HIV-1 Circulating Recombinant Forms Research Articles

Genetic characteristics of a novel HIV-1 circulating recombinant form (CRF128_07B) identified among MSM in Guangdong Province, China

ObjectiveTo obtain and investigate the genetic characteristics of four HIV-1 near full-length genome sequences (NFLGs), aiming at a description of a novel circulating recombinant form (CRF) in Guangdong China. MethodsPlasma samples were collected from HIV-1 infected MSM patients in Guangdong Province who had no epidemiological association with each other. The NFLGs were amplified with two overlapping halves and phylogenetic analyses were performed using Mega V11.0.1. Recombination analyses were comprehensively screened with the jpHMM, RIP, and BootScan analyses. Finally, the Bayesian phylogenetic analyses were performed using Beast V1.10.4 to estimate the origin time. ResultsPhylogenetic analyses revealed the four NFLGs formed a distinct monophyletic cluster distinguished from other known subtypes in the Neighbor-joining tree. Recombinant analyses revealed they shared a highly similar recombinant pattern, with the CRF07_BC backbone substituted by three subtype B segments. Subregion phylogenetic analyses confirmed them to be a novel CRF composed of CRF07_BC and subtype B, therefore, designed as CRF128_07B. According to the Bayesian phylogenetic analyses, CRF128_07B was inferred to approximately originated around 2005–2006. ConclusionsThese findings described a novel HIV-1 CRF identified from MSM in Guangdong Province. This is the first detection of a CRF comprising CRF07_BC and subtype B. The present finding highlights the urgent need for continuous molecular screening and the epidemic surveillance within the MSM populations.

Comprehensive Genetic Characterization of Four Novel HIV-1 Circulating Recombinant Forms (CRF129_56G, CRF130_A1B, CRF131_A1B, and CRF138_cpx): Insights from Molecular Epidemiology in Cyprus.

Molecular investigations of the HIV-1 pol region (2253-5250 in the HXB2 genome) were conducted on sequences obtained from 331 individuals infected with HIV-1 in Cyprus between 2017 and 2021. This study unveiled four distinct HIV-1 putative transmission clusters, encompassing 19 previously unidentified HIV-1 recombinants. These recombinants, each comprising eight, three, four, and four sequences, respectively, did not align with previously established Circulating Recombinant Forms (CRFs). To characterize these novel HIV-1 recombinants, near-full-length genome sequences were successfully obtained for 16 of the 19 recombinants (790-8795 in the HXB2 genome) using an in-house-developed RT-PCR assay. Phylogenetic analyses, employing MEGAX and Cluster-Picker, along with confirmatory neighbor-joining tree analyses of subregions, were conducted to identify distinct clusters and determine subtypes. The uniqueness of the HIV-1 recombinants was evident in their exclusive clustering within generated maximum likelihood trees. Recombination analyses highlighted the distinct chimeric nature of these recombinants, with consistent mosaic patterns observed across all sequences within each of the four putative transmission clusters. Conclusive genetic characterization identified four novel HIV-1 CRFs: CRF129_56G, CRF130_A1B, CRF131_A1B, and CRF138_cpx. CRF129_56G exhibited two recombination breakpoints and three fragments of subtypes CRF56_cpx and G. Both CRF130_A1B and CRF131_A1B featured seven recombination breakpoints and eight fragments of subtypes A1 and B. CRF138_cpx displayed five recombination breakpoints and six fragments of subtypes CRF22_01A1 and F2, along with an unclassified fragment. Additional BLAST analyses identified a Unique Recombinant Form (URF) of CRF138_cpx with three additional recombination sites, involving subtype F2, a fragment of unknown subtype origin, and CRF138_cpx. Post-identification, all putative transmission clusters remained active, with CRF130_A1B, CRF131_A1B, and CRF138_cpx clusters exhibiting further growth. Furthermore, international connections were identified through BLAST analyses, linking one sequence from the USA to the CRF130_A1B strain, and three sequences from Belgium and Cameroon to the CRF138_cpx strain. This study contributes valuable insights into the dynamic landscape of HIV-1 diversity and transmission patterns, emphasizing the need for ongoing molecular surveillance and global collaboration in tracking emerging viral variants.

