Biomedical subjects

E Domingo

Publications and source records attributed to E Domingo.

At least 127 records · Page 7Linked to original sources

PubMed1995

Direct evaluation of the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host.

The immunodominance of a major antigenic site of foot-to-mouth disease virus (FMDV) (serotype C; clone C-S8c1) in a natural host has been evaluated by serum immunoglobulin fractionation. Nineteen sera from either convalescent or vaccinated swine were fractionated by affinity chromatography using a synthetic peptide representing antigenic site A (the G-H loop of capsid protein VP1) coupled to a Sepharose matrix. Antigen-binding and neutralizing activities of serum fractions were quantitated. On average, about 57 or 27% of the virus-neutralizing activity (and about 35 or 12% of the virus-binding activity) from convalescent or vaccinated swine, respectively, corresponded to antibodies against site A. The results provide direct evidence of the important contribution of site A, and also of additional sites unrelated to site A, in the evoking of neutralizing antibodies by FMDV in a natural host. The proportion of antibodies directed to site A varied greatly among individual swine. Some animals evoked remarkably low levels of antibodies specific for site A although they were competent to raise antibodies against other antigenic sites of FMDV. Thus, the major antigenic site of FMDV shows heterogeneous dominance in a natural host. Possible implications for evolution of viral quasispecies are discussed.

Amino Acid Sequence↗

PubMed1995

Molecular evolution of aphthoviruses.

Aphthoviruses are an important group of animal pathogens. A combination of genetic and structural studies has revealed one of the main principles governing their evolution: severe limitations to variation imposed by functional and structural constraints, in conjunction with high mutation and recombination rates operating during genome replication. Evolution occurs by positive selection and random drift acting on complex quasispecies distributions. The mutant composition of a quasi-species (or mutant spectrum) is largely dictated by tolerance to nucleotide and amino acid substitutions in viral RNAs and proteins, which must remain functionally competent. We review recent evidence to support this proposal, and we suggest that similar concepts may apply to other RNA viruses as well.

Adaptation, Physiological↗

PubMed1995

The sympathetic nervous system modulates perception and reflex responses to gut distention in humans.

BACKGROUND/AIMS: Intestinal distention induces perception and gut reflexes via sympathetic and vagal pathways, but the modulatory mechanisms of such responses remain obscure. The aim of this study was to determine the effects of sympathetic nervous activity on sympathetic and vagal reflexes as well as on intestinal and somatic perception. METHODS: In 9 healthy volunteers, proximal duodenal distentions were produced in 4-mL increments and hand transcutaneous electrical nerve stimulation was produced in 3-mA increments. Increasing stimuli of 1-minute duration were randomly performed at 10-minute intervals both with and without sympathetic activation (induced by means of lower body negative pressure). Intestinal and somatic perception was scored by specific questionnaires; vagal enterogastric and sympathetic intestinointestinal relaxatory reflexes were simultaneously measured by gastric and distal duodenal barostats. RESULTS: Sympathetic activation significantly heightened perception of intestinal distention without modifying perception of somatic stimuli (perception scores increased by 41% and -2%, respectively). The reflex responses to duodenal distention significantly increased during sympathetic activation both in the stomach and in the intestine (relaxation increased by 91% and 69%, respectively; P < 0.05 for both). CONCLUSIONS: Activation of the sympathetic nervous system selectively increases visceral but not somatic sensitivity and enhances both vagally and sympathetically driven reflexes in the gut.

Adult↗

PubMed1995

Large deletions in the 5'-untranslated region of foot-and-mouth disease virus of serotype C.

Nucleotide sequences of the 5'-untranslated region (5'-UTR), at the 3'-side of the poly C tract, have been compared for 21 isolates of foot-and-mouth disease virus (FMDV) of serotype C from Europe, South America and The Philippines. A deletion of 43 nucleotides is present in the European isolates as compared with most American isolates. A larger deletion of 86 nucleotides is present in some viruses from South America and The Philippines. These deletions include the loss of one or two pseudoknot structures predicted in this region of the 5'-UTR. In addition, multiple point mutations have allowed the derivation of a phylogenetic tree which defines a grouping of isolates very similar to that derived from the capsid gene sequences of the same viruses. The study provides evidence that deletion (or addition) events must be very frequent during evolution of FMDV type C, since viruses which are phylogenetically very closely related (they belong to the same tree branch) may differ in the presence or absence of these deletions. Implications for FMDV evolution are discussed.

Animals↗

PubMed1995

[Transtracheal catheter and liquid oxygen: 5 years of experience].

