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1.
J Med Virol ; 72(1): 126-31, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14635020

ABSTRACT

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS). Several studies indicate horizontal HHV-8 transmission among children in areas where KS is endemic, but few studies have assessed acquisition of HHV-8 by children in low seroprevalence areas. Antibody screening was carried out for HHV-8 and Epstein-Barr virus (EBV) on 787 serum specimens from children living in two areas where HHV-8 is not endemic, the United States (US) and Germany, and on 184 specimens from children living in a KS-endemic area (Nigeria). For children in the US and Germany, the results showed low HHV-8 seroprevalence rates (3-4%). However, US children aged 6 months to 5 years had higher HHV-8 antibody titers than did 6-17-year-old children (P < 0.01), a finding consistent with more recent infections being detected in the younger children. Compared with seroprevalence rates and antibody titers in US and German children, those in Nigerian children were significantly higher, and seroprevalence increased with age. There was no evidence of cross-reactivity between assays for HHV-8 and EBV, despite the genetic similarity of these two herpesviruses. The data indicate that HHV-8 transmission among children where HHV-8 is not endemic occurs, but is uncommon. The findings also suggest that HHV-8 antibodies, as measured by current tests, may not persist for long periods in populations at low risk for KS and that vertical transmission is rare, although longitudinal studies are necessary to address directly these issues.


Subject(s)
Antibodies, Viral/blood , Endemic Diseases , Herpesvirus 4, Human/immunology , Herpesvirus 8, Human/immunology , Sarcoma, Kaposi/epidemiology , Adolescent , Child , Child, Preschool , Epstein-Barr Virus Infections/epidemiology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/virology , Germany/epidemiology , Humans , Infant , Nigeria/epidemiology , Sarcoma, Kaposi/immunology , Sarcoma, Kaposi/virology , Seroepidemiologic Studies , United States/epidemiology
2.
Clin Diagn Lab Immunol ; 8(5): 913-21, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11527803

ABSTRACT

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.


Subject(s)
Antibodies, Viral/biosynthesis , Herpesviridae Infections/blood , Herpesvirus 8, Human/immunology , Viral Proteins , Antibodies, Viral/metabolism , Antigens, Viral/biosynthesis , Cell Line , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Glycoproteins/analysis , Herpesviridae Infections/diagnosis , Herpesvirus 8, Human/isolation & purification , Humans , Nuclear Proteins/biosynthesis , Semliki forest virus/immunology , Viral Envelope Proteins/immunology
4.
J Clin Microbiol ; 38(6): 2174-80, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10834972

ABSTRACT

A variety of assays for the diagnosis human herpesvirus 8 (HHV-8) infection have been reported. We compared several such assays with a panel of 88 specimens from human immunodeficiency virus (HIV)-infected patients with Kaposi's sarcoma (KS) (current-KS patients; n = 30), HIV-infected patients who later developed KS (later-KS patients; n = 13), HIV-infected patients without KS (no-KS patients; n = 25), and healthy blood donors (n = 20). PCR assays were also performed with purified peripheral blood mononuclear cells (PBMCs) to confirm positive serologic test results. The order of sensitivity of the serologic assays (most to least) in detecting HHV-8 infection in current-KS patients was the mouse monoclonal antibody-enhanced immunofluorescence assay (MIFA) for lytic antigen (97%), the orfK8.1 peptide enzyme immunoassay (EIA) (87%), the orf65 peptide EIA (87%), MIFA for latent antigen (83%), the Advanced Biotechnologies, Inc., EIA (80%), and the orf65 immunoblot assay (80%). Combination of the results of the two peptide EIAs (combined peptide EIAs) increased the sensitivity to 93%. For detection of infection in later-KS patients, the MIFA for lytic antigen (100%), the orfK8.1 peptide EIA (85%), and combined peptide EIAs (92%) were the most sensitive. Smaller percentages of no-KS patients were found to be positive (16 to 56%). Most positive specimens from the current-KS and later-KS groups were positive by multiple assays, while positive specimens from the no-KS group tended to be positive only by a single assay. PCR with PBMCs for portions of the HHV-8 orf65 and gB genes were positive for less than half of current-KS and later-KS patients and even fewer of the no-KS patients. The concordance between serologic assays was high. We propose screening by the combined peptide EIAs. For specimens that test weakly positive, we recommend that MIFA for lytic antigen be done. A positive result with a titer of >/=1:40 would be called HHV-8 positive. A negative or low titer would be called HHV-8 negative. If a population has a high percentage of persons who test positive by the combined peptide EIAs, then a MIFA could be performed with the negative specimens to determine if any positive specimens are being missed. Alternatively, if a population has a low percentage that test positive, then a MIFA could be performed with a subset of the negative specimens for the same reason. As described above, only a titer of >/=1:40 would be considered HHV-8 positive.


