Naturally occurring sequence polymorphisms within HIV type 1 group O protease.

Mutations within the protease gene associated with reduced susceptibility to protease inhibitors have been well documented for HIV-1 group M subtype B strains. In contrast, limited genotypic and phenotypic information is available for the genetically diverse HIV-1 group O strains. Preexisting resistance-associated polymorphisms have the potential to contribute to a poor virological response to antiviral drug treatment in group O-infected patients. In the present study, the protease genes of 28 protease inhibitor-naive HIV-1 group O-infected patients were analyzed to identify any naturally occurring amino acid polymorphisms associated with drug resistance. Comparison of the consensus group O protease sequence with subtype B of group M indicated that both groups have almost identical sequences in the protease active site, the flap and the substrate-binding site. Analysis of the 28 individual protease sequences revealed polymorphisms at 34% of the positions within the protease gene, but no primary mutations associated with protease inhibitor resistance. In contrast, each of the strains harbored multiple secondary or accessory mutations associated with resistance to protease inhibitors in group M viruses. Residues 10I, 15V, 36I, 41K, 62V, 63T/A/K/I, 64V, 71V, and 93L were identified in most strains. The presence of multiple natural sequence polymorphisms associated with drug resistance in the protease gene of group O viruses may contribute to a more rapid emergence of drug resistance phenotype and treatment failure in group O-infected patients.

Seven human immunodeficiency virus (HIV) antigen-antibody combination assays: evaluation of HIV seroconversion sensitivity and subtype detection.

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at approximately 25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was approximately 125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.

Quantification of HIV-1 group M (subtypes A-G) and group O by the LCx HIV RNA quantitative assay.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity presents a challenge to nucleic acid-based assays with regard to sensitivity of detection and accuracy of quantification. The Abbott LCx HIV RNA Quantitative assay (LCx(R) HIV assay), a competitive RT-PCR targeting the pol integrase region, was evaluated using a panel of 297 HIV-1 seropositive plasma samples from Cameroon, Uganda, Brazil, Thailand, Spain, Argentina and South Africa. The panel included group M subtypes A-G, mosaics, and group O based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified 290 (97.6%) of the samples, including all the group O samples tested. In comparison, the Roche AMPLICOR HIV-1 MONITOR test versions 1.0 and 1.5 quantified 67.3 and 94.6% of the samples, respectively. No group O specimens were quantified by either version of AMPLICOR HIV-1 MONITOR. Seven specimens were below the detectable limits of all the three assays. The LCx HIV assay had fewer nucleotide mismatches at primer/probe binding sites as compared with both AMPLICOR HIV-1 MONITOR tests. The high degree of nucleotide conservation within the pol target region enables the LCx HIV assay to efficiently quantify the HIV-1 subtypes A-G and the most genetically diverse HIV-1, group O.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

Genetic analysis of human immunodeficiency virus in Abidjan, Ivory Coast reveals predominance of HIV type 1 subtype A and introduction of subtype G.

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.

Serotyping of HIV type 1 infections: definition, relationship to viral genetic subtypes, and assay evaluation. UNAIDS Network for HIV-1 Isolation and Characterization.

V3 serotyping refers to a system based on binding of antibody in patient sera to V3-loop peptides derived from HIV-1 env genetic subtypes. The V3x serotype represents reactivity of serum from an HIV-1-infected patient (regardless of viral genetic subtype), which reacts preferentially to a V3 peptide derived from the X subtype sequence. We have classified HIV-1 serotypes, determined the relationship between the HIV-1 V3 serotypes and viral genetic subtypes in a large study (n = 125), and evaluated the performance of three different V3 peptide-binding assays. Seven HIV-1 V3 serotypes were identified: A, B, B-Br, B-Th, C, D, and E. Serotypes B-Br and B-Th represent sera that react specifically to peptides derived from Brazilian B (B-Br, GWGR) and Thai B (B-Th, GPGQ) strains. The HIV-1 V3 B, C, and E serotypes correlated closely with their viral env genetic subtypes; 19-26 of 32 B sera (59-79%), 3-4 of 4 C sera (75-100%), and 19-22 of 23 E sera (83-96%) were identified as serotypes B, C, and E, respectively. In contrast, two major V3 serotypes were classified in A sera: A (14-18 of 36 [40-50%]) and C (12-19 of 36 [33-54%]). Similarly, two major V3 serotypes were classified in D sera: B (6-10 of 20 [30-50%]) and D (9-12 of 20 [45-60%]). Serotyping of subtype E sera showed the best concordance with genetic subtypes by all assays. Overall, HIV-1 V3 serotyping produced consistent results among three laboratories. However, HIV-1 V3 serotypes do not distinguish all HIV-1 genetic subtypes. The relative biological significance of the V3 serotypes remains to be elucidated.

HIV type 1 in Thailand, 1994-1995: persistence of two subtypes with low genetic diversity.