We studied clinical tolerance, complications, change in pulmonary function test results, arterial gasometrics, hemoglobin (Hb), 6 minute stress tests (6 wt) and subjective perception of dyspnea assessed on a visual analog scale (VAS) in a group of 18 patients (17 with obstructive disease and one with restrictive disease). These patients had previously been enrolled in a home oxygen therapy (HOT) program to deliver continuous oxygen therapy through nasal prongs, and had accepted portable oxygen therapy delivered by transtracheal catheter (TTC) from 1988 until 1993. Tolerance was good, there were no lethal complications of TTC, and excellent compliance with the prescribed HOT was achieved. Lung function test results worsened, while Hb and PaO2 improved and significant oxygen savings (50%) were achieved. The 6 wt test results had not worsened at the end of the first year but did so significantly at the end of the third year, in spite of a relative preservation of lung function. Dyspnea assessed on a VAS was not seen to worsen. We conclude that tolerance of the method was good and that no relevant complications occurred. HOT by TTC did not prevent worsening of bronchial obstruction. Oxygenation of patients was better, as shown by the decrease in Hb and the improvement in PaO2 at the end of the first year of monitoring. Changes in 6 wt showed that in order to achieve greater benefit from HOT by TTC, patients should follow a pulmonary rehabilitation program.

Aged↗

PubMed1995

Molecular characterization of human immunodeficiency virus type 1 isolates from Venezuela.

Eight HIV-1 isolates from Venezuela have been characterized by nucleotide sequencing of the entire reverse transcriptase (RT)- and surface glycoprotein (gp 120)-coding regions. Average mutant frequencies were 2.5 x 10(-2) substitutions per nucleotide (s/nt) for the RT-coding region, and 10 x 10(-2) or 6.8 x 10(-2) s/nt for the gp120-coding region, depending on whether gaps introduced for optimal alignment were or were not, respectively, considered in the calculations. Phylogenetic trees were derived by maximum-likelihood, neighbor-joining, and maximum parsimony methods. In the trees derived from both RT- and gp120-coding regions, Venezuelan isolates cluster with subtype B viruses. However, the relative position of some of the isolates is considerably different in the two trees. Unique V3 loop amino acid sequences, not represented in the current database, have been identified among the Venezuelan isolates. In addition to representing the first molecular characterization of HIV-1 from Venezuela, the extensive genetic heterogeneity observed reinforces the interest in characterizing additional HIV-1 isolates worldwide for adequate vaccine design.

Adult↗

PubMed1995

A highly divergent antigenic site of foot-and-mouth disease virus retains its immunodominance.

The ability of a highly divergent antigenic site of foot-and-mouth disease virus (FMDV) of serotype C to elicit neutralizing antibodies has been evaluated in mice and rabbits. The viruses compared, FMDV C-S8c1 and HR, differ in a single amino acid replacement in their capsid proteins, but represent two extreme antigenic specificities of the major antigenic site A of FMDV type C. Both, studies of cross-neutralization of homologous and heterologous virus, and fractionation of site A-specific antibodies by immunoaffinity chromatography suggest a similar immunodominance of antigenic site A in FMDV C-S8c1 and variant HR. This information is relevant to the formulation of synthetic peptide vaccines that ideally should consist of mixtures of peptides representing several antigenic specificities. These cocktail formulations may be required to control diseases caused by FMDV and, generally, by highly variable RNA viruses, since single specificity peptides may trigger selection of vaccine-escape viral mutants.

Amino Acid Sequence↗

PubMed1995

Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy.

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.

Amino Acid Sequence↗

PubMed1995

Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.

Animals↗

PubMed1995

Size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness.

Genetic bottlenecks are important events in the genetic diversification of organisms and colonization of new ecological niches. Repeated bottlenecking of RNA viruses often leads to fitness losses due to the operation of Muller's ratchet. Herein we use vesicular stomatitis virus to determine the transmission population size which leads to fitness decreases of virus populations. Remarkably, the effective size of a genetic bottleneck associated with fitness loss is greater when the fitness of the parental population increases. For example, for starting virus populations with low fitness, population transfers of five-clone-to-five-clone passages resulted in a fitness increase. However, when a parental population with high fitness was transferred, 30-clone-to-30-clone passages were required simply to maintain fitness values.

Animals↗

PubMed1995

Cyclic peptides as conformationally restricted models of viral antigens: application to foot-and-mouth disease virus.

Conformationally restricted cyclic peptide mimics of the antigenic site A of foot-and-mouth disease virus serotype C-S8c1 have been designed, first by comparison to the three-dimensional structure of the O1BFS serotype, later more accurately on the basis of X-ray diffraction data from a complex between a linear peptide reproducing site A and an FMDV-derived monoclonal antibody Fab fragment. A variety of cyclization strategies have been attempted, both in solution and in the solid phase, involving disulfide, side chain lactam and head-to-tail arrangements. Preliminary immunological results have shown one of the cyclic disulfide mimics to be a better immunogen than its linear counterpart.

Amino Acid Sequence↗

PubMed1994

The red queen reigns in the kingdom of RNA viruses.

Two clonal populations of vesicular stomatitis virus of approximately equal relative fitness were mixed together and allowed to compete during many transfers in vitro as large virus populations. Eventually, one or the other population suddenly excluded its competitor population, yet both the winners and losers exhibited absolute gains in fitness. Our results agree with the predictions of two major theories of classical population biology; the Competitive Exclusion Principle and the Red Queen's Hypothesis, where (in Lewis Carroll's words) "it takes all the running you can do to keep in the same place."

