Evaluation of two rapid screening assays for the detection of human immunodeficiency virus-1 infection in emergency department patients.

In order to determine the sensitivity and specificity of two rapid human immunodeficiency virus (HIV-1) assays compared with enzyme immunoassay and Western blot and to assess their potential use for routine screening in an emergency department (ED), we analyzed sera from 492 consecutive ED patients using an identity-unlinked design. Sera were analyzed for HIV-1 by standard enzyme-linked immunosorbent assay and Western blot and two rapid assays: the Abbott Testpack HIV-1 (Abbott Labs, Inc, Abbott Park, IL) and the HIV-1 Genie, (Genetic Systems, Seattle, WA). Seroprevalence of HIV-1 among 492 samples was 5.1%. Both rapid assays were easy to perform and required approximately 10 minutes per test. Sensitivity and specificity of both rapid assays were 100% and 99.8%, with positive and negative predictive values of 96.2% and 100%, respectively. It was concluded that both rapid assays showed high concordance with standard enzyme-linked immunosorbent assay and Western blot. Since the ED is often the primary care setting for many patients at risk for HIV-1, the ED may be an optimal site for routine HIV-1 screening. Rapid assay screening may provide the opportunity for timely identification of HIV-1-infected patients, allowing earlier treatment and counseling. However, ethical and practical questions regarding appropriate application of rapid HIV-1 testing in EDs still needs resolution.

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of monoclonal antibodies to JC virus and their use in the diagnosis of progressive multifocal leucoencephalopathy.

Seven monoclonal antibodies (MAbs) to polyomavirus JC were produced, and one was selected for use in immunofluorescence (IF) tests on brain material from patients with suspected progressive multifocal leucoencephalopathy (PML). The selected MAb (5.12.2) reacted by IF with JC-infected primary human foetal glial (PHFG) cell cultures (titre 1/200,000) but not with BK-infected human embryo lung (HEL) fibroblasts (titre less than 1/20). Its haemagglutination-inhibition (HI) titre was high (greater than 1/5 x 10(6)) against JC virus but low (less than 1/5) against BK virus. A diagnosis of PML was confirmed on brain biopsy or at postmortem in four patients, two of whom were also infected with human immunodeficiency virus (HIV). In one of the patients, specific detection of JC virus antigen had not been possible using our routine high titred JC and BK human sera due to interference by JC antibody present in the cerebrospinal fluid (CSF). Viral antigen was, however, detected with the MAb 5.12.2.

Four-sector tandem mass spectrometric analysis of complex mixtures of phosphatidylcholines present in a human immunodeficiency virus preparation.

A number of phosphatidylcholines have been isolated from an HIV-1/MN preparation by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by fast atom bombardment mass spectrometry (FABMS), FABMS/MS, and FABMS/MS/MS in both positive- and negative-ion modes. Negative-ion FABMS/MS with high-energy collisions was used to identify the length of the acyl groups and the degree of saturation, as well as their position on the glyceride group. FABMS/MS in the positive-ion mode was used to identify the polar head group. Negative-ion FABMS/MS/MS was used to locate positions of double bonds in acyl groups. We find that four-sector tandem mass spectrometry with high-energy collisional activation provides qualitative analysis of viral phosphatidyl lipids in considerable detail, as well as semiquantitative information. Approximate quantitation of the phosphatidylcholine content of the HIV-1/MN preparation by measuring relative peak heights of molecular ions in FABMS reveals an array of phosphatidylcholines consistent with that found in human erythrocytes, indicating the likely source of lipids in the viral membrane to be the host cell membrane.

A conformational switch is associated with receptor affinity in peptides derived from the CD4-binding domain of gp120 from HIV I.

