Biophysical characterization of GB virus C from human plasma.

Viral characterization studies were carried out on GB virus C (GBV-C) RNA positive plasma from normal human donors and from donors co-infected with GBV-C and hepatitis C virus (HCV). GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and probe hybridization in a single tube assay. Sequential filtration of GBV-C positive plasma indicated that GBV-C RNA is associated with a particle 50-100 nm in diameter. The peak of GBV-C RNA in sucrose gradients was observed at a buoyant density of 1.05-1.13 g/ml. GBV-C RNA titer was reduced following treatment with chloroform or with five detergents indicating that GBV-C has a lipid-containing envelope. Sucrose density gradients and self-forming cesium chloride gradients of detergent-treated GBV-C showed a shift in the RNA peak to heavier buoyant density only when RNase inhibitor (RNasin) and high detergent concentrations were present. The treated material was non-filterable and the RNA had a density of > 1.5 gm/ml.

Seroprevalence of GB virus C and persistence of RNA and antibody.

Exposure to GB virus C (GBV-C) was determined in several U.S. populations by both reverse-transcription-polymerase chain reaction (RT-PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell-expressed GBV-C envelope protein, E2 (GBV-C E2). Most individuals exposed to GBV-C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV-C RNA positive and GBV-C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV-C exposure was 89.2%. Serial bleed specimens tested for GBV-C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV-C E2. In other exposed individuals who tested negative for GBV-C RNA, antibodies to E2 appear to be similarly long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV-C RNA and GBV-C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV-C infection require both antibody and nucleic acid detection.

Molecular and serologic analysis in the transmission of the GB hepatitis agents.

Two flavivirus-like genomes have recently been cloned from infectious tamarin (Saguinus labiatus) serum, derived from the human viral hepatitis GB strain, which is known to induce hepatitis in tamarins. In order to study the natural history of GB infections, further transmission studies were carried out in tamarins. Reverse-transcription-polymerase chain reaction and enzyme-linked immunosorbant assays were developed for the detection of RNA and antibodies associated with the two agents, GB virus-A and GB virus-B. The infectivity of both of these agents was demonstrated in tamarins to be filterable through a 0.1 micron filter. Two distinct genomes were identified in the serum of eight of the infected tamarins, while in four tamarins, the genomes were detected independently of each other. Although specific antibodies to the GB virus-B epitopes were detected in the serum of animals inoculated with both agents or GB virus-B alone, antibodies to putative epitopes specific to GB virus-A were not detected in any of the animals. All tamarins inoculated with serum containing GB virus-B exhibited an elevation in liver enzyme levels after inoculation. Elevations of serum liver enzyme levels did not occur when GB virus-A was the only agent detected in the serum. Infection with the original infectious tamarin inoculum conferred protection from reinfection with GB virus-B but not with GB virus-A.

Frequency of cells positive for HIV-1 p24 antigen assessed by flow cytometry.

OBJECTIVE: Markers of HIV disease progression such as soluble p24 antigen detection and CD4 lymphocyte depletion are most useful in the later stages of HIV disease and are relatively insensitive as therapeutic monitors. Flow cytometric detection of HIV-1 replication in CD4 lymphocytes was evaluated for use as a marker in predicting disease progression earlier in the course of HIV disease. DESIGN: To determine whether the number of HIV-1-infected CD4 cells, as measured by p24 antigen detection, can be correlated with disease progression, we used flow cytometry to detect intracellular HIV-1 p24 in CD4 lymphocytes from HIV-1-seropositive subjects at all stages of HIV disease. METHODS: Mononuclear cells from HIV-1-seropositive subjects and uninfected control subjects were permeabilized and stained with anti-HIV-1 p24 monoclonal antibodies. The cells were then stained with a fluorescein isothiocyanate-conjugated goat antimurine immunoglobulin G followed by a phycoerythrin-conjugated monoclonal anti-CD4 antibody. The percentage of p24-positive CD4 lymphocytes was compared with absolute CD4 counts, soluble p24 detection and Walter Reed classification. RESULTS: CD4 lymphocyte absolute counts and the percentage of CD4 lymphocytes declined as the Walter Reed classification indicated disease progression. The mean percentage of p24 antigen-positive CD4 lymphocytes increased with disease progression. Only 30% of Walter Reed stage 6 subjects were soluble p24 antigen-positive, whereas 68% were cellular p24 antigen-positive. CONCLUSION: The percentage of p24 antigen-positive CD4 lymphocytes increased as HIV disease progressed. Flow cytometric quantitation of p24 antigen-positive CD4 cells is a useful method of monitoring in vivo HIV replication and disease progression.

Evaluation of a flow cytometric model for monitoring HIV antigen expression in vitro.

