[Prevention of nosocomial transmission of human immunodeficiency virus (HIV) from HIV-positive healthcare workers. Recommendations of the German Association for the Control of Viral Diseases (DVV) e.V. and the Society for Virology (GfV) e.V]. / Prävention der nosokomialen Übertragung von humanem Immunschwächevirus (HIV) durch HIV-positive Mitarbeiterinnen und Mitarbeiter im Gesundheitswesen. Empfehlungen der Deutschen Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) e.V. und der Gesellschaft für Virologie (GfV) e.V.

To the best of our knowledge, the German Association for the Control of Viral Diseases (DVV) e.V. and the Society for Virology (GfV) e.V. are the first in Europe to provide precise recommendations for the management of health care workers (HCWs) who are infected with human immunodeficiency virus (HIV). Requirements for HIV-infected HCWs need to be clearly defined. With a permanent viral burden of less than or equal to 50 copies/mL, HIV-positive HCWs are allowed to perform any surgery and any invasive procedure, as long as the infected HCW uses double-gloving, undergoes follow-up routinely by occupational medicine professionals, undergoes a quarterly examination of viral burden, and has a regular medical examination by a physician who has expertise in the management of HIV. Unrestricted professional activity is only possible with a strict compliance to take antiretroviral therapy and if the HIV-infected HCW strictly adheres to the recommended infection control procedures. Complete compliance with the recommendation almost certainly leads to no HIV transmission risk in patient care.

Human immunodeficiency virus type-1 group M quasispecies evolution: diversity and divergence in patients co-infected with active tuberculosis.

The evolution of intra-host human immunodeficiency virus type 1 (HIV-1) quasispecies prior and after treating active tuberculosis (TB) with chemotherapy in HIV-1/TB patients was assessed. Two time points HIV-1 quasispecies were evaluated by comparing HIV-1-infected patients with active tuberculosis (HIV-1/TB) and HIV-1-infected patients without tuberculosis (HIV-1/non-TB). Plasma samples were obtained from the Frankfurt HIV cohort, and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty-five clones were sequenced from each patient. Various phylogenetic analyses were performed. We found a significant increase in diversity and divergence in HIV-1/TB compared to the HIV-1/non-TB. For HIV-1/TB, the average rate of evolution of C2V5 env was higher than previous reports (2.4 × 10(-4) substitution/site/day). Two groups of HIV-1/TB were observed based on the rate of HIV-1 evolution and coreceptor usage: A fast evolving R5-tropic dominating group and a relatively slowly evolving X4 group. The results demonstrated that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV-1 may be predominant for R5 viruses.

Tissue donation and virus safety: more nucleic acid amplification testing is needed.

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

Mutations in the C-terminal region of the HIV-1 reverse transcriptase and their correlation with drug resistance associated mutations and antiviral treatment.

OBJECTIVE: replication of HIV-1 after cell entry is essentially dependent on the reverse transcriptase (RT). Antiretroviral drugs impairing the function of the RT currently aim at the polymerase subunit. One reason for failure of antiretroviral treatment is the evolvement of resistance-associated mutations in the viral genome. For RT inhibitors, almost all identified mutations are located within the polymerase; therefore, general genotyping confines to investigate this subunit. Recently several studies have shown that substitutions within the RNase H and the connection domain increase antiviral drug-resistance in vitro, and some of them are present in patient isolates. AIM: the aim of the present study was to investigate the prevalence of these substitutions and their association with mutations in the polymerase domain arising during antiretroviral treatment. MATERIAL AND METHODS: we performed genotypic analyzes on seventy-four virus isolates derived from treated and untreated patients, followed at the HIV Centre of the Johann Wolfgang Goethe University Hospital (Frankfurt/Main, Germany). We subsequently ana?lysed the different substitutions in the c-terminal region to evaluate whether there were associations with each other, n-terminal substitutions or with antiretroviral treatment. RESULTS: We identified several primer grip substitutions, but almost all of them were located in the connection domain. This is consistent with other in-vivo studies, in which especially the primer grip residues located in the RNase H were unvaried. Furthermore, we identified other substitutions in the connection domain and in the RNase H. Especially E399D seemed to be associated with an antiretroviral treatment and N-terminal resistance-delivering mutations. CONCLUSION: some of the identified substitutions were associated with antiviral treatment and drug resistance-associated mutations. Due to the low prevalence of C-terminal mutations and as only a few of them could be associated with antiviral treatment and N-terminal resistance-delivering mutations, we would not recommend routinely testing of the C-terminal RT region.

Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

The fucosyl specific lectins of Ulex europaeus and Sarothamnus scoparius. Biochemical characteristics and binding properties to human B-lymphocytes.