Identification of a HIV-1 circulating BF1 recombinant form (CRF75_BF1) of Brazilian origin that also circulates in Southwestern Europe.

The high recombinogenic potential of HIV-1 has resulted in the generation of countless unique recombinant forms (URFs) and around 120 reported circulating recombinant forms (CRFs). Here we identify through analyses of near full-length genomes (NFLG) a new HIV-1 CRF derived from subtypes B and F1. HIV-1 protease-reverse transcriptase (Pr-RT) sequences were obtained by RT-PCR amplification from plasma RNA. Near full-length genome sequences were obtained after amplification by RT-PCR in 5 overlapping fragments. Phylogenetic sequence analyses were performed via maximum likelihood. Mosaic structures were analyzed by bootscanning and phylogenetic analyses of genome segments. Temporal and geographical estimations of clade emergence were performed with a Bayesian coalescent method. Through phylogenetic analyses of HIV-1 Pr-RT sequences obtained by us from samples collected in Spain and downloaded from databases, we identified a BF1 recombinant cluster segregating from previously reported CRFs comprising 52 viruses, most from Brazil (n = 26), Spain (n = 11), and Italy (n = 9). The analyses of NFLG genomes of 4 viruses of the cluster, 2 from Spain and 2 from Italy, allowed to identify a new CRF, designated CRF75_BF1, which exhibits a complex mosaic structure with 20 breakpoints. All 4 patients harboring CRF75_BF1 viruses studied by us had CD4+ T-cell lymphocyte counts below 220/mm3 less than one year after diagnosis, a proportion significantly higher (p = 0.0074) than the 29% found in other patients studied in Spain by us during the same period. The origin of the clade comprising CRF75_BF1 and related viruses was estimated around 1984 in Brazil, with subsequent introduction of CRF75_BF1 in Italy around 1992, and migration from Italy to Spain around 1999. A new HIV-1 CRF, designated CRF75_BF1, has been identified. CRF75_BF1 is the 6th CRF of South American origin initially identified in Western Europe, reflecting the increasing relationship of South American and European HIV-1 epidemics. The finding of low CD4+ T-cell lymphocyte counts early after diagnosis in patients harboring CRF75_BF1 viruses warrants further investigation on the virulence of this variant.

Structures and immune recognition of Env trimers from two Asia prevalent HIV-1 CRFs

Structure-guided immunofocusing HIV-1 vaccine design entails a comprehensive understanding of Envs from diverse HIV-1 subtypes, including circulating recombinant forms (CRFs). Here, we present the cryo-EM structures of Envs from two Asia prevalent CRFs (CRF01_AE and CRF07_BC) at 3.0 and 3.5 Å. We compare the structures and glycosylation patterns of Envs from different subtypes and perform cross-clade statistical analyses to reveal the unique features of CRF01_AE V1 region, which are associated with the resistance to certain bNAbs. We also solve a 4.1 Å cryo-EM structure of CRF01_AE Env in complex with F6, the first bNAb from CRF01_AE-infected individuals. F6 recognizes a gp120-gp41 spanning epitope to allosterically destabilize the Env trimer apex and weaken inter-protomer packing, which in turn hinders the receptor binding and induces Env trimer disassembly, demonstrating a dual mechanism of neutralization. These findings broaden our understanding of CRF Envs and shed lights on immunofocusing HIV-1 vaccine design.

HIV-1 Circulating Recombinant Forms (CRFs) and Unique Recombinant Forms (URFs) in Israel, 2010-2018.

