Presence of human immunodeficiency virus (HIV) type 1 subtype A infection in a New York community with high HIV prevalence: a sentinel site for monitoring HIV genetic diversity in North America. Centers for Disease Control and Prevention-Bronx Lebanon HIV Serosurvey Team

To determine whether US residents are infected with subtypes of human immunodeficiency virus (HIV) type 1 other than subtype B (Western), the predominant North American subtype with a unique GPGR genetic sequence in the V3 loop, viruses from 22 HIV-infected adults were serotyped and subtyped. Twenty patients had subtype B (Western), of whom 15 had serotype B (Western), 3 had serotype A/C, 1 had serotype B (Thai), and 1 had a nontypeable serotype. Two had subtype A, both serotype A/C. Both subtype A-infected patients, only 1 of whom had been outside the United States, reported sex with persons traveling abroad, suggesting possible acquisition in the United States. Because US residents are infected with non-subtype B (Western) strains, US surveillance for HIV-1 diversity is needed to elucidate subtype-specific transmission patterns and pathogenesis and to guide evaluation and development of HIV diagnostic tests and vaccines.     

Lack of correlation between V3-loop peptide enzyme immunoassay serologic subtyping and genetic sequencing

OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.     

An isolate of human immunodeficiency virus type 1 originally classified as subtype I represents a complex mosaic comprising three different group M subtypes (A, G, and I)

Full-length reference clones and sequences are currently available for eight human immunodeficiency virus type 1 (HIV-1) group M subtypes (A through H), but none have been reported for subtypes I and J, which have only been identified in a few individuals. Phylogenetic information for subtype I, in particular, is limited since only about 400 bp of env gene sequences have been determined for just two epidemiologically linked viruses infecting a couple who were heterosexual intravenous drug users from Cyprus. To characterize subtype I in greater detail, we employed long-range PCR to clone a full-length provirus (94CY032.3) from an isolate obtained from one of the individuals originally reported to be infected with this subtype. Phylogenetic analysis of C2-V3 env gene sequences confirmed that 94CY032.3 was closely related to sequences previously classified as subtype I. However, analysis of the remainder of its genome revealed various regions in which 94CY032.3 was significantly clustered with either subtype A or subtype G. Only sequences located in vpr and nef, as well as the middle portions of pol and env, formed independent lineages roughly equidistant from all other known subtypes. Since these latter regions most likely have a common origin, we classify them all as subtype I. These results thus indicate that the originally reported prototypic subtype I isolate 94CY032 represents a triple recombinant (A/G/I) with at least 11 points of recombination crossover. We also screened HIV-1 recombinants with regions of uncertain subtype assignment for the presence of subtype I sequences. This analysis revealed that two of the earliest mosaics from Africa, Z321B (A/G/?) and MAL (A/D/?), contain short segments of sequence which clustered closely with the subtype I domains of 94CY032.3. Since Z321 was isolated in 1976, subtype I as well as subtypes A and G must have existed in Central Africa prior to that date. The discovery of subtype I in HIV-1 hybrids from widely distant geographic locations also suggests a more widespread distribution of this virus subtype, or at least segments of it, than previously recognized.     

HIV type 1 subtypes, coreceptor usage, and CCR5 polymorphism

Identification of the chemokine receptors CCR5 and CXCR4 as the major coreceptors for HIV-1 entry has greatly assisted our understanding of HIV-1 pathogenesis, transmission, and tropism. However, most of our current knowledge on coreceptor usage comes from studies using HIV-1 strains or env genes derived from the genetic subtype B predominant in North America and western Europe. In this report, the coreceptor usage of 20 primary viral isolates representative of genetic subtypes A, B, C, D, E, and group O was examined. Thirty-nine full-length CCR5 sequences from individuals of diverse geographic origins were also obtained to examine the possible effect of CCR5 polymorphism on HIV-1 subtype distribution. Our results indicate that (1) CCR5 and CXCR4 serve as the two major coreceptors for viruses belonging to HIV-1 subtypes A, B, C, D, E, and group O, whereas other chemokine receptors such as CCR2b and CCR3 play only a minor role in facilitating viral entry into stimulated PBMCs; (2) the coreceptor usage is determined by the viral phenotype rather than its genotype because all NSI strains, irrespective of their subtype classification, utilize CCR5, whereas all SI strains are able to use CXCR4; and (3) there is no geographic clustering of CCR5 polymorphism in different ethnic populations, suggesting that CCR5 diversity is not the underlying explanation for differences in the spread of different HIV-1 subtypes. Therefore, the uneven worldwide distribution of HIV-1 subtypes is more likely the result of stochastic dissemination.     