Extensive transmission of human immunodeficiency virus type 1 (HIV-1) in Thailand began in 1988, resulting in an estimated 800,000 cumulative infections by 1994. During 1994 and 1995, we collected blood specimens from 215 asymptomatic HIV-1-infected people with various risk behaviors from nine locations in all four regions of Thailand. HIV-1 subtypes and genetic heterogeneity were determined for 214 strains by a combination of direct DNA sequencing (n = 95), subtype-specific oligonucleotide probe testing (n = 201), and V3-loop peptide enzyme immunoassay (PEIA) (n = 214). All strains were either env subtype E (175; 81.8%) or B (39; 18.2%). Of the subtype B isolates, 37 (94.9%) were B' and 2 (5.1%) were more typical North American-like B strains (most subtype B strains in Thailand are part of a distinct subcluster within the subtype B branch on phylogenetic trees, termed B'; formerly Thai B or BB). Of 149 viruses from people with sexual risk behaviors from all regions, 146 (98.0%) were subtype E. Of 65 viruses from injecting drug users (IDUs), 29 (44.6%) were subtype E and 36 (55.4%) were subtype B, including 35 B' strains. There was regional variation in the proportions of subtypes E and B' among IDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene (mid-C2 to the start of the V4 region) was low (5.7% for subtype E and 3.1% for subtype B') compared with other HIV-1 group M subtypes from different countries. These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable setting for evaluating candidate HIV-1 vaccines. The mix of subtype E and B' strains among IDUs also offers the opportunity to study phenotypic differences between the two subtypes.

PIP: The extensive transmission of HIV-1 in Thailand which began in 1988 led to an estimated 800,000 cumulative infections in the country by 1994. The authors collected blood specimens during 1994 and 1995 from 215 asymptomatic HIV-1-infected people with various risk behaviors from 9 locations across Thailand. HIV-1 subtypes and genetic heterogeneity were then determined for 214 strains using a combination of direct DNA sequencing, subtype-specific oligonucleotide probe testing, and V3-loop peptide enzyme immunoassay. 175 strains were subtype E and 39 were subtype B. 37 of the subtype B isolates were B' and 2 were more typical North American-like B strains. Of 149 viruses from people with sexual risk behaviors from all regions of the country, 146 were subtype E. Of 65 viruses from IV drug users (IVDUs), 29 were subtype E and 36 were subtype B, including 35 subtype B' strains. Regional variation was observed in the proportions of subtypes E and B' among IVDUs. The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene was 5.7% for subtype E and 3.1% for subtype B'. The finding of 2 HIV-1 subtypes with low heterogeneity suggests that Thailand may be an appropriate setting in which to evaluate candidate HIV-1 vaccines. The mix of subtype E and B' strains among IVDUs will also allow the study of phenotypic differences between the 2 subtypes.

Group O human immunodeficiency virus-1 infections.

Human immunodeficiency viruses (HIV), the cause of AIDS, have remarkable genetic diversity. Among the HIV-1 viruses are the "major" (group M) HIV-1 subtypes and genetic "outliers" that have been designated as group O viruses. Group O viruses are most prevalent in parts of Africa, although they have also been reported in Europe and the United States and are associated with AIDS. Because group O viruses are so highly divergent, tests designed to detect group M viruses may be unreliable in the diagnosis of group O infection. Modification of these tests are needed to protect the safety of the blood supply.

Horizontal and vertical transmission of human immunodeficiency virus type 1 dual infections caused by viruses of subtypes B and C.

This article describes a case of horizontal (heterosexual) and subsequent vertical (mother to infant) transmission of 2 human immunodeficiency viruses type 1 (HIV-1) subtypes. Dual infection in a husband, his wife, and their child was initially detected by use of a restriction fragment length polymorphism assay of the proviral protease in peripheral blood mononuclear cells. The simultaneous presence of highly similar sets of HIV-1 subtypes B and C infecting the 3 family members was confirmed by DNA sequence analysis of pol, gag, and env genes. These data, together with available epidemiologic information, may indicate that the husband's high-risk sexual behavior was the source of dual infections. Because his wife did not report such activities, it was likely that he passed HIV-1 strains to his spouse, who subsequently transmitted them to their child.

Genetic variability of HIV type 1 in Honduras.

PIP: While Honduras is home to only 15% of Central America's population, it has 60% of the region's AIDS cases. There have been 4973 reported cases of full-blown AIDS in the country and the Health Ministry reports that more than 8000 Hondurans have been infected with HIV since the first Honduran case was diagnosed in 1985. 995 people with AIDS have since died. The authors conducted an investigation to determine which HIV-1 subtype is present in Honduras and the degree of genetic variation among HIV-1 strains by analyzing viral nucleotide sequences from the envelope region of HIV-1 isolates obtained from the two most affected regions of the country. They determined the predominant HIV-1 subtype among 27 HIV-1-infected patients attending sexually transmitted disease clinics in San Pedro Sula and Tegucigalpa by sequencing and analyzing the C2V3 regions of the envelope glycoprotein gp 120. Genomic DNA was isolated from patients' peripheral blood mononuclear cells. Phylogenetic analysis determined that all 27 Honduran sequences clustered with known subtype B sequences.^ieng

HIV type 1 subtypes in Malaysia include B, C, and E.

