Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.     

Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.     

The changing molecular epidemiology of HIV type 1 among northern Thai drug users, 1999 to 2002

CRF01_AE and subtype B have dominated the HIV-1 epidemic in Thailand since 1989. We reported a new circulating recombinant form of HIV-1, CRF15_01B, as well as other unique CRF01_AE/B recombinants among prevalent HIV infections in Thailand. We sought to study this challenging molecular picture through assessment of subtypes among recent HIV-1 seroconverters in northern Thai drug users. A total of 847 HIV-1 seronegative drug users (342 IDU and 505 non-IDU) were enrolled, from 1999 to 2002, in a prospective study; 39 HIV-1 incident cases were identified and characteristics were collected. The overall HIV-1 incidence rate was 2.54/100PY, but it was 10.0/100PY among male IDU. HIV was strongly associated with injection history; 38 of 39 seroconverters gave a history of IDU. A near full-length genome of HIV-1 was recovered by PCR amplification and sequenced from peripheral mononuclear cell extracted DNA of 38 seroconverters. Phylogenetic analysis revealed that 33 (86.8%) were CRF01_AE and 5 (13.2%) were CRF01_AE/B recombinants. These recombinants had different structure but shared some common breakpoints, indicating an ongoing recombination process. Recombinant infection increased with year of sampling (0 to 57.1%). The molecular epidemiology of HIV-1 among drug users in northern Thailand has thus entered a new era. CRF01_AE remains predominant while pure subtype B is becoming rare, and now a substantial component of the epidemic. These findings support the need for CRF01_AE and subtype B components in clade-matched vaccine strategies for Thai phase III trials. Ongoing molecular surveillance of circulating HIV-1 strains is imperative for the evaluation of HIV vaccine efficacy.     

Heterosexually acquired CRF01_AE/B recombinant HIV type 1 found in Thailand

CRF01_AE and subtype B are circulating in Thailand and the strains have become intermixed in some high-risk groups, establishing the possibility of intersubtype recombination. The first such recombinant, mostly B with gp120 from CRF01_AE, was recently identified. Here we report a heterosexually acquired recombinant of different structure, with most of the genome from CRF01_AE but almost the entire envelope from subtype B. Surveillance by V3 serotype and genotype in multiple regions, followed by full-genome sequencing, was used to identify this strain. Pending vaccine trials in Thailand require knowledge of the presence of such strains in the population, and these recombinants provide valuable reagents for the laboratory evaluation of cross-subtype immunity. Studies are underway to determine whether either recombinant is circulating widely.     

Molecular Detection of HIV-1 Subtype B,CRF01_AE,CRF33_01B,and Newly Emerging Recombinant Lineages in Malaysia

A molecular genotyping assay for human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia is difficult to design because of the high level of genetic diversity. We developed a multiplex real-time polymerase chain reaction (PCR) assay to detect subtype B, CRF01_AE, CRF33_01B, and three newly described circulating recombinant forms, (CRFs) (CRF53_01B, CRF54_01B, and CRF58_01B). A total of 785 reference genomes were used for subtype-specific primers and TaqMan probes design targeting the gag, pol, and env genes. The performance of this assay was compared and evaluated with direct sequencing and phylogenetic analysis. A total of 180 HIV-infected subjects from Kuala Lumpur, Malaysia were screened and 171 samples were successfully genotyped, in agreement with the phylogenetic data. The HIV-1 genotype distribution was as follows: subtype B (16.7%); CRF01_AE (52.8%); CRF33_01B (24.4%); CRF53_01B (1.1%); CRF54_01B (0.6%); and CRF01_AE/B unique recombinant forms (4.4%). The overall accuracy of the genotyping assay was over 95.0%, in which the sensitivities for subtype B, CRF01_AE, and CRF33_01B detection were 100%, 100%, and 97.7%, respectively. The specificity of genotyping was 100%, inter-subtype specificities were > 95% and the limit of detection of 10³ copies/mL for plasma. The newly developed real-time PCR assay offers a rapid and cost-effective alternative for large-scale molecular epidemiological surveillance for HIV-1.     

HIV type 1 isolates from 200 untreated individuals in Ho Chi Minh City (Vietnam): ANRS 1257 Study. Large predominance of CRF01_AE and presence of major resistance mutations to antiretroviral drugs

HIV-1 isolates from 200 untreated patients recruited in 2001 and 2002 in the south part of Vietnam and particularly in Ho Chi Minh City were sequenced in the RT, protease, and env genes. Out of 200 isolates 198 belonged to CRF01_AE while only one subtype B and one intersubtype (B-CRF01_AE) recombinant could be observed. Of the isolates 6.5% had major resistance mutations to antiretroviral drugs.     

