Phylodynamics of HIV-1 from a phase-III AIDS vaccine trial in North America.

In 2003, a phase III placebo-controlled trial (VAX004) of a candidate HIV-1 vaccine (AIDSVAX B/B) was completed in 5,403 volunteers at high risk for HIV-1 infection from North America and the Netherlands. A total of 368 individuals became infected with HIV-1 during the trial. The envelope glycoprotein gene (gp120) from the HIV-1 subtype B viruses infecting 349 patients was sequenced from clinical samples taken as close as possible to the time of diagnosis, rendering a final data set of 1,047 sequences (1,032 from North America and 15 from the Netherlands). Here, we used these data in combination with other sequences available in public databases to assess HIV-1 variation as a function of vaccination treatment, geographic region, race, risk behavior, and viral load. Viral samples did not show any phylogenetic structure for any of these factors, but individuals with different viral loads showed significant differences (P = 0.009) in genetic diversity. The estimated time of emergence of HIV-1 subtype B was 1966-1970. Despite the fact that the number of AIDS cases has decreased in North America since the early 90s, HIV-1 genetic diversity seems to have remained almost constant over time. This study represents one of the largest molecular epidemiologic surveys of viruses responsible for new HIV-1 infections in North America and could help the selection of epidemiologically representative vaccine antigens to include in the next generation of candidate HIV-1 vaccines.

Modeling of CCR5 Recognition by HIV-1 gp120: How the Viral Protein Exploits the Conformational Plasticity of the Coreceptor.

The chemokine receptor CCR5 is a key player in HIV-1 infection. The cryo-EM 3D structure of HIV-1 envelope glycoprotein (Env) subunit gp120 in complex with CD4 and CCR5 has provided important structural insights into HIV-1/host cell interaction, yet it has not explained the signaling properties of Env nor the fact that CCR5 exists in distinct forms that show distinct Env binding properties. We used classical molecular dynamics and site-directed mutagenesis to characterize the CCR5 conformations stabilized by four gp120s, from laboratory-adapted and primary HIV-1 strains, and which were previously shown to bind differentially to distinct CCR5 forms and to exhibit distinct cellular tropisms. The comparative analysis of the simulated structures reveals that the different gp120s do indeed stabilize CCR5 in different conformational ensembles. They differentially reorient extracellular loops 2 and 3 of CCR5 and thus accessibility to the transmembrane binding cavity. They also reshape this cavity differently and give rise to different positions of intracellular ends of transmembrane helices 5, 6 and 7 of the receptor and of its third intracellular loop, which may in turn influence the G protein binding region differently. These results suggest that the binding of gp120s to CCR5 may have different functional outcomes, which could result in different properties for viruses.

Improved prediction of coreceptor usage and phenotype of HIV-1 based on combined features of V3 loop sequence using random forest.

HIV-1 coreceptor usage and phenotype mainly determined by V3 loop are associated with the disease progression of AIDS. Predicting HIV-1 coreceptor usage and phenotype facilitates the monitoring of R5-to-X4 switch and treatment decision-making. In this study, we employed random forest to predict HIV-1 biological phenotype, based on 37 random features of V3 loop. In comparison with PSSM method, our RF predictor obtained higher prediction accuracy (95.1% for coreceptor usage and 92.1% for phenotype), especially for non-B non-C HIV-1 subtypes (96.6% for coreceptor usage and 95.3% for phenotype). The net charge, polarity of V3 loop and five V3 sites are seven most important features for predicting HIV-1 coreceptor usage or phenotype. Among these features, V3 polarity and four V3 sites (22, 12, 18 and 13) are first reported to have high contribution to HIV-1 biological phenotype prediction.

Distinct sequence patterns characterize the V3 region of HIV type 1 gp120 from subtypes A and C.

The known sequences of HIV-1 viruses have been categorized into subtypes based on the phylogenetic partitioning of their env and gag gene sequences. The env gene encodes the protein gp120, which contains five sequence- variable regions (V1 to V5), of which the V3 loop is of central importance to viral infectivity. The V3 loop consensus sequences of HIV-1 subtype A and C viruses are similar, and more similar to one another than the V3 consensus sequences of any other two HIV-1 subtypes. However, using a position-specific statistical comparison, we found that the V3 region of these two subtypes is statistically distinct (p = approximately 0.0). (The p-value calculated to the lowest limit of representation on the computer used to run the calculation. This lowest limit was 10(16). Although theoretically a p-value cannot be equal to 0.0, the p-value for the comparisons in question can be intuitively considered to be extremely small, or approximately 0.0.).

