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RNA viruses replicate as complex mutant spectra termed viral quasispecies. The frequency of each individual genome in a mutant spectrum depends on its rate of generation and its relative fitness in the replicating population ensemble. The advent of deep sequencing methodologies allows for the first-time quantification of haplotype abundances within mutant spectra. There is no information on the haplotype profile of the resident genomes and how the landscape evolves when a virus replicates in a controlled cell culture environment. Here, we report the construction of intramutant spectrum haplotype landscapes of three amplicons of the NS5A-NS5B coding region of hepatitis C virus (HCV). Two-dimensional (2D) neural networks were constructed for 44 related HCV populations derived from a common clonal ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells in the absence of external selective pressures. The haplotype profiles consisted of an extended dense basal platform, from which a lower number of protruding higher peaks emerged. As HCV increased its adaptation to the cells, the number of haplotype peaks within each mutant spectrum expanded, and their distribution shifted in the 2D network. The results show that extensive HCV replication in a monotonous cell culture environment does not limit HCV exploration of sequence space through haplotype peak movements. The landscapes reflect dynamic variation in the intramutant spectrum haplotype profile and may serve as a reference to interpret the modifications produced by external selective pressures or to compare with the landscapes of mutant spectra in complex in vivo environments. IMPORTANCE The study provides for the first time the haplotype profile and its variation in the course of virus adaptation to a cell culture environment in the absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer haplotype distribution with an ample basal platform and a lower number of protruding peaks. The results suggest an inferred intramutant spectrum fitness landscape structure that offers potential benefits for virus resilience to mutational inputs.

Human immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD41 T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD41 homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD41 and CD81 T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD41 T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR1CD381 CD41 and CD81 T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD41 T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD41 T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV1 infection. Importance: The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD41 T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD41 T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis. This research was supported by a Gilead Fellowship (grant GLD15/0298) and La Caixa Foundation (grant LCF/PR/PR16/11110026). M.C.-L. is a Beatriu de Pinós postdoctoral fellow (grant BP 00075) supported by the Government of Catalonia’s Secretariat for Universities and Research of the Ministry of Economy and Knowledge. J.G.P. was supported by the ISCIII (grant CP15/00014). E.J.-M. was funded by Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041); Acción Estratégica en Salud; Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008–2011; and Instituto de Salud Carlos III. E.J.-M. was cofunded by European Regional Development Fund/European Social Fund (FEDER) “Investing in your future.” J.M.-P. is supported by the Spanish Ministry of Science and Innovation (grant PID2019-109870RB-I00). J.G.P. and M.C.-L. designed the study, supervised experiments and data. J.G.P., M.C.-L., and A.K. contributed to data interpretation. M.C.-L., R.P., E.J.-M., M.P., and C.C. performed experiments, analyzed, and interpreted the data. J.D. carried out the clinical follow-up and patient identification. M.C.-L., D.O., M.P., and C.C. performed data analysis. M.C.-L., A.K., M.P., C.L.-G., B.C., J.M.-P., and J.G.P. performed manuscript writing, critical revision, and discussion. We declare no conflict of interest. Sí

The understanding of HIV-1 pathogenesis and clinical progression is incomplete because of the variable contribution of host, immune and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has concentrated many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller’s individuals contemporary to LTNPs or recent, named Old and Modern progressors. We analyzed the Env expression, the fusion and cell-to-cell transfer capacities as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4 and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs presenting poor viral functions and shorter sequences were associated with viremic control and the non-progressor clinical phenotype, whereas functional Envs were associated with the lack of virological control and progressor clinical phenotypes. These correlations support the central role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.IMPORTANCEThe role of the virus in the pathogenesis of HIV-1 infection has not been investigated in isolates from individuals with different progression rates. In this work, we studied the properties of the envelope glycoprotein complex (Env) in individuals with different progression rates to elucidate its role in pathogenesis. We estimated the Env expression, the CD4 binding, the fusion and cell-to-cell viral transfer capacities that affect the infectivity of the viral Envs in recombinant viruses. The Envs from individuals which control viral replication and lack clinical progression (LTNP-ECs and vLTNPs) showed lower functional capacities than from subjects with clinical progression (Old and Modern). The functional increase of the Envs characteristics was associated with an increase in viral infectivity and in increased length of variable loops and the number of glycosylation sites of the Env (gp120/SU). These results support the concept that viral characteristics contribute to viral infection and pathogenesis.

Following the success of HIV-1 antiviral treatment that maintains undetectable levels of viral replication and lack of clinical progression, the design of an HIV-1 cure for patients became the next objective. The success of the treated individuals together with the identification of subjects that spontaneously control the clinical progression for long periods, such as long-term non-progressors (LTNPs) and particularly LTNP Elite Controllers (LTNP EC) have shed hope for the feasibility of a potential cure. Although a successful cure has not been attained yet, these patients have provided critical information on the mechanisms involved in the clinical control such as host genetic factors, as well as strong immune responses against the virus. Less attention has been paid to virological factors, particularly the association of the genetic variability and the control of viral infection. Considering all these studies, it has become clear that a combination of several host, immune and viral factors is needed to attain control of the viral replication control and the non-progressor clinical phenotype. Because this control can be reached through different combinations of factors, this group of individuals is not homogenous. As HIV-1 cure has been shown to be extremely difficult to achieve, a more feasible objective is the functional cure of the viral infection. After the analysis of multiple studies on the mechanisms of control in LTNP EC, we found subjects with various host protective factors and prolonged viral control. These subjects present a complete lack of evolution after more than 20-30 years of infection, stable levels of CD4+ cells (>400-500 cells/μl), a strong immune response, and no signs of clinical progression. We propose that individuals with these characteristics could have attained a functional cure of the HIV-1 infection.

Fitness landscapes reflect the adaptive potential of viruses. There is no information on how fitness peaks evolve when a virus replicates extensively in a controlled cell culture environment. Here we report the construction of Self-Organized Maps (SOMs), based on deep sequencing reads of three amplicons of the NS5A-NS5B-coding region of hepatitis C virus (HCV). A two-dimensional neural network was constructed and organized according to sequence relatedness. The third dimension of the fitness profile was given by the haplotype frequencies at each neuron. Fitness maps were derived for 44 HCV populations that share a common ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells. As the virus increased its adaptation to the cells, the number of fitness peaks expanded, and their distribution shifted in sequence space. The landscape consisted of an extended basal platform, and a lower number of protruding higher fitness peaks. The function that relates fitness level and peak abundance corresponds a power law, a relationship observed with other complex natural phenomena. The dense basal platform may serve as spring-board to attain high fitness peaks. The study documents a highly dynamic, double-layer fitness landscape of HCV when evolving in a monotonous cell culture environment. This information may help interpreting HCV fitness landscapes in complex in vivo environments.IMPORTANCEThe study provides for the first time the fitness landscape of a virus in the course of its adaptation to a cell culture environment, in absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer fitness distribution with an ample basal platform, and a lower number of protruding, high fitness peaks. This landscape structure offers potential benefits for virus resilience to mutational inputs.