Genotyping HIV-1 and HCV strains by a combinatorial DNA melting assay (COMA)

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) strains can be genetically classified into genetic lineages known as genetic types or subtypes according to phylogenetic analyses of complete or partial nucleotide sequences of their genomes. The genetic classification of HIV-1 and HCV strains has important implications for the development of globally effective vaccines and for the management of patients. MATERIALS AND METHODS: A new method, termed combinatorial DNA melting assay (COMA), allows rapid accessing of comparative genetic information between related DNA sequences, making it possible to rapidly and accurately genotype unknown HIV-1 and HCV strains. COMA is mainly based on the differential melting properties of long DNA heteroduplexes. Combinatorial arrays of DNA heteroduplexes are formed when captured PCR-amplified reference DNA with known nucleotide sequences are combined with solution-phase complementary and antigenically labeled DNA with unknown sequences. Genetic divergence between the known and the unknown sequences is inferred as the experimentally derived melting curves of the two strands of the DNA heteroduplexes increasingly diverge. RESULTS: COMA was successfully applied to the genetic classification of HIV-1 and HCV strains into phylogenetic lineages or subtypes. CONCLUSIONS: Use of this assay should accelerate current efforts to understand the global molecular epidemiology of HIV-1 and HCV and may extend to the genetic characterization of other genetically diverse infectious pathogens associated with numerous diseases.     

Genetically related human immunodeficiency virus type 1 in three adults of a family with no identified risk factor for intrafamilial transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son's virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.     

HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.     

Correlation of genetic and serologic approaches to HIV-1 subtyping in Thailand

The aim of this study was to compare the performance of differential polymerase chain reaction (PCR) typing and peptide enzyme-linked immunosorbent assay (V3-EIA) for human immunodeficiency virus type 1 (HIV-1) subtyping in Thailand using heteroduplex mobility assay (HMA) as the reference standard. Paired peripheral blood mononuclear cells (PBMC) and sera were collected from 38 HIV-1 seropositive persons in Thailand. HMA was done by standard methods; differential PCR employs primer pairs that differentially amplify either subtype E or B. V3-EIA used peptides specific for subtypes E or B. Thirty-two cases (84%) were found by HMA to be infected with subtype E: and six with (16%) subtype B. The results obtained with differential PCR were 100% concordant with those of HMA; V3 EIA correctly predicted the subtype in 95% (36 of 38). Six samples that molecularly subtyped as E were repeatedly dual reactive by screening V3-EIA, but these resolved to subtype E using an antigen-limiting EIA. Two samples were serologically nontypeable because of overall low levels of V3 antibody. Using HMA as the standard, differential PCR was shown to subtype HIV-1 reliably from patient PBMC samples. V3-EIA correctly predicted HIV-1 subtype in most (95%) of our cases. Because of the less rigorous sampling requirements, specimen processing, and logistical and technical requirements of serotyping compared with molecular techniques, it appears to be practical for screening purposes in a field environment. Samples that cannot be definitively subtyped serologically should undergo differential PCR and antigen-limiting V3 EIA. These approaches to HIV-1 subtyping should be used in complementary fashion in Thailand, where subtypes B and E are currently known to cocirculate.     

Cross-reactions between the cytotoxic T-lymphocyte responses of human immunodeficiency virus-infected African and European patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Subtype G and multiple forms of A/G intersubtype recombinant human immunodeficiency virus type 1 in Nigeria

Multiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994-1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion of gag. Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found.     

Molecular cloning and characterization of viruses isolated from chimpanzees with pathogenic human immunodeficiency virus type 1 infections

We have previously described the development of AIDS in a chimpanzee (C499) infected with human immunodeficiency virus type 1 (HIV-1) and the subsequent pathogenic HIV-1 infection in another chimpanzee (C455) transfused with blood from C499 (F. J. Novembre et al., J. Virol. 71:4086-4091, 1997). In the present study, two virus isolates were derived from these animals: HIV-1JC from peripheral blood mononuclear cells (PBMC) of C499, and HIV-1NC from plasma of C455. These virus isolates were used to generate two infectious molecular clones, termed HIV-1JC16 and HIV-1NC7 (JC16 and NC7, respectively). Comparative analyses of the sequences of the two clones showed that they were highly interrelated but distinct. Based on heteroduplex mobility assays, JC16 and NC7 appear to represent dominant viruses in the uncloned stock population. Compared with amino acid sequences of the parental viruses HIV-1SF2, HIV-1LAV-1b, and HIV-1NDK, JC16 and NC7 showed a number of differences, including insertions, deletions, and point mutations spread throughout the genome. However, insertion/deletion footprints in several genes of both JC16 and NC7 suggested that recombination between SF2 and LAV-1b could have occurred, possibly contributing to the generation of a pathogenic virus. Comparative in vitro analyses of the molecular clones and the uncloned stocks of HIV-1JC and HIV-1NC revealed that these viruses had strikingly similar replicative abilities in mitogen-stimulated PBMC and in macrophages. Compared to the SF2 and LAV-1b isolates of HIV-1, HIV-1JC and HIV-1NC isolates were more similar to LAV-1b with respect to the ability to replicate in mitogen-stimulated PBMC and macrophages. These viruses should prove to be useful in mapping determinants of pathogenesis.     

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

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.     