A 15-residue region within the CD4-binding domain of gp120 from HIV I was identified with use of folding algorithms as conserving the potential for forming a particular secondary structure throughout 11 sequenced HIV strains. The region chosen has a potential for forming both beta-sheet and alpha-helix; the helical form would be amphipathic with the five hydrophobic residues all totally or functionally conserved. Five peptides were synthesized corresponding to this region in strain LAV and the strain most highly divergent from it in primary structure (Z3) plus three additional peptides with critical substitutions in the LAV sequence. The conformation of these five peptides was examined under various conditions with circular dichroism, and the results were compared with the ability of each peptide to bind to a CD4-expressing strain of HeLa cells (HeLa T4). In solution, the unmodified peptides exhibit a bistable structure, existing as beta-sheet in dilute buffer and converting to alpha-helix under more apolar conditions. The transition is reversible and sharp, occurring at a particular point in the polar/apolar gradient with virtually no intermediate state. The ability to undergo this bistable flip is closely associated with binding ability, amino acid substitutions that eliminate binding ability also eliminating the switch, and vice versa. The transition thus may reflect conformational changes occurring in this region of gp120 as it binds to the CD4 receptor.

HIV-1 seropositivity and mortality at University Hospital, Kinshasa, Zaire, 1987.

This study examines the impact of HIV-1 infection and AIDS on 500 of 563 consecutive deaths at University Hospital, Kinshasa, Zaire, in late 1987. HIV-1 seroprevalence was 31% for the entire population and 43% for the 247 adults. Forty-two (38%) of the 110 HIV-1-seropositive adult deaths occurred in those between the ages of 25 and 34 years. The mean age of death for seropositives was 36 years, 7.5 years less than seronegative deaths. AIDS and AIDS-associated diagnoses such as cryptococcal meningitis, chronic diarrhea and pneumonia accounted for 42% of all adult deaths and 74% of all HIV-1-seropositive adult deaths. Seventeen per cent of 50 sera initially negative by enzyme-linked immunosorbent assay (ELISA) were ultimately found to be HIV-1-seropositive by Western blot or p24 antigen testing. The data indicate that HIV-1 infection and AIDS contribute significantly to adult mortality in Kinshasa population and that sensitivity of ELISA tests decreases in terminal HIV-1 infection.

Frequency of nonparenteral occupational exposures to blood and body fluids before and after universal precautions training.

PURPOSE: During annual periods before and after Universal Precautions training, we compared the frequency of health care workers' self-reported cutaneous exposures to blood and various body substances from any patient and from patients presumed infected with human immunodeficiency virus type 1 (HIV-1). SUBJECTS AND METHODS: Self-reported cutaneous exposures to blood, sputum, urine, feces, and other body substances were evaluated separately in 559 workers during the first survey and 269 workers during the second. RESULTS: Mean annual blood exposures decreased from 35.8 to 18.1, and mean annual exposures to all substances decreased from 77.8 to 40.0 (p less than 0.001 for both determinations). Two matched analyses of a subset of 200 participants who completed both surveys had similar results. Reported exposures to blood, presumably infectious blood, sputum, presumably infectious sputum, and urine were significantly decreased. Participants were tested for antibodies to HIV-1; no participant reporting cutaneous exposures acquired HIV-1 infection. The upper bound for the 95% confidence interval for the risk of HIV-1 infection associated with a single cutaneous exposure was 0.04% for blood presumed to contain HIV-1 and 0.02% for any body substance presumed to contain HIV-1. CONCLUSIONS: These data suggest that Universal Precautions training significantly decreased but did not eliminate cutaneous exposures to blood and body substances. The results further suggest that the risk for HIV-1 infection associated with cutaneous exposures is substantially lower than the risk associated with parenteral exposures.

Neopterin and beta 2-microglobulin as prognostic indices in human immunodeficiency virus type 1 infection.