From the seeds of the gorse, Ulex europaeus and of the broom, Sarothamnus scoparius L-fucosyl-specific lectins were isolated by affinity chromatography on L-fucosyl-epoxy-Sepharose. The lectins showed similarities in their molecular weights, amino acid composition, carbohydrate content and in the finger-prints of their tryptic peptides. The fluorescein-labeled lectins of both seeds attached especially to the plasma membranes of human B-lymphocytes.

Enterovirus RNA sequences in sera of schoolchildren in the general population and their association with type 1-diabetes-associated autoantibodies.

Type 1 diabetes (T1D) is an autoimmune disease linked with genetic factors as well as with environmental triggers, such as virus infections, but the aetiology is still unclear. The authors analysed serum from autoantibody-positive (n=50) and autoantibody-negative (n=50) schoolchildren as well as children newly diagnosed with T1D (n=47; time from diagnosis, median 5 days, interquartile range 1-12 days) for the presence and frequency of enterovirus (EV) and adenovirus sequences. The autoantibody-positive and -negative groups were part of the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population, which represents a general population without T1D first-degree relatives. There was no significant seasonality of sampling in any of the three groups investigated. EV RNA sequences were detected in 10 of 50 (20%) autoantibody-positive children and in 17 of 47 (36%) children newly diagnosed with T1D, but only in two of 50 (4%) of the age- and sex-matched controls (P<0.05, P<0.001). Characterization of the EV amplicons by direct sequencing revealed high homology with coxsackievirus B group. For adenovirus we found no data to support an association with T1D. The data support the hypothesis that different enteroviruses may be aetiologically important as a trigger and/or accelerating factor in the process of T1D development.

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

A new second-generation anti-HIV-1 enzyme immunoassay using recombinant envelope and core proteins.

In a multicenter collaborative study a new second-generation HIV-1 antibody enzyme immunoassay (Abbott recombinant HIV-1 EIA) using Escherichia coli-expressed recombinant p24 and p41 proteins as solid-phase antigens was compared with the first-generation H9 cell-line-based Abbott HIV-1 EIA. The results of the confirmatory assays (Western blot, immunofluorescence), combined with clinical information, were used as the reference standard for the detection of HIV-1 antibodies in 10,676 random blood donor serum specimens, in a panel of 840 specimens from symptomatic and asymptomatic patients and a total of 63 serial blood specimens from 23 people at risk. With fresh blood donor sera, the specificity of the first-generation assay ranged between 99.54 and 99.76% (95% confidence limits, CL) compared with 99.81-99.95% (95% CL) for the second-generation EIA. With panel specimens the recombinant HIV-1 EIA achieved an overall sensitivity of 100% and a specificity range of 98.3-99.7% (95% CL); the corresponding sensitivity and specificity ranges observed for the first-generation EIA were 98.0-99.5% (95% CL) and 94.3-96.8% (95% CL), respectively. The improved sensitivity for the second-generation assay was confirmed by testing serial samples from seroconverting patients. The use of recombinant proteins eliminated non-specific reactions due to class II human leukocyte antigen (HLA)-directed antibodies.

Recombinant peptides derived from the env-gene of HIV-2 in the serodiagnosis of HIV-2 infections.

We produced recombinant envelope-derived peptides of HIV-2 for use in a diagnostic enzyme-linked immunosorbent assay (ELISA) or Western blot by expressing several restriction enzyme fragments from the env gene of HIV-2 in the bacterial fusion vector pEX-3. On Western blots, 17 out of 18 anti-HIV-2-positive sera available to us reacted strongly with those recombinant peptides which were derived from, or extended into, the transmembrane protein of HIV-2. In contrast, recombinant peptides derived from the external envelope glycoprotein were only weakly recognized by two sera. We observed a cross-reactivity of some human sera containing antibodies to HIV-1 with the HIV-2 peptides derived from the transmembrane protein of HIV-2. In spite of this cross-reactivity, a serological distinction between anti-sera to HIV-1 and HIV-2 can be attempted by simultaneous testing in ELISA on recombinant peptides derived from the transmembrane protein of HIV-1 and HIV-2.

HIV-1 and HIV-2 isolates differ in their ability to activate the complement system on the surface of infected cells.