Subject(s)
Herpesviridae Infections/diagnosis , Herpesvirus 8, Human/isolation & purification , Polymerase Chain Reaction/methods , Sarcoma, Kaposi/diagnosis , Serologic Tests/methods , Algorithms , Antigens, Viral/isolation & purification , Evaluation Studies as Topic , Fluoroimmunoassay , HIV Infections/complications , Herpesviridae Infections/complications , Humans , Immunoenzyme Techniques , Male , Sarcoma, Kaposi/complications , Sensitivity and Specificity , Viral Proteins/isolation & purification
5.
AIDS ; 14(3): 213-23, 2000 Feb 18.
Article in English | MEDLINE | ID: mdl-10716496

ABSTRACT

OBJECTIVES: To determine the molecular nature of HIV-1 quasispecies and their evolution, in vivo over time, in an American cohort of 22 homosexual men [four rapid progressors (RP), 15 slow progressors (SP) and three long-term non-progressors (LTNP)], infected with HIV-1 between 1982 and 1983, and to assess the possible role of the HIV-1 V2 region extension in HIV disease progression. DESIGN: Genetic and phylogenetic analyses of the V3 region and the nef gene clones over time from uncultured peripheral blood mononuclear cells (PBMC) of American patients with varying HIV disease progression rates. METHODS: Proviral DNA from longitudinally collected uncultured PBMC were subjected to PCR amplification in the nef gene and env V2 and V3 regions, followed by cloning, sequencing and phylogenetic analysis to establish evolutionary relationships between HIV-1 strains over time. RESULTS: Analysis of multiple viral clones showed nef gene deletions/insertions in 10 out of 15 SP, along with the coexistence of intact and defective nef gene lineages in the same individual over time, whereas these nefgene abnormalities were absent from HIV-1 strains from LTNP. Increasing quasispecies diversity in HIV-1 strains, over time, abrogation of a V3 region N-linked glycosylation site in > 60% of the clones, and, importantly, an extended V2 region were unique features of HIV-1 strains from SP and LTNP. CONCLUSIONS: The V2 region extension was unique to only SP and LTNP, and so may have a role in slow progression or non-progression of HIV disease. Increasing genetic diversity in HIV-1 strains in SP and LTNP correlated with the immunocompetent status of the host.


Subject(s)
HIV-1/isolation & purification , Amino Acid Sequence , Base Sequence , Cohort Studies , DNA Primers , Disease Progression , Genes, nef , HIV-1/genetics , HIV-1/physiology , Homosexuality, Male , Humans , Male , Molecular Sequence Data , Sequence Homology, Amino Acid , Virus Replication/physiology
7.
J Infect Dis ; 180(4): 1033-42, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10479128