ECRIS integrated in the Spanish AIDS Research Network., [Introduction] HIV‐controllers have the ability to spontaneously maintain viraemia at low or undetectable levels in the absence of antiretroviral treatment. Furthermore, HIV‐controllers seem to have a superior capacity to spontaneously clear hepatitis C virus (HCV) compared to non HIV‐controllers. Some of these subjects eventually lose HIV‐controller status (transient controllers), whereas some HIV‐controllers show a persistent natural HIV control (persistent controllers). We aimed to analyse whether persistent controllers have superior capacity to spontaneously clear HCV compared to transient controllers., [Methods] We recruited HIV‐controllers from January 1981 up to October 2016 with available antibodies to HCV (anti‐HCV) data (n = 744). Factors associated with HIV spontaneous control in relation to HCV status were analysed in persistent and transient HIV‐controllers with anti‐HCV positive (n = 202 and n = 138 respectively) in comparison with 1700 HCV positive non HIV‐controllers recruited from January 1981 up to March 2018, bivariate and multivariate analyses, following a logistic regression model, were applied. In addition, the factors related to the loss and time to lose HIV‐controller status were explored (n = 744) using Log rank test and Kaplan–Meier curves, in this case the multivariate analysis consisted in a Cox regression model., [Results] A higher frequency of HCV spontaneous clearance was found in persistent HIV‐controllers (25.5%) compared to non‐controllers (10.2%). After adjusting for potential confounders, as sex, age, HIV transmission risk, CD4+ T‐cell nadir and time of follow‐up, HCV clearance was independently associated with persistent HIV spontaneous control (p = 0.002; OR (95% CI) = 2.573 (1.428 to 4.633)), but not with transient spontaneous control (p = 0.119; 1.589 (0.888 to 2.845)). Furthermore, persistent HIV‐controllers were more likely to spontaneously clear the HCV in comparison with transient controllers (p = 0.027; 0.377 (0.159 to 0.893). Finally, not to lose or lengthen the time of losing this control was independently associated with HCV spontaneous clearance (p = 0.010; 0.503 (0.297 to 0.850)., [Conclusions] This study shows an association between spontaneous persistent HIV‐control and HCV spontaneous clearance. The study findings support the idea of preserved immune mechanisms in persistent HIV control implicated in HCV spontaneous clearance. These results highlight persistent HIV‐controllers but not transient controllers as a good model of functional HIV cure., Research funding: Instituto de Salud Carlos III. Grant Numbers: CPII014, /00025, FI14, /00431, PI12, /02283, PI16, /00684, PI19, /01127. Red Temática de Investigación Cooperativa en SIDA. Grant Numbers: RD12, /0017/0029, RD12, /0017/0031, RD16, /0025/0020, RD16, /0025/0013. Consejería de Salud y Bienestar Social of Junta de Andalucía through the Nicolás Monardes program. Grant Number: C‐0032/17. The Spanish Ministry of Education. Grant Number: FPU13/02451. MINECO. Grant Number: 2016‐77894‐R. RIS‐RETIC. Grant Numbers: RD06, /006/0036, RD12, /0017/0028

In the search for new antiviral therapies against human immunodeficiency virus type 1 (HIV-1), several cellular targets are being investigated. Ataxia telangiectasia and Rad3-related protein (ATR) has been implicated in HIV-1 replication, namely during retroviral DNA integration. We studied the effect of the ATR inhibitor ETP-46464 on HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and in the persistently HIV-1-infected cell line H61-D. After treatment with ETP-46464, a significant decrease in virus production was observed in both cell systems. Quantification of viral DNA forms in the acutely infected PBMCs suggests that inhibition could take place in the early phase of the viral life cycle before viral DNA integration. Moreover, after treatment of H61-D cells with 3'-azido-3'-deoxythymidine (AZT), which blocks new reverse transcription events, ETP-46464 decreased viral production, suggesting that inhibition of viral replication occurred in the late phase of the life cycle after viral DNA integration. A decrease in virus production after transfection of 293T cells with an HIV-1 infectious molecular clone also suggested that the effect of ETP-46464 is exerted at a post-integration step. We propose that ETP-46464 produces its inhibitory effect on HIV-1 replication by acting in both the early and late phases of the retroviral replication cycle. Thus, ATR could represent a new target for inhibition of HIV-1 replication.

In untreated HIV-1-infected individuals, viremia is positively associated with disease progression. However, some viremic non progressors (VNPs) individuals show paradoxical high CD4+ T cell counts. HIV-1 envelope glycoprotein complex (Env) is a major cytopathic determinant in viral replication; therefore, we have deeply characterized Env function in this rare clinical phenotype. Full-length Env clones isolated from individuals with Viral Load (VL) > 10,000 copies/mL classified as VNPs (n = 15) or rapid progressors (RPs, n = 17) were geno- and phenotypically analyzed by determining diversity, expression, CD4 binding/signaling, fusogenicity, infectivity and autophagy induction. Selected Env clones from VNPs and RPs (n = 32) showed similar expression, fusion and infection abilities. Env clones from both groups showed similar affinity for CD4 during cell-to-cell transmission and consistently induced similar levels of CD4 signaling, measured by α-tubulin acetylation. Moreover, we demonstrate for the first time that primary Env clones from VNP and RP induce autophagy in uninfected cells and that this feature correlated with fusogenic capacity but was unrelated to disease progression. In conclusion, our data suggest that Env clones from VNP individuals are fully functional. Therefore, the paradoxical CD4+ T cell count stability coexisting with high levels of viral replication is unrelated to Env function. This work is supported by Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” RD12/0017/0002, RD12/0017/0028, RD12/0017/0034, RD16/0025/0011, RD16/0025/00XX and RD16/0025/0041 as part of the Plan Nacional R + D+ I and cofunded by Spanish “Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER)”. Work in JB’s lab is supported by the Spanish Ministry of Health Project Number EC11-043 (to JB and AV-F), by the CNRS PICS program (to MB-P), J.B. and C.C. are researchers from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the Health Department of the Catalan Government/Generalitat de Catalunya and ISCIII grant numbers PI14/01307 (to JB) and PI15/01053 (to CC). Work in CL-G’s lab was supported by grants SAF (2010-17226) and (2016-77894-R) from MINECO (Spain) and FIS (PI 13/02269, ISCIII). A.V-F’s Lab is supported by the European Regional Development Fund (ERDF), SAF2015-64118-R (MINECO, Spain) “Fundación CajaCanarias” BIO29, UNLL10-3E-783 (ERDF and “Fundación CajaCanarias”). D.M-A and S.M-H are funded by Fundación CajaCanarias BIO29, SAF2011-24671-FPI, “TESIS2017010116- and TESIS2015-010038-Programa Predoctoral de Formación del Personal Investigador, Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund-fellowships. R.C-R is funded by RIS-RETIC RD16/0025/0011 and J.E-H is funded by the Cabildo Tenerife “Agustin de Betancourt” 2017 Program. Sí

