Immunoperoxidase Stain of Measles Antigen in Tissue Culture

A specific electron microscopy staining technique for measles antigen has been developed by using Vero cells infected with a subacute sclerosing panencephalitis (SSPE) measles virus strain and fixed in glutaraldehyde or formaldehyde. Peroxidase-labeled antibody was prepared according to the method of Avrameas (4). Sera from SSPE patients with high measles antibody titer as well as normal human sera with and without measles antibody were used. With both fixatives, specific labeling was obtained on the surface of infected cells, on the budding site, and on complete viral particles. The cell membrane staining sometimes had a patchy distribution in that the reaction was most intense on the surface projections in front of each nucleocapsid. This suggests modification of the cell membrane in association with the nucleocapsids. In contrast, no label was detected on the membranes of the cells during the latent period from penetration through maturation of the virus. In formaldehyde-fixed cultures, cytoplasmic inclusions were stained, and this label was located on the "fuzzy" material around the nucleocapsids. The smooth type of nucleocapsids, mainly seen in the nucleus, were never labeled. These findings suggest that the antigenic nature of the "fuzzy" nucleocapsids in the cytoplasm may be different from that of the "smooth" nucleocapsids. The immunoperoxidase method gives good resolution of viral antigenic sites at high magnifications under electron microscopy and may be of value in studies on the immunopathogenesis of SSPE and other chronic viral infections.     

Fine Structure of Cellular Inclusions in Measles Virus Infections

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus.     

Latency of Human Measles Virus in Hamster Cells

A latent system employing measles virus (Schwarz strain) was developed in hamster embryo fibroblasts (HEF). Measles virus-specific antigen was detected by immunofluorescence in 30 to 50% of HEF cells, and these cells released infectious virus when co-cultivated with a susceptible monkey cell line, BSC-1 cells. No infectious virus could be detected in the cells when measures were taken to exclude passage of viable latent cells onto the indicator BSC-1 cells. Infectious center assays demonstrated that about 1 in 10 of the latently infected cells in the population could release infectious virus. Infectious virus appeared within 6 hr after co-cultivation of the HEF cells with BSC-1 cells, as compared to 24 hr required for normal replication of measles virus in the BSC-1 cells. Furthermore, labeling of progeny virus ribonucleic acid (RNA) by using tritiated uridine, and inhibition of RNA or protein synthesis by 5-azacytidine or cycloheximide suggested that neither additional RNA nor protein synthesis is required after co-cultivation of the cells to effect early virus release. It can therefore be postulated that there is a block at a late step in virus replication in the latently infected hamster cells. The most obvious site would concern maturation of infectious virions at the cell membrane.     

Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Morphogenesis of Type 2 Parainfluenza Virus Examined by Light and Electron Microscopy

The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration.     

Rescue of a positive stranded RNA virus from antigen negative neuroblastoma cells.

Single cell clones of latently infected mouse neuroblastoma cells were isolated from a culture chronically infected with mouse hepatitis virus in the presence of an antiviral antibody. These cell clones did not produce infections virus or exhibit viral cytopathic effects during cultivation at 32, 37, or 39°C. Infectious virus was isolated from single cell clones via fusion with permissive cells using polyethylene glycol, but not after fusion with inactivated Sendai virus or following treatment with metabolic inhibitors. One cell clone (S-3) from which virus was rescued was negative for viral antigen by immunofluorescence. The S-3 cell clone and no demonstrable virus antigen by complement-fixation tests using cytoplasmic extracts or virus-specified proteins detectable by polyacrylamide gel electrophoresis. The rescued viruses exhibited a temperature dependent growth defect at 32°C and have been classified as cold sensitive mutants. This study suggests that a complete genome of a positive stranded RNA virus can remain latent in infected cells without the expression of detectable virus antigen.     

Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.     

Growth of Rio Bravo Virus in Cell Cultures

The growth of Rio Bravo virus (RBV) in eight cell culture systems was studied. Highest yields of virus were produced in BHK-21 (C13), L, and Vero cell lines, but L cells were resistant to low doses of virus. LLC-MK(2), HeLa, and human embryo skin cells produced moderate amounts of virus, but FL amnion and primary chick embryo fibroblasts supported little virus growth. Virus was rapidly inactivated by exposure to pH values below 7.0. Single-cycle growth in BHK-21, L, and LLC-MK(2) cell monolayers was characterized by a latent period of about 12 hr followed by rapid virus production that peaked at 36 to 48 hr. Vero cell cultures can remain chronically infected with RBV for more than 100 days. Such cultures show evidence of cell destruction, and their supernatant fluids contain virus at 10(4) to 10(5) log(10) per ml.     