Emergence of a New HIV Type 1 CRF01_AE Variant in Guangxi, Southern China

Abstract The distribution of HIV-1 subtypes and genetic characterization of CRF01_AE in Guangxi, southern China were identified. The distribution of HIV-1 genotypes based on gag, pol, and partial env sequences (n=349) was as follows: CRF01_AE (66.5%), CRF08_BC (19.2%), CRF07_BC (7.2%), URF (4.6%), subtype B (1.7%), and subtype B' (0.9%). CRF01_AE predominated in all geographic regions and risk populations and there were multiple introductions of CRF01_AE strains in Guangxi. We found a peculiar CRF01_AE monophyletic lineage distinct from other CRF01_AE viruses, and we designated it "CRF01_AE-v" for convenience. CRF01_AE-v circulating in both heterosexuals and injecting drug users (IDUs) had accounted for 39.7% of CRF01_AE. It showed a selective advantage in the Guangxi population and formed its own characteristic compared with all the CRF01_AE references. Our results suggested that CRF01_AE-v was a new variant of CRF01_AE and it might lead to a new epidemic in Guangxi.     

Identification of subtype B, multiple circulating recombinant forms and unique recombinants of HIV type 1 in an MSM cohort in China

To study HIV-1 genetic diversity among HIV-positive men who have sex with men (MSM) in China, a cohort consisting of HIV-positive MSM was established in 2005 and was monitored every 2 years. In 2007, 44 HIVpositiveMSM subjects were genotyped, and the results showed HIV-1 subtype B decreased from 77.5% to41.9%, but non-B subtypes increased rapidly represented by CRF01_AE from 3.7% to 30.2% compared to 2005.In addition, one case of CRF07_BC was first identified in this population, which mainly circulated among HIV-1-infected injection drug users (IDUs) in China. There were 11 unique recombinant forms (URFs) consisting ofa recombination of CRF01_AE with subtype B or CRF07_BC. The subtype-specific phylogenic tree analysis showed that the genetic distance within subtype B group viruses was larger than the genetic distance within the CRF01_AE group (p 0.001). Of the identified viruses in the Chinese MSM population, over 80% of subtype B viruses might originate from the United States and Brazil, and over 85% of the CRF01_AE viruses mightoriginate from Thailand. In addition, epidemic study data showed that some of the HIV-1-infected MSM had foreign sexual partners (13.6%) and heterosexual activities (43.2%). The patients infected with HIV-1 URF viruses had more heterosexual encounters (54.5%) and more sexual partners (72.7%) compared to those infected with subtype B (44.4%; 33.3%) and CRF01_AE (23.1%; 69.2%) viruses. Taken together, we suspected that the genetic complexity of HIV-1 viruses identified in Chinese MSM populations was more likely a result of multiple introductions of viruses from the general population infected with HIV-1 through IDUs or heterosexual transmission.     

High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. DESIGN: A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. METHODS: HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. RESULTS: Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. CONCLUSION: The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.     

Identification of a Novel HIV Type 1 Circulating Recombinant Form (CRF52_01B) in Southeast Asia

Abstract Thirty HIV-1 URF_01AE/ B' complete or nearly full-length genome sequences sampled within Southeast Asia were obtained from the Los Alamos HIV Sequence Database. Phylogenetic and recombinant analyses revealed that three sequences indeed displayed the identical recombinant structure. Of note, the three subjects, harboring novel CRF01_AE/B recombinants, did not have apparent epidemiological linkage. They fulfilled the criteria for the designation of a new circulating recombinant form (CRF) and constituted the 52nd CRF identified in the worldwide HIV-1 pandemic. In this chimera, two short subtype B segments were inserted into a backbone of CRF_01AE. The breakpoints corresponded to HXB2 nucleotide positions 2930, 3251, 8521, and 9004 approximately. This CRF is the first one identified by neatening and analyzing the sequences already presented in the Los Alamos HIV Sequence Database. This indicates that we should pay attention not only to explicit subtype sequences but also to those classified as a unique recombinant form (URF) so far.     

Molecular epidemiology of HIV Type 1 in preparation for a Phase III prime-boost vaccine trial in Thailand and a new approach to HIV Type 1 genotyping

To characterize HIV-1 genotypes in candidate populations for a prime-boost phase III vaccine trial in Thailand, specimens from prevalent and incident HIV-1 infections from a family planning clinic population in Rayong Province and a community cohort in Chon Buri Province, collected from 1998 to 2001, were genotyped. A new multiregion hybridization assay, MHAbce, capable of distinguishing HIV-1 CRF01_AE, subtype B, and subtype C and their recombinants, was developed and applied to prevalent infections. Most incident and selected prevalent infections were studied by complete genome sequencing. By MHAbce, 168 of 194 prevalent infections were genotyped. Of these, 90.5% were CRF01_AE, 2.4% were subtype B, and 7.2% showed discordant or dual probe reactivity, indicative of recombination or dual infection, respectively. Among 23 incident infections, 20 were sequenced. Eighteen CRF01_AE, one subtype B, and one CRF01/B recombinant strains were seen. Two CRF01/B and one CRF01/C recombinant were identified among selected prevalent infections. These results indicate that incident and prevalent HIV-1 infections in Rayong and Chon Buri during 1998-2001 were 90% CRF01_AE, 3% subtype B, and 7% either recombinant or dual. This study frames the genetic diversity of HIV-1 in these cohorts in their preparatory phase for the ongoing ALVACHIV (vCP1521) prime, AIDSVAX B/E boost, phase III vaccine trial and will provide a benchmark for interpretation and analysis.     

