Ultrastructural localization of rotavirus antigens using colloidal gold.

Colloidal gold was used to localize six of the ten known proteins of the simian rotavirus SA11 within infected cells by ultrastructural immunocytochemistry. Monospecific or monoclonal antibodies to selected structural and nonstructural proteins were the primary antisera. The major outer capsid glycoprotein, VP7, was associated with nonenveloped particles, with particles de-enveloped by Triton X-100 and with both nuclear and cytoplasmic inclusions. The protease-sensitive outer capsid protein, VP3, was also found on nonenveloped and de-enveloped particles. The major inner capsid protein, VP6, was accessible to antibodies on some of the nonenveloped particles (presumably single-shelled particles) and on the de-enveloped particles. A monospecific antibody to the gene 11 product, believed to be a precursor to a minor structural protein, VP9, reacted strongly with viroplasmic inclusions. Virus particles were weakly labeled by this antibody. NS35, a nonstructural SA11 protein, was found only in the viroplasms. NS29, a nonstructural glycoprotein, was localized to the cytoplasmic side of the endoplasmic reticulum membrane and to the inside of enveloped virus particles. These data support the hypothesis that NS29 facilitates budding of the virus particles and acquisition of the outer capsid layer.

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.

Identification of rotavirus particle types.

Negative-contrast electron microscopy of purified rotavirus particles reveals two particle types: single-shelled and double-shelled particles. The relationship of these particle types, seen by negative staining, to the enveloped and various types of nonenveloped particles seen in thin sections of virus-infected cells was determined. Thin-section and negative-contrast electron microscopic analyses were performed on cell lysates from simian rotavirus. SA11-infected cells and on highly purified double- and single-shelled particles. In thin sections, double-shelled particles appeared as smooth-edged ovals containing dense nucleoids, whereas single-shelled particles had ragged edges and threads of material extending from their centers. The majority of nonenveloped particles seen in thin sections of infected cells were identified as double-shelled particles. Enveloped particles showed typical membrane structure and were observed rarely in crude rotavirus stocks, although they constitute about 10% of the particles within infected cells. It is hypothesized that the enveloped form is a transient one and the envelope is lost in the endoplasmic reticulum of the host cells. Finally, the 50-55 nm type IV particles seen within lysosome-like bodies in infected cells were identified as subviral particles formed from input virions.

Effects of tunicamycin on rotavirus morphogenesis and infectivity.

The functions of the two rotavirus glycoproteins were investigated by using tunicamycin and a variant of SA11 rotavirus having nonglycosylated VP7. Results showed that glycosylation of VP7 is not required for normal viral morphogenesis and infectivity and suggested that the nonstructural glycoprotein is involved in assembly of the outer capsid.

Localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry.

Virus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, mol. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.

In vivo and in vitro ultrastructural alterations induced by human immunodeficiency virus in human lymphoid cells.

The ultrastructural alterations induced by human immunodeficiency virus (HIV) in human lymphoid cells have been evaluated. Electron microscopic examination of peripheral blood mononuclear cells (PBMC) from 14 male homosexuals with confirmed acquired immunodeficiency syndrome (AIDS) or AIDS-related complex revealed that tubuloreticular inclusions were present in 5-15% of the cell sections from each case. In 5 of 14 cases, cylindrical confronting lamellae were found in 1-2% of the cell sections. No retrovirus-like particles or surface membrane alterations were detected. Neither of these structural alterations was observed in control PBMC obtained from six HIV-seronegative, hepatitis B virus surface antigen (HBsAg)-positive carriers or in 11 healthy subjects. When primary cultures of CD4+-enriched lymphocytes were infected in vitro with HIV, tubuloreticular inclusions could be detected in 3-10% of the cell sections, but no cylindrical confronting lamellae-like structures were found. In contrast, neither of these alterations were seen in uninfected or HIV-infected H9-HT continuous cell lines. These in vivo and in vitro studies indicate that there is an association between the appearance of the tubuloreticular inclusions and cytopathic HIV infection, although no correlation between cytopathic changes and active viral replication was observed at the single cell level. Further studies will be required to establish the mechanism(s) of formation of the tubuloreticular inclusions and to determine their prognostic potential.

Electron microscopy procedure influences detection of rotaviruses.

Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses.

Rotaviruses code for two types of glycoprotein precursors.

Rotaviruses are nonenveloped viruses that code for two glycoproteins: a structural glycoprotein (VP7) and a nonstructural glycoprotein (NS29). The precursor to VP7 (37K) was shown to contain a 1.5K cleavable signal sequence. The 37K precursor was authentically processed (signal sequence cleaved and the polypeptide "core" glycosylated) when synthesized in a cell-free system supplemented with dog pancreatic microsomes. Similar experiments were performed with the nonstructural glycoprotein precursor (20K); however, the 20K precursor contained an integral (noncleavable) signal sequence. Both precursors were inserted into membranes cotranslationally and both glycosylated products underwent posttranslational oligosaccharide processing. The results suggest a morphogenetic scheme for the simian rotavirus SA11.

Cytomegalovirus antigen within human arterial smooth muscle cells.

Arterial tissues from carotid artery plaques or from punch-biopsy samples of uninvolved areas of the aorta were removed from 132 patients with atherosclerosis during blood-vessel surgery. Cells morphologically identical to smooth muscle cells were cultured from 26 to 126 plaque samples and from 6 of 6 punch-biopsy samples. Immunofluorescence tests of these cells showed that more than 25% of the cell cultures from both types of sample contained antigens of human cytomegalovirus (CMV) but not of herpes simplex virus type 1 or type 2. Replicating CMV was not detected by electron microscopy in the antigen-positive cells, suggesting that the artery walls may be a site of CMV latency.

Fusion as a mediator of cytolysis in mixtures of uninfected CD4+ lymphocytes and cells infected by human immunodeficiency virus.

We describe an unusual type of cytopathology in which uninfected CD4+ (helper/inducer) cells (cells expressing the human leukocyte antigen CD4) interact with cells persistently infected with the human immunodeficiency virus (HIV). Prior antigenic stimulation was not required, since CD4+ cells taken either from healthy persons without anti-HIV antibodies or from individuals with anti-HIV antibodies were capable of inducing cytolysis. Neither CD8+ (suppressor/cytotoxic) nor CD16+ (natural killer) cells mediated the reaction. Light microscopic and autoradiographic studies revealed that, prior to cytolysis, multinucleated giant cells were formed from fusions between HIV-infected cells and large numbers of uninfected CD4+ lymphocytes. These data may explain the paradox that exists in vivo in which a dramatic depletion of CD4+ lymphocytes occurs in the presence of a small number of HIV-infected CD4+ cells. These new insights into the pathogenesis of acquired immunodeficiency syndrome (AIDS) may lead to future therapeutic strategies.

High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis.

157 caucasian male patients undergoing vascular surgery for atherosclerosis and a matched control group of patients with high cholesterol levels were screened for antibodies to cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV1) and type 2 (HSV2), indicative of persistent infection. The prevalence of CMV antibodies was higher in the surgical group than in the control group (90% and 74%, respectively), and a significantly greater percentage (p less than 0.001) of surgical cases than controls had high titres of CMV antibodies (57% and 26%, respectively). Small but not significant differences in antibodies to HSV1 were observed, and there were no differences in HSV2 antibody titres. For each virus there was no correlation between antibody titre and blood levels of total cholesterol or triglycerides. It is suggested that periodically activated virus may have a role in the pathogenesis of atherosclerosis.

Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.