Replication of human immunodeficiency virus in two T-cell lines and their application as antigens for immunofluorescence

Two T-cell lines, TALL-1 and CCRF-CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF-CEM, but, as the antigen for IF, TALL-1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.     

Combined Fluorescent-Antibody and Electron Microscopy Study of Marek''s Disease Virus-Infected Cell Culture

Duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cultures infected with Marek's disease virus were studied by combined fluorescent antibody and electron microscopy techniques. In both DEF and CEF cultures, cells containing immunofluorescent (IF) antigen also contained herpesvirus particles; conversely, cells lacking this antigen lacked herpesvirus particles. Two morphologically distinct IF antigens were detected in the cytoplasm. (i) A granular antigen in the perinuclear region was brightly stained with the conjugated antibody. This antigen was composed of a granular mass of osmiophilic material and did not contain virions. (ii) A diffuse antigen, present throughout the cytoplasm of infected cells, was less brightly stained. The area of the cell with the highest concentration of this antigen contained small vesicles, folded membranes, and fine electron-dense granules. Naked virions were occasionally seen in these areas. A diffuse nuclear IF antigen was occasionally seen in infected cells. This antigen was often separated from the nuclear membrane and the nucleolus by a clear margin. The intranuclear IF antigen was composed of a fine granular aggregate and naked herpesvirus particles which were randomly distributed throughout the nucleus. Viral capsids in antibody-treated cells were coated with fine filamentous material.     

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.     

Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup.

An antigenic determinant common to the major capsid polypeptide (VP1) of simian virus 40 (SV40) and polyoma virus is described. Antisera prepared against intact viral particles reacted only with cells infected with the homologous virus by immunofluorescence tests (IF). However, antisera prepared against disrupted SV40 particles reacted in IF with both polyoma- and SV40-infected permissive cells. The cross-reaction with polyoma was localized to VP1 by the following evidence. (i) The IF cross-reaction was inhibited by preincubation of the antiserum with purified SV40 VP1; (ii) purified radiolabeled polyoma VP1 was precipitated by the cross-reactive serum, and this reaction was inhibited by unlabeled SV40 VP1; (iii) other antisera prepared against purified SV40 VP1 or polyoma VP1 reacted in IF with both SV40- and polyma-infected permissive cells. These cross-reacting antisera also reacted in IF with permissive cells infected with BK virus, rabbit kidney vacuolating virus, and the stumptailed macaque virus, suggesting that all members of the polyoma-SV40 subgroup share a common antigenic determinant located in their major capsid polypeptides.     

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

Location of Ferritin-Labeled Concanavalin A Binding to Influenza Virus and Tumor Cell Surfaces

Concanavalin A (Con-A) was linked to ferritin with glutaraldehyde and chromatographed on Sepharose 6B to separate unconjugated Con-A and ferritin from covalently cross-linked molecules. Ehrlich ascites tumor cells were infected with WSA influenza virus, stained at intervals with the ferritin-labeled Con-A and examined by electron microscopy. The surfaces of most mature viruses were specifically stained, providing direct evidence that influenza viruses maturing in this cell type have exposed Con-A receptor sites. The ferritin cores of the staining reagent were found at an average distance of 21.3 nm from the virus membrane and 10.8 nm from the uninfected cell membrane. This finding was interpreted to mean that the population of Con-A receptor sites on influenza virus particles is located at an average distance from the virus membrane twice that of the population of Con-A receptor sites found on uninfected cells. The structural elements of viral membranes can provide a reliable means for evaluating electron microscopy staining reagents, thereby enhancing their usefulness as probes for the study of membrane relationships.     

Herpes-Type Virus of the Frog Renal Adenocarcinoma: I. Virus Development in Tumor Transplants Maintained at Low Temperature

Development of the herpes-type virus of the frog kidney tumor was investigated by electron microscopy and high-resolution autoradiography in eyechamber transplants of tumor maintained at 7.5 C for up to 27 weeks. Virus particles were first detected at 10 weeks in nuclei containing aggregates of dense granular material. The initial incorporation of a pulse of (3)H-thymidine into these aggregates indicated that they contained newly synthesized viral deoxyribonucleic acid. Capsids enclosing doubleshelled cores were labeled with (3)H-thymidine before capsids with dense cores, and intermediate core forms were observed, suggesting that the double-shelled core transforms into the dense core. Particles with dense cores were observed while being enveloped by budding through the inner membrane of the nuclear envelope, and subsequently while being unenveloped in passing through the outer membrane into the cytoplasm. Virus particles within the cytoplasm acquired fibrillar coats and budded into vesicles, from which they were released, in enveloped form, at the cell surface. Tubular forms and particles considerably smaller than virus particles were regularly encountered in infected nuclei, and the relationship of these forms to virus replication is discussed.     

