María Sierra

IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Identification of a newly characterized HIV-1 BG intersubtype circulating recombinant form in Galicia, Spain, which exhibits a pseudotype-like virion structure

Summary: We recently reported the finding of phylogenetically related HIV‐1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full‐length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV‐1 circulating recombinant form (CRFI4_BG), the first reported to originate in Western Europe.

Identification of a novel HIV-1 complex circulating recombinant form (CRF18_cpx) of Central African origin in Cuba.

Background:Analysis of partial pol and env sequences have indicated a high diversity of HIV-1 genetic forms in Cuba, including two potential novel circulating recombinant forms (CRF): Upol/Henv and Dpol/Aenv. Objectives:To determine whether Upol/Henv recombinant viruses from Cuba, detected in 7% of samples, represent a novel HIV-1 CRF, and to identify non-Cuban viruses related to this recombinant form. Methods:Near full-length genome amplification was carried out by nested polymerase chain reaction in four overlapping DNA segments of two epidemiologically unlinked viruses in uncultured peripheral blood mononuclear cells. The sequences were analysed phylogenetically. Recombinant structures and phylogenetic relationships were analysed by bootscanning and by maximum likelihood. Searches for related viruses in databases were initially based on sequence homology and sharing of signature nucleotides. Results:Both Cuban viruses clustered uniformly in bootscans all along the genome with each other and with a virus from Cameroon, CM53379, indicating that all three represent the same recombinant form. Their genome comprised multiple segments clustering with subtypes A1, F, G, H and K, as well as segments failing to cluster with recognized subtypes. The newly defined CRF, designated CRF18_cpx, was phylogenetically related in partial segments to CRF13_cpx, CRF04_cpx and 36 additional viruses, most of them from Central Africa. One of the viruses from Cameroon, sequenced in the near full-length genome, was a CRF18_cpx/subtype G secondary recombinant. Conclusions:A novel HIV-1 complex circulating recombinant form (CRF18_cpx) has been identified that is circulating in Cuba and Central Africa.

Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19_cpx) in Cuba.

Circulating recombinant forms (CRFs) represent a substantial proportion of HIV-1 isolates in the global pandemic. Characterization of HIV-1 genetic forms, including CRFs, may be relevant to studies on molecular epidemiology, recombination, superinfection, vaccine development, and antiretroviral therapy. This study analyzes near complete genomes of 4 epidemiologically unlinked viruses from Cuba, originally characterized as D/A intersubtype recombinants in pol and env segments. The genomes of 3 viruses exhibited virtually coincident mosaic structures, with multiple segments of subtypes A, D, and G and uniform phylogenetic clustering with each other along the genome. These results allow us to define a new CRF (CRF19_cpx). The 4th analyzed Cuban virus was recombinant between CRF19_cpx and CRF18_cpx (which also circulates in Cuba). CRF19_cpx exhibited homology to an AG intersubtype recombinant virus from Cameroon (CM53392) along approximately 5 kb and clustered with a subtype D virus from Gabon (G109) in gag. Four other viruses from central or west Africa were also phylogenetically related to CRF19_cpx in env fragments. These results allow us to define CRF19_cpx as a second novel CRF of African origin circulating in Cuba, to identify putative representative viruses of its parental strains, and to characterize a unique CRF18/CRF19 recombinant virus.

High prevalence of unique recombinant forms of HIV-1 in Ghana: molecular epidemiology from an antiretroviral resistance study

Background:In Ghana, programs to expand antiretroviral access are being implemented. In this context, the dynamic genetic evolution of HIV-1 requires continuous surveillance, particularly when diverse genetic forms co-circulate. Methods:Phylogenetic and antiretroviral resistance analyses of HIV-1 partial pol sequences from plasma RNA samples from 207 Ghanaian individuals were performed. Results:66% of infections were CRF02_AG, whereas 25% were unique recombinant forms (URFs). All 52 URFs were characterized by bootscanning. CRF02_AG was parental strain in 87% of URFs, forming recombinants with genetic forms circulating in minor proportions: CRF06_cpx, sub-subtype A3, CRF09_cpx and subtypes G and D. Two triple recombinants (CRF02_AG/A3/CRF06_cpx and CRF02_AG/A3/CRF09_cpx) were identified. Antiretroviral resistance analyses revealed that six individuals, five of which were antiretroviral drug-experienced, harbored mutations conferring high level of resistance to reverse transcriptase inhibitors. No major resistance mutations were identified in the protease, although insertions of one and three amino acids were detected. Conclusions:The high frequency of URFs detected probably reflects a significant incidence of coinfections or superinfections with diverse viral strains, which increases the genetic complexity of the HIV-1 epidemic in West Africa. Monitoring of HIV-1 drug resistance might provide data on the implications of intersubtype recombination in response to antiretrovirals.

HIV type 1 intersubtype recombinants during the evolution of a dual infection with subtypes B and G.

ABSTRACT The aim of this study was to characterize the HIV-1 intersubtype recombinant forms generated during the follow-up of a dual natural infection with subtypes B and G. Near full-length sequences from plasma and peripheral blood mononuclear cell (PBMC) compartments were analyzed and the biological characteristics of their derived primary isolates studied. Different mutations were detected in V1, V2, and V3 sequences from primary isolates but not in sequences from plasma RNA or PBMC DNA. The HIV-1 near full-length sequence from the first collected plasma was of subtype G and the presence of subpopulations of subtypes B and G was observed with subtype-specific primers for protease and reverse transcriptase segments. Subsequent sequences from plasma, PBMCs, and primary isolates were obtained during a follow-up of 6 years; all of them were BG recombinants and showed identical intersubtype breakpoints between subtypes B and G in pol and nef. The env sequence from all primary isolates harbored a unique insert of subtype B. Specific primers for the V3 loop identified fluctuating subtype B and/or subtype G sequences either from plasma RNA or PBMC DNA.

Oligonucleotide Ligation Assay for Detection of Mutations Associated with Reverse Transcriptase and Protease Inhibitor Resistance in Non-B Subtypes and Recombinant Forms of Human Immunodeficiency Virus Type 1

ABSTRACT The oligonucleotide ligation assay is a genotypic assay for the detection of resistance-associated mutations to reverse transcriptase and protease inhibitors in human immunodeficiency virus type 1 subtype B. This assay has been modified and developed for non-B subtypes and recombinant strains and has been evaluated with sequencing, resulting in a more sensitive assay than sequencing for non-B subtypes.