ABSTRACT

Persons who were human immunodeficiency virus type 1 (HIV-1)-infected but who remained persistently seronegative (HIPS) on HIV-1 antibody tests were examined through AIDS case surveillance. Six such individuals (HIPS-1 to -4, -7, and -9) were examined to determine whether their persistent seronegativity was attributable to immune dysfunction or infection with atypical HIV. Of the 6, 4 had antibody titers to at least 1 other common pathogen. In vitro stimulation of peripheral blood mononuclear cells from HIPS-4 and HIPS-7 with pokeweed mitogen or phosphorothioate oligodeoxynucleotide (direct B cell mitogen) did not produce HIV-1-specific antibody. Reconstitution experiments with recombinant interleukin (rIL)-4 and rIL-12 also had no impact on antibody production. Virus isolates from HIPS-4 and -9 were R5X4-tropic, whereas HIPS-7 was CCR5-tropic only. Sequence analysis of long terminal repeat, p24, and env gp41 did not reveal any specific mutation, and phylogenetic analysis confirmed that all 6 virus specimens were HIV-1 subtype B. These data suggest that the lack of a detectable antibody response in these patients may be the result of immune dysfunction.


Subject(s)
B-Lymphocytes/immunology , Genes, nef , HIV Envelope Protein gp41/genetics , HIV Infections/immunology , HIV Infections/virology , HIV Seronegativity , HIV-1/genetics , Amino Acid Sequence , Antibody Formation , B-Lymphocytes/drug effects , Base Sequence , Consensus Sequence , Epitopes/chemistry , HIV Antibodies/blood , HIV Envelope Protein gp41/immunology , HIV-1/isolation & purification , Humans , Interleukin-12/pharmacology , Interleukin-4/pharmacology , Lymphocyte Activation , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/pharmacology , Recombinant Proteins/pharmacology , Sequence Alignment , Thionucleotides
8.
Virology ; 261(1): 106-19, 1999 Aug 15.
Article in English | MEDLINE | ID: mdl-10441559

ABSTRACT

To study human herpesvirus 8 (HHV-8) transmission between individuals and in populations, we developed a system for genetic fingerprinting of HHV-8 strains based on variation in the HHV-8 K1, glycoprotein B (gB), and glycoprotein H (gH) genes. Using this system, we sequenced nearly the entire K1 gene (840 bp); two segments of the gB gene (open reading frame 8), totaling 813 bp; and a 702-bp segment of the gH gene (open reading frame 22) from blood and tissue samples obtained from 40 human immunodeficiency virus-infected and noninfected individuals, including those with Kaposi's sarcoma, primary effusion lymphoma, or Castleman's disease. The specimen collection was assembled from individuals living in diverse geographical locations, including the United States, Australia, New Zealand, Uganda, and Zambia. As reported by others, K1 was the most variable gene, with up to 16% variation at the nucleotide sequence level and up to 32% variation at the amino acid sequence level. Despite this extensive sequence variation, the K1 amino acid sequence contained 14 conserved cysteine sites, suggesting a conserved tertiary structure. gB and gH sequences were highly conserved, in most cases differing by <0.6% in pairwise comparisons. K1 was the most useful gene for strain discrimination, but the other genes enabled the discrimination of strains with identical K1 sequences. Individuals from diverse geographic locations were infected with four different HHV-8 genotypes; strains did not strictly segregate by continent of origin. The majority of HHV-8 strains from the United States and Europe were relatively closely related, whereas some strains identified from Uganda and Australia were phylogenetically distant. Genotype I strains were the most common and were found on three continents. Identical sequences were found in specimens obtained from different body sites and at different times from the same individual.


Subject(s)
AIDS-Related Opportunistic Infections/virology , Herpesviridae Infections/virology , Herpesvirus 8, Human/classification , Lymphoma, AIDS-Related/virology , Sarcoma, Kaposi/virology , Africa , Amino Acid Sequence , Asia , Australia , DNA Fingerprinting , DNA, Viral/chemistry , DNA, Viral/genetics , Genotype , HIV Infections/virology , Herpesvirus 8, Human/genetics , Humans , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , United States , Viral Envelope Proteins/genetics , Viral Proteins/genetics
9.
J Clin Microbiol ; 37(5): 1298-301, 1999 May.
Article in English | MEDLINE | ID: mdl-10203474

ABSTRACT

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.