Effect of Host Cell on Distribution of a Lysosomal Enzyme During Virus Infection

The time of appearance of a lysosomal enzyme, beta-glucuronidase, in the medium of cells infected with either measles virus or echovirus 6 varied with the host cell system. Replication and release of virus preceded leakage of beta-glucuronidase from green monkey kidney cells. In contrast, extracellular enzyme appeared before replication and release of virus in human amnion cells. Hydrocortisone depressed enzyme leakage but did not retard replication of measles virus or viral-induced cytopathology. The intracellular distribution of beta-glucuronidase in uninfected and measles virus-infected cells was also studied. Measles virus infection altered the position of particulate-bound beta-glucuronidase in linear sucrose gradients prior to substantial release of this enzyme intra- and extracellularly. At early stages in infection, most of the cell-associated virus banded with particulate-bound enzyme in the middle of the gradient. As infection progressed, separation of measles virus infectivity from enzyme activity occurred, and intracellular virus was recovered near the meniscus of sucrose gradients.     

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

Immune responses during measles infection in immunosuppressed Rhesus monkeys.

Rhesus monkeys immunosuppressed with horse anti-human thymocyte gamma-globulin (ATG) were infected with measles and simultaneously inoculated with sheep erythrocytes (SRBC), a thymus-dependent antigen, and with pneumococcal polysaccaride type III (SSS-III), a thymus-independent antigen. ATG treatment alone suppressed SRBC antibody production, had no effect on SSS-III antibody production, and effectively eliminated circulating T cells compared to nonsuppressed monkeys. ATG treatment of measles-infected monkeys resulted in delayed virus clearance and delayed antibody production compared to nonsuppressed infected monkeys. After cessation of ATG treatment, measles antibodies and T cells reached normal levels, and measles virus was eliminated. Thus, immune clearance of measles virus is T cell-dependent, but the relative roles of cellular- and humoral-mediated immunity in vivo could not be clearly separated. Also, measles infection was associated with a decreased T cell mitogen responsiveness of circulating lymphocytes but not of lymph node lymphocytes, suggesting an altered circulating pattern of the cells responsible for delayed hypersensitivity. Also, measles infection had no effect on T-dependent antibody production to SRBC.     

Cat Interferon inhibits Feline Leukaemia Virus Infection in Cell Culture

TRANSMISSION of feline leukaemia can be accomplished with tissue extracts from cases which occur naturally¹. Virus particles which are morphologically indistinguishable from the murine and avian C-type viruses are present in cats with the transmitted disease². Feline leukaemia virus (FeLV) replicates in cat cell cultures³ and infected cells are demonstrable by the indirect immunofiuorescent antibody test which detects FeLV group-specific antigen as granular punctate fluorescence in the cytoplasm of acetone fixed cells⁴; this method allows easy quantitation of the antiviral effect of interferon. We report the production and assay of feline interferon using the fluorescent antibody test with FeLV infected cat cell cultures.     

树类-细小病毒的初步研究

从树肾细胞培养物中分离出3株病毒,嗜在对数分裂期的TL细胞上复制,产生细胞病变和血凝素抗原,能凝集豚鼠和小鼠红细胞。交互血凝抑制实验表明3株病毒同属一个血清型。免疫酶染色显示抗原首先出现于细胞核。电镜观察负染标本病毒形态近似圆形和六角形,无胞膜,直径约30nm。血清学检查大多数树血清中含有该病毒抗体,证明是树的一种潜在病毒。初步鉴定为类细小病毒。     

Electron Microscopy of Measles Virus Replication

Replication of measles virus in HeLa cells was examined by electron microscopy with ultrathin sectioning and phosphotungstic acid negative staining methods. The cytoplasmic inclusion bodies consisted of masses of helical nucleocapsid which was similar in structure to the nucleocapsid found in measles virions. The cytoplasmic helical nucleocapsid appeared to align near the HeLa cell membrane, and the membrane differentiated into the internal membrane of the viral envelope and the outer layer of the short projections. The viral particles were released by a budding process involving incorporation into the viral envelope of membrane which was contiguous to but morphologically altered from the membrane of the HeLa cells. The intranuclear inclusion bodies were composed of tubular structures similar to those found in the cytoplasmic inclusion bodies. These structures aggregated to crystalline arrangement. The relationship between nuclear inclusion body and replication of measles virus was not clear.     

Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacteriol. 92:1792-1804. 1966.-After the development of marked cytopathic effects in HeLa cultures infected with the Edmonston strain of measles virus, renewed cell growth occurred, and HeLa sublines persistently infected with measles virus were obtained. Persistent infection has occurred in a large fraction of the cells of infected clonal lines for more than 300 to 500 cell generations during a period of 6 years. One mechanism by means of which infection was maintained in the clonal lines is transmission of virus or viral subunits from cell to cell at division. Continued subculture of the persistently infected populations resulted in the virtual disappearance of cytopathic effects, a marked decrease in the amount of extracellular virus, and alterations in the cytopathogenicity of virus recovered from persistently infected populations. The intracellular virus-host cell events in late passages of the infected clonal lines appeared to be similar to those in cells of primary infected cultures at early stages of infection, as judged by the pattern of viral immunofluorescence and the very low incidence of cells with intranuclear inclusion bodies. Cultures of the persistently infected clonal lines were highly resistant to super infection by parent Edmonston virus. Cultures of one of these clonal lines were just as susceptible as normal HeLa cultures to vaccinia, herpes simplex, and polio type 2 viruses, and a simian agent, with a possible low degree of resistance to the simian agent.     

Immunofluorescence and Cytochemical Studies of Visna Virus in Cell Culture

Sequential morphological changes occurring in sheep choroid plexus cells infected with visna virus were studied by direct immunofluorescence, acridine orange, and hematoxylin and eosin staining methods. Specific immunofluorescence was first detected in the perinuclear cytoplasm of solitary cells 24 hr after infection. As the infection progressed, viral antigen appeared in an increasing number of cells, and rounded globular cells with long slender processes harboring intense fluorescence were seen. Nuclear fluorescence was not observed in infected monolayers. Polykaryocytes formed within 6 hr after inoculation due to the direct cell-fusing effect of the virus inoculum did not show specific fluorescence. Viral antigen was found, however, in the cytoplasm of multinucleated giant cells in cover slips harvested after new infective virus had been released, and later in the course of infection circular fluorescent inclusions were seen in the cytoplasm of polykaryocytes. Comparable eosinophilic inclusions were observed in hematoxylin and eosin preparations, and acridine orange staining of infected monolayers demonstrated similar inclusions which fluoresced with the color characteristic of single-stranded nucleic acid and were susceptible to digestion with ribonuclease. Visna virus appears to be a ribonucleic acid virus which replicates in the cytoplasm.     

Further Studies on the Effect of 5-Iododeoxyuridine on Polyoma Virus Multiplication in Mouse Cells

5-Iododeoxyuridine (IUDR) inhibited production of infectious polyoma virus in mouse embryo cells and mouse kidney cells in culture. Deoxythymidine reversed its effect. IUDR did not inactivate infectivity of free virus particles. IUDR did not prevent adsorption and penetration of polyoma virus to cells. The events sensitive to IUDR treatment occurred at around 20 hours after infection. The cytopathic effects of polyoma virus, including emergence of DNA containing-inclusions in the nucleus, were observable in infected cells in which viral replication was completely arrested by IUDR. It was shown by fluorescent antibody technique in infected mouse embryo cells and by complement fixation test in infected mouse kidney cells that IUDR inhibited completely the synthesis of viral antigen. No virus-like particles were demonstrated in the IUDR-treated infected-mouse kidney cells by electron microscope examinations.     

Modulation of Immune System Function by Measles Virus Infection: Role of Soluble Factor and Direct Infection

Measles virus infection can result in a variety of immunologic defects. We have begun studies to determine the basis for the lack of immune responsiveness to antigen and mitogen following infection. Here we present data showing that Epstein-Barr virus-transformed B-cell lines infected with measles virus produce a soluble factor that can inhibit antigen-specific T-cell proliferation and inhibit the proliferation of uninfected B cells. The soluble factor was neither interleukin-10, transforming growth factor β, nor alpha/beta interferon. B cells infected with measles virus or treated with the soluble factor were unable to present antigen to T cells in a manner that supported antigen-specific proliferation. This could represent one mechanism of how measles virus limits T-cell expansion. However, we found that once CD4⁺ or CD8⁺ T cells were activated, their cytolytic activity was intact whether infected with measles virus or treated with soluble factor. Thus, while slow to be generated these cytoxic cells could participate in viral clearance.