Biomedical subjects

E Domingo

Publications and source records attributed to E Domingo.

At least 37 records · Page 2Linked to original sources

PubMed2001

Evidence for positive selection in the capsid protein-coding region of the foot-and-mouth disease virus (FMDV) subjected to experimental passage regimens.

We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (d(N)/d(S)) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our analyses also identified a subregion of VP3, part of the fivefold axis of FMDV particles, that also appeared to be subjected to positive selection of amino acid replacements. From these results, we can conclude that under the restrictive conditions imposed either by the presence of the monoclonal antibodies, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in some capsid-coding regions can be positively selected toward an increase of those mutants with a higher capability to infect the cell.

Aphthovirus↗

PubMed2001

Contingent neutrality in competing viral populations.

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.

Animals↗

PubMed2001

Efficient virus extinction by combinations of a mutagen and antiviral inhibitors.

The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 10(6)-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.

Animals↗

PubMed2001

A single amino acid substitution in nonstructural protein 3A can mediate adaptation of foot-and-mouth disease virus to the guinea pig.

The genetic changes selected during the adaptation of a clonal population of foot-and-mouth disease virus (FMDV) to the guinea pig have been analyzed. FMDV clone C-S8c1 was adapted to the guinea pig by serial passage in the animals until secondary lesions were observed. Analysis of the virus directly recovered from the lesions developed by the animals revealed the selection of variants with two amino acid substitutions in nonstructural proteins, I(248)-->T in 2C and Q(44)-->R in 3A. On further passages, an additional mutation, L(147)-->P, was selected in an important antigenic site located in the G-H loop of capsid protein VP1. The amino acid substitution Q(44)-->R in 3A, either alone or in combination with the replacement I(248)-->T in 2C, was sufficient to give FMDV the ability to produce lesions. This was shown by using infectious transcripts which generated chimeric viruses with the relevant amino acid substitutions. Clinical symptoms produced by the artificial chimeras were similar to those produced by the naturally adapted virus. These results obtained with FMDV imply that one or very few replacements in nonstructural viral proteins, which should be within reach of the mutant spectra of replicating viral quasispecies, may result in adaptation of a virus to a new animal host.

Adaptation, Physiological↗

PubMed2001

Prevalence and genetic heterogeneity of the reverse transcriptase T69S-S-X insertion in pretreated HIV-infected patients.

The emergence of resistance to antiretroviral drugs represents one of the main reasons for treatment failure in HIV-infected persons. Resistance to multiple nucleoside analogues may result from rearrangements in the HIV pol gene, in particular one insertion of two amino acids at position 69. Herein, we examined the prevalence of this resistant genotype and its genetic heterogeneity in a group of 475 healthy pretreated HIV-positive subjects in Spain. Only 4 (0.8%) carried the codon 69 insertion. It was always found coupled to the T69S mutation. The extra amino acids were S-S in 2 subjects and S-G in the other 2. The presence of the insert was seen only in subjects previously exposed to AZT monotherapy for at least 6 months.

Anti-HIV Agents↗

PubMed2001

Frequency and clinical significance of myocardial ischemia detected early after coronary stent implantation.