[Objectives]: The long-term non-progressors (LTNPs) are a heterogeneous group of HIV-positive individuals characterized by their ability to maintain high CD4+ T-cell counts and partially control viral replication for years in the absence of antiretroviral therapy. The present study aims to identify host single nucleotide polymorphisms (SNPs) associated with non-progression in a cohort of 352 individuals., [Methods]: DNA microarrays and exome sequencing were used for genotyping about 240 000 functional polymorphisms throughout more than 20 000 human genes. The allele frequencies of 85 LTNPs were compared with a control population. SNPs associated with LTNPs were confirmed in a population of typical progressors. Functional analyses in the affected gene were carried out through knockdown experiments in HeLa–P4, macrophages and dendritic cells., [Results]: Several SNPs located within the major histocompatibility complex region previously related to LTNPs were confirmed in this new cohort. The SNP rs1127888 (UBXN6) surpassed the statistical significance of these markers after Bonferroni correction (q = 2.11 × 10−6). An uncommon allelic frequency of rs1127888 among LTNPs was confirmed by comparison with typical progressors and other publicly available populations. UBXN6 knockdown experiments caused an increase in CAV1 expression and its accumulation in the plasma membrane. In vitro infection of different cell types with HIV-1 replication-competent recombinant viruses caused a reduction of the viral replication capacity compared with their corresponding wild-type cells expressing UBXN6., [Conclusions]: A higher prevalence of Ala31Thr in UBXN6 was found among LTNPs within its N-terminal region, which is crucial for UBXN6/VCP protein complex formation. UBXN6 knockdown affected CAV1 turnover and HIV-1 replication capacity., This study was partially supported by the Spanish AIDS Research Network (RIS), funded by Instituto de Salud Carlos III and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (projects RD12/0017/0015 and RD16CIII/0002/0001, Plan Estatal de I+D+I 2013-2016), the French Government's Investissement d'Avenir programme, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (no. ANR-10-LABX-62-IBEID), the Spanish Government's FIS-ISCIII (PI16CIII/00034) and Sara Borrell Postdoctoral Programme (CD12/00515 to F.D.F.) and the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 681137.

The HIV-1 epidemic in India is caused mainly by subtype C viruses that are transmitted sexually and by injecting drug use. The state of Tamil Nadu in Southern India has an HIV-1 median prevalence of 16.8% among injecting drug users, 6.6% in men who have sex with men, and 4.6% in female sex workers. In the rural district of Namakkal, a prevalence >3% was detected among antenatal women. The goal of this study was to determine the HIV-1 molecular epidemiology in Tamil Nadu. Blood samples were collected from 40 high-risk HIV-seropositive individuals from Chennai and Namakkal. HIV-1 subtype was determined by envelope nucleotide sequencing. Among the samples studied, 85% were subtype C, however, a cluster of subtype A samples (12.5%) and one subtype E recombinant form CRF01_AE (close to the Thailand strains) were detected. The average genetic distance of subtype C samples from Chennai and Namakkal were 9.44 ± 0.77% and 11.8 ± 0.7%, respectively indicating an evolved epidemic. This pilot study confirmed that subtype C was predominant in these regions but an outbreak of subtype A was detected in Namakkal. These results stress the importance of periodic monitoring of circulating HIV-1 subtypes in South India.

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2 '-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2 '-difluoro-2 '-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment. © 2011 Elsevier Inc., Fundacion para la Investigacion y Prevencion del SIDA en España (FIPSE); Red Temática Cooperativa de Investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social; MICINN; Fundación Ramón Areces

The study on the evolutionary consequences of an RNA viral population's fluctuations can be approached by in vitro experiments. This work describes the fitness recovery of HIV-1 after 20 large-population passages in 10 debilitated clones. The serial passages promoted an increase in viral fitness. In addition, we detected a significant number of mutations fixed in the complete genome consensus sequence of the final viral populations. Among the mutations, events of convergent evolution with important phenotypic characteristics occurred in several independent clones. One common change, V35I, in the nuclear localization signal of the p17 protein appeared in four viruses of three different lineages. Other common alterations mapped in position E196K of the reverse transcriptase or in position S316K of the V3 loop of the gp120 residue that is associated with the X4/R5 phenotype. Together with this mutational analysis, we studied the quasispecies heterogeneity of the initial and final viruses, revealing that fitness increase correlated with an augmentation in the genetic heterogeneity of viral quasispecies. However, while heterogeneity was mostly composed of synonymous ( dS ) mutations in the first 10 passages performed, at passage 21 it switched to nonsynonymous ( dN ) substitutions, with significant differences in dN − dS values between passages 11 and 21. In summary, the HIV-1 in vitro fitness recovery depicts a multiphase process occurring first by generation of mutations followed by fixation of the beneficial ones, depicting a classical Darwinian process.

RNA viruses replicate as complex mutant spectra, also termed ‘mutant clouds’, known as viral quasispecies. While this is a widely observed viral population structure, it is less known that a number of biologically relevant features of this important group of viral pathogens depend on (or are strongly influenced by) the complexity and composition of mutant spectra. Among them, fitness increase or decrease depending on intrapopulation complementation or interference, selection triggered by memory genomes, pathogenic potential of viruses, disease evolution and the response to antiviral treatments. Quasispecies represent the recognition of complex behavior in viruses, and it is an oversimplification to equate such a population structure with the classic polymorphism of population biology. Darwinian principles acting on genome collectivities that replicate with high error rates provide a unique population structure prone to flexible and largely unpredictable behavior.

We studied viral evolution in three HIV-1 ancestral patients from a group of LTNPs; although some minor sequences showing viral evolution were detected in all patients, the extremely low viral evolution of their viruses was shown by the phylogenetic analysis of the env sequences. Complete nucleotide sequencing of viral DNA showed the major presence of deletions. In two patients, deletions of 1088 and 228 nucleotides mapped to 5′ LTR–gag region; in the other, a 247 nucleotide deletion was positioned in pol gene up to the vif ORF. These deleted genomes became dominant during follow up. Patient's viruses displayed 13 common mutations in conserved residues, from the 5′ LTR to the nef gene. These mutations provided evidence of a common origin. Regarding host characteristics, one patient had HLA B2705/B5801; another B1402/B5701; whereas a third showed B3901/B4402 and was Δ32-CCR5 heterozygous. These HIV controllers presented a combination of deleted viral genomes and host protective factors.