Subject(s)
DNA, Viral/analysis , Herpesvirus 8, Human/genetics , Polymerase Chain Reaction , Semen/virology , Humans , Male , Reproducibility of Results , Sensitivity and Specificity
10.
AIDS ; 12(13): F137-43, 1998 Sep 10.
Article in English | MEDLINE | ID: mdl-9764773

ABSTRACT

OBJECTIVE: To study coreceptor usage of sequential primary HIV-1 isolates in a longitudinal follow-up cohort of HIV-1-infected men to understand its contribution to pathogenesis of HIV disease. DESIGN: Viral coreceptor usage of sequential primary isolates from HIV-1-infected individuals was examined at various timepoints and data was compared with CD4 cell counts, rates of disease progression and beta-chemokine production. METHODS: Fifty-eight sequential primary isolates were obtained from four rapid progressors, six late progressors, and three long-term nonprogressors (LTNP) and their coreceptor usage was examined by infection of peripheral blood mononuclear cells (PBMC) from donors with wild-type or non-functional CC-chemokine receptor (CCR)-5, and by infection of GHOST4 cells expressing CD4 and various chemokine receptors [CCR-1-CCR-5, CXC-chemokine receptor (CXCR)-4, BOB/GPR15, BONZO/STRL33]. Production of RANTES and macrophage inflammatory protein (MIP)-1beta was examined using unstimulated or phytohemagglutinin (PHA)-stimulated PBMC isolated from these individuals at multiple timepoints during infection. RESULTS: A switch from single CCR-5 coreceptor usage to multiple coreceptor usage occurred in all four rapid progressors and three out of six late progressors. In addition to the commonly used coreceptors CXCR-4, CCR-5, and CCR-3, some of the viruses isolated from patients in the terminal stage of infection also used CCR-1, CCR-2b, CCR-4, and BOB as coreceptors. The emergence of viral variants capable of utilizing multiple coreceptors generally preceded CD4 cell decline to < 200 x 10(6)/l and correlated with the onset of AIDS. In contrast, three LTNP maintained exclusive usage of CCR-5 over a period of 7-12 years post-infection. Endogenous production of RANTES and MIP-1beta by PBMC from LTNP was not significantly different from rapid and late progressors. However, PHA-driven production of both chemokines was significantly higher in LTNP, suggesting that in vivo activating stimuli might curtail HIV replication by inducing these chemokines. CONCLUSIONS: Viral variants capable of utilizing a broad range of coreceptors correlated with HIV-1 disease progression. In contrast, LTNP maintain exclusive usage of CCR-5 and produce higher levels of beta-chemokines. Thus, both viral and host determinants leading to the emergence of viral variants capable of using an expanded range of coreceptors may be likely determinants of disease progression.


Subject(s)
HIV Seropositivity/virology , HIV-1/pathogenicity , Homosexuality, Male , Receptors, G-Protein-Coupled , Receptors, HIV/metabolism , Adaptation, Physiological , Cell Line , Chemokine CCL4 , Chemokine CCL5/metabolism , Chemokines, CC/metabolism , Cohort Studies , Disease Progression , GTP-Binding Proteins/metabolism , HIV-1/metabolism , Humans , Macrophage Inflammatory Proteins/metabolism , Male , Receptors, CCR1 , Receptors, CCR2 , Receptors, CCR3 , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Receptors, Chemokine/metabolism , Receptors, Cytokine/metabolism , Receptors, Peptide/metabolism
12.
J Clin Microbiol ; 36(6): 1574-7, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9620379

ABSTRACT

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi's sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , Epitope Mapping , Herpesvirus 8, Human/immunology , Immunodominant Epitopes , AIDS-Related Opportunistic Infections/diagnosis , Amino Acid Sequence , Antigens, Viral/genetics , Female , Fluorescent Antibody Technique , Herpesvirus 8, Human/genetics , Humans , Immunoenzyme Techniques , Male , Oligopeptides/genetics , Oligopeptides/immunology , Open Reading Frames/genetics , Sarcoma, Kaposi/diagnosis
13.
AIDS Res Hum Retroviruses ; 14(4): 305-9, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9519891

