Biomedical subjects

Cecilio López-Galíndez

Publications and source records attributed to Cecilio López-Galíndez.

11 recordsLinked to original sources

PubMed2006

A dual superinfection and recombination within HIV-1 subtype B 12 years after primoinfection.

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.

Amino Acid Sequence↗

PubMed2005

A subset of human immunodeficiency virus type 1 long-term non-progressors is characterized by the unique presence of ancestral sequences in the viral population.

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 microl(-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 beta2 microglobulin (beta2m) 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 Delta32-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 beta2m 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.

CD4-CD8 Ratio↗

PubMed2005

Few mutations in the 5' leader region mediate fitness recovery of debilitated human immunodeficiency type 1 viruses.

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 tRNA3Lys-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.

5' Untranslated Regions↗

PubMed2004

Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity.

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.

Animals↗

PubMed2004

Long-term suppression of plasma viremia with highly active antiretroviral therapy despite virus evolution and very limited selection of drug-resistant genotypes.

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 (naïve) 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.

Adult↗

PubMed2004

Co-existence of recent and ancestral nucleotide sequences in viral quasispecies of human immunodeficiency virus type 1 patients.

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.

Amino Acid Sequence↗

PubMed2003

Genetic analysis of culture-negative UNAIDS subtype C samples.

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.

Adult↗

PubMed2002

Analysis, quantification, and evolutionary consequences of HIV-1 in vitro recombination.

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.

Coculture Techniques↗

PubMed2002

Frequency-dependent selection in human immunodeficiency virus type 1.

Genetic variation is the main evolutionary strategy adopted by RNA viruses and retroviruses. Evolution operates through competition between different individuals in the same environment, resulting in the imposition of the fittest variant. The process of competition could be affected by various factors, including the frequency of the different competing individuals. In order to investigate this aspect, individual virus populations derived from a human immunodeficiency virus type 1 isolate were studied at different competing proportions. The dynamics of variant imposition in each competition experiment permitted the detection of frequency-dependent selection (FDS); i.e. the imposition of variants is related to their biological fitness, which is also affected by the proportions at which they compete. The existence of FDS in different viruses with RNA genomes would indicate a general mechanism favouring genetic heterogeneity.

Adaptation, Physiological↗

PubMed2002

In vitro analysis of human immunodeficiency virus type 1 resistance to nevirapine and fitness determination of resistant variants.

Nevirapine-resistant variants were generated by serial passages in MT-2 cells in the presence of increasing drug concentrations. In passage 5, mutations V106A, Y181C and G190A were detected in the global population, associated with a 100-fold susceptibility decrease. Sequence analysis of biological clones obtained from passage 5 and subsequent passages showed that single mutants, detected in first passages, were progressively replaced in passage 15 by double mutants, correlating with a 500-fold increase in phenotypic resistance. Fitness determination of single mutants confirmed that, in the presence of nevirapine, every variant was more fit than wild-type with a fitness order Y181C>V106A>G190A>wild-type. Unexpectedly, in the absence of the drug, the Y181C resistant mutant was more fit than wild-type, with a fitness gradient Y181C>wild-type >G106A>or=V190A. Using a molecular clone in which the Y181C mutation was introduced by in vitro mutagenesis, the greater fitness of the Y181C mutant was confirmed in new competition cultures. These data exemplify the role of resistance mutations on virus phenotype but also on virus evolution leading, occasionally, to resistant variants fitter than the wild-type in the absence of the drug.

Adaptation, Physiological↗

PubMed2001

Different evolutionary patterns are found within human immunodeficiency virus type 1-infected patients.

In order to study the evolution in vivo of human immunodeficiency virus type 1 (HIV-1) in patients with normal clinical evolution, six individuals were selected from a group of 46 patients followed for 1 to 4 years. Patients were selected not by clinical progression characteristics but on the basis of virus genetic variability, as analysed by heteroduplex mobility assay and RNase A mismatch cleavage method. Two patients displayed a homogeneous virus population, two showed very heterogeneous quasispecies and two presented two distinct variants within the virus population. Virus quasispecies were studied by nucleotide sequencing of the C2-fusion domain of the env gene. Virus evolution was approached by analysing the distribution of genetic distances, calculation of divergence and heterogeneity as well as the K(a)/K(s) ratio and by the construction of the phylogenetic trees. Three patients displayed the same tree topology, characterized by the presence of independent clades supported by high bootstrap values, whereas this pattern was not present in the other three patients. In the three patients displaying independent clades, a recombination analysis was carried out between distinct subpopulations and recombinant variants were identified. In one patient of this group, different selective pressures were detected in distinct virus clades, measured by their corresponding K(a)/K(s) ratios, revealing that different evolutionary forces are occurring at the same time within the same patient. These results show that multiple evolutionary patterns can be found in typical HIV-1-infected patients.

Acquired Immunodeficiency Syndrome↗