The first molecular epidemiology study of human immunodeficiency virus type 1 (HIV-1) in Panama has been performed with plasma samples from 66 AIDS patients infected by different transmission routes and obtained from distinct locations. All samples were amplified by RT-PCR and sequenced in gag (p17) and env (C2–C4) genes. Phylogenetic analyses revealed that 64 (97%) of the samples belong to subtype B. We also identified the presence of two CRF, one CRF12_BF and one CRF02_AG. The most notable feature of the subtype B epidemic in Panama was the large genetic distance among isolates with a mean of 15.2% but reaching up to 31.3 % in env, indicating an epidemic with a long period of evolution.

Human immunodeficiency virus type 1 (HIV-1) viruses with C/S/D/E at 215 codon of the reverse transcriptase (RT) have been associated with the T215Y/F HIV-1 resistant viruses transmission to naïve patients. The uncommon T215V mutation was detected in DNA proviral samples of a treatment-naïve patient. Virus T215V was obtained after cloning the patient-RT into a molecular clone. Wild-type (T215) and resistant (T215F) clones, were obtained in T215V clone by "in vitro" site-directed mutagenesis. Phenotypic resistance against AZT, ddI and d4T, replication kinetics and the selection of resistance mutations were estimated "in vitro". The T215V virus replicated as efficiently as the wild-type (T215) without antivirals and in the presence of AZT or ddI. The T215V virus showed higher replicative capacity than the wild-type and a 4.3-fold increase in the IC(50) values in cultures with d4T. Selection of resistance mutations was not observed in viral quasispecies of the T215V virus after serial passages in culture in presence of increasing concentrations of AZT, ddI or d4T. This work demonstrates that the T215V mutation decreases the susceptibility of HIV-1 to d4T, and consequently, it could compromise the response to regimens containing this antiviral.

The objective of the present study was to determine if natural suppression of plasma viremia below the detection limit of commercial assays (50-80 copies HIV-1 RNA/ml) can contain the HIV-1 evolution. HIV-1 quasispecies complexity in PBMC DNA was assessed in the env gene at two time points in 14 long-term nonprogressors (LTNPs). Sequence changes consistent with viral evolution was found in all patients with a median plasma RNA viral load100 copies/ml. Evidence of low-level viral evolution was detected in two of four patients with intermittent viremia and a median plasma HIV-1 RNA load of80 copies/ml. No significant evolution was observed in the three LTNPs with persistent viral suppression below the detection limit. Overall, a significant positive correlation (p0.001) was observed between viral evolution and plasma RNA viral load in the LTNPs analyzed. These results suggest that the detection limit of ultrasensitive viremia assays could represent an important threshold below which intrahost HIV-1 evolution does not occur.

The understanding of the evolutionary processes underlying HIV-1 fitness recovery is fundamental for HIV-1 pathogenesis, antiretroviral treatment and vaccine design. It is known that HIV-1 can present very high mutation and recombination rates, however the specific contribution of these evolutionary forces in the "in vitro" viral fitness recovery has not been simultaneously quantified. To this aim, we analyzed substitution, recombination and molecular adaptation rates in a variety of HIV-1 biological clones derived from a viral isolate after severe population bottlenecks and a number of large population cell culture passages. These clones presented an overall but uneven fitness gain, mean of 3-fold, respect to the initial passage values. We found a significant relationship between the fitness increase and the appearance and fixation of mutations. In addition, these fixed mutations presented molecular signatures of positive selection through the accumulation of non-synonymous substitutions. Interestingly, viral recombination correlated with fitness recovery in most of studied viral quasispecies. The genetic diversity generated by these evolutionary processes was positively correlated with the viral fitness. We conclude that HIV-1 fitness recovery can be derived from the genetic heterogeneity generated through both mutation and recombination, and under diversifying molecular adaptation. The findings also suggest nonrandom evolutionary pathways for in vitro fitness recovery.

To analyze superinfection in an HIV-1-infected patient showing high-risk practices, viral quasispecies were analyzed in pol and env genes in several plasma samples. Phylogenetic analysis in the reverse transcriptase fragment in pol gene identified a single virus in the first 3 samples analyzed, but 12 years after primoinfection, 3 different viral strains were detected in the patient quasispecies. This result suggests a superinfection with 2 HIV-1 strains, one of which showed the T215Y + M184V resistance mutations. The analysis in the env gene confirmed the existence of 3 different strains in the viral population, one of them a recombinant. This study illustrates that events of superinfection and recombination contribute to the viral genetic variability observed in HIV-1-infected individuals.

Repeated bottleneck passages of RNA viruses result in fitness losses due to the accumulation of deleterious mutations. In contrast, repeated transfers of large virus populations result in exponential fitness increases. Human immunodeficiency virus type 1 (HIV-1) manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells. An analysis of the mutations associated with fitness loss in four debilitated clones revealed mutation frequencies in gag that were threefold higher than those in env . We now show an increase in the fitness of the debilitated HIV-1 clones by repeated passages of large populations. An analysis of the entire genomic nucleotide sequences of these populations showed that few mutations, from two to seven per clone, mediated fitness recovery. Eight of the 20 mutations affected coding regions, mainly by the introduction of nonsynonymous mutations (75%). However, most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5′ untranslated leader region of the genome, and more specifically, in the primer binding site (PBS) loop. Two of the viruses incorporated the same mutation in the primer activation signal in the PBS loop, which is critical for the tRNA 3 Lys -mediated initiation of reverse transcription. Moreover, 25% of the mutations observed were reversions. This fact, together with the presence of a large proportion of nonsynonymous replacements, may disclose the operation, during large population passages, of strong positive selection for optimal HIV-1 replication, which seems to be primarily affected by binding of the tRNA to the PBS and the initiation of reverse transcription.

HIV-1 evolution and the possible emergence of mutations associated with resistance to antiretroviral inhibitors have been evaluated in a cohort of sixty-three patients successfully treated with highly active antiretroviral therapy (HAART). The patients under effective HAART were recruited in three different hospitals in Spain, and none of them had been treated (naive) before entering this study. HIV-1 RNA levels, CD4+, and CD8+ T-cell counts were determined, and nucleotide sequences of proviral regions encoding protease and reverse transcriptase (RT) were obtained for longitudinal blood samples spanning a mean follow-up period of 88 weeks. Phylogenetic reconstructions and calculations of genetic distances among the different sequences of each patient were performed. All except one of the patients under study showed an early and sustained decrease in plasma HIV-1 RNA to levels that were below 200 copies/ml. The plasma viral decline paralleled a significant increase in the CD4+ T-lymphocyte counts. Amino acid sequence analyses revealed the occurrence of mutations associated with antiretroviral resistance in nine patients (14.3%) during HAART treatment, that in some cases could be attributed to excess G to A transitions. In six of the nine patients, the mutations conferred resistance to inhibitors administered in the treatment regime, although the mutations did not result in treatment failure. Sequence comparisons revealed viral evolution during the period of treatment in 47.5% of the patients. The results indicate successful suppression of HIV-1 under HAART for extended time periods, indistinguishable for patients in which evidence of virus evolution could or could not be documented. J. Med. Virol. 73:350–361, 2004. © 2004 Wiley-Liss, Inc.