ABSTRACT

The relevance of a TNF-alpha promoter polymorphism, a G-to-A polymorphic sequence at position-308, was examined to test whether variant alleles of TNF-alpha affect susceptibility to infection with HIV-1 and progression to AIDS. Analysis of specimens from cohorts of HIV-1 positive homosexual men demonstrated that 3 of the 32 (9.4%) HIV-1-infected long-term nonprogressors (LTNPs) were homozygous for the uncommon TNF-2 allele compared with 3 of the 196 (1.5%) HIV-1-seronegative blood donors and uninfected homosexual men (p < 0.05). There was no difference in heterozygosity among HIV-1-seropositive or -seronegative groups, although some of the seropositive men heterozygous for the TNF2 genotype were also heterozygous for CCR5delta32. However, no significant association was found between TNF genotypes and time of survival, CD4 slopes, or viral loads when seroincident (n = 109) and seroprevalent cases (n = 442) from the Chicago MACS were analyzed. Functional analysis of lymphocytes from the seronegative group revealed no difference in endogenous or mitogen-induced TNF-alpha production, as well as susceptibility to in vitro HIV-1 infection between different TNF-genotype donors. These data suggest that TNF genotypes do not play a direct role in HIV-1 disease progression; however, they could potentially be part of a multigenic linkage that may be involved in delaying progression to AIDS.


Subject(s)
HIV Infections/genetics , HIV Infections/immunology , HIV-1 , Polymorphism, Genetic , Tumor Necrosis Factor-alpha/genetics , Acquired Immunodeficiency Syndrome/etiology , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/immunology , Alleles , Cohort Studies , Genotype , HIV Seronegativity/genetics , HIV Seronegativity/immunology , HIV Seropositivity/genetics , HIV Seropositivity/immunology , Heterozygote , Homosexuality, Male , Homozygote , Humans , In Vitro Techniques , Lymphocytes/immunology , Male , Promoter Regions, Genetic , Receptors, CCR5/genetics , Tumor Necrosis Factor-alpha/biosynthesis
14.
AIDS Res Hum Retroviruses ; 12(15): 1443-56, 1996 Oct 10.
Article in English | MEDLINE | ID: mdl-8893052

ABSTRACT

HIV-1 produces more than 20 mRNAs encoding the viral proteins. We have used a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) approach to determine HIV-1 transcriptional patterns during the course of viral infection in unstimulated peripheral blood mononuclear cells (PBMCs) from different patients. Several sets of PCR primers, used in parallel reactions, allowed the amplification and specific detection of almost all individual HIV-1 transcripts. We investigated the transcriptional profile in two individuals during primary acute and early chronic infection. In these individuals, HIV-1 mRNA expression was elevated at the first time points examined and declined over time. In addition, we performed a detailed study of HIV-1 expression in several individuals over a minimum of 7 years following seroconversion. We found that long-term asymptomatic individuals had undetectable or low levels of the three classes of HIV-1 transcripts (unspliced, singly spliced, and multiply spliced). Individuals who demonstrated disease progression showed either a general increase in the amount of expression of all transcripts or elevated levels of unspliced transcripts in late-stage disease. The splicing pattern in each patient was conserved over the years and differed among the different individuals. No evidence of major changes in the splicing pattern was found during disease progression within the same individual. Thus, HIV-1 transcriptional patterns are viral strain specific rather than disease stage specific. These results indicate that high-level expression of any class of HIV-1 transcripts is associated with clinical progression. Our analysis also demonstrates the importance of using more than one set of primers to evaluate HIV-1 RNA expression, since virus in patient PBMCs showed sequence heterogeneity in conserved regions.


