Genetic characterization of HIV type 1 from migrant workers in three South African gold mines

The phylogenetic relationships between 44 HIV-1 isolates from 43 infected subjects employed by three adjacent South African gold mines were investigated. The patients were migrant workers originating from rural areas of South Africa and the neighboring countries of Lesotho, Botswana, Swaziland, and Mozambique. Proviral HIV-1 DNA was subtyped using a heteroduplex mobility assay (HMA) based on the 700-bp V3-V5 region of the env gene. DNA sequence analysis was used to confirm the subtype designation and to determine phylogenetic relationships between isolates. All 44 HIV-1 isolates were identified as env subtype C using both HMA and phylogenetic analysis. These isolates did not show a distinct phylogenetic relatedness based on the geographic origins of the migrant workers or show close homology to other subtype C sequences from southern Africa or India. However, five clusters of closely related sequences were identified, mainly involving miners of disparate geographic origins, suggesting possible epidemiological linkage in these few cases. The characteristic tetrapeptide sequence, GPGQ, at the tip of the V3 loop of subtype C viruses was conserved in the predicted amino acid sequences of most isolates. The heterogeneity of HIV-1 sequences among migrant workers in a mining cohort suggests multiple introductions of HIV-1 subtype C into this population that are not apparently linked to the geographic origins of the patients.     

HIV-1 genetic subtype A/B recombinant strain causing an explosive epidemic in injecting drug users in Kaliningrad

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of the virus strain(s) causing an outbreak of HIV-1 infection in the Kaliningrad province of the Russian Federation and to investigate the relationship of this outbreak to some other emerging HIV-1 epidemics in the countries of the former Soviet Union. DESIGN: A molecular epidemiological investigation was conducted in the city of Kaliningrad amongst individuals recently diagnosed as HIV-1-positive. Samples were also collected from neighbouring Lithuania and from the Ukraine. METHODS: Incident and population data was collected from official health statistics in Kaliningrad. A standardized questionnaire was administered to newly diagnosed individuals to assess risk factors for HIV-1 infection. For genotyping, two regions of the virus (env C2-V3 and gag NCp7) were directly sequenced. RESULTS: The number of newly diagnosed individuals testing seropositive for HIV-1 infection in Kaliningrad rose from less than one per month to more than 100 per month during the period of July-October 1996. A total of 1335 new infections were identified between 1 July 1996 and 30 June 1997. The main reported risk factor for HIV-1 infection (80%) was injecting drug use, in particular with a locally produced opiate. Sequence analysis of patient viruses in Kaliningrad (n = 50) showed that the epidemic was caused by a highly homogenous HIV-1 strain, recombinant between the genetic subtypes A and B. Comparison with subtype A strains prevalent amongst injecting drug users (IDU) in the Ukraine showed that one of these strains was the direct subtype A parent of the epidemic A/B recombinant strain in Kaliningrad. CONCLUSIONS: The HIV-1 epidemic in Kaliningrad probably started from a single source, with rapid spread of the virus through the IDU population. The origin of the epidemic strain is a recombination event occurring between the subtype A strain virus prevalent among IDU in some southern CIS countries, and a subtype B strain of unknown origin.     

Evolution of envelope sequences from the genital tract and peripheral blood of women infected with clade A human immunodeficiency virus type 1

The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.     

Diversification of subtype E human immunodeficiency virus type 1 env in heterosexual seroconverters from Northern Thailand

The C2-V3 region of the human immunodeficiency virus (HIV)-1 env was determined from 15 northern Thailand seroconverters between 1993 and 1995. Similar sequences were also determined from 18 seroconverting injection drug users in Baltimore. All seroconverters from northern Thailand were infected with subtype E HIV-1 on the basis of env sequences. Intersubject viral DNA distances increased from 2.3% in asymptomatic HIV-1-infected subjects characterized between 1990 and 1992 to 7.8% in these more recent seroconverters from Thailand. On the other hand, sequences from 18 seroconverters from Baltimore had a mean intersubject distance of 13.2%. The genetic diversity within HIV-1 subtype E in seroconverters in Thailand has increased significantly but is still less than that observed in HIV-1 from seroconverters in the United States, where the epidemic of HIV-1 infection is more mature. These results suggest that continued monitoring of the molecular epidemiology of HIV-1 infection in Thailand will be important for HIV vaccine development and evaluation.     