OBJECTIVE: To analyse the ability of different HIV-1 and HIV-2 isolates to activate the complement system. DESIGN: H9 cells chronically infected with various HIV isolates and the corresponding purified viruses were tested for complement activation. To identify the molecules responsible for complement activation on the surface of infected cells, the expression of complement inhibitors/regulators and viral proteins on the cell surface was analysed. METHODS: C3 deposition on the cell surface and the expression of viral and cellular antigens were determined by flow cytometry analysis. Complement activation by purified viruses was measured using a complement consumption assay and a C1 activation assay. RESULTS: H9 cells infected with different HIV-1 and HIV-2 isolates showed varying degrees of complement activation on the cell surface, ranging from strong activation and deposition of large amounts of C3 to no increased C3 deposition compared to uninfected cells. The C3 deposition was eliminated by EDTA and reduced in the presence of EGTA. In contrast, all purified viral isolates tested activated the complement system in a comparable manner. While the expression of MCP, DAF and CD59 was not modified after infection with different viral isolates, the reaction of the infected cells with a monoclonal antibody (3D6) directed against a gp41 epitope (amino acids 601-620) was found to correlate with the complement activation on the cell surface. CONCLUSIONS: Some HIV-1 as well as HIV-2 isolates activate the complement system on the surface of infected cells independent of anti-HIV antibodies, while other isolates fail to do so. Complement activation on the cell surface is mediated by the alternative and, to a lesser extent, the classical pathway. The differences in complement activation on the cell surface are not caused by a modified expression of membrane-bound complement inhibitors/regulators. C3 deposition on the cell surface correlates with the expression of an epitope lying within the major complement activating domain of gp41 (amino acids 591-620). These results suggest a role of gp41 for complement activation on HIV-infected cells as has been described previously for purified HIV.

Multicenter evaluation of a new recombinant enzyme immunoassay for the combined detection of antibody to HIV-1 and HIV-2.

A newly developed recombinant antigen-based anti-HIV-1/HIV-2 enzyme immunoassay (Abbott Recombinant HIV-1/HIV-2 EIA) was evaluated against a second generation anti-HIV-1 EIA (Abbott Recombinant HIV-1 EIA). Five thousand and twenty-nine sera from European blood donors and 403 sera from central African blood donors were used in the evaluation, along with four panels and one cohort. The panels included 99 'problem' sera, 733 sera with antibodies to HIV-1 from asymptomatic people and from patients at different disease stages, 25 serial bleeds from five plasmapheresis donors seroconverting for antibodies to HIV-1, and 202 sera with antibodies to HIV-2 collected from healthy and diseased people of European or west African origin. In addition, 734 sera collected from a west African cohort were tested. Using Western blot as the reference standard, the specificity obtained by the recombinant anti-HIV-1 EIA (HIV-i EIA) was 99.90% [99.81-99.99%; 95% confidence limits (95% CL)] with European blood donor sera; 99.50% (98.78-100%) with Central Africa blood donor sera; 92.93% (87.78-98.08%) with 'problem' sera and 99.43% (98.87-100%) with sera from a west African cohort. Using the same samples, the recombinant anti-HIV-1/HIV-2 EIA (HIV-1/HIV-2 EIA) yielded a specificity of 99.84% (99.73-99.95%), 99.50% (98.78-100%), 95.96% (92.00-99.92%) and 98.58% (97.69-99.47%), respectively. All 776 Western blot-confirmed anti-HIV-1 sera were reactive in both EIAs, and the EIA-reactive samples from seroconverting plasma donors were always observed for both assays in the same serial bleed. For HIV-2, the HIV-1 EIA yielded an overall sensitivity of 75.83% (69.93-81.72%) compared with 99.53% (98.58-100%) for HIV-1/HIV-2 EIA. The addition of a recombinant env-protein of HIV-2 to the recombinant env and core proteins of HIV-1 on the solid phase of HIV-1 EIA improved the detection of anti-HIV-2 while preserving the assay's overall specificity and sensitivity for the detection of anti-HIV-1.

Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A.

After incubation of H9 cells infected with human immunodeficiency virus (HIV) with pepstatin A at 10(-4) M for 2, 4, or 11 days, the culture medium contained significantly less HIV core antigen (p24) than controls without pepstatin A and no or only borderline activity of reverse transcriptase was detected. In addition, after pepstatin A treatment no infectious HIV at 2 or 4 days and only minimal amounts at 11 days were detectable in the culture medium.

Carrier bound synthetic oligopeptides in ELISA test systems for distinction between HIV-1 and HIV-2 infection.

A series of synthetic carrier bound oligopeptides derived from corresponding regions of the core and envelope proteins of HIV-1 and HIV-2 were used in enzyme-linked immunoabsorbent assays (ELISA) for serodiagnosis of HIV-1 and HIV-2 infected individuals. The combination of peptides from regions either conserved or highly variable between the two virus types allowed the identification of HIV infection in general and the differentiation between HIV-1 and HIV-2. No specific reaction was found in seronegative individuals. The use of peptides bound to the same polystyrene carrier as in peptide synthesis allowed the establishment of a highly specific and sensitive test system without the risk of unspecific cross-reaction due to contamination with bacterial or cellular protein material.

Phase I/II trial with fozivudine tidoxil (BM 21.1290): a 7 day randomized, placebo-controlled dose-escalating trial.