Subject(s)
HIV-1/genetics , Leukocytes, Mononuclear/virology , RNA Splicing , RNA, Messenger/chemistry , RNA, Viral/chemistry , CD4 Lymphocyte Count , Disease Progression , Humans , Polymerase Chain Reaction
15.
J Virol ; 70(8): 5236-45, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8764033

ABSTRACT

An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37 degrees C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or (Ca2+)- or (Mg2+)-dependent cofactor. The mechanism of inactivation cannot be explained by agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.


Subject(s)
HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , Animals , Antigen-Antibody Reactions , Cell Line , HIV Antibodies/therapeutic use , HIV Infections/therapy , Humans , Mice
16.
N Engl J Med ; 335(4): 233-41, 1996 Jul 25.
Article in English | MEDLINE | ID: mdl-8657239

ABSTRACT

BACKGROUND: If Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma, serologic evidence of infection should be present in patients before the disease develops. METHODS: Using an immunoblot assay for two latent nuclear antigens of KSHV, we tested serum samples from homosexual male patients with the acquired immunodeficiency syndrome (AIDS) with and without Kaposi's sarcoma (HIV-infected men with hemophilia), HIV-seronegative blood donors, and HIV-seronegative patients with high titers of antibodies against Epstein-Barr virus (EBV). Serial serum samples obtained from patients with Kaposi's sarcoma before the diagnosis of the disease were tested for evidence of seroconversion. RESULTS: Of 40 patients with Kaposi's sarcoma, 32 (80 percent) were positive for antibodies against KSHV antigens by the immunoblot assay, as compared with only 7 of 40 homosexual men (18 percent) without Kaposi's sarcoma immediately before the onset of AIDS. Of 122 blood donors, 22 EBV-infected patients, and 20 HIV-infected men with hemophilia, none were seropositive. When studied by the immunoblot assay over a period of 13 to 103 months, 21 of the 40 patients with Kaposi's sarcoma (52 percent) seroconverted 6 to 75 months before the clinical appearance of Kaposi's sarcoma. The median duration of antibody seropositivity for KSHV-related latent nuclear antigens before the diagnosis of Kaposi's sarcoma was 33 months. CONCLUSIONS: In most patients with kaposi's sarcoma and AIDS, seroconversion to positivity for antibodies against KSHV-related nuclear antigens occurs before the clinical appearance of Kaposi's sarcoma. This supports the hypothesis that Kaposi's sarcoma results from infection with KSHV.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , Herpesviridae/immunology , Nuclear Proteins/immunology , Sarcoma, Kaposi/virology , Acquired Immunodeficiency Syndrome/complications , HIV Infections/complications , HIV Seronegativity , Hemophilia A/complications , Herpesviridae/isolation & purification , Humans , Immunoblotting , Male , Sarcoma, Kaposi/etiology , Sensitivity and Specificity
18.
Lancet ; 346(8990): 1601-2, 1995 Dec 16.
Article in English | MEDLINE | ID: mdl-7500753

ABSTRACT

We explored a possible route of transmission of Kaposi's-sarcoma-associated herpes virus (KSHV) with nested and unnested PCR techniques. We looked for KSHV DNA sequences in semen of HIV-positive homosexual men and HIV-negative healthy semen donors. With unnested primers we found KSHV sequences in 21 of 33 (64%) homosexual men and in none of 30 healthy donors. With a nested PCR assay, 30 of 33 (91%) specimens from the homosexual men and 7 of 30 (23%) specimens from healthy donors had detectable KSHV sequences. Over 5 years of follow-up, 13 of 30 KSHV-positive homosexual men (43%) developed KS compared with none of the 3 KSHV-negative homosexual men.