Genotypic characterization of human immunodeficiency virus type 1 in Greece. Multicentre Study on HIV-1 Heterogeneity

The HIV-1 subtype distribution in 83 HIV-1-seropositive individuals living in Greece was investigated by using the heteroduplex mobility assay (HMA), DNA sequencing, and phylogenetic analysis. The results revealed that partial HIV-1 gp120 sequences from 71 (86%) patients were subtype B, 5 (6%) were subtype A, 4 were subtype D (5%), 2 (2%) were subtype C, and 1 (1%) was subtype I. The subtype I isolate was documented in an intravenous drug user. A high prevalence (90-100%) of B isolates among intravenous drug users, hemophiliacs, and homosexual men was observed, in contrast to heterosexuals, among whom non-B subtypes seemed to be common (42.9%, p < 0.001). Among the Greek population subtype B is the most frequent (94%), in contrast to the high prevalence (57%) of non-B isolates found in emigrants living in Greece (p < 0.001). A heterosexual transmission case of subtype D in a Greek individual not traveling abroad was also documented. The broad HIV-1 diversity in Greece may be explained by population movements, such as migration and traveling.     

Nucleotide sequence and restriction fragment-length polymorphism analysis of human T-cell lymphotropic virus type II (HTLV-II) in southern Europe: evidence for the HTLV-IIa and HTLV-IIb subtypes

Human T-cell lymphotropic virus type II (HTLV-II) has been subtyped into two major groups, IIa and IIb, according to molecular studies involving env gene sequencing. Subsequently, this retrovirus was further subclassified by examining the long terminal repeat (LTR), the most divergent genomic region. Sequence analysis and restriction fragment-length polymorphism (RFLP) applied to the LTR region identified either four or five groups within the IIa subtype (depending on the restriction enzyme sets used) and six within the IIb subtype. In this study, we analyzed the LTR sequences of 29 samples obtained from HTLV-II-infected individuals living in Spain and Italy, which included 24 injecting drug users (IDUs), three blood donors, and two subjects at risk for HIV/HTLV infection. Sequence analysis and phylogenetic analysis of 720 base pairs of the LTR performed in 10 Spanish samples showed that all of these samples belonged to IIb subtype, with a divergence of 7.5% and 1.66% compared with MoT (IIa) and NRA/G12 (IIb) isolates, respectively. RFLP analysis demonstrated the presence of the IIb 4-subtype restriction pattern in 26 samples, a IIb5-subtype pattern in one Italian IDU, and a IIa0-subtype pattern in two Italian samples (blood donors), according to W.M. Switzer's nomenclature. This is the first report of the presence of IIb5 in Southern Europe and IIa0 among Italian blood donors. RFLP correlated with nucleotide sequence and phylogenetic data obtained in this study, demonstrating the ability of the RFLP method to predict the phylogroup of HTLV-II-infected samples.     

Extensive diversification of human immunodeficiency virus type 1 subtype B strains during dual infection of a chimpanzee that progressed to AIDS

A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiency virus type 1 (HIV-1), one (HIV-1(SF2)) that replicates poorly and one (HIV-1(LAV-1b)) that replicates efficiently in chimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086-4091, 1997). Nucleotide sequence and phylogenetic analyses of the C2 to V5 region of env (C2-V5env) in proviral DNA from peripheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One of these populations appeared to be a recombinant in env, having the V3 loop from HIV-1(SF2) and the V4-V5 region from HIV-1(LAV-1b); the other population had evolved from HIV-1(LAV-1b). In addition to C2-V5env, the entire p17gag and nef genes were sequenced; however, based on nucleotide sequences and phylogeny, whether the progenitor of the p17gag and nef genes was SF2 or LAV-1b could not be determined. Compared to the two original viruses, the divergence of all clones of C2-V5env ranged from 9.37 to 20.2%, that of p17gag ranged from 3.11 to 9.29%, and that of nef ranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5env for C-499, the maximum diversities in C2-V5env in proviruses from two chimpanzees infected with HIV-1(LAV-1b) for 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1 populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induced disease and (ii) recombination between two subtype B strains occurred even though the second strain was inoculated 15 months after the first one. Furthermore, evaluation of env genes from three chimpanzees infected with the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viral burdens, manifestations of disease, and/or dual infection.     