Human Immunodeficiency Virus type 1 (HIV-1) because of high mutation rates, large population sizes, and rapid replication, exhibits complex evolutionary strategies. For the analysis of evolutionary processes, the graphical representation of fitness landscapes provides a significant advantage. The experimental determination of viral fitness remains, in general, difficult and consequently most published fitness landscapes have been artificial, theoretical or estimated. Self-Organizing Maps (SOM) are a class of Artificial Neural Network (ANN) for the generation of topological ordered maps. Here, three-dimensional (3D) data driven fitness landscapes, derived from a collection of sequences from HIV-1 viruses after "in vitro" passages and labelled with the corresponding experimental fitness values, were created by SOM. These maps were used for the visualization and study of the evolutionary process of HIV-1 "in vitro" fitness recovery, by directly relating fitness values with viral sequences. In addition to the representation of the sequence space search carried out by the viruses, these landscapes could also be applied for the analysis of related variants like members of viral quasiespecies. SOM maps permit the visualization of the complex evolutionary pathways in HIV-1 fitness recovery. SOM fitness landscapes have an enormous potential for the study of evolution in related viruses of "in vitro" works or from "in vivo" clinical studies with human, animal or plant viral infections. Work in CNM is supported by grant SAF 2007-61036 and 2010-17226 from MINECO Spain, by grants 36558/06, 36641/07, 36779/08, 360766/09 from FIPSE Spain, and in part by the RETIC de Investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias (FISss). Work in UCM is supported by grants BFU 2009-12895-C02-02 MICINN and BFU2012-39816-C02-02 from MINECO (Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

To characterize the viruses responsible for the HIV-1 epidemic in Spain, we genetically characterized 79 samples obtained from Spanish patients with different risk practices (injecting drug users and male homosexuals) in two regions (Madrid and Navarra). Genetic characterization was carried out by nucleotide sequencing in the C2-V3-C3 region and by phylogenetic analysis. All samples were of subtype B except one that clustered with clade F. Because no segregation of samples was determined according to the risk group of patients nor to their geographic origin, the Spanish samples analyzed constitute a single group of viruses. These data, along with the starlike topology of the phylogenetic tree, support the existence of a single introduction of HIV-1 subtype B in Spain. The mean genetic distance among subtype B sequences was of 13.9%+/-0.06% (range, 5%-25%), suggesting an epidemic of long evolution. Analysis of sequences in relation to isolation dates revealed an increase in the heterogeneity of the nucleotide sequences with time. According to these data, a divergence rate of 0.49%+/-0.11% per year was calculated for the Spanish samples during the period analyzed.

HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (p, eLife digest Developing treatments or vaccines for HIV is challenging because the genetic makeup of the virus is constantly changing in an effort to outwit the human immune system. Moreover, the immune system is highly variable as a result of the long-standing co-evolution of humans and microbes. Each individual will try to oppose the invading virus in a unique way, forcing the virus to acquire specific mutations that can be interpreted as the genetic signature of this one-against-one battle. To explore the influence of co-evolution on HIV, Bartha et al. took samples of both human and viral genomes from 1071 individuals infected with HIV, the AIDS virus, and used genotyping and sequencing technology to obtain a comprehensive description of the genetic variation in both. Computational techniques were then used to search for links between variants in the human DNA sequences and variants in the viral sequences. The most common type of genetic variation found in the human genome is a single nucleotide polymorphism, or SNP for short: a SNP is produced when a single nucleotide – an A, C, G or T – is replaced by a different nucleotide. Bartha et al. found that SNPs within the human DNA sequences in their study were linked to variations in 48 amino acids in HIV. Moreover, all these SNPs were found within a group of genes known as the HLA (human leukocyte antigen) system, which encodes for proteins that play a vital role in the immune response. This work identified the areas of the human genome that put pressure on the AIDS virus, and the regions of the virus that serve to escape human control. The approach developed by Bartha et al. allows the interactions between a microbe and a human host to be studied by looking at the genome of the microbe and the genome of the infected person. It also differentiates host-induced mutations that limit the capacity of the virus to do harm from those that are tolerated by the pathogen. A similar strategy could be used to study other infectious diseases. DOI: http://dx.doi.org/10.7554/eLife.01123.002

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor-elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC. METHODS: For DI detection, nucleotide sequences in env gene from viruses from 20 LTNP-EC were analyzed by maximum likelihood. Epidemiological and clinical parameters and host factors of patients were also studied. RESULTS: DI was detected in 4 (20%) of the 20 LTNP-EC, of which 3 maintained the elite controller status. CD4 T-cell counts were not different between single and DI patients although higher CD8 T-cell counts were observed in DI patients, and, consequently, the CD4/CD8 ratios were lower in LTNP-EC DI patients. CONCLUSIONS: Prevalence of HIV-1 DIs in LTNP-EC is similar to other groups of HIV-1 patients; in addition, DI was not associated with loss of disease control in the patients. These DI LTNP-EC patients showed, in comparison with single infected patients, higher numbers of CD8 T cells and lower CD4/CD8 ratios. Copyright © 2013 by Lippincott Williams & Wilkins.

Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70's-early 80's). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients. Work in CNM is supported by grants [SAF 2007-61036 and 2010-17226] from MICINN Spain, by grants [36558/06, 36641/07, 36779/08, 360766/09] from Fundación para la investigación y prevención del SIDA en España (FIPSE) Spain, and in part by the RETIC de Investigación en SIDA [RD06/006/0036] of the Fondo de Investigaciones Sanitarias (FISss). Work in H12 de Octubre was financed by FIPSE [36749], and ISCIII-FIS [PI080806 and PI1101580] to RD. The HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by the Instituto de Salud Carlos III, Spanish Health Department (Grant n° RD06/006/0035) and FIPSE. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Sí

We have analyzed by the RNase-A mismatch method 35 isolates from four WHO-sponsored vaccine evaluation sites as a secondary laboratory of the WHO Network for HIV Isolation and Characterization. The application of an estimator for the establishment of genetic distances based on the RNase-A digestion patterns in combination with the phylogenetic analysis has allowed us to construct a tree with five well defined groups of viruses. Because the clustering with known reference strains, samples from Brazil could be grouped as subtype B and the majority of those from Thailand were subtype E. Some of the samples from Uganda were classified as subtype D. Isolates from Rwanda and some from Uganda were identified as subtype A viruses. These results coincide with data obtained by heteroduplex mobility assay and nucleotide sequencing in env regions. The RNase-A mismatch method combined with phylogenetic analysis permitted the primary genetic classification of 33 of 35 samples from the WHO Network.