BACKGROUND AND OBJECTIVES: A Phase I dose-escalating trial with single doses of fozivudine tidoxil (BM21.1290; FZD), in vitro, and experimental in vivo data indicated that further investigations with this compound were warranted. Advantages of fozivudine tidoxil are the direct delivery of zidovudine monophosphate intracellularly and high concentrations in lymphatic tissues. Our objectives were to evaluate safety, tolerability and efficacy of fozivudine tidoxil in human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: In a Phase I/II dose-escalating trial, three doses of fozivudine tidoxil (400, 800 or 1200 mg/day) were administered for 1 week. The study was randomized and placebo controlled. Inclusion criteria were HIV infection, CD4 count > 100 cells/mm3 and informed consent. The exclusion criteria were active opportunistic infection and prior antiretroviral treatment. RESULTS: The tolerability of fozivudine tidoxil was excellent in all dose groups. No treatment discontinuations were necessary. Steady-state pharmacokinetics did show slightly higher concentrations as compared with levels after the first dose (20%). Viral load reduction was most pronounced in the 1200 mg/day dose group (-0.64 log). No viral load reduction was seen in the placebo group. CONCLUSIONS: The zidovudine conjugate fozivudine tidoxil is safe and well tolerated at the three doses tested. Based on the differences in molecular weights, the 1200 mg dose is roughly equivalent to 400 mg zidovudine.

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.

Correlation of antibodies to LAV/HTLV III in hemophiliacs with the use of virus-inactivated clotting factors.

The retrovirus LAV/HTLV III, highly likely to be responsible for the acquired immunodeficiency syndrome (AIDS) in some recipients of blood products, can be inactivated by chemical and/or heat treatment, so the use of virus-inactivated factor VIII and factor IX preparations for treating hemophilia A and B has become important. We examined hemophilic children and found that those children treated since 1979 with virus-inactivated preparations did not develop antibodies against LAV/HTLV III. In contrast, 77% of patients treated with conventional factor VIII or factor IX preparations had antibodies against this virus.

Reduced CD21 (CR2) and CD54 (ICAM-1) expression in MT2 cells with HIV-1 or HIV-2 strains.

Alterations in the expression of cell-surface receptors have been reported in HIV-infected cells for CD4, CD25 (IL-2 receptor), CD2, CD3 and CD8 and CD26. In the present study we provide evidence that CD21 is down-regulated in the human T-lymphoblastoid cell line MT2 after infection with HIV-1 and -2 isolates. The same effect was observed with ICAM-1 (CD54). CD21 expression was monitored by means of fluorescence intensity, its functional ability to bind to C3d and by quantitative measurement of CD21-antigen in supernatants and cell lysates using an immunoassay. In addition, the decrease of CD21 and ICAM-1-specific mRNA suggests a mechanism at a transcriptional level. Our data suggest that HIV might have a direct influence on the receptor expression.

Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959.

During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.

New crown motif of an HIV-1 V3 loop sequence from a Ugandan AIDS patient.

PIP: HIV-1 V3 loop sequences from Ugandan patients include motifs from subtypes A, B, and D. To characterize further HIV isolates, V3 loop sequences were amplified from HIV-1 isolated in 1987 from peripheral blood mononuclear cells (PBL) of three patients with full-blown AIDS from Kampala, Uganda. The PBL were separated by Ficoll Paque gradients and cocultivated with noninfected donor lymphocytes for two weeks. The HIV was then transferred to HUT-78 cells. From extracted DNA of the permanently-infected HUT-78 cells, nested polymerase chain reaction (PCR) was conducted, with V3 loop sequencing performed directly upon PCR fragments derived from two independent DNA preparations and on cloned fragments. Isolates MVP-9801, -9802, and -9803 show 35.6%, 32.4%, and 29.7% nucleotide sequence divergence from the ELI subtype D sequence; 31.5%, 25.7%, and 18.9% divergence from the Z2Z6 subtype D sequence; and 21.9%, 12.2%, and 12.2% divergence from the subtype D consensus sequence. All three deduced amino acid sequences fit into the subtype D consensus sequence rather than into other V3 loop sequences described for Ugandan subtype A isolates. MVP-9802 and MVP-9803 contain the GSGQA pentapeptide motif at the tip of the V3 loop, while MVP-9801 contains GGRA. This may be explained by a deletion of proline codon between the codons for the two glycine residues. The authors believe that this deletion has not been previously reported. They also note that the deletion does not appear to be associated with a growth difference in vitro or with a difference in pathogenicity in vivo. The immunogenic implications of this altered V3 loop crest remain unclear. The Western blot profiles for the gp160, gp120, and gp41 proteins of the three Ugandan isolates manifest normal molecular weights.^ieng