[Percutaneous revascularization in women. Risks and results of different techniques of angioplasty].
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Adult↗
Biomedical subjects
E Domingo
Publications and source records attributed to E Domingo.
RNA virus quasispecies: significance for viral disease and epidemiology.
The experimental evidence available for animal and plant RNA viruses, as well as other RNA genetic elements (viroids, satellites, retroelements, etc.), reinforces the view that many different types of genetic alterations may occur during RNA genome replication. This is fundamentally because of infidelity of genome replication and large population sizes. Homologous and heterologous recombination, as well as gene reassortments occur frequently during replication of retroviruses and most riboviruses, especially those that use enzymes with limited processivity. Following the generation of variant genomes, selection, which is dependent on environmental parameters in ways that are poorly understood, sorts out those genome fits enough to generate viable quasispecies. Chance events can also be destabilizing, as illustrated by recent results on fitness loss and other phenotypic changes accompanying bottleneck transmission. Variation, selection, and random sampling of genomes occur continuously and unavoidably during virus evolution. Evolution of RNA viruses is largely unpredictable because of the stochastic nature of mutation and recombination events, as well as the subtle effects of chance transmission events and host/environmental factors. Among environmental factors, alterations resulting from human intervention (deforestation, agricultural activities, global climatic changes, etc.) may alter dispersal patterns and provide new adaptive possibilities to viral quasispecies. Current understanding of RNA virus evolution suggests several strategies to control and diagnose viral diseases. The new generation of chemically defined vaccines and diagnostic reagents (monoclonal antibodies, peptide antigens, oligonucleotides for polymerase chain reaction amplification, etc.) may be adequate to prevent disease and detect some or even most of the circulating quasispecies of any given RNA pathogen. However, the dynamics of viral quasispecies mandate careful consideration of those reagents to be incorporated into diagnostic kits. Broadening diagnosis without jeopardizing specificity of detection will be challenging. There is a finite probability (impossible to quantify at present) that a defined vaccine may promote selection of escape mutants or a particular diagnostic kit may fail to detect a viral pathogen. Of particular concern are the potential long-term effects of weak selective pressures that may initially go unnoticed. Variant viruses resulting from evolutionary pressure imposed by vaccines or drugs may insidiously and gradually replace previous quasispecies. The great potential for variation and phenotypic diversity of some important RNA virus pathogens (human immunodeficiency virus, the hepatitis viruses, the newly recognized human hantaviruses, etc.) has become clear. Prevention and therapy should rely on multicomponent vaccines and antiviral agents to address the complexity of RNA quasispecies mutant spectra.(ABSTRACT TRUNCATED AT 400 WORDS)
Biological Evolution↗
[A surgical alert in coronary angioplasty in 1994].
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Angioplasty, Balloon, Coronary↗
[An evolutionary study at 5 years of left ventricular systolic function in patients with an uncomplicated infarct].
INTRODUCTION AND OBJECTIVES: The aim of the present study was to compare the left ventricular ejection fraction in a first uncomplicated acute myocardial infarction before hospital discharge (initial ejection fraction) and 5 years later (late ejection fraction) and to evaluate the factors that may influence their possible changes. PATIENTS AND METHODS: Eighty-three patients (mean age: 52.2 +/- 7.5, range: 35-65 years) who had a first uncomplicated infarction were evaluated. Between 10 and 15 days after the acute episode, thallium perfusion scintigraphy, radionuclide ventriculography and coronary arteriography were carried out. The ventricular function was reevaluated after 5 years with radionuclide ventriculography. The population was classified according to the localization of infarction (anterior or inferior) and to the initial ejection fraction (< 30%, 30-50% and > 50%). The factors derived from predischarge exercise test, perfusion scintigraphy, radionuclide ventriculography and coronary arteriography that might have had an influence on ejection fraction changes were evaluated with bivariate and multivariate analysis. RESULTS: 1) left ventricular ejection fraction of anterior infarctions was lower than in inferior infarctions, both during the subacute phase (35.1 +/- 12.9 vs 48.1 +/- 12.1; p < 0.001) and after 5 years (41.3 +/- 15.1 vs 47.6 +/- 12.8; p = 0.006); 2) a significant increase in ejection fraction after 5 years was found in patients with anterior infarction (35.1 +/- 12.9 vs 41.3 +/- 15.1; p < 0.001), particularly in those with initial ejection fraction between 30-50% (38.8 +/- 5.9 vs 44.8 +/- 11.2; p < 0.001), and 3) no variable, either clinical or derived from exercise test, perfusion scintigraphy, radionuclide ventriculography or coronary arteriography, discriminated between the patients in whom ejection fraction decreased by more than 5% during follow-up nor those with anterior infarction and initial ejection fraction between 30-50% in whom ventricular function was improved after 5 years. CONCLUSIONS: Left ventricular ejection fraction of anterior infarctions shows a significant improvement after 5 years, particularly in the subgroups with initial ejection fraction between 30-50%. Predictive factors of such improvement have not been identified.