A study of the genetic variability of herpes simplex virus (HSV) type 1 from recurrent lesions and clinical reinfections was done using restriction endonuclease analysis and the RNase A mismatch cleavage method. Comparative genetic analyses of HSV-1 recurrent isolates from 1 patient and of HSV-1 isolates from different anatomic areas (vagina and lip) from another patient showed differences only in the glycoprotein B gene but not in the thymidine kinase gene even though the viruses had the same restriction endonuclease pattern. These results suggest the RNase A mismatch cleavage method is useful for epidemiologic studies of DNA viruses.

We have determined the nucleotide sequence of the HIV-1 vif gene in viruses obtained from symptomatic patients of distinct risk groups in Madrid. The genetic diversity among the isolates was estimated in 4.6% (+/- 1.4 standard deviation), a similar value to that obtained for the gag gene 3.9% (+/- 0.8 standard deviation) and env 4.1% (+/- 1 standard deviation) (Rojas et al., Virus Res 31, 331-342, 1994). Amino acid sequence analysis revealed the presence of hypermutable residues at positions 101 and 167, close to antigenically relevant sequential epitopes (comprising amino acids 87-94 and 172-178). Phylogenetic analysis supports the existence of two virus lineages circulating preferentially within different risk groups.

The nucleotide sequences of two pol gene regions (codons 41 to 108 and 181 to 219 of reverse transcriptase) of 60 human immunodeficiency virus type 1 genomes obtained directly from primary lymphocytes from infected individuals are reported. In addition, the mutant spectra of several quasispecies have been sampled by repetitive sequencing of molecular clones representing the same pol genomic regions. Average mutation frequencies ranged from 1.6 x 10(-2) to 3.4 x 10(-2) substitutions per nucleotide for independent samples (relative to their consensus nucleotide sequence) and from 3.6 x 10(-3) to 1.1 x 10(-2) substitutions per nucleotide for individual quasispecies distributions. Several mutations leading to amino acid substitutions related to loss of sensitivity to reverse transcriptase inhibitors have been identified in samples from patients not subjected to antiretroviral therapy. Mutation frequencies in the codons previously identified as involved in resistance to reverse transcriptase inhibitors were very similar to the average mutation frequencies in the pol region analyzed. Thus, the finding of mutations related to drug resistance (even in the absence of positive selection by the corresponding drugs) is the expected consequence of the statistical distribution of mutations along the pol gene. The presence of such critical amino acid replacements in human immunodeficiency virus type 1 populations underscores the importance of viral quasispecies as reservoirs of phenotypic virus variants and has a number of implications for AIDS control.

BACKGROUND: CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. METHODS: We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay

OBJECTIVE: To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. METHODOLOGY AND PRINCIPAL FINDINGS: We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. CONCLUSIONS: The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC. This work was supported by grants SAF 2007-61036 and SAF 2010-17226, by Fundacion para la Investigacion y Prevencion del SIDA en España (FIPSE) grants 36558/06, 36641/07, 36779/08, 360766/09 and 360737/09, and by the United States National Institute of Health NIH-NIAID contract N01-AI-30024. Support by the Red Temática Cooperativa de Investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss) is appreciated. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Human immunodeficiency virus type 1 (HIV-1) infection with more than one strain, termed dual infection (DI), encompasses both co and superinfection (SI). Co-infection is the acquisition of two viral strains during primo-infection, whereas SI is the infection with a second heterologous strains after primary infection or during the course of an established infection. The first evidence that support HIV-1 DI, is the existence of numerous circulanting recombinant forms, because recombination requires that a single cell is infected by two viruses. Recombination could arise inter-subtype (where viruses differ by ~30% in the viral envelope sequence) and intra–subtype (where viruses differ by only ~10% in the envelope). The first case of a intersubtype SI in a man, infected with a CRF01-AE recombinant form, who became superinfected with a subtype B strain was described by (Jost et al., 2002). Similar inter subtype cases have been reported by (Ramos et al., 2002). Intrasubtype cases, where the first and the second strain belong to the same B subtype, have been described (Altfeld et al., 2002; Pernas et al., 2006).

BACKGROUND: Various patterns of HIV-1 disease progression are described in clinical practice and in research. There is a need to assess the specificity of commonly used definitions of long term non-progressor (LTNP) elite controllers (LTNP-EC), viremic controllers (LTNP-VC), and viremic non controllers (LTNP-NC), as well as of chronic progressors (P) and rapid progressors (RP). METHODOLOGY AND PRINCIPAL FINDINGS: We re-evaluated the HIV-1 clinical definitions, summarized in Table 1, using the information provided by a selected number of host genetic markers and viral factors. There is a continuous decrease of protective factors and an accumulation of risk factors from LTNP-EC to RP. Statistical differences in frequency of protective HLA-B alleles (p-0.01), HLA-C rs9264942 (p-0.06), and protective CCR5/CCR2 haplotypes (p-0.02) across groups, and the presence of viruses with an ancestral genotype in the "viral dating" (i.e., nucleotide sequences with low viral divergence from the most recent common ancestor) support the differences among principal clinical groups of HIV-1 infected individuals. CONCLUSIONS: A combination of host genetic and viral factors supports current clinical definitions that discriminate among patterns of HIV-1 progression. The study also emphasizes the need to apply a standardized and accepted set of clinical definitions for the purpose of disease stratification and research. Funding: CLG is supported by grant SAF 2007-61036, by Fundacion para la Investigacion y Prevencion del SIDA en Espana (FIPSE) grant 36558/06, 36641/07, 36779/08, 360766/09 and in part by the Red Tematica Cooperativa de Investigacion en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss). AT is supported by the Swiss National Science Foundation. This work has been partially financed within the framework of the Swiss HIV Cohort Study (SHCS) funded by the Swiss National Science Foundation (grant no#33CSC0-108787) and by the SHCS research foundation. The funders had no role in study design, data collection and analysisis, decision, or preparation of the manuscript. Sí

[Background] Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages., [Principal Findings] After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p, [Conclusions] Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture., Work in the Centro Nacional de Microbiología was supported by grants, SAF 2005/03833, SAF 2007/61036 by the Plan Nacional del SIDA and in part by the Red Temática Cooperativa de investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss) and Fundación para la Investigación y la Prevención del SIDA en España (FIPSE) grant 36558/06, 36641/07, 36779/08, 36641/07 and 360766/09. Work in the Centro de Biología Molecular “Severo Ochoa” was supported by grant BFU 2008-02816/BMC from Ministerio de Ciencia e Innovación, FIPSE 36558/06 and 360766/09 and Fundación Ramón Areces. Centro de Investigaciones Biomédica en Red (CIBER): enfermedades hepáticas y digestivas (ehd) is funded by Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis. © 2010 Elsevier Ltd.