The antigenic tip GPGRAFY of the V3 loop on HIV-1 gp120: genetic variability and subtypes.

V3 loop on HIV-1 gp120 is tightly correlated with syncytium formation, coreceptor usage, virus infectivity and antibody neutralization. The antigenic tip GPGRAFY with its flanking sequence has a conserved secondary structure, and is the target of neutralizing antibodies. We analyzed its genetic variability in 30096 M-group isolates and 269 O-group isolates. Subtype-related restricted mutations were observed, which could help to identify subtypes.

Increasing genotypic and phenotypic selection from the original genomic RNA populations of HIV-1 strains LAI and MN (NM) by peripheral blood mononuclear cell culture, B-cell-line propagation and T-cell-line adaptation.

OBJECTIVE: To address the question of whether T-cell-line adaptation of the original LAI and MN (NM) HIV-1 populations biased the interpretation of the intraindividual and population-wide virus distributions. PATIENTS AND METHODS: HIV-1 genomic RNA coding for the gp120 C2V3 region was obtained from serum samples of patients LAI and MN and compared to the proviral DNA derived from simultaneously sampled peripheral blood mononuclear cells (PBMC) as well as B-and T-cell lines. RESULTS: Two (10%) of 20 clones of HIV-1 LAI RNA and none of 16 clones of the HIV-1 MN RNA carried syncytium-inducing (SI)-determining amino-acid changes. HIV-1 LAI RNA formed on SI and two non-SI (NSI) phylogenetic clusters. The HIV-1 LAI DNA in PBMC included both SI and NSI clones but lacked one NSI cluster and contributed NSI clones to the SI cluster as well as an SI clone to the NSI cluster, indicating the existence of an intermediate SI/NSI genotype. In vitro culture using either primary cells or B-cell lines yielded only SI clones, distributed over two SI/NSI mixed clusters. Long-term propagation in T-cell lines further restricted the clonality and yielded SI clones belonging to only one cluster. On the population level, HIV-1 LAI, MN and BRU sequences all clustered according to the individual host and apart from each other and separate from the epidemiological controls without notable influence of SI/NSI distinction or cell-culture adaptation. CONCLUSION: Our data demonstrate a selection bias during the cell-line adaptation of HIV-1 strains LAI and MN with more impact on phenotypic than on genotypic characteristics.

Diversity of the V3 region of HIV in Paris, France.

OBJECTIVE: To carry out, within France, a large-scale molecular epidemiological investigation on the principal neutralizing determinant of HIV-1, located in the third variable region (V3) of the envelope protein. Such investigations are of the utmost importance in the identification and monitoring of the distribution and spread of different viral strains internationally. DESIGN: Using polymerase chain reaction (PCR), we examined the genetic variation of the V3 region sequences of 28 HIV-infected patients from Paris, France. RESULTS: Comparison of the Parisian V3 loop sequences with other published data indicates that the range of diversity in France is included within that of a large group that contains sequences from North America, the rest of Europe, Japan, India and Africa. Variability appears to be lower in the V3 loop than in its flanking regions. Five out of the six putative N-linked glycosylation sites show preferential alterations to charged amino acids. We report two motifs at the tip of the loop that have not been described previously. CONCLUSIONS: The structural homogeneity and the wide geographic representation of the major V3 group suggests that a common strategy could be applied to a large proportion of isolates in the development of a broad-spectrum HIV vaccine.

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.

Correlation between HIV sequence evolution, specific immune response and clinical outcome in vertically infected infants.

OBJECTIVE: To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1. DESIGN: Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3). METHODS: Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time. RESULTS: Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of children's sera to react with synthetic peptides representing the autologous virus sequence was determined. CONCLUSION: Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

The impact of immigration on env HIV-1 subtype distribution among heterosexuals in the Netherlands: influx of subtype B and non-B strains.