Subject(s)
HIV Infections/complications , Herpesviridae/isolation & purification , Homosexuality, Male , Sarcoma, Kaposi/virology , Semen/virology , DNA, Viral/genetics , HIV Infections/virology , HIV Seronegativity , Herpesviridae/genetics , Humans , Male , Polymerase Chain Reaction , Retrospective Studies , Sarcoma, Kaposi/etiology , Sequence Analysis, DNA
19.
Pediatr Infect Dis J ; 14(6): 527-35, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7667059

ABSTRACT

We investigated children with CD4+ T lymphocytopenia to determine the magnitude and public health impact of this condition and to investigate possible causes. Children < 13 years old with CD4+ T lymphocyte counts below age-adjusted cutoffs (age < 24 months, 1000 cells/microliters; age > or = 24 months, 300 cells/microliters) or < 20% on 2 separate measurements were considered to have CD4+ T lymphocytopenia. We solicited information from clinicians and public health departments on these children and their families and collected blood for immunologic and retroviral testing. We identified 18 children (10 boys; 14 African-Americans) with a median age of 10 months at their first low CD4+ T lymphocyte measurement. Three children had had opportunistic infections and two still had low CD4+ T lymphocyte counts 5 and 7 years later. Of the 11 children born to human immunodeficiency virus (HIV)-infected mothers 7 were asymptomatic. Specimens from all children were negative for HIV and human T lymphotropic virus antibodies and negative for HIV by culture or polymerase chain reaction. Among 12 families interviewed no other HIV-seronegative family or household member had illnesses suggestive of immunosuppression. We conclude that negative retroviral tests and lack of illness among their family members do not support the hypothesis that a retrovirus causes CD4+ T lymphocytopenia among these children.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
HIV Seronegativity/immunology , Opportunistic Infections , Retroviridae Infections , T-Lymphocytopenia, Idiopathic CD4-Positive , Antibodies, Bacterial/analysis , CD4 Lymphocyte Count , Child , Child, Preschool , Female , HIV Infections/immunology , HIV Infections/transmission , Humans , Immunophenotyping , Infant , Infectious Disease Transmission, Vertical , Male , Opportunistic Infections/diagnosis , Opportunistic Infections/immunology , Retroviridae Infections/diagnosis , Retroviridae Infections/epidemiology , Retroviridae Infections/immunology , Retroviridae Infections/physiopathology , Serologic Tests , T-Lymphocytopenia, Idiopathic CD4-Positive/diagnosis , T-Lymphocytopenia, Idiopathic CD4-Positive/epidemiology , T-Lymphocytopenia, Idiopathic CD4-Positive/etiology , T-Lymphocytopenia, Idiopathic CD4-Positive/immunology
20.
J Infect Dis ; 171(5): 1210-6, 1995 May.
Article in English | MEDLINE | ID: mdl-7538549

ABSTRACT

In an ultrasensitive assay for reverse transcriptase (RT), an in vitro-transcribed heteropolymeric RNA sequence was used as a template and polymerase chain reaction (PCR) amplification with Southern blot hybridization served as a detection system for the cDNA reaction product. The assay, called Amp-RT, detected 9 tested retroviruses in unconcentrated culture supernatants diluted 10(2)- to 10(5)-fold. A comparative analysis using human immunodeficiency virus type 1 (HIV-1) revealed that Amp-RT was 100,000 times more sensitive than the standard RT assay, 10,000 times more sensitive than p24 antigen capture and branched DNA assays, and 100 times more sensitive than RT-PCR or TCID50 assays. Analysis of serum specimens from 42 HIV-1-infected persons by Amp-RT showed that 36 samples (85.7%) were RT-positive. In contrast, 41 serum specimens from persons seronegative for HIV-1 and human T lymphotropic virus types I and II were all Amp-RT-negative.


Subject(s)
HIV Infections/diagnosis , HIV-1/isolation & purification , Polymerase Chain Reaction/methods , RNA-Directed DNA Polymerase/blood , Animals , Base Sequence , CD4 Lymphocyte Count , DNA, Complementary/biosynthesis , DNA, Viral/blood , Encephalomyocarditis virus/genetics , HIV Core Protein p24/blood , HIV Infections/immunology , HIV Infections/virology , HIV Reverse Transcriptase , HIV-1/enzymology , Humans , Male , Molecular Sequence Data , RNA, Viral/genetics , Retroviridae/isolation & purification , Sensitivity and Specificity , Templates, Genetic
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