The great majority of individuals with human immunodeficiency virus type 1 (HIV-1) infection presents with no signs or symptoms, or only lymphadenopathy. To initiate prophylactic measures in time it is necessary to establish risk criteria. CD4+ cell counts are significant predictors. Supplementary methods to improve the predictive information of CD4+ cell counts are still required. In addition, CD4+ cell counting is laborious, expensive, and restricted to specialized laboratories. Thus, there is also a place for more easily performed laboratory tests with similar predictive value as CD4+ cell counts. Neopterin and beta 2-microglobulin levels proved to be significant predictors of AIDS risk in HIV-1 seropositives. The predictive value of both parameters is equal to CD4+ cell counts and both markers are significant joint predictors in addition to CD4+ cell counts. Measurement of the parameters is done in serum (neopterin and beta 2-microglobulin) or urine (neopterin) specimens which reduces the risk of HIV-1 transmission compared to handling of whole-blood samples as it is required for cell counting. Although more studies are needed, especially in developing countries and in persons receiving zidovudine, it can be recommended to use neopterin and beta 2-microglobulin as additional marker to estimate AIDS risk in HIV-1 seropositive individuals. Moreover, both markers may be useful for this purpose without CD4+ cell counts if cell counting is not available.

Structural comparison of the external glycoproteins of human and simian immunodeficiency virus.

The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.

Change in size of the envelope glycoprotein of a human immunodeficiency virus 1 (HIV 1) strain.

A strain of HIV 1 (PA 40), isolated from a patient with AIDS, showed a size variation of its external glycoprotein. This glycoprotein had an estimated molecular weight of 105 Kd and differed from that of both HIV 1 IIIb and HIV 2 Rod strains. The protein was derived from a bigger (140 Kd) precursor, detectable only in the infected cells and could incorporate labeled glucose in its prosthetic portion. The change in size of the external glycoprotein may be the result of envelope sequence variations since the unglycosylated form of the envelope precursor of PA 40 strain, detected in tunicamycin treated cells, was smaller than that of the HIV 1 IIIb strain. The different size of the external glycoprotein is a further aspect of the variability of the biological characteristic of HIV 1 strains and might be correlated with the emergence of more virulent variants which arose during the progression of the clinical disease.

Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses.

The effects of C-terminal and internal deletions on the synthesis, transport, biological properties, and antigenicity of the human immunodeficiency virus type 1 envelope protein were determined. A family of recombinant vaccinia viruses that express N-terminal overlapping env proteins of 204, 287, 393, 502 (full-length gp120), 635, 747, and 851 (full-length gp160) amino acids was constructed. All of the proteins were detected in intra- and extracellular forms which differed in the extent of glycosylation. The 747- and 851-amino-acid proteins were cleaved, were expressed on the surface of infected cells, and bound CD4. The 635-amino-acid env protein was cleaved inefficiently, and both the precursor and product were secreted, indicating absence of the transmembrane sequence. The 635- as well as the 502-amino-acid protein, which was also largely secreted, could still bind CD4. Unexpectedly, the 393-amino-acid protein was anchored in the plasma membrane, but neither it nor smaller proteins bound to soluble CD4. When amino acids at the gp120-gp41 junction were deleted, proteolytic cleavage of gp160 did not occur. Nevertheless, gp160 was inserted into the plasma membrane and bound soluble CD4. The predominant conserved B-cell epitopes were mapped to gp41 and the C terminus of gp120, whereas cytotoxic T-cell epitopes were distributed throughout the length of the glycoproteins.

Fetal human immunodeficiency virus type 1 infection of different organs in the second trimester.

Human immunodeficiency virus type 1 (HIV-1) infection in utero was examined by isolating the virus and detecting the HIV-1 DNA sequence from different fetal tissues. The brain, thymus, lung, liver, spleen, and placenta tissues from fetuses (10-23 weeks of gestation) born to HIV-1-infected asymptomatic mothers were examined. HIV-1 was isolated from 2 of 7, 1 of 7, and 1 of 7 cocultures of splenic, thymic, and trypsin-resistant cells from the liver and placenta, respectively, with peripheral blood mononuclear cells; 20-30% and 40-60% of splenic and of thymic cells were CD4+ lymphoid cells and 40-80% of trypsin-resistant cells were mononuclear phagocytes. The HIV-1 DNA sequence was detected in 4 of 7, 3 of 7, 1 of 7, 1 of 7, 2 of 7, and 2 of 6 samples from the spleen, thymus, brain, lung, liver, and placenta, respectively, using the polymerase chain reaction. In one case, the intensity of the HIV-1 DNA sequence appeared to be correlated with the success of viral isolation. We indicate that fetal HIV-1 infection may frequently occur in the second trimester and the cells responsible for the infection may be CD4+ lymphoid cells and mononuclear phagocytes.