Synthesis and anti-HIV properties of new hydroxyquinoline-polyamine conjugates on cells infected by HIV-1 LAV and HIV-1 BaL viral strains

To find new derivatives that block different virus strains entry in cells bearing specific surface receptors represent an interesting challenge for medicinal chemists. Here, we report the synthesis and the anti-HIV properties of a new series of analogues based on the introduction of quinoline moiety on various polyamine backbones, including polyazamacrocycles. Three compounds 7, 8, and 10 of this series were found active on PBMCs cells infected by HIV-1 LAV or by HIV-1 BaL, in contrast the well-known reference compound 1a (AMD 3100) was found only active on HIV-1 LAV strain.     

Morphological and immunological confirmation of the presence of chlamydiae in the gut tissues of the chesapeake bay clam,Mercenaria mercenaria

The original electron microscopic identification by other investigators in 1977 of chlamydiae in the gut tissues of the Chesapeake Bay hard clam (Mercenaria mercenaria) is corroborated and further supported by evidence ofChlamydia-specific immunofluorescence (IF). Our electron microscopy demonstrated that gut tissue cells were heavily infected with chlamydiae in all stages of development but the intrachlamydial phage-like particles reported in 1977 were not seen. Tissue sections stained with IF reagents were strongly positive, and IF was blocked in varying degrees with chlamydial antisera produced in a goat and a turkey. The IF-positive tissue sections contained intracytoplasmic inclusions that stained darkly with Lugol's iodine (indicating the presence of glycogen) while IF-negative tissues had little if any iodinestaining material. Furthermore, electron micrographs of chlamydiae-containing phagosomes showed numerous rosettes of electron-dense particles typical of glycogen. The presence of iodine-positive phagosomes with electron dense rosettes suggests that the organisms are glycogen-producing chlamydiae biochemically related toChlamydia trachomatis. Repeated attempts to cultivate chlamydiae from the clam tissues in cell cultures and laboratory animals failed.     

Morphogenesis of Avian Infectious Bronchitis Virus and a Related Human Virus (Strain 229E)

Avian infectious bronchitis virus (IBV) and strain 229E, a virus recently recovered from patients with colds, have been shown to possess a similar distinctive morphology in negatively stained preparations. An electron microscopic study of the morphogenesis of IBV in the chorioallantoic membrane and of strain 229E in WI-38 cells was performed. In infected cells, round electron-dense particles 82 mmu in diameter were observed to form by a process of budding from membranes of the endoplasmic reticulum and cytoplasmic vesicles. The particles in IBV-infected cells were similar in size and shape to those in strain 229E-infected cells but showed certain differences in internal structure. The evidence that the particles represent virions and the implications of these findings in the classification of this virus group are discussed.     

在昆虫细胞中同时表达蓝舌病毒VP3与VP7蛋白可装配成核心样颗粒

将蓝舌病毒（ＢＴＶ）１３型Ｓ７与Ｌ３基因同时插入杆状病毒双表达载体ｐＥａｓｔＢａｃＤｕａｌ,获得重组杆状病毒ｒｖＢａｃＢＴＶＰ３７。该病毒在昆虫细胞中同时高水平表达ＢＴＶ１３ ＶＰ３与ＶＰ７蛋白,可以高效自动装配出２０面体的６０￣７０ｎｍ空心颗粒。分析表明,所获颗粒为空心的ＢＴＶ核心样颗粒（ＣＬＰ）,其成分为ＶＰ３与ＶＰ７,不含ＢＴＶ其它任何蛋白与核酸。这种装配需要ＶＰ３与ＶＰ７的共同参与,二者缺     

RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts.     

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.