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.     

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.     

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.     

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.     

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.     

Characterization of complete HIV type 1 genomes from non-B subtype infections in U.S. military personnel

Infections with non-B HIV-1 subtypes are rare in the United States, but comprise a significant percentage of infections among U.S. military personnel. Risk behavior while on overseas deployment correlates with non-B infection in this population. Extensive genetic characterization will be required to define HIV-1 diversity, and to effectively evaluate requirements for HIV-1 vaccines and other prevention strategies in this group. From 1997 to 2000, 520 recent seroconverters, identified through routine HIV-1 testing in the U.S. active military force, volunteered for a prospective study. V3 loop serology or partial genome sequencing identified 28 non- B subtype infections; 14 were studied by full genome sequencing and phylogenetic analysis. Five strains were CRF01_AE. Four of these clustered with CM240 from Thailand, and one clustered with African CRF01_AE. Four strains were CRF02_AG, prevalent in West and West Central Africa. Two strains were subtype C. One strain was a unique recombinant between CRF01_AE and subtype B, and another was a complex unique recombinant between subtype A and D. The final strain was a member of a complex circulating recombinant first identified in Senegal, CRF09_cpx, incorporating subtypes A, F, G, and an unclassified genome. This diversity of non-B subtype HIV-1 strains, encompassing three globally prevalent non-B strains and including rare or even possibly unique strains, illustrates the breadth of U.S. military exposure while deployed and sets the bar higher for breadth of cross-subtype protection to be afforded by an HIV-1 vaccine.     

Genetic characterization of CRF01_AE full-length human immunodeficiency virus type 1 sequences from Fujian, China

HIV-1 CRF01_AE is the main prevalent HIV-1 recombinant strain in China and the dominant strain in Fujian as well; therefore, obtaining and understanding the genetic characterization of this clade become important. To date only a few full-length HIV-1 CRF01_AE genomes from China have been sequenced. We reported 13 full-length HIV-1 CRF01_AE isolate sequences from Fujian, China. DNA was extracted, the 5' and 3' halves of each genome were separately amplified, and the PCR products were cloned and sequenced. The phylogenetic analysis of the 13 sequences showed that they clustered with HIV-1 CRF01_AE isolates; these isolates were separated into a few distinct subgroups and were dispersed among the Thailand isolates, and the intersample diversity among Fujian isolates was significantly high. The high gene diversity among Fujian HIV-1 CRF01_AE isolates indicates that the HIV-1 CRF01_AE viruses in Fujian may be transmitted from multiple introductions. The work is essential for the design and development of an effective AIDS vaccine for China.     

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.     

Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.     

New HIV type 1 CRF01_AE/B recombinants displaying unique distribution of breakpoints from incident infections among injecting drug users in Thailand

The goals of this study were to identify and characterize recombinant human immunodeficiency virus type 1 (HIV-1) genomes among incident infections in a prospective cohort study of injecting drug users (IDUs) in Bangkok, Thailand. Through cross-sectional, comparative phylogenetic analysis of the protease and env (C2-V4) gene regions, subtype discordance was observed in HIV-1 sequences from 4 of 111 IDUs (3.5%). Near-full-length HIV-1 genome sequences of the four strains revealed that in all four, the gp120 sequences clustered with a CRF01_AE prototype, while the remainder of the genomes displayed distinct mosaic patterns, with multiple breakpoints between HIV-1 CRF01_AE and subtype B-like regions. Two of the four HIV-1 recombinant strains displayed a nearly identical mosaic structure, suggesting the possible emergence and spread of a potentially new circulating recombinant form of HIV-1. Further characterization of these and other recombinant genomes through long-term follow-up will be important in understanding the generation of viral diversity and escape from the hosts immune responses. This information will be especially important for vaccine development.     

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.     

Identification of new CRF51_01B in Singapore using full genome analysis of three HIV type 1 isolates

A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form, which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B recombinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1 transmission in Singapore.     

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.     

Frequency of HIV-1 dual subtype infections, including intersubtype superinfections, among injection drug users in Bangkok, Thailand

OBJECTIVES: To estimate the frequency and incidence of dual HIV-1 subtype infections, including superinfections, among recent seroconvertors from a cohort of injection drug users (IDUs). METHODS: A total of 1209 HIV-negative IDUs were followed in a prospective cohort study at 15 methadone clinics in Bangkok, Thailand. After 2308 person-years (PY) of follow-up, 133 seroconverted to HIV-1, of which approximately 20% were subtype B and 80% were CRF01_AE (formerly called subtype E). Specimens from 126 individuals were available at time of first seropositive test and specimens from 80 of these 126 individuals were also available more than 12 months later. For each infected participant, we calculated the amount of time to superinfection, loss to follow-up, or to the closest visit more than 12 months after the time of initial seropositivity. RESULTS: Of all 126 seroconverters seen at the time of the first seropositive test result, there was no apparent case of concurrent dual subtype infection detected despite 2301 PY of observation. Overall, the incidence of superinfection was 2.2 per 100 PY [95% confidence interval (CI), 0.3-7.8]. The 1-year incidence of CRF01_AE superinfection following subtype B primary infection was 3.9 per 100 PY (95% CI, 0.1-21.9) and the incidence of subtype B superinfection following CRF01_AE primary infection was 1.5 per 100 PY (95% CI, 0.04-8.3). CONCLUSIONS: Determination of the frequency and incidence of dual HIV-1 subtype infection demonstrates that HIV-1 superinfection is not uncommon in a population with high HIV-1 incidence with more than one circulating strain.