Animals↗

PubMed1994

Rapid cell variation can determine the establishment of a persistent viral infection.

Evidence for a mechanism of initiation of viral persistence in which the cell, and not the virus, plays a critical role has been obtained using the important animal pathogen foot-and-mouth disease virus (FMDV). We have developed a virulence assay consisting of quantification of the ability of virus to kill cells and of cells to divide in the presence of virus and to initiate a carrier state. Cells were cured of FMDV at early times following a cytolytic infection of BHK-21 monolayers with FMDV. When cured cells were subjected to the virulence assay they showed an increased ability to survive a second infection by FMDV but not by other RNA viruses. This altered phenotype was maintained as a stable genetic trait. When the virus present in such early surviving cells was used to infect BHK-21 cells, it proved to be as virulent as the initial cytolytic FMDV and, furthermore, its ability to kill BHK-21 cells increased upon replication in the surviving cells. Both the level of genetic heterogeneity and the rate of evolution of FMDV were similar to those previously documented during acute and persistent FMDV infections. The results suggest that, in contrast to most other viral systems, the critical element in the establishment of a persistent infection of BHK-21 cells with FMDV is the ability of the host cells to vary genetically and phenotypically, which promotes selection of cells with increased resistance to virus. The possible relevance of this mechanism to viral persistence in vivo is discussed.

Animals↗

PubMed1994

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND: Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS: The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION: In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment against foot-and-mouth disease virus and of its complex with the main antigenic site peptide.

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot-and-mouth disease virus (FMDV) C-S8c1 and its complex with a peptide, corresponding to the major antigenic site of FMDV (VP1 residues 136-150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 A resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 A, b = 89.12 A, c = 64.04 A, and beta = 112.9 degrees and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 A resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 A, b = 60.67 A, c = 143.45 A, and beta = 95.4 degrees. Density packing considerations indicate that there are two Fab molecules in the asymmetric unit.

Amino Acid Sequence↗

PubMed1994

Antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites.

Antigenic variation in a major discontinuous site (site D) of foot-and-mouth disease virus (FMDV) of serotype C has been evaluated with neutralizing monoclonal antibodies. Isolates representing the major evolutionary sublines previously defined for serotype C were compared. Extensive variation, comparable to that of continuous epitopes within the hypervariable immunodominant site A (the VP1 G-H loop), was found. The amino acid sequences of the complete capsids of three antigenically highly divergent FMDVs (C1 Haute Loire-Fr/69, C5 Argentina/69, and C3 Argentina/85) have been determined and compared with the corresponding sequences previously determined for seven additional type C viruses. Differences in antigenicity are due to a very limited number of substitutions of surface amino acids accessible to antibodies and located within antigenic sites previously identified on FMDV. A significant number of residues at these positions were also replaced in monoclonal antibody escape mutants. Depending on the variants compared, replacements within site A or at site D, or at both sites, contributed significantly to their antigenic differences. Examples of divergence mediated by a few amino acid replacements were found among FMDVs of Europe and South America. The results suggest that within a serotype of FMDV, antigenically highly divergent viruses can arise in the field by very limited sequence variation at exposed key residues of each of several antigenic sites.

Amino Acid Sequence↗

PubMed1994

Subclonal components of consensus fitness in an RNA virus clone.

Most RNA virus populations exhibit extremely high mutation frequencies which generate complex, genetically heterogeneous populations referred to as quasi-species. Previous work has shown that when a large spectrum of the quasi-species is transferred, natural selection operates, leading to elimination of noncompetitive (inferior) genomes and rapid gains in fitness. However, whenever the population is repeatedly reduced to a single virion, variable declines in fitness occur as predicted by the Muller's ratchet hypothesis. Here, we quantitated the fitness of 98 subclones isolated from an RNA virus clonal population. We found a normal distribution around a lower fitness, with the average subclone being less fit than the parental clonal population. This finding demonstrates the phenotypic diversity in RNA virus populations and shows that, as expected, a large fraction of mutations generated during virus replication is deleterious. This clarifies the operation of Muller's ratchet and illustrates why a large number of virions must be transferred for rapid fitness gains to occur. We also found that repeated genetic bottleneck passages can cause irregular stochastic declines in fitness, emphasizing again the phenotypic heterogeneity present in RNA virus populations. Finally, we found that following only 60 h of selection (15 passages in which virus yields were harvested after 4 h), RNA virus populations can undergo a 250% average increase in fitness, even on a host cell type to which they were already well adapted. This is a remarkable ability; in population biology, even a much lower fitness gain (e.g., 1 to 2%) can represent a highly significant reproductive advantage. We discuss the biological implications of these findings for the natural transmission and pathogenesis of RNA viruses.

Animals↗

PubMed1994

Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.

Animals↗