The effects of incremental submaximal exercise on circulating leukocytes in physically active and sedentary males and females

The presence of HIV-1 DNA sequence variants of pediatric AIDS patients was investigated in a two-stage polymerase chain reaction (PCR) using nested primers for the first and second (V1-V2) hypervariable regions of the proviral envelope (gp 120) gene (env1). Gel electrophoresis analysis yielded amplified DNA bands which exhibited length variations which were characteristic for each child. The PCR products were cloned and the resulting clones demonstrated inserts of different lengths in a patient and between patients. Analysis of five clones from two different patients at the level of DNA sequencing indicates an extreme heterogeneity in the V1-V2 region. DNA maximum similarity between all of the isolated clones ranged between 69 to 96%. Comparison between DNA sequences of the isolated clones and HIV-1 strains of other parts of the world was also performed. The highest percentage of similarity that was found with known HIV variants included the following strains: HIVADA, HIVJFL, HIVSW42, HIVSWB83, HIVTRA2, HIVWMJ22. The sequences also showed a high degree of similarity to the clade B virus HIVSF162. The analyzed HIV-1 sequences demonstrated the expected high degree of variation in the V1-V2 region of the envelope gene and in some cases that the variation between isolates from the same patient may exceed the variation between the individual clones and the reference HIV-1 strains.     

Characterization of gag and pol sequences from long-term survivors of human immunodeficiency virus type 1 infection

We previously identified a group of 10 long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection. Extensive biological analysis revealed that some of these individuals do well, at least in part, because they possess weakened or attenuated viruses. Also, previously, to determine the genotype associated with the attenuated phenotype in vivo, we characterized nef, vif, vpr, vpu, env, and LTR in our cohort of LTS. In this study, we analyzed gag and pol genes derived from eight individuals in our cohort. For each subject multiple full-length gag and pol clones were obtained for analysis. In most cases, the sequences derived from the LTS had an intact open reading frame. At the protein level, there were no discernible differences between the sequences derived from LTS and those derived from patients with AIDS. Thus, no common defect in gag and pol was found in our cohort. One individual (subject SF), however, had only Gag-defective proviral sequences (10 of 10) in his peripheral blood mononuclear cells. Furthermore, longitudinal studies of the samples collected from SF over a 2-year period showed that all p17 gag clones sequenced (24 of 24) were defective due to G-to-A hypermutations. This viral defect in Gag may provide the molecular basis for this individual's extremely low viral load and long-term asymptomatic state. These results, together with previous findings in our LTS cohort, reinforce the notion that it is unlikely that a single common viral genetic determinant accounts for the lack of disease progression in all cases. Multiple host and viral factors undoubtedly contribute to the well-being of LTS of HIV-1 infection.     

HIV infection and HIV-1 clades among pregnant women in Harare, Zimbabwe

Official figures indicate that at least 1 million of Zimbabwe's 11 million population are infected with HIV, while the most recent survey results indicate that about 24% of apparently healthy women in the country are HIV seropositive. 60 (29.1%) of 206 pregnant women attending Edith Opperman and Budiriro clinics in Harare who were screened for infection with HIV were found to be HIV-1 seropositive. 66.6% of the HIV-1-infected women were infected with subtype C, 48.3% with subtype A, and 33.3% with subtype B. 45% of the infected were infected with subtypes A and C, 10% with A and D, 30% with B and C, and 6.6% with subtypes A, B, C, and D. 2 samples (3.3%) were nonreactive. The high prevalence rate of HIV-1 infection in this study population points to the urgent need to implement more aggressive approaches to controlling HIV/AIDS among women in Zimbabwe.     

Phylogenetic and serological characterization of two Ugandan HIV-1 isolates

HIV-1 isolates Ug06 and Ug23 were established in culture from peripheral blood mononuclear cells (PBMCs) of Ugandan subjects. The isolates were studied for phylogenetic and serological relationships with each other and with the laboratory strains, HTLV-IIIB and HIV-1MN. The results suggest that the Ugandan isolates are related to different subgroups of African viruses with 17.3% of genetic distance between UG06 and the U455 provirus (Uganda); and 12.6% of genetic distance between UG23 and the JY1 provirus (Zaire). Analysis of the predicted amino acid sequences for Ug06 and Ug23 showed marked sequence heterogeneity in the V3 region and CD4-binding site. A conserved amino acid sequence was identified in the C-terminal immunodominant region of the envelope glycoprotein gp120. The isolates were compared in virus-neutralization experiments with HTLV-IIIB and HIV-1MN stocks, using panels of Western blot-positive North American and Ugandan sera. The North American serum samples showed broad neutralizing activity against both of the Ugandan isolates. However, the Ugandan serum panel demonstrated strain-specific activity against either Ug06 or Ug23. Furthermore, the African serum specimens showed higher prevalence and titers of neutralizing activity against the HIV-1MN stock as compared with HTLV-IIIB.     

Modular distribution of vasoactive intestinal polypeptide in the rat barrel cortex: changes induced by neonatal removal of vibrissae

The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo.