Full-length HIV-1 Sequences Research Articles

Recombination Analysis of Near Full-Length HIV-1 Sequences and the Identification of a Potential New Circulating Recombinant Form from Rakai, Uganda.

The Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium has been vital in the generation and examination of near full-length HIV-1 sequences generated from Sub-Saharan Africa. In this study, we examined a subset (n = 275) of sequences from Rakai, Uganda, collected between August 2011 and January 2015. Sequences were initially screened with COMET for subtyping and then evaluated using bootscanning and phylogenetic inference. Among 275 sequences, 38.6% were subtype D, 19.3% were subtype A, 2.9% were subtype C, and 39.3% were recombinant. The recombinants were structurally diverse in the number of breakpoints observed, the location of recombinant segments, and represented subtypes, with AD recombinants accounting for the majority of all recombinants (29.8%). Within the AD subpopulation, we identified a potential new circulating recombinant form in five individuals where the polymerase gene was subtype D and most of env was subtype A (D-A junctures at HXB2 6760 and 8709). While the breakpoints were identical for the viruses from these individuals, the viral fragments did not cluster together. These results suggest selection for a viral strain where properties of the subtype A and subtype D portions of the virus confer a survival advantage. The continued study of recombinants will increase our breadth of knowledge for the genetic diversity and evolution of HIV-1, which can further contribute to our understanding toward a universal HIV-1 vaccine.

Molecular characterization of full-length Tat in HIV-1 subtypes B and C.

HIV-1Tat (trans-acting activator of transcription) plays essential roles in the replication through viral mRNA and genome transcription from the HIV-1 LTR promoter. However, Tat undergoes continuous amino acid substitutions. As a consequence, the virus escapes from host immunity indicating that genetic diversity of Tat protein in major HIV-1 subtypes is required to be continuously monitored. We analyzed available full-length HIV-1 sequences of subtypes B (n=493) and C (n=280) strains circulating worldwide. We observed 81% and 84% nucleotide sequence identities of HIV-1 Tat for subtypes B and C, respectively. Based on phylogenetic and mutation analyses, global diversity of subtype B was apparently higher compared to that of subtype C. Positively selected sites, such as positions Ser68 and Ser70 in both subtypes, were located in the Tat-transactivation responsive RNA (TAR) interaction domain. We also found positively selected sites in exon 2, such as positions Ser75, Pro77, Asp80, Pro81 and Ser87 for both subtypes. Our study provides useful information on the full-length HIV-1 Tat sequences in globally circulating strains.

Phylogenetic Characterization of Six Full-Length HIV-1 Subtype C Molecular Clones from Three Patients: Identification of Rare Subtype C Strains Containing Two NF-κB Motifs in the Long Terminal Repeat

Molecular surveillance is the backbone of HIV-1 vaccinology. Full-length HIV-1 sequences are useful tools that can provide a better understanding of the epidemiology in a given region. A limited number of full-length HIV-1 sequences are available from India, where >95% of the HIV infections are due to HIV-1 subtype C (HIV-1C), which is distinct from the prototype African HIV-1C. In this study, we sequenced six full-length clones isolated from three patients. Extensive phylogenetic analyses of the full-length viral sequences using bioinformatic tools identified a separate cluster of Indian strains, thus confirming the distinct phylogenetic identity of the Indian HIV-1C. Notably, the long terminal repeat (LTR) of two of the six molecular clones contained only two NF-κB binding sites. The sequences also displayed features characteristic of HIV-1C including a Tat dicysteine motif, a shortened Rev open reading frame, and a predicted CCR5 coreceptor tropism for gp120 of three of the proviral sequences.

Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals

The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB.

Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance

Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes; TaqMan ®-probes) of variable genomes, such as HIV-1, can result in underestimation of viral copy numbers due to mismatches in the FH-probe's target sequences. Therefore both target conservation and physical properties of FH-probes, such as melting temperature, baseline fluorescence and secondary structure, should be considered in design of FH-probes. Analysis of a database of 1242 near full-length HIV-1 sequences with a novel computational tool revealed that the probability of target and FH-probe identity decreases exponentially with FH-probe length. In addition, this algorithm allowed for identification of continuous sequence stretches of high conservation, from which FH-probes with global HIV-1 clade coverage could be chosen. To revise the prerequisites of physical FH-probe function, properties of 30 DNA and 21 chimeric DNA locked nucleic acid (DLNA) HIV-1 FH-probes were correlated with their performance in qPCR. This identified the presence of stable secondary structures within FH-probes and the base composition and thermal stability of the 5′ proximal end as novel predictors of FH-probe performance. Thus, empirically validated novel principles of FH-probe design regarding conservation and qPCR-performance were identified, which complement and extend current rules for FH-probe design.

Rare HLA Drive Additional HIV Evolution Compared to More Frequent Alleles

HIV-1 can evolve HLA-specific escape variants in response to HLA-mediated cellular immunity. HLA alleles that are common in the host population may increase the frequency of such escape variants at the population level. When loss of viral fitness is caused by immune escape variation, these variants may revert upon infection of a new host who does not have the corresponding HLA allele. Furthermore, additional escape variants may appear in response to the nonconcordant HLA alleles. Because individuals with rare HLA alleles are less likely to be infected by a partner with concordant HLA alleles, viral populations infecting hosts with rare HLA alleles may undergo a greater amount of evolution than those infecting hosts with common alleles due to the loss of preexisting escape variants followed by new immune escape. This hypothesis was evaluated using maximum likelihood phylogenetic trees of each gene from 272 full-length HIV-1 sequences. Recent viral evolution, as measured by the external branch length, was found to be inversely associated with HLA frequency in nef (p < 0.02), env (p < 0.03), and pol (p < or = 0.05), suggesting that rare HLA alleles provide a disproportionate force driving viral evolution compared to common alleles, likely due to the loss of preexisting escape variants during early stages postinfection.

Full-Length HIV Type 1 Proviral Sequencing of 10 Highly Exposed Women from Nairobi, Kenya Reveals a High Proportion of Intersubtype Recombinants

Phylogenetic analysis has revealed that the current HIV/AIDS pandemic consists of a multitude of different viral clades and recombinant viruses. The predominant circulating HIV-1 clade in Kenya is A1; however, Kenya borders countries where different subtypes are prominent, making Kenya a likely location for recombination. Previous studies have reported significant differences in the proportions of sequences in Kenya that are intersubtype recombinants. Studies that performed sequence-based typing on multiple HIV-1 genomic regions or full-length sequences found higher rates of recombination than those that examined a single gene or gene fragment. In this study, we describe full-length HIV-1 proviral sequence-based genotyping after limited peripheral blood mononuclear cell (PBMC) coculture. Ten subjects from a highly exposed cohort located in Nairobi, Kenya were examined. Pairwise comparison found minimal difference between sequences generated directly from patient PBMC DNA compared to sequences from cocultured PBMC DNA. Of the 10 full-length HIV-1 sequences examined, five were nonrecombinant clade A1, while the other five were unique intersubtype recombinants. Although this frequency of recombination is higher than previously described in Kenya, this finding is in agreement with previous full-length sequence data. Interestingly, although all the nonrecombinant sequences were clade A1, not all the recombinant sequences contained a clade A1 sequence.

HIV type 1 subtypes among blood donors in the Mbeya region of southwest Tanzania.

HIV-1 is endemic in Tanzania where three different subtypes, A, C, and D, have been identified. Information on HIV-1 genetic diversity is crucial to define requirements for an effective vaccine, in regions where HIV-1 vaccine trials are planned. To define the subtype distribution of HIV-1 in the Mbeya region of southwest Tanzania, peripheral blood mononuclear cells (PBMC) and plasma were obtained from 36 discarded HIV seropositive blood units. Multiregion hybridization assay (MHA) was performed on both PBMC DNA and plasma RNA to determine the subtype distribution. Twenty virtually full-length HIV-1 sequences were amplified from the extracted DNA, sequenced, and phylogenetically analyzed. Subtype distribution determined by all three assays was comparable. More than 50% of the samples analyzed were subtype C, followed by a high proportion of subtype C-containing intersubtype recombinants. Based on this work, subtype C appears to be the prevalent subtype in southwest Tanzania, followed by a high proportion of intersubtype recombinants.

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants,

Objective: To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. Design: Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. Methods: Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. Results: Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. Conclusions: In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.

ERRATUM Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants William E. Dowling, Bohye Kim, Carl J. Mason, K. Monique Wasunna, Uzma Alam, Lynne Elson, Deborah L. Birx, Merlin L. Robb, Francine E. McCutchan and Jean K. Carr

ERRATUM Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants William E. Dowling, Bohye Kim, Carl J. Mason, K. Monique Wasunna, Uzma Alam, Lynne Elson, Deborah L. Birx, Merlin L. Robb, Francine E. McCutchan and Jean K. Carr

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants

To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.

Genetic variability of HIV type 1 in Kenya.

The AIDS epidemic is a rapidly growing problem in Nairobi where the seroprevalence in pregnant women increased from 4% in 1988 to over 10% in 1991. 22 HIV-1-seropositive pregnant women and 1 HIV-1-infected baby (K88) attending the Pumwani Maternity Hospital of Nairobi between 1990 and 1992 were studied as part of a cohort study of maternal risk factors in mother-to-child transmission. A 250-base pair (bp) fragment of the env gene encoding C2V3 was amplified mostly from DNA isolated from primary peripheral blood mononuclear cells and subsequently sequenced. The 23 newly determined HIV env sequences were aligned with 23 previously known sequences of HIV-1 isolates of diverse geographical origin and the sequence of the HIV-1-related chimpanzee isolate SIVcpz-gab on the basis of primary structure. Distance calculation tree construction and bootstrap analysis were realized with the software package TREECON. In the tree 8 major branches could be observed containing sequences representative of 8 different subtypes A B C D E F G and H besides the outlier group O. 19 of 23 Kenyan isolates clustered with D687 Z321 U455 and SF170 which were members of genetic subtype A. Phylogenetic analyses favored positioning of K976 as a divergent A subtype strain. For 4 strains (K29 K88 K98 and K112) the subtype A classification based on the gag gene was also observed on the basis of phylogenetic analysis of the C2V3 coding part of the env gene. The predicted amino acid sequence of the V3 region for these strains was also presented. The finding that among 23 HIV-1 isolates collected in Nairobi 19 were classified in subtype A versus 3 in subtype D together with a much larger variation between subtype A strains as compared to subtype D strains suggests an earlier introduction of a subtype A strain multiple introductions of subtype A strains and/or faster diversification of subtype A strains as compared to subtype D strains.