Use of monoclonal antibodies for the detection and quantitation of HIV1 core protein p25: comparative evaluation of in vitro HIV1 infection by immunofluorescence, antigen capture ELISA and reverse transcriptase assays.

A two-site enzyme linked immunosorbent assay (ELISA) was developed to detect and quantify the HIV1 core protein p25 in the cell-free supernatant from virus-infected CEM cell culture, and compared with other assays. The assay, based on a sandwich method, employs two monoclonal antibodies (mAb) directed against different epitopes on the p25 core protein of HIV1, one used for p25 antigen capture and the other as a biotinylated probe. This immunoassay is sensitive enough to detect as little as 30 pg/ml of recombinant p25, the range of sensitivity of commercial kits, and therefore compares favourably with the conventional reverse transcriptase assay. Moreover, several hundred assays can be monitored quite conveniently by this simple ELISA procedure, which represents a useful tool for detection of HIV1 replication in microculture systems and rapid screening of antiretroviral agents using the reference strain HIV1-BRU as a model system.

[The biological properties of the HIV isolated from a virus carrier living in the Byelorussian SSR]. / Biologicheskie svoistva VICh, vydelennogo ot virusonositelia, prozhivaiushchego v BSSR.

Biological properties of an AIDS agent first isolated from a native citizen in the USSR are presented. The source of the virus was a young Byelorussian woman who in the near past had had sexual contacts with a citizen from one of the Central Africa countries. The isolate is thought to be of HIV-I type. It replicated perfectly in many continuous lymphocyte lines and had HIV-characteristic morphology. The protein spectrum of the isolate was gp120, gp41, p65/51, p55, p32, p24, p17. Reverse transcriptase activity was detected in the culture fluid of the virus-containing cell cultures. The isolate was designated HIV-IZ.

The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers.

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization.

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Identification of the most significant amphipathic helix with application to HIV and MHV envelope proteins.

Amphipathic helices, which play important roles in protein structure, occur in a wide variety of lengths. Yet existing methods employ fixed window lengths. We present a hierarchical procedure that identifies the Q most significant amphipathic helices regardless of length. Since the observed hydrophobicities are not normally distributed, test statistics usually employed for least-squares regression are inappropriate for assessing statistical significance of amphipathic helices. We show that an adjusted F statistic provides a good test. An application to the envelope protein of HIV finds an unexpected long amphipathic helix in gp41.

Update on the virology of AIDS

This article looks at the history, development, progress and research of the Human Immunodeficiency Virus (HIV) which causes AIDS. The author reports of the ongoing research into a vaccine for HIV, he examines the viral life cycle and indicates the points at which the virus can be attacked, and classifies antiviral strategies

[The characteristics of the interaction of the proteins comprising the virions of the human immunodeficiency virus type 1]. / Osobennosti vzaimodeistviia belkov v sostave virionov virusa immunodefitsita cheloveka tipa 1.

Treatment of virions of human immunodeficiency virus type 1 (HIV-1) with ionic and nonionic detergents (NP-40, octylglucoside, sodium deoxycholate) exerted an effect on the virus uncommon for enveloped viruses: instead of solubilization, both glycoproteins (gp120 and gp41) were found in subviral particles, whereas the core protein p24 was found in the supernatant fluid after the removal of subviral particles by centrifugation. The matrix protein p17 and unprocessed molecules of the precursor protein p55 were associated with subviral particles. The above data confirm the proposed model of the HIV-I structural organization according to which glycoproteins are incorporated into the isometric matrix formed by protein p17. Our data indicate that the core protein p24 is not incorporated into the matrix and not associated with nucleocapsid proteins.