OBJECTIVE: To examine the epidemiological factors influencing the distribution and spread of HIV-1 subtypes among heterosexuals in the Netherlands. METHOD: A nationwide serosurveillance in 21 HIV/AIDS centres from 1997 to 1999 involved 200 individuals for whom the mode of HIV transmission was heterosexual contact or unknown. HIV-1 subtypes were determined by phylogenetic analysis of env V3 sequences and correlated with sociodemographic characteristics of the subjects and their sexual partners. RESULTS: HIV-1 subtype B infection occurred in 121 subjects (60%). Non-B subtypes were identified in 31 (A), 24 (C), 10 (D), six (E), four (F) and three (G) individuals; one had an unclassified subtype. The proportion of subtype B was about 60% in four of the six regions of the Netherlands, but in the Northwest and Southwest regions these proportions were 76% and 46%, respectively. The Surinamese and Antilleans, large immigrant groups, were all infected with subtype B, as were almost all individuals with an unknown source. The proportions of non-B viruses did not change significantly over time in Amsterdam, where subtyping was available from 1988 onward, but a shift in the various subtype B strains was observed, suggesting introductions of new subtype B strains in Amsterdam. CONCLUSION: To date, HIV-1 non-B subtypes in the Netherlands are still found predominantly among heterosexuals with an epidemiological link with sub-Saharan Africa. Despite continuing introductions of non-B subtypes, the B/non-B distribution has been stable over time, most likely as a result of introductions of subtype B strains from Caribbean and South American countries.

Using phylogenetic analysis to trace HIV-1 migration among western European injecting drug users seroconverting from 1984 to 1997.

OBJECTIVE: To reconstruct the epidemiological relationships of the HIV epidemics among injecting drug users (IDU) in western Europe. METHODS: HIV env V3 sequences of and epidemiological data were obtained from 145 IDU who seroconverted in three sequential periods: 1984-1988, 1989-1992 and 1993-1997. The sequences were phylogenetically analysed and examined for signature patterns characteristic of northern European IDU, including the conserved GGC codon in the V3 loop. RESULTS: Subpopulations of genetically related HIV strains were observed in Italy, France, Scotland and Spain, in contrast to the Netherlands, Austria and Switzerland. This difference between the two groups of countries suggests that the HIV epidemics amongst IDU in the latter group was caused by multiple virus introductions. In Edinburgh and the surrounding area, most IDU were infected with the same GGC strain over the 12-year study period. The epidemic among IDU in north-western Europe started with GGC viruses, whereas in south-western Europe non-GGC viruses predominated. This geographical separation has faded during the course of the epidemic, most likely because of virus exchange among IDU populations.

gp120 sequences from HIV type 1 subtype C early seroconverters in India.

A limited number of full-length gp120 sequences are currently available for subtype C HIV-1 from India. Sequence data from HIV-1 subtype C in early seroconverter stage virus are also very limited. With the objective of identifying the sequence variation in early seroconverters, we compared Indian subtype C gp120 sequences obtained from six early seroconverters presented in this study with non-Indian subtype C sequences from other parts of the world obtained from the Los Alamos database and subtype C potential vaccine candidate sequences. All these samples were collected within a few weeks of seroconversion and hence they represent gp120 sequences of currently circulating viral strains in India. The phylogenetic tree indicated that the Indian sequences compared here clustered together within the C clade. The seroconverter sequences presented in the study would surely help in identifying the immunogenic epitopes and could be utilized further for developing effective prophylactic strategies against HIV-1 subtype C for India.

HIV type 1 envelope sequences from seroconverting patients in Barbados.

PIP: Because of the prevalence of leptospirosis in Barbados, patients who present to the hospital with febrile illnesses are routinely screened for Leptospira infection and their sera are stored for future reference. While the majority of patients are infected with Leptospira, some are not. Since some symptoms of acute HIV-1 illness are similar to those of leptospirosis, patient records were reviewed to identify patients whose clinical symptoms may have been due to HIV-1 infection. 10 HIV-1-positive patients originally hospitalized during 1990-94 were identified whose medical histories suggested the occurrence of acute HIV-1 illness at the time of Leptospira testing. Stored sera from those patients were then tested for the presence of HIV-1 p24 antigen and by Western blotting. Evidence of acute HIV-1 infection was considered to be a positive p24 test or a characteristic Western blot profile occurring at or shortly before the time of seropositivity for HIV-1 antibody. The authors determined the sequence of viral RNA from the 12 remaining sera samples from 8 patients, including paired samples drawn at 3- or 4-day intervals from 4 people. The Barbados patient variants aligned more closely with HIV-1 clade B reference strains than with the other subtypes. 2 variants, however, align separately from the classic B subtype and somewhat closer to variants from clades A and C. The Venezuelan isolate, although different from the patient sequences, is also separate from the other B variants.^ieng

HIV type 1 V3 serotyping of Tanzanian samples: probable reasons for mismatching with genetic subtyping.