Monitoring HIV-1 circulating recombinant forms (CRFs) and unique recombinant forms (URFs) is important for disease surveillance. Recombination may affect prevention efforts and interfere with the diagnosis and treatment of HIV-1 infection. Here, we characterized the epidemiology of HIV-1 CRFs and URFs in Israel. Partial pol sequences from treatment naïve patients diagnosed in 2010–2018 were assessed using the recombinant identification program (RIP), the recombinant detection program (RDP5), and using the maximum-likelihood phylogenetic method, using 410 reference sequences obtained from the Los Alamos database. CRFs and URFs were identified in 11% (213/1940) of all sequenced cases. The median age at diagnosis was 38 (30–47) years, 61% originated from Israel, and 82% were male. The most common were CRF02_AG (30.5%), CRF01_AE (16.9%), and the more complex forms CRF01_AE/CRF02_AG/A3 (10.8%) and B/F1 (7%). A significant increase in their overall proportion was observed in recent years (8.1% in 2010–2012, 20.3% in 2016–2018, p < 0.001). This increase was most prominent in individuals carrying CRF02_AG (2.5% in 2010–2015, 9.8% in 2016–2018, p < 0.001). Men who have sex with men (MSM) was the most common risk group; however, those infected with the secondary recombinant CRF02_AG/A6 were mainly injecting drug users (IDUs). The most common resistance mutations were K103N (5/213, 2.3%) and E138A (18/213, 8.5%) in the reverse transcriptase. Only E138A was more frequent in the recombinants compared with the classic subtypes and was significantly associated with a specific secondary CRF, CRF02_AG/A4. We concluded that CRFs and URFs were mainly detected in Israeli-born MSM and that an increase in the overall proportion of such HIV-1 sequences could be observed in more recent years.

Identification of two novel HIV-1 circulating recombinant forms of CRF111_01C and CRF116_0108 in southwestern Yunnan, China

ABSTRACT Yunnan, the region hardest hit by HIV/AIDS in China, is also an area with the most abundant HIV-1 genetic diversity. A large number of novel HIV-1 circulating recombinant forms (CRFs) and unique recombinants were identified among injection drug users in Yunnan; however, few were found among sexual contacts. Here, we obtained 15 near full-length genome sequences (NFLGs) from HIV-1 seropositive heterosexual contacts in Yunnan who received antiretroviral therapy during the period from 2014 to 2016. Phylogenetic analysis showed that six NFLGs belonged to CRF01_AE (n = 3) and CRF106_cpx (n = 3), and the other nine sequences were novel inter-subtype recombinants. Of the recombinants, two novel CRFs (CRF111_01 C (n = 4) and CRF116_0108 (n = 4)) and one CRF106_cpx variant (n = 1) were identified. CRF111_01 C had a CRF01_AE backbone with seven subtype C fragments inserted into the gag, pol, vif, env, nef and 3ʹLTR regions. CRF116_0108 had a CRF08_BC backbone with a CRF01_AE fragment inserted into the pol, tat, rev, vif, vpr, vpu and env regions. Phylogeographic analyses estimated that CRF111_01 C and CRF116_0108 originated approximately 1995.7–1998.6 and 1991.7–1993.7, respectively. These identifications of two novel HIV-1 CRFs highlighted the importance of continuous surveillance in heterosexual contacts and other high-risk groups in this region and the surrounding regions.

Identification of CRF66_BF, a New HIV-1 Circulating Recombinant Form of South American Origin

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 110 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n = 4), Spain (n = 3), Paraguay (n = 1), Brazil (n = 1), and Italy (n = 1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17gag, integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF “family” clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.

Identification of a newly emerging HIV-1 CRF111_01C comprising CRF01_AE and C in Yunnan, China: Genetic characterization and recombinant history.

