Identification of new CRF43_02G and CRF25_cpx in Saudi Arabia based on full genome sequence analysis of six HIV type 1 isolates

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.     

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 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.     

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.     

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.     

Near full-length genome characterization of three additional HIV type 1 CRF13_cpx strains from Cameroon

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genomes of three candidate HIV-1 CRF13_cpx strains originating in Cameroon, 04CM-173-9, 04CM-632-28, and 02CM-A1394. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed similar genomic structures with identical breakpoint positions compared to the three available CRF13_cpx sequences. The candidate and reference sequences formed a distinct cluster well separated from other group M subtypes and had a mosaic structure derived from subtypes A1, G, J, and CRF01_AE. The similarity in genomic composition and position of recombination breakpoints suggest that these isolates share a common ancestor. The epidemiological significance of CRF13_cpx strains in Cameroon is unknown; however, the availability of three additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.     

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.     

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 indonesian variants of CRF33_01B: Near-full length sequence analysis

Cocirculation of subtype B and CRF01_AE in Southeast Asia has led to the establishment of new recombinant forms. In our previous study, we found five samples suspected of being recombinants between subtype B and CRF01_AE, and here, we analyzed near full-length sequences of two samples and compared them to known CRFs_01B, subtype B, and CRF01_AE. Five overlapped segments were amplified with nested PCR from PBMC DNA, sequenced, and analyzed for genome mosaicism. The two Indonesian samples, 07IDJKT189 and 07IDJKT194, showed genome-mosaic patterns similar to CRF33_01B references from Malaysia, with one short segment in the 3' end of the p31 integrase-coding region, which was rather more similar to subtype B than CRF01_AE, consisting of unclassified sequences. These results suggest gene-specific continuous diversification and spread of the CRF33_01B genomes in Southeast Asia.     

Heterosexual transmission of novel CRF01_AE and subtype B recombinant forms of HIV type 1 in northern Thailand

The increased proportion of CRFO1_AE/subtype B recombinant infections among injecting drug users raised a concern that such recombinant forms may also spread in a heterosexual population in Thailand. Using the BootScan method, we reanalyzed pol gene sequences among 114 heterosexually infected individuals in northern Thailand, who were tested for a drug-resistance genotype between July 2000 and July 2001. Two individuals were suspected of carrying a recombinant HIV-1. Thus we analyzed a nearly full-length HIV genome in the two individuals and their spouses. An identical recombinant form of CRF01_AE and subtype B was found in one couple, indicating that this recombinant virus was heterosexually transmitted. Interestingly, this recombinant form had multiple breakpoints in the core protein of Gag and both infected individuals had a high CD4+ cell count without antiretroviral therapy. CRFO1/subtype B recombinant forms exist in a heterosexual population in northern Thailand. Some recombinant virus may be associated with a slow rate of HIV disease progression.     

Near full-length genome analysis of HIV type 1 CRF02.AG subtype C and CRF02.AG subtype G recombinants

HIV-1 CRF02.AG strains are prevalent in west and west-central Africa, suggesting a longstanding presence of these subtype A/G recombinants in the global epidemic. Cocirculation of CRF02.AG strains with other group M subtypes may give rise to HIV-1 recombinants constituting a mosaic genome comprising fragments of three different subtypes. We report on the genetic analysis of the near-full-length genomes of such recombinants (VI1035 and VI1197) as well as CRF02.AG strains in Belgian individuals. VI1035 and VI1197 may be the result of successful "second-generation" recombinations of HIV-1 strains CRF02.AG with, respectively, subtype C (VI1035) and G (VI1197) strains in a dually infected individual.     

Identification of a new circulating recombinant form of HIV type 1, CRF11-cpx, involving subtypes A, G, J, and CRF01-AE, in Central Africa

In this study, we characterized three full-length genome sequences with a similar mosaic structure from epidemiologically unlinked individuals from Cameroon (97CM-MP818) and the Central African Republic (99CF-MP1298 and 99CF-MP1307). Phylogenetic and recombinant analysis confirmed that the three strains had a similar complex recombinant genome, which we can designate now as CRF11-cpx. This new CRF was composed of successive fragments of subtype A, G, J, and CRF01-AE. The previously reported GR17 virus from a Greek patient infected in the Democratic Republic of Congo (DRC) has a similar structure and should be considered as the prototype strain of CRF11-cpx. This new CRF circulates in Cameroon, Central African Republic, Gabon, and DRC, although the exact prevalences remain to be determined.     

Near full-length genomic characterization of a novel HIV type 1 CRF07_ BC/CRF08_ BC recombinant strain from Yunnan, China

Yunnan province was considered the HIV-1 epicenter of China, where many subtypes and CRFs of HIV-1 were circulating. CRF07_BC and CRF08_BC were two of the main circulating subtypes that caused more than 90% of the HIV-1 infections in intravenous drug users (IDUs) in this district. The cocirculation of these two CRFs in the same area and population predicted the emergence of new second-generation recombinants. This study presented a near full-length genomic analysis of a novel HIV-1 recombination (09YN072) involving CRF07_BC and CRF08_BC. The analyses of the sequence of 09YN072 showed that two CRF07_BC segments were inserted into the CRF08_BC backbone. The discovery of the novel recombinant strain complicates the HIV-1 epidemic in Yunnan, China, as well as the development of effective vaccines to limit the spread of HIV-1 in 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.     

Near full-length genome characterization of an HIV type 1 CRF25_cpx strain from Cameroon

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genome of one candidate HIV-1 CRF25_cpx strain originating in Cameroon, 06CM-BA-040. Viral RNA was extracted from plasma, and the genome was obtained using RT-PCR amplification to generate 10 overlapping fragments. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed that 06CM-BA-040 had a genomic structure consistent with two available CRF25_cpx reference sequences. The CRF25_cpx mosaic composition consisted of nine segments derived from subtypes A and G as well as unclassified (U) regions. Subtype G and CRF25_cpx clusters diverged from each other with long branch lengths but were distinct from other known subtypes with high bootstrap support (94%). The epidemiological significance of CRF25_cpx strains is unknown; however, the availability of additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.     

Near full-length genome characterization of an HIV type 1 CRF05_DF virus from Spain

We report the near full-length sequence characterization of a HIV-1 DF intersubtype recombinant virus from Spain, X492, directly amplified from peripheral blood mononuclear cells' DNA. This isolate shares an identical mosaic structure and exhibits consistent phylogenetic clustering along the genome with VI961, a previously characterized DF recombinant virus. By contrast, VI1310, which may represent the same recombinant form as VI961 (CRF05_DF), is only partially homologous to VI961 and X492. Of three additional DF recombinant viruses previously characterized in gag-pol, only one, VI1267, clusters uniformly with VI961 and X492; the other two branch separately in a segment of pol. These results allow us to define an HIV-1 circulating recombinant form (CRF05_DF), characterized in near full-length genomes of two isolates (VI961 and X492) and in partial gag-pol sequences of a third virus (VI1267). Three other reported DF recombinant viruses, including the fully sequenced VI1310, exhibit incomplete homology to VI961 and X492.     

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.