Background: Although HIV-1 CRF12_BF and CRF38_BF are two epidemiologically important recombinant lineages circulating in Argentina and Uruguay, little is known about their population dynamics.Methods: A total of 120 "CRF12_BF-like" and 20 "CRF38_BF-like" pol recombinant sequences collected in Argentina and Uruguay from 1997 to 2009 were subjected to phylogenetic and Bayesian coalescent-based analyses to estimate evolutionary and demographic parameters.Results: Phylogenetic analyses revealed that CRF12_BF viruses from Argentina and Uruguay constitute a single epidemic with multiple genetic exchanges among countries; whereas circulation of the CRF38_BF seems to be confined to Uruguay. The mean estimated substitution rate of CRF12_BF at pol gene (2.5 × 10-3 substitutions/site/year) was similar to that previously described for subtype B. According to our estimates, CRF12_BF and CRF38_BF originated at 1983 (1978-1988) and 1986 (1981-1990), respectively. After their emergence, the CRF12_BF and CRF38_BF epidemics seem to have experienced a period of rapid expansion with initial growth rates of around 1.2 year-1 and 0.9 year-1, respectively. Later, the rate of spread of these CRFs_BF seems to have slowed down since the mid-1990s.Conclusions: Our results suggest that CRF12_BF and CRF38_BF viruses were generated during the 1980s, shortly after the estimated introduction of subtype F1 in South America (~1975-1980). After an initial phase of fast exponential expansion, the rate of spread of both CRFs_BF epidemics seems to have slowed down, thereby following a demographic pattern that resembles those previously reported for the HIV-1 epidemics in Brazil, USA, and Western Europe. Fil: Bello, Gonzalo. Instituto Oswaldo Cruz; Brasil Fil: Aulicino, Paula. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Laboratorio de Biología Celular y Retrovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ruchansky, Dora. Servicio Nacional de Laboratorios de Salud Publica; Uruguay Fil: Guimarães, Monick L.. Instituto Oswaldo Cruz; Brasil Fil: Lopez Galindez, Cecilio. Instituto de Salud Carlos III; España Fil: Casado, Concha. Instituto de Salud Carlos III; España Fil: Chiparelli, Hector. Servicio Nacional de Laboratorios de Salud Publica; Uruguay Fil: Rocco, Carlos Alberto. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Laboratorio de Biología Celular y Retrovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mangano, Andrea María Mercedes. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Laboratorio de Biología Celular y Retrovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sen, Luisa. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Laboratorio de Biología Celular y Retrovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morgado, Mariza G.. Instituto Oswaldo Cruz; Brasil

Recombination has been shown to be an important force in HIV-1 evolution. Recombination contributes to an increase in genetic variation and hinders vaccine design efforts. Several molecular epidemiology studies in South America described the circulation of subtypes B, F, and C as well as several B/F1 recombinants. This study performed by nucleotide sequencing in at least two genes of 89 samples from Uruguay has shown a complex HIV-1 epidemic characterized by the cocirculation of subtype B, and subtype C strains, together with an important group of BF1 recombinant viruses, including isolates similar to CRF12_BF. In addition we identified a new circulating recombinant form, designated CRF38_BF1, which was dominant in the recombinant virus group.

To investigate cellular factors involved in HIV-1 chronic infection, three cell lines chronically infected with the same HIV-1 viral isolate (s61) were studied by cDNA microarray analysis. Two T cell lines, H61 and M61, showed the characteristics of a persistent infection whereas U61 cell line displayed a latent infection pattern. Analysis of genes with altered expression in the three cell lines revealed evidence of apoptosis control by up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes. In addition, cell cycle control was affected in the two persistent T cell lines particularly through the down-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Moreover, each cell line showed specific characteristics, like in M61 cells, genes related with cellular activation and with cell migration and motility. In U61 cells, genes associated with immune response were activated. Genes with altered expression in our experiments, and not previously related with HIV such as ANXA 1 or CFLAR were detected and validated. This work revealed that different cell mechanism such as control of apoptosis and cell cycle are important for "in vitro" HIV-1 chronic infections, and discovered new genes previously not related with HIV-1 replication.

Three persistently infected cell lines (H61, M61, and U61) were established by infection with an HIV-1 isolate (s61) of two T cell lines, H9 and MT-4, and the promonocytic U937-2. In H61, 35% of cells expressed viral antigens yielding low virus titers and a majority of mature particles. M61 showed viral expression in every cell but with the frequent generation of immature particles. In U61, 1% of cells displayed viral expression, which increased after cell activation, indicating a latent infection. Nucleotide sequences of the complete provirus from the persistent cell lines revealed extremely high mutation rates in accessory genes and non-coding regions from 1.1 to 2.8 x 10(-2), whereas in structural genes they ranged from 3.2 to 9.8 x 10(-3). Ten nonsynonymous mutations were shared by all persistent proviruses including five strong amino acid changes in the env gene (related to the NSI phenotype) and in vpr and tat genes; other alterations were in accessory genes and two in the USF and c-Myb motifs in LTR. Truncated vpr and vpu proteins were found specifically in H61 and in vif in M61. This comprehensive study disclosed the role of the cell on the HIV-1 persistence pattern as well as common and specific mutations in the virus.

Human immunodeficiency virus 1 (HIV-1) dual infections are considered important because they have been related to AIDS progression. We identified dual infections in 2 patients with long-term, nonprogressive HIV-1 disease; the first patient was diagnosed as being already coinfected, on the basis of the first sample analyzed, but a previous superinfection could not be excluded; the second patient was diagnosed as having a superinfection, on the basis of the 9-year difference between the viral dating of the 2 strains. Dual infections occur in patients with long-term, nonprogressive disease, with no immediate clinical manifestations. Such occurrences could indicate a general phenomenon in natural HIV-1 infections.

Mature enzymes encoded within the human immunodeficiency virus type 1 (HIV-1) genome (protease (PR), reverse transcriptase (RT) and integrase (IN)) derive from proteolytic processing of a large polyprotein (Gag-Pol). Gag-Pol processing is catalyzed by the viral PR, which is active as a homodimer. The HIV-1 RT functions as a heterodimer (p66/p51) composed of subunits of 560 and 440 amino acid residues, respectively. Both subunits have identical amino acid sequence, but p51 lacks 120 residues that are removed by the HIV-1 PR during viral maturation. While p66 is the catalytic subunit, p51 has a primarily structural role. Amino acid substitutions affecting the stability of p66/p51 (i.e. F130W) have a deleterious effect on viral fitness. Previously, we showed that the effects of F130W are mediated by p51 and can be compensated by mutation T58S. While studying the dynamics of emergence of the compensatory mutation, we observed that mutations in the viral PR-coding region were selected in HIV clones containing the RT substitution F130W, before the imposition of T58S/F130W mutations. The PR mutations identified (G94S and T96S) improved the replication capacity of the F130W mutant virus. By using a trans-complementation assay, we demonstrate that the loss of p66/p51 heterodimer stability caused by Trp130 can be attributed to an increased susceptibility of RT to viral PR degradation. Recombinant HIV-1 PRs bearing mutations G94S or T96S showed decreased dimer stability and reduced catalytic efficiency. These results were consistent with crystallographic data showing the location of both residues in the PR dimerization interface.