New human immunodeficiency virus (HIV)-1 circulating recombinant forms (CRFs) have recently emerged and disseminated rapidly in China; in total 38 CRFs have been identified thus far. Yunnan province shares its border with Myanmar, and is regarded as a "hotspot" for the occurrence of new HIV-1 recombinations; more than half of novel CRFs reported in China have been first documented in Yunnan province. In the present study, based on the information available on four existing near-full-length genome (NFLG) sequences, combined with data on four other closely related sequences obtained via Basic Local Alignment Search Tool (BLAST) analysis, NFLG/subregion phylogenetic, bootscanning, and time to the most recent common ancestor (TMRCA) analyses were performed. Phylogenetic analysis revealed that the eight strains demonstrated the formation of a distinct monophyletic branch with a bootstrap value of 100%. Strains in this branch were distantly related to all known HIV-1 CRFs; it was temporarily named CRF111_01C. Bootscanning analysis revealed that CRF111_01C consisted of a CRF01_AE backbone and four inserted subtype C segments. Remarkably, CRF111_01C shared six mosaic fragment identities with the previously identified CRF100_01C. Furthermore, CRF111_01C may be deemed a potential second-generation CRF consisting of CRF100_01C and C. Coalescent Bayesian analyses revealed that the TMRCA of CRF111_01C was approximately the period 1999-2002. The emergence of such second-generation recombinants highlights that continuous molecular screening is necessary to carefully monitor the evolutionary dynamics of HIV-1 epidemics.

The first third-generation HIV-1 circulating recombinant form (CRF114_0155) identified in central China.

A novel circulating recombinant form (CRF) was identified in eight HIV-1-infected patients without direct epidemiological relationships in Henan Province, Central China. Recombination analysis indicated that the genome of this novel CRF comprises five segments: three inherited from CRF01_AE cluster-4 and two from CRF55_01B. Therefore, the CRF was designated CRF114_0155. It is not only the first novel CRF identified in Henan Province but also the first third-generation CRF of HIV-1 and the first CRF descendant of CRF55_01B. Bayesian inference of phylogeny dated the most recent common ancestor of the CRF114_0155 cluster to 2010. The emergence of CRF114_0155 reflects that the genotype constitution of HIV-1 has become more complex and that stricter intervention measures should be implemented in central China.

Identification of CRF89_BF, a new member of an HIV-1 circulating BF intersubtype recombinant form family widely spread in South America

Circulating recombinant forms (CRFs) contribute substantially to the HIV-1 pandemic. Among 105 CRFs described in the literature, 16 are BF intersubtype recombinants, most of South American origin, of which CRF12_BF is the most widely spread. A BF recombinant cluster identified in Bolivia was suggested to represent a new CRF_BF. Here we find that it belongs to a larger cluster incorporating 39 viruses collected in 7 countries from 3 continents, 22 of them in Spain, most from Bolivian or Peruvian individuals, and 12 in South America (Bolivia, Argentina, and Peru). This BF cluster comprises three major subclusters, two associated with Bolivian and one with Peruvian individuals. Near full-length genome sequence analyses of nine viruses, collected in Spain, Bolivia, and Peru, revealed coincident BF mosaic structures, with 13 breakpoints, 6 and 7 of which coincided with CRF12_BF and CRF17_BF, respectively. In a phylogenetic tree, they grouped in a clade closely related to these CRFs, and more distantly to CRF38_BF and CRF44_BF, all circulating in South America. These results allowed to identify a new HIV-1 CRF, designated CRF89_BF. Through phylodynamic analyses, CRF89_BF emergence was estimated in Bolivia around 1986. CRF89_BF is the fifth CRF member of the HIV-1 recombinant family related to CRF12_BF.

Identification of HIV-1 subtypes and drug resistance mutations among HIV-1-infected individuals residing in Pontianak, Indonesia.

The present study investigated the HIV-1 subtype classification in addition to prevalence of drug resistance mutations (DRMs) in antiretroviral therapy (ART)-experienced and ART-naïve residents of Pontianak, West Kalimantan, Indonesia. Whole blood samples collected from 30 HIV-1-infected individuals, comprising 19 ART-experienced and 11 ART-naïve individuals, were subjected to RNA and DNA extraction, followed by HIV-1 genes amplification and sequencing analysis. HIV-1 subtyping was classified on viral pol genes encoding reverse transcriptase (RT gene) and protease (PR gene) accompanied by the env and gag genes. DRMs in the RT and PR genes were also analyzed. CRF01_AE was identified as the predominant circulating recombinant form (CRF) of HIV-1 in both ART-experienced and ART-naïve individuals. In addition, CRF02_AG, subtype B, recombinant virus expressing CRF01_AE and subtype B viral genomic fragments, also recombinant virus containing CRF01_AE and CRF02_AG genomic fragments were also identified. Acquired drug resistance (ADR) was identified in 28.5% of ART-experienced individuals, while no transmitted drug resistance was identified in ART-naïve individuals. This study identified CRF01_AE as the most predominant HIV-1 CRF distributing in Pontianak, Indonesia. The prevalence of ADR is considered to be high; thus, further surveillance is needed in this region.

First Detection of a Circulating Recombinant Form of HIV-1 CRF01_AE/08_BC (CRF105_0108) with Drug-Resistant Mutations in Sichuan, China.

In this study, we identified a novel circulating recombinant form (CRF) of HIV (CRF105_0108) in a total of five patients with no obvious epidemiological linkage in Sichuan, China. Four strains were found in this study, and another strain (XC2014EU01) was reported in a previous study. The phylogenetic analysis of the five near full-length genomes showed that a CRF08_BC segment was inserted into the CRF01_AE backbone with one recombinant breakpoint in the pol region, which is the first HIV-1 CRF of CRF01_AE and CRF08_BC. Three genetic sequences had drug-resistant mutations, E138Q and V179D, indicating that there were low resistance levels to efavirenz (EFV) and nevirapine (NVP) in Liangshan Yi Autonomous Prefecture. CRF105_0108 increases the diversity and complexity of the prevalent HIV-1 CRFs in Sichuan. Simultaneously, drug-resistant mutations in this CRF may influence the efficacy of highly active antiretroviral treatments (ARTs) in Liangshan Yi Autonomous Prefecture.

Genetic characterization and recombinant history of a novel HIV-1 circulating recombinant form (CRF101_01B) identified in Yunnan, China

The high frequency of multiple HIV-1 recombinant events among the B, C and CRF01_AE were constantly occurring in Yunnan China. Here, we characterized a novel HIV-1 circulating recombinant form (CRF) consisting of CRF01_AE and subtype B (CRF101_01B) from three epidemiologically unlinked individuals. Phylogenetic analysis based on near full length genome (NFLG) sequences revealed that CRF101_01B formed a distinct monophyletic cluster supported by a high bootstrap value of 100%, distantly related to all known HIV-1 CRFs. CRF101_01B had a CRF01_AE backbone with two B segments inserted, respectively, in the gag and pol region. Further, subregion tree analysis showed that CRF01_AE backbone and subtype B segment inserted originated from a Thailand lineage. In addition, our study found that CRF101_01B originated around the year 1996–1998. This findings described a novel HIV-1 CRF, and highlighted the importance of continual monitoring of genetic diversity and complexity of HIV-1 strains in Yunnan, China.

HIV prevalence and phylogenetic characteristics among entry travelers in Xishuangbanna prefecture, Yunnan province, between 2003 and 2012.