PIP: Peripheral blood mononuclear cell specimens were collected from 13 HIV-1-infected IV drug users in Kuala Lumpur, Malaysia, as well as one HIV-infected baby, between 1992 and 1993. DNA was then amplified by nested polymerase chain reaction and a 345-bp fragment of the C2V3 region of the env gene was sequenced. 11 of the 14 Malaysian sequences clustered with the B' subtype, one different from the typical subtype B US strains HIVMN and HIVSF2. Two sequences grouped in the C subtype and had sister taxa closer to the Indian C subtype sequences than those from Zambia. The sequence from the infant was identified as a subtype E virus, grouped more closely with subtype E strains from Thailand than subtype E viruses from the Central African Republic.^ieng

Surveillance for human immunodeficiency virus type 1 group O infections in the United States.

BACKGROUND: Reports that the human immunodeficiency virus type 1 (HIV-1) group O variants are not reliably detected by some commercial diagnostic tests have raised concerns about the sensitivity of existing screening tests, especially with regard to blood safety. Although it is unlikely that these divergent strains are prevalent in North America, systematic, continuous surveillance is needed to monitor the potential spread of HIV variants into that region. STUDY DESIGN AND METHODS: Stored serum samples (n = 1072) from both high- and low-risk population groups at several sites in the United States and Puerto Rico were tested by peptide enzyme immunoassays specific for the prototypic HIV-1 group O strains, MVP5180 and ANT70. RESULTS: None of the 1072 samples examined had peptide reactivity that was consistent with HIV-1 group O infection. CONCLUSION: While no evidence of specific HIV-1 group O (MVP5180 or ANT70) infection was found in this study, the sensitivity of current tests has not been fully evaluated against the wide range of genetic variation of HIV. Therefore, it is important to continue active surveillance for HIV-1 and HIV type 2 strains, to characterize any divergent strains, and to judiciously modify tests to correct for any deficiencies in sensitivity.

Identification of single and dual infections with distinct subtypes of human immunodeficiency virus type 1 by using restriction fragment length polymorphism analysis.

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic. As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections caused by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infections caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes. To address this need, we have developed a genetic method based on restriction fragment length polymorphism (RFLP) to screen for these two types of infections within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples. A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis. The viral regions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classification of HIV-1 strains to well defined subtypes B, D, F, and A/C is done by sequential endonuclease restriction analysis of a PCR amplified-protease gene followed by analysis of the p24 gag region. The electrophoretic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel. In infections caused by viruses of a singular subtype, a single restriction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of different digestion patterns are observed in infected individuals. Using this methodology we have screened for genetic variations in HIV-1 proviral DNA from thirty-three Brazilian samples. Our RFLP procedure classified thirty-two samples as single infections caused by viruses of subtypes B (31) and F (1), and one sample as dual infection caused by distinct viral strains. Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirmed our RFLP findings in single infections, and demonstrated the existence of two distinct HIV-1 strains of subtypes F and D in a patient which lymphocytes showed the simultaneous presence of two different digestion patterns. As up to now, single infections caused by subtype D variants were not identified in Brazil, our data provide the first evidence of subtype D HIV-1 in this country. Because sequencing of HIV proviral DNA is not particularly practical for large-scale molecular epidemiological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple infections caused by HIV-1 strains of distinct subtypes. We believe that this information is crucial for both evaluation of the HIV-1/AIDS pandemic and intervention strategies.

The emerging genetic diversity of HIV. The importance of global surveillance for diagnostics, research, and prevention.

The discovery of highly divergent strains of human immunodeficiency virus (HIV) not reliably detected by a number of commonly used diagnostic tests has underscored the need for effective surveillance to track HIV variants and to direct research and prevention activities. Pathogens such as HIV that mutate extensively present significant challenges to effective monitoring of pathogens and to disease control. To date, relatively few systematic large-scale attempts have been made to characterize and sequence HIV isolates. For most of the world, including the United States, information on the distribution of HIV strains among different population groups is limited. We describe herein the implications resulting from the rapid evolution of HIV and the need for systematic surveillance integrated with laboratory science and applied research. General surveillance guidelines are provided to assist in identifying population groups for screening, in applying descriptive epidemiology and systematic sampling, and in developing and evaluating efficient laboratory testing algorithms. Timely reporting and dissemination of data is also an important element of surveillance efforts. Ultimately, the success of global surveillance network depends on collaboration and on coordination of clinical, laboratory, and epidemiologic efforts.

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.