Adult↗
Use of substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot-and-mouth disease virus.
Antigenic site A of foot-and-mouth disease virus (FMDV) is an exposed, mobile loop which includes a central, highly conserved Arg-Gly-Asp tripeptide (RGD, VP1 residues 141-143 in serotype C) thought to be part of the cell attachment site. We have analyzed the contribution of RGD to the interaction of site A with antibodies by incorporating selected amino acid replacements at RGD into synthetic peptides representing site A, and analyzing the reactivity of substituted peptides with site A-specific monoclonal antibodies (MAbs). Replacement of Arg-141, Gly-142 or Asp-143 by alanine resulted in the loss of one, three and five epitopes, respectively, out of seven epitopes probed. Other replacements resulted in the loss of even larger numbers of epitopes, suggesting that the amino acids of the RGD region are either directly involved in interaction with antibodies or that they exert an important influence on the interaction of surrounding residues with antibodies. Thus, we explored the ability of tandem repeats of the RGDL sequence (corresponding to FMDV C-S8c1) to evoke neutralizing antibodies in rabbits and guinea pigs. Neutralizing activity was generally low but with a broad specificity for different FMDV serotypes and variants. Significant decreases in neutralizing titers were observed with boosting, suggesting a possible suppression of those anti-peptide antibodies which may also be directed to cellular RGD sequences. The results point to an involvement of RGD in the antigenic structure of site A, and open the possibility that broadly neutralizing antibodies might be induced by tandem repeats of the critical, conserved domain.
Amino Acid Sequence↗
Cyclic disulfide model of the major antigenic site of serotype-C foot-and-mouth disease virus. Synthetic, conformational and immunochemical studies.
A cyclic disulfide peptide representing antigenic site A of foot-and-mouth disease virus (FMDV) strain C-S8c1 (residues 134 to 155 of viral protein 1 (VP1) with Tyr136 and Arg153 replaced by cystine; TTCTASARGDLAHLTTTHACHL) was synthesized by solid phase methods. Formation of the cyclic disulfide was carried out by air oxidation of the fully deprotected and reduced bis-cysteine precursor, under high dilution conditions. The identity of the cyclic peptide was confirmed by both physical and enzymatic methods. A conformational study of the cyclic peptide and of its linear parent structure (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1 of FMDV C-S8c1) by circular dichroism in the presence of a structure-inducing solvent showed the cyclic disulfide analog to adopt lower levels of alpha-helix than its linear counterpart. In competitive ELISA assays both peptides reacted with similar affinity against a representative panel of neutralizing monoclonal antibodies directed towards antigenic site A. Thus, a high inherent flexibility of this loop may preclude a conformational restriction strong enough to alter recognition by anti-virus antibodies.
Amino Acid Sequence↗
Viral genetic variation: hepatitis B virus as a clinical example.
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Genetic Variation↗
The two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities.
Initiation of protein synthesis on the foot-and-mouth disease virus RNA occurs at two sites, thus, two forms of the leader protein, termed Lab and Lb, are produced. Plasmids have been constructed which encode these proteins either together or individually. Plasmids encoding the Lab protein alone express a modified form of this protein in which the second methionine residue, which corresponds to the first amino acid of Lb, is changed to an alternative residue. Four different mutant forms of the Lab sequence were made. Each of the plasmids was introduced into a mammalian cell transient expression system which allowed the determination of the known activities of the L proteins. It was shown that the Lb protein and each of the modified Lab proteins were capable of cleaving the L/P1 junction in trans. Furthermore, each of these proteins induced the cleavage of the p220 component of the cap-binding complex (eIF-4F) producing inhibition of cap-dependent translation. These results indicate that the two species of L have the same functions.
Aphthovirus↗
Rat myocardial tissue lipids and their effect on ventricular electrical activity: influence on dietary lipids.
OBJECTIVE: The aim was to investigate the effect of diets with different lipid content on rat myocardial tissue lipid composition and their possible influence on myocardial electrical activity. METHODS: 60 male Sprague-Dawley rats were randomised in three dietary feeding groups. Half the animals were used for the myocardial lipid study and the other half for the ventricular refractory period and ventricular conduction velocity measurements. Synthetic diets of low fat, high fat (predominantly lard fat), and high fat plus marine oil, the last two with cholesterol, were supplied ad libitum for five weeks. After 2-propanol myocardial lipid extraction, lipid fractions were separated by thin layer chromatography and their esterified fatty acids by gas-liquid chromatography. Ventricular refractory period was obtained according to the extrastimulus technique and maximum conduction velocity by ventricular pacing. RESULTS: The experimental diets induced marked changes in fatty acid composition of myocardial phospholipids and in esterified cholesterol content. The high fat group showed a significant decrement in oleic and linoleic acids, with an increment in arachidonic and docosahexaenoic acids in their phospholipid composition. This dietary group had the highest esterified cholesterol content. These changes were related to lowering of maximum ventricular paced heart rate and lengthening of ventricular refractory period, and were partly corrected by marine oil supplement. CONCLUSIONS: Saturated fat diets cause profound changes in myocardial fatty acyl composition which are linked to sustained differences in myocardial electrical activity. These changes can be partly corrected by a moderate fish oil supplement.