Xishuangbanna is a popular tourist destination in southern Yunnan Province, bordering with Vietnam and Laos, and Myanmar. There are more than 2 million cross-border travelers annually entering China at two land ports, Daluo and Mohan. To clarify the characteristics of HIV-1 prevalence among entry travelers, a retrospective epidemic investigation was conducted. Between 2003 and 2012, 196 among 91 432 entry travelers were detected to be HIV-1-positive, accounting for infection rate of 0.21%. Eighty-one gag-pol gene sequences were obtained for phylogenetic analyses and subsequent recombination analysis. It was shown that the most prevalent HIV-1 subtype in this population was circulating recombinant form (CRF) 01_AE (42, 51.86%), followed by CRF08_BC (17, 20.99%), CRF07_BC (4, 4.94%), B' (2, 2.47%), C (2, 2.47%), other recombinants (9, 11.11%), and undefined subtype (5, 6.17%). HIV-1 genotype distribution among travelers entering at two land ports is different, CRF01_AE and CRF08_BC accounted for a larger percentage among individuals from Daluo, whereas the proportions of subtype C and undefined URFs were larger among individuals from Mohan. The undefined subtype indicates the occurrence of novel HIV-1 subtype or CRF. This finding is useful for developing of AIDS prevention strategies.

Identification of Two New HIV-1 Circulating Recombinant Forms (CRF87_cpx and CRF88_BC) from Reported Unique Recombinant Forms in Asia

The on-going generation of HIV-1 intersubtype recombination has led to new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in Asia. In this study, we evaluated whether previously reported URFs were actually CRFs. All available complete or near full-length HIV-1 URF sequences from Asia were retrieved from the HIV Los Alamos National Laboratory Sequence database, and phylogenetic, transmission cluster, and bootscan analyses were performed using MEGA 6.0, Cluster Picker 1.2.1, and SimPlot3.5.1. According to the criterion of new CRFs, two new HIV-1 CRFs (CRF87_cpx and CRF88_BC) were identified from these available URFs. CRF87_cpx comprised HIV-1 subtypes B, C, and CRF01_AE, and CRF88_BC comprised subtypes B and C. HIV Blast and bootscan analysis revealed that besides the three representative strains, there were two additional CRF87_cpx strains. Furthermore, we defined seven dominant URFs (dURF01-dURF07), each of which contained two strains sharing same recombination map and can be used as sequence references to facilitate the finding of new potential CRFs in future. These results will benefit the molecular epidemiological investigation of HIV-1 in Asia.

Identification of an HIV-1 BG Intersubtype Recombinant Form (CRF73_BG), Partially Related to CRF14_BG, Which Is Circulating in Portugal and Spain.

HIV-1 exhibits a characteristically high genetic diversity, with the M group, responsible for the pandemic, being classified into nine subtypes, 72 circulating recombinant forms (CRFs) and numerous unique recombinant forms (URFs). Here we characterize the near full-length genome sequence of an HIV-1 BG intersubtype recombinant virus (X3208) collected in Galicia (Northwest Spain) which exhibits a mosaic structure coincident with that of a previously characterized BG recombinant virus (9601_01), collected in Germany and epidemiologically linked to Portugal, and different from currently defined CRFs. Similar recombination patterns were found in partial genome sequences from three other BG recombinant viruses, one newly derived, from a virus collected in Spain, and two retrieved from databases, collected in France and Portugal, respectively. Breakpoint coincidence and clustering in phylogenetic trees of these epidemiologically-unlinked viruses allow to define a new HIV-1 CRF (CRF73_BG). CRF73_BG shares one breakpoint in the envelope with CRF14_BG, which circulates in Portugal and Spain, and groups with it in a subtype B envelope fragment, but the greatest part of its genome does not appear to derive from CRF14_BG, although both CRFs share as parental strain the subtype G variant circulating in the Iberian Peninsula. Phylogenetic clustering of partial pol and env segments from viruses collected in Portugal and Spain with X3208 and 9691_01 indicates that CRF73_BG is circulating in both countries, with proportions of around 2–3% Portuguese database HIV-1 isolates clustering with CRF73_BG. The fact that an HIV-1 recombinant virus characterized ten years ago as a URF has been shown to represent a CRF suggests that the number of HIV-1 CRFs may be much greater than currently known.

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A–D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3×104pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively.The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p<0.0001) with the data obtained from quantitative real time PCR.This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field.