HIV-1 evolution in the envelope gene (env) was analyzed in four asymptomatic antiretroviral therapy naïve patients with typical and slow disease progression rates. In typical progressors, viral populations were monophyletic and two distinct evolutionary patterns were observed. In one patient, HIV-1 evolution displayed a strong temporal structure similar to the consistent pattern previously described. In the other, viral evolution displayed a lack of temporal structure with no increase in genetic heterogeneity and divergence over time. In slow progressors, several clades were observed in viral populations. However, analysis within the major sub-population revealed the same two evolutionary patterns described for typical progressors. Synonymous and non-synonymous substitution rate analyses indicated that positive selection was the major force driving HIV-1 evolution in viral populations with temporal structure, while evolution in viral populations with an atemporal structure was dominated by genetic drift and purifying selection. These results support the existence of distinct patterns of env evolution in untreated HIV-1-infected patients.

Retrovirus are unique because they present two complete copies of the genomic RNA in each virion. It is believed that only one proviral DNA is formed from the two genomic RNAs. To check this hypothesis, we constructed two deleterious HIV-1 variants in gag gene which upon transfection in Cos-1 cells were able, by complementation, to form heterozygous viruses, used to infect MT4 cells in a plaque assay. Analysis of the proviral DNA of the eight plaques obtained indicated that five were recombinants between the two deleterious mutants. Three other plaques showed three bands corresponding to the reverse transcription of both strands of one heterozygous virion and to the recombination of the two genomes. These results demonstrate that the two genomic RNAs in HIV-1 heterozygous virions could be used in the generation of new viruses. This mechanism permits the recovery of deleterious mutants and enhances the evolutive potential of HIV-1.

Within human immunodeficiency virus type 1 (HIV-1)-infected patients, there are those who have been infected for more than 10 years with a CD4+ cell count of >500 cells μl−1 and who remain asymptomatic without antiretroviral therapy; these patients are designated long-term non-progressors (LTNPs). In a set of 16 LTNPs, viral dating, DNA viral load, quasispecies heterogeneity and antibody (Ab) titres against gp160 and β 2 microglobulin (β 2m) were determined. Plasma viral RNA and CD4+ and CD8+ T-cell numbers were estimated in more than three samples per patient. Host genetic characteristics, such as Δ32-CCR5 genotype and human leukocyte antigen (HLA) genotype and supertypes, and clinical–epidemiological factors were evaluated. Dating of global populations and of DNA and RNA viral quasispecies identified two subsets of patients: one displaying only ancestral sequences and the other displaying predominantly modern sequences. The ancestral patients displayed a significant reduction in RNA and DNA viral loads, quasispecies heterogeneity, CD8+ cell number, anti-gp160 Ab titres and β 2m level, and they were also associated with better use of safe-sex practices and higher presence of the HLA sB58 supertype than the modern subset. Viral dating has therefore permitted the segregation of LTNPs into two subsets that show very different virological, immunological, host and clinical–epidemiological characteristics. Moreover, whereas the modern subset displayed low levels of virus replication, the ancestral group displayed not only a very limited virus replication, often to undetectable levels, but also very slow or arrested viral evolution, maintaining the close relationship of the viral population to the transmitted virus.

The human immunodeficiency virus type 1 (HIV-1) viral set point has been associated with the rate of disease progression and with the level of HIV-specific immune response. The analysis of the possible association between viral set point and quasispecies heterogeneity has important consequences in the understanding of HIV-1 in vivo evolution. In this study, we analyzed the association between intrapatient viral diversity and RNA viral load in 16 antiretroviral therapy-naive HIV-1-infected patients at a single time point, during the disease free period. Patients were separated into low and high viral load groups according to plasma RNA values. HIV-1 quasispecies complexity was assessed in the C2-V5 env region. The average intrapatient quasispecies heterogeneity in both groups was not significantly different (t-test, P > 0.05). However, while within the low viral load group both synonymous and non-synonymous mutations contribute to the variation observed, in the heterogeneity observed in the high viral load group there was an increase in the contribution of the non-synonymous mutations. Thus, this study show that although intrapatient quasispecies heterogeneity is not associated with viral set point in HIV-1 infection, some differences exist between the two groups in the pattern of mutation accumulation.

In human immunodeficiency virus type 1 (HIV-1) infection, the presence of divergent nucleotide sequences within a quasispecies has been associated with double infections or samples from different times or from different tissue compartments. The authors analysed HIV-1 proviral quasispecies from PBMC of three untreated Spanish patients displaying highly divergent nucleotide sequences without evidence of double infection. The origin of these nucleotide sequences was determined by phylogenetic analysis and by dating of the different groups using a genetic divergence versus sampling year plot from a set of Spanish samples. By their short genetic distance to the node of the patient's HIV-1 phylogenetic tree and by their early date of origin, close to the seroconversion time, some groups of sequences were considered ancestral. The presence within HIV-1 quasispecies of ancestral sequences, dated up to 10 years earlier than present ones, has important consequences for in vivo viral evolution, in the pathogenesis and treatment of HIV-1 infection.

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.

To help in the vaccine development, WHO-UNAIDS launched a program for the isolation and characterization of subtype C viruses, the most prevalent HIV-1 subtype in the world. Isolates were obtained from Brazil, China, India, Israel, and South Africa, countries in which these strains are circulating. In this study we genetically characterized a set of samples displaying the culture-negative phenotype by sequencing the nucleotides of three genomic regions: the p17 region of the gag gene, the C2V3C3 fragment of the env gene, and the nef gene. The association of the culture-negative phenotype with the nef gene was studied, and we found a significant accumulation of gene alterations. Except for one B/C recombinant from India, the samples studied formed a monophylogenetic subtype C clade, although samples from Brazil formed a statistically significant, independent subcluster in two of the three genes analyzed.

HIV-1 recombination was studied in vitro by viral cocultivation of four combinations of strains of subtypes B, D, and F. Viral cocultivations were performed in MT-4 cells and maintained for 22 days. The parental and recombinant forms were quantified by a specific PCR system in an env fragment of 2500 nucleotides. On day 5, there was a close correlation between the proportion of recombination and the genetic distance between strains. In three of the four viral combinations studied, a steady increase in the proportion of recombinant genomes was observed over time. This rise coincided with the progressive loss of one of the parental strains, resulting in less diverse viral populations. Nucleotide sequencing of biological recombinant clones from the B/D cocultivation revealed a higher number of recombination events in pol than in env gene, and an increasing number of crossovers per clone with time.