UNLABELLED: A high number (30%-50%) of reversible defects have been detected early after coronary balloon angioplasty. Inadequate luminal enlargement despite a good angiographic appearance has been suggested as a possible mechanism of these perfusion abnormalities, and some reports have shown better coronary flow reserve after coronary stent implantation than after balloon dilatation. The primary objective of this study was to evaluate the frequency of early ischemic defects detected by maximal exercise (plus dipyridamole) with (99m)Tc-tetrofosmin SPECT after successful coronary angioplasty with stent implantation. A secondary objective was to determine the prognostic value of these early ischemic defects. METHODS: Thirty patients without previous myocardial infarction who successfully underwent 1-vessel coronary angioplasty with stent implantation were studied. Maximal-exercise (99m)Tc-tetrofosmin myocardial SPECT, with simultaneous dipyridamole if exercise was suboptimal, was performed at 6 +/- 1 d (mean +/- SD) after percutaneous transluminal coronary angioplasty. At 8 +/- 3 mo, all patients were followed up clinically, and 77% of them underwent follow-up angiography. RESULTS: The percentage of stenosis decreased from 68.5% +/- 12.6% of luminal diameter to 9.3% +/- 8.8% after stent implantation, and minimal luminal diameter increased from 0.89 +/- 0.36 mm to 2.85 +/- 0.45 mm. Mild-to-moderate reversible myocardial defects in the territory of the dilated artery were detected in 5 patients (17%), with no angiographic or procedural differences occurring between them and patients without ischemic defects. At follow-up, the target lesion revascularization rates depending on the presence or absence of early ischemic defects were 40% and 8%, respectively (P = 0.18). Angiographic restenosis occurred in 3 of 4 patients who had early ischemic defects and underwent follow-up angiography and in 3 of 19 patients who had no early ischemic defects and underwent follow-up angiography (restenosis rate, 75% and 16%, respectively; P < 0.05). CONCLUSION: Coronary angioplasty with stent implantation is associated with a 17% rate of ischemic defects early after the procedure. Patients with early myocardial perfusion defects after coronary stent implantation had a high rate of restenosis.

Angioplasty, Balloon, Coronary↗

PubMed2000

Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance.

The 3'-azido-3'-deoxythymidine (AZT)-resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3'-terminal nucleotide from AZT- terminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT.

Amino Acid Sequence↗

PubMed2000

Viruses at the edge of adaptation.

How vulnerable is the line that separates adaptation from extinction? Viruses, in particular RNA viruses, are well known for their high rates of genetic variation and their potential to adapt to environmental modifications (Drake and Holland, 1999; Domingo et al., 2000). Yet, fitness variations-both increases and decreases-can be spectacularly rapid, and the simple genetic stratagem of forcing virus multiplication to go through repeated genetic bottlenecks can induce fitness losses, at times near viral extinction. New information has been recently obtained on the two sides of the survival line: the edge of adaptation and the edge of extinction.

Adaptation, Physiological↗

PubMed2000

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy.

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.

Adult↗

PubMed2000

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Amino Acid Substitution↗

PubMed2000

Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity.

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10(6)-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.

Amino Acid Substitution↗

PubMed2000

Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1.

Repeated bottleneck passages result in fitness losses of RNA viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations per genome, with dominance of A --> G and G --> A transitions (57% of all mutations) and 49% nonsynonymous replacements. One clone-but not three sibling clones-showed an overabundance of G --> A transitions, evidencing the highly stochastic nature of some types of mutational bias. The distribution of mutations along the genome was very unusual in that mutation frequencies in gag were threefold higher than in env. Particularly striking was the complete absence of replacements in the V3 loop of gp120, confirmed with partial nucleotide sequences of additional HIV-1 clones subjected to repeated bottleneck passages. The analyses revealed several amino acid replacements that have not been previously recorded among natural HIV-1 isolates and illustrate how evolution of an RNA virus genome, with regard to constant and variable regions, can be profoundly modified by alterations in population dynamics.

Base Sequence↗

PubMed2000

Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Amino Acid Sequence↗

PubMed2000

Memory in viral quasispecies.

Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint.

Animals↗

PubMed2000

[Diagnostic accuracy of tomographic myocardial imaging for evaluation of aortocoronary graft patency].

