Identification of a novel circulating recombinant form (CRF) 36_cpx in Cameroon that combines two CRFs (01_AE and 02_AG) with ancestral lineages of subtypes A and G

An array of CRFs have been identified in Cameroon, the most notable being CRF02_AG. HIV-1 in the East Province of Cameroon is particularly diverse: in a recent study, we found a high proportion of unique recombinant forms (URFs). Herein we describe the analysis of the full-length sequences of two of these URFs, which, after preliminary analysis of gag, pol, and env fragments, appeared to be a novel CRF. This novel strain, CRF36_cpx, contains fragments that can be assigned to the CRF01_AE, CRF02_AG, and subtype A and G radiations. Forty percent of the genome can be classified as CRF02_AG, including regions in gag, pol, env, and the accessory genes. Twenty-seven percent is CRF01_AE, comprising the majority of gag, the beginning of env, and the end of env into the 3' LTR. Twenty percent of the genome can be assigned to subtype A, with segments in pol and env. The remaining 13% of the sequence is classifiable as subtype G, in pol and vpu. The subtype A and G lineages formed by the CRF36_cpx sequences are unique and appear ancestral in nature. CRF36_cpx is both the first to combine more than one CRF and the first to include fragments of CRF02_AG. The ancestral sequences present in CRF36_cpx represent a link to extinct strains, and, potentially, insight into the evolution of HIV-1.     

HIV-1 CRF09_cpx circulates in the North West Province of Cameroon where CRF02_AG infections predominate and recombinant strains are common

This study describes the HIV-1 genetic diversity that currently circulates in Bamenda, the provincial capital of the North West province of Cameroon. Phylogenetic analysis of the protease (pro) gene of 20 HIV-1-seropositive individuals identified 11 (55%) CRF02_AG, one D, one F2, one J, and four (20%) unclassifiable strains. Interestingly, the remaining two (10%) samples, 02CMNYU3072 and 03CMNYU3224, originating from epidemiologically unlinked individuals, were classified as CRF09_cpx, representing the first reported cases of this complex circulating recombinant form (CRF) in Cameroon. Additional analysis of the C2V5 portion of the envelope (env) gene confirmed the CRF09_cpx identity of these isolates and classified the remaining isolates as CRF02_AG (n = 12, 63%), subtype D (n = 2, 11%), subtype F2 (n = 2, 11%), and subtype A1 (n = 1). In combination, the pro and env subtyping results revealed three (16%) isolates with discordant subtypes including J( pro )CRF02_AG( env ), CRF02_AG( pro )D( env ), and CRF02_AG( pro )F2( env ). In conclusion, this study highlights the presence of HIV-1 CRF09_cpx in Cameroon and identifies three possible intersubtype recombinants (ISRs) containing CRF02_AG in a town where CRF02_AG infections predominate, and stresses the commonness of HIV-1 recombinant strains in a region where broad genetic diversity exists.     

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.     

Infectious DNA clone of HIV type 1 A/G recombinant (CRF02_AG) replicable in peripheral blood mononuclear cells

We constructed an infectious DNA clone of the HIV-1 A/G recombinant 97GH-AG2, which was isolated in Ghana in 1997 and was classified originally as subtype A. By phylogenetic and recombination breakpoint analyses, p97GH-AG2 was grouped in the circulating form of A/G recombinants (CRF02_AG) and was found to contain the least amount of subtype G-derived region among the known CRF02_AG HIV-1 DNAs. This result suggests that CRF02_AG may be a predominant form in Ghana. Virions produced by transfection of p97GH-AG2 into 293T cells grew in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs). 97GH-AG2 also replicated efficiently in CCR5-expressing HeLa cells, MAGIC5, but only weakly in the parent MAGI cells, indicating that 97GH-AG2 uses mostly CCR5 as a coreceptor. Isolation of the first HIV-1 (CRF02_AG) DNA clone that replicates in PBMCs will accelerate the molecular analysis of this subtype.     

No difference in clinical progression between patients infected with the predominant human immunodeficiency virus type 1 circulating recombinant form (CRF) 02_AG strain and patients not infected with CRF02_AG,in Western and West-Central Africa: a four-year prospective multicenter study

To compare human immunodeficiency virus (HIV) type 1 disease progression in patients infected by the predominant strain circulating recombinant form (CRF) 02_AG in western and west-central Africa and in patients infected by other strains, a prospective multicenter cohort study was conducted in Cameroon and Senegal. Among the 335 patients, a broad HIV-1 group M subtype diversity was observed in the envelope V3-V5 region, but strain CRF02_AG predominated in both Cameroon and Senegal (61.2% and 62.9%, respectively; P<.8). Multivariate analyses showed no difference between patients infected by CRF02 strains and those infected by other strains in terms of survival (adjusted hazards ratio [HR], 1.16; 95% confidence interval [CI], 0.76-1.78; P=.5), clinical disease progression (HR, 0.79; 95% CI, 0.50-1.25; P=.3), or square root CD4 cell decline (regression coefficient, -0.01; 95% CI, -0.82 to 0.81; P=.9). This study suggests that the predominance of HIV-1 CRF02_AG strain in western and west-central Africa should have no major clinical consequences.     

Emergence of complex and diverse CRF02-AG/CRF06-cpx recombinant HIV type 1 strains in Niger,West Africa

On the basis of partial env and gag subtyping, we documented that the majority of HIV-1 strains circulating in Niger were CRF02-AG (54.3%) or CRF06-cpx (18.1%) and that 9% of the samples were possible recombinants between CRF02 and CRF06. To determine in more detail the precise structure of these viruses we sequenced the full-length genomes for three such strains (97NE-003, 00NE-036, and 00NE-095). From the bootscan and phylogenetic tree analysis it is evident that the new viruses are the result of recombination events between CRF02-AG and CRF06-cpx strains. Importantly, each virus had a different complex recombinant structure with multiple breakpoints, leading to viruses with complex mosaic patterns.     

Development and application of a high-throughput HIV type 1 genotyping assay to identify CRF02_AG in West/West Central Africa

In West/West Central Africa, CRF02_AG is the most prevalent HIV-1 strain and circulates in the milieu of rare subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs). The molecular complexity of HIV-1 epidemics in this region and the need to extensively sample large populations, such as in the case of vaccine trials, pose seemingly conflicting requirements between full-genome sequencing and high-throughput low-resolution assays. Here we describe the development and evaluation of a multiregion hybridization assay (MHAcrf02) for the efficient genotyping of CRF02_AG in West/West Central Africa. Subtype A, G, and CRF02_AG-specific fluorescent probes were designed flanking five recombination breakpoints in CRF02_AG and were used in real-time PCRs. A panel representing West/West Central African HIV-1 genetic diversity was evaluated by MHAcrf02. The sample set, previously characterized by full-genome sequencing, included CRF02_AG and CRF02_AG-containing recombinants (n = 28), other subtypes, CRFs, and URFs (n = 34). DNA from peripheral blood mononuclear cells, cocultures, and plasmids was used as template. When the patterns of probe reactivity were evaluated. CRF02_AG was identified with a 100% specificity and sensitivity. In conclusion, MHAcrf02 will permit more efficient characterization of HIV-1 in West/West Central Africa, where CRF02_AG is an important strain. Together with other regional genotyping assays MHAcrf02 will contribute to the development of a global picture of HIV-1 diversity and geographic distribution, providing a strong foundation for intervention, including vaccine development.     

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.     

Circulating recombinant form (CRF) 37_cpx: an old strain in Cameroon composed of diverse, genetically distant lineages of subtypes A and G

HIV-1 in Cameroon is genetically diverse, but is predominated by the circulating recombinant form (CRF) 02_AG, which cocirculates among an array of other CRFs, unique recombinant forms (URFs), and all group M subtypes. In particular, our studies of HIV-1 diversity in the East Province found a high proportion of URFs and second generation recombinants (SGRs), suggesting this region of Cameroon may be a breading ground for new CRFs. Herein we present the full-length sequence analysis of one such CRF, composed primarily (66%) of unique, distant lineages of subtypes A and G in alternating regions throughout the genome. This CRF also combines segments in pol and env genes possessing intrasubtype distance (<15%) to the CRF01_AE and CRF02_AG radiations. The genomic composition of this strain comprising gene segments of subtypes A and G as well as CRF01_AE and CRF02_AG defines this strain as a circulating SGR (CSGR), and the 37th CRF to be identified. Furthermore, more than half of CRF19_cpx, a CRF identified in Cuba, clusters with CRF37_cpx, and the clear genetic distance among the viruses in this cluster suggests this strain has been in circulation since the early days of the epidemic. The genetically distant segments comprising CRF37_cpx, which were found to cluster outside the crown groups of previously described viruses, may represent a link to very rare or extinct strains, and, potentially, to understanding the evolutionary history of HIV-1 in this region.     

Characterization of an old complex circulating recombinant form, CRF27_cpx, originating from the Democratic Republic of Congo (DRC) and circulating in France

Full-length genomes were characterized for two samples, 02CD-LBR024 from the Democratic Republic of Congo (DRC) and 04FR-CD-KZS from France, that formed a separate subcluster with a previously characterized env subtype E isolate from DRC with a recombinant structure different from CRF01-AE. Since the three viruses are clearly epidemiologically unlinked and share the same complex recombinant structure, they represent a circulating recombinant form, designated as CRF27-cpx. The recombination pattern involves six different HIV-1 subtypes (A, E, G, H, J, and K) and a small unclassified fragment. The genetic distances are relatively high, indicating that CRF27-cpx evolved over a long time. Their prevalence is low (0.75%) and remained stable over time in the DRC. The existence of the 04FR.CD.KZS virus, in a patient who recently seroconverted in France, confirmed that these strains now circulate outside the DRC. Continuous monitoring of HIV-1 strains thus remains important to allow early identification of the introduction of new variants.     

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.     

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.     

Subtypes A,C, G,and recombinant HIV type 1 are circulating in Bangladesh

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.     

Identification of two CRF11-cpx genomes and two preliminary representatives of a new circulating recombinant form (CRF13-cpx) of HIV type 1 in Cameroon

Recombination is an efficient mechanism of HIV-1 to generate genetic variability. Some of the recombinant forms of HIV-1 that have been described are of epidemic importance, and they are referred to as circulating recombinant forms (CRFs). In this study, we characterized four HIV-1 isolates from Cameroon that had previously been classified as subtype J in the protease gene and as subtype A in the env C2-V5 region. Analyses of the nearly complete genomes revealed that two of the samples (95CM-1816 and 96CM-4496) had the same recombinant structure as CRF11-cpx. The two remaining samples (96CM-1849 and 96CM-4164) constituted a new recombinant virus variant with genomic regions identified as subtypes A, G, J, and CRF01-AE. This mosaic virus structure was found in two individuals who had no direct epidemiological relationship, and may thus represent a new CRF. The fragments that were classified as subtype J in the new recombinant form were more related to subtype J regions of the CRF11-cpx sequences than to the reference strains of subtype J. The complex structures of CRF11-cpx and our new recombinant form, which are both the result of at least four recombination events, exemplify the coming difficulties in characterizing the internal relationships and origins of future recombinant HIV-1 strains. The new recombinant structure has been designated CRF13-cpx in the Los Alamos HIV Sequence Database.     

HIV type 1 CRF13_cpx revisited: identification of a new sequence from Cameroon and signal for subsubtype J2

A nearly full-length genome sequence of an HIV-1 isolate originating from Cameroon, 02CM.3226MN, was found to cluster together with previously reported CRF13 sequences 96CM-4164 and 96CM-1849. Similarity plotting, bootscanning, breakpoint analysis, and phylogenetic trees confirmed similar genomic structures with almost identical breakpoint positions among these three isolates. Thus, CRF13 now fulfills the HIV-1 nomenclature requirements. A X2 analysis across all three genomes simultaneously was applied to more accurately determine breakpoints and address the uncertainty in such estimates. Some fragments were found to be difficult to classify, as indicated by a low branching index (BI), due to limited knowledge about parental and reference subtype sequences. One fragment with low BI association to reference subtype J sequences (BI = 0.27, cut-off for subtype classification >0.55) was found to be closer to J fragments of CRF11 similar to the way that A1-A2 and F1-F2 subsubtypes associate. This suggests that subtype J may need to be reclassified into subsubtypes J1 and J2. The CRF13 genome consists of fragments from subtypes A1, G, and both J1 and J2 as well as CRF01 and one region that was left unclassified.