Extraction of pacemaker and implantable cardioverter defibrillator leads

To determine the mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses the placenta into the fetal blood, 12 matched samples of serial maternal blood, term placentas, and infant blood obtained from a cohort of pregnant women in Cameroon identified as predominantly infected by subtype A viruses were studied. HIV-1 env sequences were detected by polymerase chain reaction (PCR) in both chorionic villi and enriched trophoblastic cells of all 12 placentas but at variable rates of detection. Heteroduplex mobility assay analysis showed the presence of multiple HIV-1 env quasispecies in sequential maternal peripheral blood mononuclear cell samples, but only a small number of env variants were found in chorionic villi and enriched trophoblastic cells. These data indicate that HIV-1 env sequences are always present in term placentas of seropositive women, contrasting with the low frequency at which infection is diagnosed by PCR in neonates with tat, gag, and env primers. Maternal HIV-1 variants appear to undergo a strong negative selection by different cell populations within the placental villi.     

Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement

Human immunodeficiency virus type 1 (HIV-1) M group strains have been assigned to date to nine distinct genetic subtypes, designated A through I, according to phylogenetic analyses of nucleotide sequences of their env or gag genes. Whether there is any relationship between phylogenetic subtypes and the neutralization serotypes is not clear, yet defining the nature of any such relationship by mathematical means would be of major importance for the development of globally effective HIV-1 vaccines. We have therefore developed a quantitative method to analyze serum neutralization of HIV-1 isolates and to identify HIV-1 neutralization serotypes. This method involves calculations of the neutralization index, N(i), a newly defined parameter derived from plots generated from in vitro neutralization assays, calculations of pairwise serum-virus vector distances, and cluster analyses. We have applied this approach to analyze three independent neutralization matrices involving primary HIV-1 strains and sera from genetic subtypes A, B, C, D, E, F, and I. Detailed serum and HIV-1 isolate cluster analyses have shown that in general, the identified neutralization serotypes do not directly correlate with HIV-1 genetic subtypes. These results suggest that neutralization serotypes do not during natural HIV-1 infection are not governed by antibodies directed against simple epitopes within gp120 monomers. A significant proportion (28%) of 1,213 combinations of sera and HIV-1 isolates caused serum-dependent infectivity enhancement [negative N(i) values] rather than neutralization. We also noted that negative N(i) values tended to correlate better with certain HIV-1 isolates rather than with HIV-1-positive sera. Syncytium-inducing variants of HIV-1 were slightly more likely than non-syncytium-inducing variants to undergo serum-dependent infectivity enhancement, although the latter variants could clearly be susceptible to enhancement.     

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 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.     

Molecular characterization of human T-lymphotropic virus, type 1 (HTLV-1) found in Kuwait: close similarity with HTLV-1 isolates originating from Mashhad, Iran