INTRODUCTION AND OBJECTIVES: Conventional ergometry has limitations in the evaluation of coronary artery bypass graft patency. The aim of the present study was to determine the efficacy of exercise single photon emission tomography with 99mTc-compounds for the diagnosis of coronary artery bypass grafts disease. PATIENTS AND METHODS: The state of sixty-seven coronary artery bypass grafts (31 with mammary artery, 36 with safein vein; 35 to left descending anterior artery, 15 to circumflex and 17 to right coronary artery) were analyzed retrospectively in 38 patients (mean age 63+/-8.7; 35 men). The time that elapsed between the coronary surgery and the exercise tomography was 9,7 years. In 16 cases, exercise tomography was performed with 99mTc-MIBI and in the 22 remaining with 99mTc-tetrofosmin. In 6 cases, dipiridamol was administred simultaneously during an insufficient exercise test. RESULTS: Sensitivity (73.1%), specificity (93%), positive predictive value (86.3%), negative predictive value (84.4%), global value (85%), positive likelihood ratio (10.4) and negative likelihood ratio (0.29) of exercise tomography were significantly (p<0.01) better than those obtained with the exercise test alone (53.8%, 43.6%, 38.9%, 58. 6%, 47.7%, 0.95 and 1.06, respectively). CONCLUSIONS: Exercise tomography with 99mTc-compounds is a highly effective for the detection of coronary artery bypass grafts disease.

Coronary Artery Bypass↗

PubMed2000

Uveal melanoma model with metastasis in rabbits: effects of different doses of cyclosporine A.

OBJECTIVE: To compare the toxicity and efficacy of different doses of cyclosporine A (CsA) in a rabbit model of uveal melanoma. METHODS: We used four experimental groups: control, no CsA; group 1, 15 to 10 mg/kg/day; group 2, 15 mg/kg/day; and group 3, 20 mg/kg/day. The MKT-BR cell line was implanted in the choroid. All animals underwent ophthalmoscopic evaluation; the animals were weighed and blood levels of CsA, blood urea nitrogen (BUN), creatinine and alanine aminotransferase (ALT) were measured weekly. Necropsies and histologic study were performed to detect intraocular tumors and metastasis. RESULTS: A difference in survival rates was found between groups 2 and 3 (p = 0.0042). Differences were observed in the mean BUN and creatinine levels (p < 0.001 and p < 0.003, respectively) between groups but not in the ALT. Intraocular tumors were detected ophthalmoscopically in 50%, 65%, and 70% of the animals in groups 1, 2, and 3 respectively, and histologically in 70%, 90%, and 100% of the same groups. Lung metastases were found in 26.8% of animals with intraocular tumors. Differences were observed in mean CsA blood levels between animals with and without histologically demonstrated uveal tumors (p = 0.001) but not in animals with or without metastasis. CONCLUSIONS: Different doses of CsA affect survival, tumor development and renal toxicity. Metastatic disease is independent of CsA dose and the subsequent CsA blood levels. A blood level of CsA ranging from 500 to 1000 ng/ml and doses of 10 to 15 mg/kg/day may effectively develop this model of uveal melanoma.

Alanine Transaminase↗

PubMed2000

[Quasispecies and molecular evolution of viruses].

Ribonucleic acid (RNA) viruses evolve as complex distributions of genetically different but closely related variants termed viral quasispecies. The precise genome of a quasispecies cannot be defined, since the consensus genome is an average of many variants. The dynamics of quasispecies has considerable implications for the understanding of the adaptability and pathogenic potential of viruses, and in addition, for the design of preventive and therapeutic measures for the diseases caused by these viruses. The authors summarise current knowledge on the structure of quasispecies, and the biological implications of this structure.

Adaptation, Biological↗

PubMed1999

Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase.

The highly conserved Phe160 residue is located in the "palm" subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside triphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs having Tyr, Trp, Ile, Ala or Gln instead of Phe160 were obtained by site-directed mutagenesis. F160Y and F160W retained substantial DNA polymerase activity, whereas the catalytic efficiency of nucleotide incorporation of mutants F160I, F160A and F160Q was less than 10 % that of the wild-type RT, using poly(rA).oligo(dT)20 as the template-primer. The low catalytic efficiency of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-type RT in nucleotide insertion assays carried out with heteropolymeric DNA/DNA template-primers. However, nucleotide affinity was two- to sixfold reduced in the case of mutant F160W. Fidelity assays revealed similar misinsertion and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral clone, mutations F160I, F160A and F160Q rendered non-viable virus. The importance of Phe160 for polymerase function and viral replication could be mediated by its interaction with Tyr115, as suggested by the analysis of the available crystal structures of HIV-1 RT.

Amino Acid Substitution↗