HIV-1 V3 serotyping is used to classify immunodeficiency viruses on the basis of antibody binding to V3 peptides derived from env genetic subtypes. Although it shows a reasonable overlap, it has been reported to be distinct from viral genetic subtypes. The aim of this study is to determine the feasibility of HIV-1 serotyping to predict genetic subtypes in an East African setting, where multiple HIV-1 subtypes have coexisted for many years. HIV-1 genetic subtypes of 86 AIDS patients in Mbeya Town, southwest Tanzania, were determined, using env nucleic acid sequencing as the basis for comparison. Those data were compared with V3 serotyping results obtained by four different methodologies. Four HIV-1 genetic subtypes were identified, including A (25, 29%), C (47, 55%), D (13, 15%), and G (1, 1%). The sensitivity and specificity of those serotyping assays varied considerably: sensitivity for genetic subtype A (40-48%), C (52-96%), and D (9-31%); and specificity for genetic subtype A (77-95%), C (46-63%), and D (97-100%). We further tried to identify reasons for the discrepancies between serotyping results and genetic subtypes. By means of logistic regression analysis three amino acid residues within the V3 loop (positions 12, 13, and 19; V, H, and A for serotype A, I, R, and T for serotype C) were found to be most important for antibody binding; a deviation from the subtype-specific amino acids was highly related to mismatched results. In addition, we have shown that phenetic analysis of V3 amino acid sequence data could be used to predict the majority of V3 serotypes (93-94%). Our data demonstrated that for the majority of specimens HIV-1 V3 serotyping results closely match the subtype of the analyzed sample as revealed by the V3 loop amino acid sequence. However, our data demonstrate that HIV-1 serotyping is not sufficiently accurate to predict genetic subtypes in Tanzania, where subtypes A, C, D, and G are circulating. This was due to highly similar amino acid sequences throughout the prevalent genetic subtypes, which caused the inability of HIV-1 V3 serotyping to differentiate subtype A from C as well as D from C. Instead, the serotyping results reflect the frequency distribution of V3 serotypes. To investigate HIV-1 genetic subtypes in population-based studies in this African setting additional or modified algorithms are needed.

PIP: HIV-1 V3 serotyping is used to classify immunodeficiency viruses on the basis of antibody binding to V3 peptides derived from env genetic subtypes. Findings are reported from a study conducted to determine whether HIV-1 serotyping could be effectively used to predict genetic subtypes in an East African setting, where multiple HIV-1 subtypes have coexisted for many years. The HIV-1 genetic subtypes of 86 people with AIDS in Mbeya Town, southwest Tanzania, were determined, using env nucleic acid sequencing as the basis for comparison. Those data were then compared with V3 serotyping results obtained by analysis with tests manufactured by Behring and the Pettenkofer Institute, tests conducted by St. Mary's Hospital Medical School, tests conducted by Georg-Speyer-Haus, and tests conducted by Universite Francois Rabelais. The following HIV-1 genetic subtypes were identified: 25 cases of A (29%), 47 of C (55%), 13 of D (15%), and 1 of G (1%). The sensitivity and specificity of the serotyping assays varied considerably. These data indicate that HIV-1 serotyping is not accurate enough to predict genetic subtypes in Tanzania. This conclusion was reached based upon the highly similar amino acid sequences throughout the prevalent genetic subtypes, which caused the inability of HIV-1 V3 serotyping to differentiate subtype A from C as well as D from C. The serotyping results instead reflect the frequency distribution of V3 serotypes.

HIV type 1 V3 domain serotyping and genotyping in Gauteng, Mpumalanga, KwaZulu-Natal, and Western Cape Provinces of South Africa.

More than 20.8 million people are living with HIV/AIDS in sub-Saharan Africa, with southern Africa the worst affected area and accounting for one of the fastest growing AIDS epidemics worldwide. Samples from 81 patients, including 25 from KwaZulu-Natal, 26 from Gauteng, 5 from Mpumalanga, and 25 from Western Cape Province, were serotyped using a competitive V3 peptide enzyme immunoassay (cPEIA). Viral RNA was also isolated from serum and the V3 region amplified by reverse transcriptase polymerase chain reaction (RT-PCR) to obtain a 240-bp product for direct sequencing of 29 samples. CLUSTAL W was used to make multiple sequence alignments. Distance calculation, tree construction methods, and bootstrap analysis were done using TREECON. Subtype C-like V3 loop sequences predominate in all provinces tested in South Africa. Discordant sero- and genotype results were observed in one patient only. The correlation between sero- and genotyping was 96% (24 of 25) in KwaZulu-Natal and 100% in Gauteng and Mpumalanga. In Western Cape Province 18% of patients were identified as sero/genotype B and 82% as sero/genotype C. Our data show that results of the second-generation V3 cPEIA correlated well with V3 sequencing and would be a rapid and affordable screening test to monitor the explosive southern African HIV-1 epidemic.