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.     

Identification of CRF34_01B, a second circulating recombinant form unrelated to and more complex than CRF15_01B, among injecting drug users in northern Thailand

In Thailand, the circulating HIV-1 strains include CRF01_AE, subtype B, and their recombinants. Genotyping and full-genome sequencing had previously identified circulating recombinant form CRF15_01B within a cohort of 347 HIV-1-infected individuals enrolled in the Opiate Users Research (OUR) study in northern Thailand. Using an improved MHAbce in six to eight genome regions and archived OUR serum samples, seven strains were identified with a new and complex 01/B recombinant pattern in common, different from that of CRF15_01B. Complete sequencing of three strains, amplified from serum as overlapping half-genomes, confirmed their common recombinant structure, mostly CRF01_AE, but with segments of subtype B in pol and gp41, plus a region of frequent 01/B crossovers in pol. OUR strains 1969P, 2275P, and 2478P were from individuals without direct epidemiological linkage and thus establish CRF34_01B. More comprehensive HIV-1 prevention and treatment programs in IDU can help to limit the growing complexity of HIV-1 strains in Thailand.     

A new circulating recombinant form,CRF15_01B,reinforces the linkage between IDU and heterosexual epidemics in Thailand

HIV-1 subtype B and CRF01_AE have been in circulation in Thailand and Southeast Asia for more than a decade. Initially separated by risk group, the two strains are increasingly intermixed, and two recombinant strains of essentially reciprocal structure have been recently reported. Here we identify additional CRF_01B recombinants and provide the evidence that HIV-1 strains now pass freely between the two high-risk populations. HIV isolates that showed discordance between CRF01_AE and subtype B in multi-region genotyping assays were selected for the study. They were drawn from 3 different cohorts in Thailand representing different risk behaviors and demographic characteristics: a drug user cohort in the north, a family planning clinic attendee cohort in the southeast, and a cohort study of the mucosal virology and immunology of HIV-1 infection in Thailand. The DNA from these isolates was PCR amplified to recover the full HIV-1 genome and subjected to sequencing and phylogenetic analysis. We establish that one particular CRF_01B recombinant, with the external envelope of subtype B and the rest of the genome from CRF01_AE, is circulating widely in Thailand. Termed CRF15_01B (also referred to as CRF15), the strain was primarily heterosexually transmitted, although injecting drug use (IDU) also played a role. In aggregate data from the studies, CRF15 constituted 1.7% of all HIV-1 infections (95% confidence interval 0.5-4.4%) and was dispersed widely in the country. The previously separate heterosexual and IDU epidemics have apparently been bridged by a new CRF. The entry of CRF15 into the mainstream of the epidemic signals new complexity in the long stable molecular picture in Thailand. These recombinants must be considered in ongoing or projected efficacy evaluations of HIV-1 vaccines and antiviral therapies.     

On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan Province of China

OBJECTIVES: To investigate the molecular epidemiology of HIV in China's Yunnan Province, where the initial HIV-1 outbreak among injecting drug users (IDU) occurred in 1989, and to analyse the genesis and interrelationship of the epidemic with that in surrounding areas. DESIGN: A molecular epidemiological investigation was conducted among IDU in three prefectures in Yunnan Province, including Wenshan (east), Honghe (southeast) and Dehong (west). METHODS: Thirty-nine specimens were collected from consenting IDU in 2000-2001. The nucleotide sequences of 2.6 kb gag-RT and 340 base pair (bp) env (C2/V3) regions were determined. Phylogenetic tree and recombination breakpoint analyses were performed. RESULTS: The circulating recombinant form (CRF), CRF08_BC, predominated in east Yunnan near Guangxi Province (89% in Wenshan and 81% in Honghe), whereas it was not detected in Dehong (0/14) in the west. In contrast, 71% (10/14) of the Dehong isolates were unique recombinant forms (URF), mostly between subtypes B' (Thailand variant of subtype B) and C, with distinct profiles of recombination breakpoints. The subtype B' accounts for the remaining 29% (4/14) of Dehong isolates. Interestingly, two Honghe isolates (2/16) shared some of the precise B'/C recombination breakpoints with CRF07_BC. CONCLUSION: New recombinant strains are arising continually in west Yunnan near the Myanmar border. Some appeared to be secondary recombinants derived from CRF07_BC that had further recombined with other strains. The uneven distribution of subtypes, CRF and URF, suggests the presence of independent transmission networks and clusters among IDU in Yunnan.