 TUMORIGENESIS BY IMMUNODIFFUSION TECHNIQUES

The use of the agar gel diffusion technique has established the presence of three distinct antigenic reactions in the sera of Ad. 12 tumor-bearing hamsters. Only one of these antigens is directly demonstrable in the tumor. This "tumor" antigen is also formed during early stages of the infectious cycle in tissue culture cells. Other antigens present in the tumor, but only demonstrable indirectly with the use of antibody-containing serum of tumored hamsters, are the classical type-specific C antigen, and a new antigen, termed D. Of ninety-eight Ad. 12 tumored hamster sera, six reacted in gel diffusion with virus and tumor preparations, and thirty-one with tumor only. Sera which reacted in gel diffusion with viral antigen uniformly bad neutralizing antibody and high titers of CF antibody against viral and tumor antigens; however, many sera with comparable antibody titers did not react with the virus in gel diffusion. Sera which reacted in gel diffusion only with tumor antigen also had high CF antibody titers, but there was no correlation with neutralizing antibody.     

PERSISTENCE OF GROUP A STREPTOCOCCAL CELL WALLS RELATED TO CHRONIC INFLAMMATION OF RABBIT DERMAL CONNECTIVE TISSUE

Antibodies specific for the mucopeptide and group-specific C polysaccharide antigens of Group A streptococcal cell walls were prepared by acid dissociation of immune precipitates, and labeled with either fluorescein or ¹²⁵I. Employing both fluorescent and radioautographic procedures the persistence of the antigens was followed in skin sites injected with cell wall fragments. Both antigens persisted within macrophages for at least 54 days in those animals which developed no chronic tissue response. In animals which did develop chronic nodular lesions the concentration of antigen decreased as the inflammatory process subsided. Lesion activity was thus associated with the presence of cell wall material. The fate of these antigens was also determined following the intradermal injection of intact Group A streptococcal cells. Cell wall antigens persisted in the tissue site considerably longer than morphologically identifiable streptococci, indicating that cell wall fragments are released during dismantling of streptococci in phagocytic cells.     

CENTRIFUGATION AND ULTRAFILTRATION STUDIES ON ALLANTOIC FLUID PREPARATIONS OF INFLUENZA VIRUS

A synthetic density gradient technique has been applied to the study of the PR8 and Lee strains of influenza virus in the angle centrifuge. The method counteracted convective disturbances and permitted about a fiftyfold improvement in clearing supernatant fluids of virus. Sedimenting boundaries of infective virus particles, hemagglutinin, and complement-fixing antigen were obtained in the angle centrifuge and correlated with boundaries observed optically in the ultracentrifuge. The sedimentation constant of infective Lee virus particles is approximately 800 Svedberg units, while that of PR8 virus is only about 700. On the assumption of spherical shape, these values correspond to approximate diameters of 85 and 80 mµ respectively. These values agree with those obtained by filtration with graded collodion membranes. The concentration of primary virus particles in untreated allantoic fluid preparations of PR8 or Lee virus is of the order of 0.01 per cent. The primary infective particles are identical with the hemagglutinin and the complement-fixing antigen to a large extent. However, allantoic fluid preparations of PR8 virus also show a slightly inhomogeneous group of particles with an average sedimentation constant of 460 S, which are adsorbed by and eluted from red blood cells yet appear to be non-infective. In addition the virus preparations contain a small amount of "soluble antigen" which sediments more slowly than the virus and is not adsorbed by red blood cells. This soluble antigen is probably associated with material which was observed optically in the ultracentrifuge to sediment at rates ranging from very low values up to that characteristic of the primary virus boundary. This distribution of rate makes it seem likely that the material represents disintegrated virus particles. Calculations based on the experimental results obtained indicate that of the order of 10 influenza virus particles are required to produce infection of chick embryos or mice with the PR8 virus. While a comparable number is required with Lee virus for infection of chick embryos, about 10,000 particles are necessary for infection of mice. The ratio of hemagglutinin to red blood cells required to produce 50 per cent agglutination with dilute virus suspensions in the standard test is roughly 1.