Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).     

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.     

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.     

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.     

Maintaining low HIV type 1 env genetic diversity among injection drug users infected with a B/C recombinant and CRF01_AE HIV type 1 in southern China

HIV-1 outbreaks in Guangxi Province, southern China were initiated from two separate border cities in 1996 and 1997. Drug users in Pingxiang City, which borders Vietnam, were infected with CRF01_AE HIV-1, and drug users in Baise City, which borders Yunan Province, were infected with a novel B/C recombinant HIV-1. Since 1997, HIV-1 has been rapidly spreading in Guangxi, including its capital city Nanning. Survey data indicated that HIV-1 prevalence among IDUs in new outbreak regions increased 8 to 42% within 1 year. The B/C recombinants obtained from five separate regions in Guangxi, which span a 4-year time frame, were remarkable for their low intersubject env V3 diversity, less than 0.2%. Similarly, the CRF01_AE from IDUs over a 3-year time frame had low intersubject env V3 diversity of less than 1.6%. Different patterns of sequence variations in the V3 and V4 regions were observed for the B/C recombinant and the CRF01_AE HIV1. The rapid spreading of homogeneous HIV-1 strains in Guangxi may have important implications for HIV transmission as well as vaccine development and evaluation.     

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.     

Isolation and characterization of replication-competent molecular DNA clones of HIV type 1 CRF01_AE with different coreceptor usages

We have isolated replication-competent molecular clones of HIV-1 circulating recombinant form CRF01_AE with different coreceptor usages. After lambda phage cloning of unintegrated circular proviral DNAs derived from a CRF01_AE strain (HIV-1NH1), isolated in Japan, the infectious molecular clone, designated p93JP-NH1, was reconstituted. 93JP-NH1 showed an X4 and R5 phenotype in NP2 cell-based coreceptor utilization assays and exerted robust replication in human T cell lines, including MT2, M8166, and PM1 cells, whereas it propagated modestly in peripheral blood mononuclear cells. The CRF01_AE molecular clone with R5 phenotype (p93JP-NH2env) was then constructed by replacing the env gene of p93JP-NH1 with that of a nearly isogenic CRF01_AE R5 strain isolated from an epidemiologically linked case. The phylogeny and recombination break-point analysis confirmed that these clones shared an A/E recombinant structure similar to that of the prototype CRF01_AE strain, CM240. These replication-competent CRF01_AE molecular clones with different coreceptor usages would be useful tools for the study of CRF01_AE, one of the most prevalent strains in Asia.     

我国男男性行为人群中HIV-1病毒亚型分布及其演变

男男性行为人群(MSM)仍是我国HIV感染的高危人群,欧美B亚型、CRF01_AE和CRF07_BC亚型是其人群中流行的主要病毒亚型。近年来,MSM中HIV-1亚型的流行趋势发生改变,欧美B亚型所占比例显著下降,CRF01_AE和CRF07_BC亚型所占比例则明显增加,并且出现新的重组亚型。运用分子生物学技术分析我国男男性行为人群中HIV-1病毒亚型分布和变化趋势,可有助于阐明MSM人群中HIV-1的流行规律,追踪传染来源和确定相关危险因素,并为MSM人群精准干预提供重要的参考依据。     

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.     

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.     

Importation of multiple HIV type 1 strains into West Papua, Indonesia (Irian Jaya).

HIV-1 from 16 sexually transmitted disease clinic patients in Timika, West Papua, Indonesia was amplified by RT-PCR and subtyped by a combination of envelope and gag region heteroduplex mobility analysis (HMA) and direct PCR DNA sequencing. HMA showed the presence of 14 subtype E (CRF01_AE) and 2 subtype B HIV-1. Phylogenetic analysis of a 540-bp V3-V4 region of gp120 showed that 9 of 10 CRF01_AE variants clustered tightly with a median distance of 1.3% (range, 0.5 to 2.2%) whereas 1 CRF01_AE variant diverged significantly from the others (median distance, 10.7%; range, 10.1 to 11.8%). One subtype B virus envelope was typical of United States/European strains whereas the other appeared to be related to Thai subtype B' variants. These results reflect the independent introduction of multiple HIV-1 strains into West Papua, with the rapid spread in the majority of infected patients tested of a single strain of HIV-1E (CRF01_AE).     

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.     

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.