Human T-lymphotropic virus, type 1 (HTLV-1) infection was detected in two unrelated Kuwaiti patients with tropical spastic paraparesis (HAM/TSP) and in the asymptomatic mother of one of them. The family roots of these patients were traced to the Najaf region of Iraq. The DNA sequence of three PCR-amplified fragments (env, 512 bp; pol, 140 bp; LTR, 704 bp) was determined for each of Kuwaiti HTLV-1 isolates (KUW-1,2,3). All three Kuwaiti HTLV-1 were identical in env and pol fragments and virtually identical in LTR. Two rare substitutions were found in the env and pol fragments. They were shared only with two isolates from Reunion Island (substitution in env), and two isolates from India and the Caribbean (substitution in pol). The sequences of env and pol fragments of the Middle Eastern HTLV-1 isolates were not available. However, the comparison of Kuwaiti isolates with representative Middle Eastern HTLV-1 was possible for the LTR fragment. The phylogenetic analysis of LTR sequences of KUW and 34 other HTLV-1 isolates has shown that Kuwaiti HTLV-1 belongs to a cosmopolitan "a" subtype of HTLV-1 and tends to cluster together with HTLV-1 originating from the Mashhad region of Iran. These results suggest that common origin of Mashhadi and Kuwaiti (Najafi) HTLV-1 and the possibility of another pocket of HTLV-1 infection in the Middle East, located in the Najaf region of Iraq.     

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.     

Genetic characterization of viral quasispecies in blood and cervical secretions of HIV-1- and HIV-2-infected women

We evaluated cervical samples from 11 HIV-1- and 25 HIV-2-infected individuals. The rate of viral shedding was 36.4% for HIV-1 and 16% for HIV-2, after repeat PCRs. We sequenced multiple clones of the C2-C3 env region from cervical secretions and PBMC samples from three HIV-2-infected individuals, and the C2-V3 env region from four HIV-1-infected individuals. In most cases, phylogenetic analysis showed that the viral sequences from blood and genital secretions were intermingled and subclusters did not segregate according to sample site. In rare cases, however, tissue-specific sequences were observed, suggesting a complex relationship between quasispecies in the two sites where preferential transmission of HIV variants may be due to multiple factors.     

Intravascular ultrasound. Three-dimensional applications and forward viewing

Variation in HIV-1 genomic RNA was studied in seroconversion samples from mother-child pairs from a Rwandan cohort. The mothers (n = 8) were heterosexually infected and their children (n = 6) were vertically infected by breast milk. Five of the children seroconverted within the same 3-month period as did their mothers. Highly homogeneous subtype A V3 and p17gag sequence populations were observed in three mother-child pairs, one of the two nontransmitting mothers, and one child (mean nucleotide distances 0 to 0.9%). Heterogeneous populations of subtype A V3 and p17gag sequences were found in one mother and a mother-child pair (1.4 to 2.8% for V3, 1.0 to 1.9% for p17). The second nontransmitting mother was infected with a heterogeneous AV1-V3/Cp17-p24 recombinant virus population (3. 8% for V3, 2.4% for p17). Finally, in one woman subtype C V3 sequences were observed, in addition to highly homogeneous subtype A V3 and p17gag sequence populations, also found in the child. Coexistence of subtype AV1-V3 and CV1-V3 env sequences in the mother was confirmed in a follow-up sample. The gag gene of both the maternal and the child's virus population represented an A/C recombinant sequence (Ap17/Cp24). An infection with subtype CV1-V3/p17-p24 was found upon testing of three additional participants of the mother-child cohort, indicating that subtype C is present in Rwanda. In conclusion, heterogeneity, coinfection, and intersubtype recombinants are not uncommon in primary HIV-1 infections in Rwanda.     

Serological properties of HIV-1 isolates from the focus of an epidemic in the Gomel region of the Belarus Republic (1996)

Immunoreactivity of sera obtained in 1996 from HIV-1-infected intravenous narcomaniacs from an epidemic focus in Gomel region, Belarus, is studied with a panel of 10-16-component synthetic peptides simulating apical epitope of surface glycoprotein gp120 V3 loop of HIV-1 variants. Comparative analysis of resultant spectra with representative immunoreactivity spectra of sera from a sampling of HIV-1-positive sera collected in 1986-1997 in different regions of the former USSR demonstrated a high homogeneity of immunoreactivity spectra of sera from Gomel region. Serotypes of HIV-1 A/C variants circulating in populations of intravenous narcomaniacs in the focus in Gomel region in 1996 and in Southern and South-Eastern Ukraine in 1995-1996 are similar. This confirms a previous conclusion about the prevalence of A/C serotype among intravenous narcomaniacs in the former USSR.     

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.