A Novel Drug-Resistant HIV-1 Circulating Recombinant Form CRF76_01B Identified by Near Full-Length Genome Analysis.

HIV-1 CRF01_AE and subtype B (B) have dominated and their different circulating recombinant forms (CRFs) have emerged in East and Southeast Asian countries. Here, we report a novel drug-resistant HIV-1 CRF. Five independent recombinant specimens exhibiting discordant subtype results for the gag, pol, and env sequences were isolated. These recombinants had the CRF01_AE (gag p17)/B (pol PR-RT and IN)/CRF01_AE (env C2-V3) pattern similar to CRF69_01B. Sequence analysis of four near full-length HIV-1 genomes revealed a unique phylogenetic cluster distinct from previously reported CRFs. Of the four recombinants, three shared an identical mosaic structure including seven breakpoints in the gag, pol, vif, and env regions, designated CRF76_01B. The one remaining recombinant had additional recombination breakpoints in the vpu region and exhibited another unique recombinant form composed of CRF76_01B and B. These findings provide important insight into the transmission dynamics of HIV-1 in Asia that may be important for its effective prevention.

Identification of a Novel HIV Type 1 Circulating Recombinant Form (CRF65_cpx) Composed of CRF01_AE and Subtypes B and C in Western Yunnan, China

A novel HIV-1 circulating recombinant form (CRF) designated CRF65_cpx was recently characterized from three epidemiologically unlinked individuals infected through heterosexual contact in western Yunnan province of China. This is the first complex mosaic HIV-1 CRF, consisting of contributions from three or more different subtypes, identified in China. An additional full-length genome sequence with identical recombinant breakpoints was found among a previously reported recombinant strain from a man who had sex with a man in Anhui province of East Central China. The breakpoint analysis of the recombinants showed a complex genome organization composed of parental subtypes B' (Thailand variant of subtype B), C, and CRF01_AE, with 13 recombination breakpoints observed in almost all structure genes of HIV-1. The generation of complex recombinant forms is likely due to cocirculation of multiple lineages of HIV-1 strains in high-risk populations in western Yunnan.

High frequency of HIV-1 infections with multiple HIV-1 strains in men having sex with men (MSM) in Senegal

Circulating and unique recombinant HIV-1 strains continue to be identified and their number increases over time, suggesting that co-infection with multiple HIV-1 is frequent. In this study we analyzed to what extent dual infections with different HIV-1 variants occur in a population group with high risk behaviour, high HIV-1 prevalence and in an area where multiple HIV-1 subtypes and Circulating Recombinant Forms (CRFs) co-circulate. We studied 69 MSM with our recently developed multi-region hybridization assay (MHA), based on fluorescent probe detection for eight common variants circulating in West and West Central Africa. At least 11 (15.9%) of the 69 patients were simultaneously infected with two different HIV-1 subtypes and/or CRFs. Among the 29 samples identified as subtype C by MHA in gag, 15 (57.7%) reacted with both C1 and C2 probes. Sequence analysis suggests that the majority of the samples reactive with C1 and C2 probes are most likely infected with two different subtype C clades. Single genome amplification and DNA dilutions confirmed dual infection with subtype D and C for MSM1193, triple infection with two different C subtype strains and one CRF02_AG strain in MSM1157 and showed that MSM3017 is at least co-infected with CRF06_cpx and CRF02_AG and another strain that could not be classified. Comparison of all subtype C sequences from the MSM population and from the general population from this and previous studies confirmed the intermixing of HIV-1 variants between low-risk women and high-risk men as shown by the intermixing of subtype C variants from MSM1157 and a female patient (02SN-HALD478). Comparison of dual infection rates between the general population and MSM in Senegal, show also clearly the importance of high HIV prevalence and high risk behavior in dual infections and subsequent intermixing of HIV-1 variants which can lead to emergence and spread of new recombinants (CRFs).