Animals↗
New observations on antigenic diversification of RNA viruses. Antigenic variation is not dependent on immune selection.
Recent results have revealed novel features in the process of antigenic diversification of FMDV. (i) Antigenic variation is not necessarily the result of immune selection. (ii) Single, critical amino acid replacements may either have a minor effect on antigenic specificity or cause a drastic antigenic change affecting many epitopes on an antigenic site. (iii) The effect of such a critical replacement may be suppressed by additional substitutions at neighbouring sites. (iv) Antigenic diversification does not necessarily involve net accumulation of amino acid substitutions over time. We review evidence that some of these features apply also to other riboviruses and retroviruses. A model is proposed to relate antigenic variation without immune selection to the quasispecies structure of RNA virus populations.
Antigenic Variation↗
Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.
Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.
Cell Line↗
Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.
Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.
Amino Acid Sequence↗
Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.
We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.
Acquired Immunodeficiency Syndrome↗
Many-trillionfold amplification of single RNA virus particles fails to overcome the Muller's ratchet effect.
We showed earlier that transfers of large populations of RNA viruses lead to fitness gains and that repeated genetic bottleneck transfers result in fitness losses due to Muller's ratchet. In the present study, we examined the effects of genetic bottleneck passages intervening between population passages, a process akin to some natural viral transmissions, using vesicular stomatitis virus as a model. Our findings show that the pronounced fitness increases that occur during two successive population passages cannot overcome the fitness decreases caused by a single intervening genetic bottleneck passage. The implications for natural transmissions of RNA viruses are discussed.
Genetic Variation↗
A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.
Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.
Animals↗
[The usefulness of Doppler echocardiography in the preoperative assessment of valvulopathies. A comparison with the hemodynamic and surgical findings].
The Doppler echocardiography and cardiac catheterization studies of all patients who underwent valvular surgery in a three-year period were reviewed to assess the correlation between the estimated severity of valvular disease by both methods. Two-hundred and thirty-five patients (group I: 140 male, age 58 +/- 12; 95 female, age 60 +/- 13) underwent both studies within 6 months. There was agreement on estimation of severity of valve lesions in 140 of 162 patients with aortic valve disease (93% of stenosis, 82% of regurgitations and 79% of mixed lesions), in 58 of 80 patients with mitral valve disease (83% of stenosis, 76% of regurgitations and 33% of mixed lesions) and in 10 of 16 patients with prosthetic valve disfunction. The correlation between both methods was significantly lower in mixed mitral lesions than in the remaining native valve lesions (p < 0.05). Significant disagreement occurred in 4 cases of aortic valve disease, four of mitral valve disease and five of prosthetic disfunction. When disagreement was present, Doppler often underestimated the severity of the disease. Disagreement was more frequent in patients with combined aortic and mitral disease. According to the surgical conclusions cardiac catheterization provided a diagnostic profit in the assessment of the disease severity in 8, 11 and 22% of cases of aortic and mitral valve disease and prosthetic valve disfunction, respectively. Coronary artery disease was present in 19% of patients who underwent coronary arteriography. One-hundred and two patients (group II: 44 m, 48 +/- 15; 58 f, 53 +/- 11) underwent surgery without previous cardiac catheterization.(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗
Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase.
The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10(-3)) were observed at this (guanine) site. We sought evidence for RNA 3'-->5' exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[1-thio]triphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.
Base Sequence↗
Rapid fitness losses in mammalian RNA virus clones due to Muller's ratchet.
Muller's ratchet is an important concept in population genetics. It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms. In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair of genetic damage. Experimental support for Muller's ratchet has previously been obtained in protozoa and in a tripartite RNA bacteriophage. We now show clear evidence that Muller's ratchet can operate on a nonsegmented nonrecombining pathogenic RNA virus of animals and humans. We did genetic bottleneck passages (plaque-to-plaque transfers) of vesicular somatitis virus (VSV) and then quantitated relative fitness of the bottleneck clones by allowing direct replication competition in mixed infections in cell culture. We document variable fitness drops (some severe) following only 20 plaque-to-plaque transfers of VSV. In some clones no fitness changes (or only insignificant changes) were observed. Surprisingly, the most regular and severe fitness losses occurred during virus passages on a new host cell type. These results again demonstrate the extreme genetic and biological variability of RNA virus populations. Muller's ratchet could have significant implications for variability of disease severity during virus outbreaks, since genetic bottlenecks must often occur during respiratory droplet transmissions and during spread of low-yield RNA viruses from one body site to another (as with human immunodeficiency virus). Likewise, the lower-probability generation of increased-fitness clones during repeated genetic bottleneck transfers of RNA viruses in nature might also affect disease pathogenesis in infected individuals and in host populations. Whenever genetic bottlenecks of RNA viruses occur, enhanced biological differences among viral subpopulations may result.
Animals↗