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.

Dynamics of the HIV-1 subtype distribution in the Swedish HIV-1 epidemic during the period 1980 to 1993.

We have investigated the distribution of the major subtypes, or clades (A to E), in the Swedish human immunodeficiency virus type 1 (HIV-1) epidemic between the years 1980 to 1993, using gp120 V3 domain serotyping and genotyping. Until 1984 the Swedish HIV-1 epidemic was almost exclusively caused by HIV-1 subtype B. During the late 1980s and the early 1990s an increase in nonsubtype B infections (mainly subtypes A and C) was observed. Nonsubtype B infections constituted approximately 30% of the newly diagnosed HIV-1 cases in 1993. In the analyzed material we also noted a substantial increase in the proportion of HIV-1 infections caused by heterosexual transmission. The present data suggest that the subtype distribution in the Swedish HIV-1 epidemic may well continue to change with time, which may have implications for preventive research and vaccine development strategies.

Genetic diversity and phylogenetic analysis of human immunodeficiency virus type 1 subtypes circulating in French Guiana.

We investigated the characterization of different HIV-1 subtypes present in French Guiana by use of three different methods. Serological methods were used for the initial screening, which were then confirmed by the heteroduplex mobility assay (HMA). The V3 env region was subsequently sequenced for phylogenetic analysis, to confirm the subtype of the samples, and to assign a subtype to samples that gave results that were difficult to interpret or discordant by serology or HMA. A total of 221 HIV-1 seropositive samples were typed; 110 of them were confirmed by HMA and 16 were sequenced. Of the 221 samples tested 210 patients (95%) were found to be infected with subtype B, 10 (4.5%) were infected with subtype A, and one patient was infected with subtype F. Phylogenetic analysis demonstrated that the strains from French Guiana were closely related to the subtype A and B subtypes, and that one strain was closely related to an F subtype (100% bootstrap value). Four strains from French Guiana clustered in the subtype A (99% bootstrap value) and the other strains were associated with subtype B (100% bootstrap value). The geographic position of French Guiana suggested that HIV-1 was probably introduced into the country via several routes, and thus the pattern of the HIV-1 epidemic might evolve in the near future.

Survey of reverse transcriptase from the heterosexual epidemic of human immunodeficiency virus type 1 CRF01_AE in Thailand from 1990 to 2000.

Genetic diversity of the HIV-1 envelope gene has shown a steady increase over time in the Thai and other regional epidemics. A serial survey of subtype CRF01_AE polymerase gene (RT) diversity in Thailand was performed, using 48 novel and 15 reported sequences covering the period 1990--2000. These sequences were gathered from individuals whose sole risk factor for infection was heterosexual contact. By contrast to envelope, diversity was low and, despite a 40% increase early in the epidemic, has remained static since 1996. These results indicate that epidemic HIV-1 may be constrained within defined limits of genetic diversity at least in some genomic regions.

Full-length sequences of two CRF01_ae (subtype e) HIV type 1 isolates from 1995 samples of patients with sexually transmitted diseases in Thailand.

We isolated two CRF01_AE human immunodeficiency virus type 1 (95TNIH022 and 95TNIH047) from the 1995 blood samples derived from asymptomatic carriers in Ubonratchatani province of northeastern Thailand. Both isolates can replicate in peripheral blood mononuclear cells, but not in several T cell lines examined. The full-length sequences recovered from proviruses in infected cells by long-range polymerase chain reaction were determined. Phylogenetic analyses of these sequences at individual genes showed them to be closely related to those of reported CRF01_AE HIV-1, such as 1990 isolate CM240 and 1993 isolate 93TH253. Two isolates in this study also showed a similar pattern of CRF01_AE mosaicism and a similar structure at the long terminal repeat, i.e., a copy number of NF-kappaB binding sites, sequence at the TATA box, and the putative secondary structure of stem-loop in the transactivation response region. Our results showed that 1995 Thai E isolates could contribute to our understanding of the epidemiology, pathogenesis, and diagnostics of HIV-1 CRF01_AE and further to vaccine development.