Using flow cytometry, monoclonal antibodies to the HIV proteins p24, gp41 and p17 were evaluated for their ability to detect HIV antigens associated with HIV-infected T cells. Mixtures containing varying ratios of HIV-infected and uninfected cells were subjected to analysis with these monoclonal antibodies. In most cases, the monoclonal antibodies identified the correct ratio of HIV-infected cells to uninfected cells in the mixtures tested. An HIV anti-p24 monoclonal antibody was selected for further studies. Flow cytometric analysis was performed on various populations of cells including uninfected, acutely infected and chronically infected cells. Based on cell population fluorescence intensity three distinct regions were identified. In the first region were cells having low level fluorescence that were considered negative for HIV antigens, a profile detected in uninfected cells, and in the majority of cells in the first days following acute HIV infection. In the second region were those cells exhibiting strong fluorescence such as chronically infected cells or acutely infected cells several days after infection. A third region was identified containing cells that were intermediate in fluorescence intensity. Cells exhibiting intermediate intensity fluorescence appeared to have low concentrations of HIV p24 antigen associated with them either through viral adsorption and uptake or through low level virus expression. These intermediate region cells appeared in the early stages following acute infection, and also when chronically infected cells and uninfected cells were permeabilized together, suggesting a 'leaching' of HIV proteins from highly infected cells to uninfected cells. This leaching type of phenomenon could present problems in determining gating parameters for positive cells since uninfected cells that have associated HIV antigens exhibit higher fluorescence intensity than uninfected cells.

Altered lymphocyte phenotypes and proliferative responses in chimpanzees infected with hepatitis C virus.

Peripheral blood mononuclear cells (MNC) from three chimpanzees infected with hepatitis C virus (HCV) and from two uninfected animals were analyzed by monoclonal antibody phenotyping using flow cytometry. Significant differences in numbers of MNC's expressing cluster designation (CD) phenotypes CD4, CD14, CD19, and CD45RA were found. Additionally, significant differences in MNC proliferation in response to mitogens were also found. This altered proliferative capacity and cellular phenotype profile may be important markers in studying the pathogenesis of chronic HCV disease.

The effect of interleukin 4 (BSF-1) on infection of peripheral blood monocyte-derived macrophages with HIV-1.

The effects of various cytokines were examined in an in vitro model of human immunodeficiency virus type 1 (HIV-1) infection of human peripheral blood monocyte-derived macrophages (MDM). Monocytes were obtained from blood of normal donors by Ficoll/hypaque gradient centrifugation and adherence. These cells were allowed to mature in the presence of varying concentrations of cytokines. After five days in culture, cells were harvested, counted, and inoculated with S5G7, an HTLV-IIIB subclone. The cells were replated in the presence of the same concentrations of cytokines. Culture supernatants were sampled over 28 days for p24 antigen (Ag) as measured by Ag capture assay. In repeat experiments, the following observations were made: 1. MDM from some donors could be infected only in the presence of tumor necrosis factor-alpha (TNF-alpha), granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 4 (IL-4); 2. The effect of GM-CSF was variable; TNF alpha also enhanced HIV replication above controls; 3. IL-4 was the most potent enhancer of HIV-1 replication in MDM of the cytokines tested, inducing p24 Ag levels 75-230 times those seen in control cultures run simultaneously. This effect was dose dependent. Ag production was not observed until Day 14 postinfection in most experiments. Multinucleated giant cell formation was observed only in the presence of IL-4.

Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis.

Serum and plasma samples were collected from blood donors who were confirmed positive for antibodies to HIV-1 in the United States, and from blood donors and individuals in West Africa and Portugal who were positive for antibodies to HIV-1, HIV-2, or both. Western blots and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) radioimmunoprecipitation assays (RIPA) utilizing native HIV-1 and HIV-2 proteins were performed on these specimens to determine the ability of these procedures to discriminate between HIV-1 and HIV-2 infections. Extensive serologic cross reactivity between HIV-1 and HIV-2 p24 was found in both populations. Antibody reactivity to the envelope protein gp120 was able to discriminate 20 of 20 (100%) U.S. specimens as HIV-1 infections. In specimens from West Africa and Portugal, Western blot and RIPA were in complete agreement on 33 of 42 samples (78.6%). Among these 33 specimens, 10 were found to be reactive for antibodies to HIV-1 only, 10 were reactive to HIV-2 only, and 13 were considered to be dually reactive, having antibodies reactive with both HIV-1 gp120 and HIV-2 gp120. Nine of the 42 specimens were discordant by Western blot and RIPA classification, being dually reactive by one procedure and reactive with only one viral gp120 by the other technique. Because of the serological cross reactivities between HIV-1 and HIV-2, in certain populations it is difficult to ascertain whether an individual is infected with HIV-1, HIV-2, a new viral type, or whether the individual is infected simultaneously with multiple viruses. More specific tests such as viral isolation or molecular probes may be necessary to distinguish between infections with these viruses in certain populations.

Effects of growth temperature on the ingestion and killing of clinical isolates of Listeria monocytogenes by human neutrophils.

In this study, we compared three human isolates (F5380, Scott A, and Murray B) and one laboratory strain (EGD) of Listeria monocytogenes for their resistance to ingestion and killing by human neutrophils. We observed no substantial difference in killing among these strains when they were grown at 37 degrees C. Because it is likely that listerial growth occurs at lower temperatures during food-borne outbreaks of listeriosis, we also compared these strains after they were grown at 22 and 4 degrees C. A general reduction in the ability of human neutrophils to kill L. monocytogenes was observed as the temperature at which the listeriae were grown decreased. Growth at 4 degrees C significantly decreased the killing of all four strains of L. monocytogenes by human neutrophils; two strains (EGD and F5380) were more resistant to killing than were the other two strains (Scott A and Murray B). No obvious relationship was noted between the chemiluminescence response of neutrophils to opsonized listeriae and the ability of the neutrophils to kill listeriae in vitro. Growth at 4 degrees C, however, significantly increased the resistance of L. monocytogenes to killing by hydrogen peroxide.