Foot-and-mouth disease virus lacking the VP1 G-H loop: the mutant spectrum uncovers interactions among antigenic sites for fitness gain.

The Arg-Gly-Asp (RGD) triplet found in the G-H loop of capsid protein VP1 of foot-and-mouth disease virus (FMDV) is critically involved in the interaction of FMDV with integrin receptors and with neutralizing antibodies. Multiplication of FMDV C-S8c1 in baby hamster kidney 21 (BHK-21) cells selected variant viruses exploiting alternative mechanisms of cell recognition that rendered the RGD integrin-binding triplet dispensable for infectivity. By constructing chimeric viruses, we show that dispensability of the RGD in these variant FMDVs can be extended to surrounding amino acid residues. Replacement of eight amino acid residues within the G-H loop of VP1 by an unrelated FLAG marker yielded infectious virus. Evolution of FLAG-containing viruses in BHK-21 cells generated complex quasispecies in which individual mutants included amino acid replacements at other antigenic sites of FMDV. Inclusion of such replacements in the parental FLAG clone resulted in an increase of relative fitness of the viruses. These results suggest structural or functional connections between antigenic sites of FMDV and underscore the value of mutant spectrum analysis for the identification of fitness-promoting genetic modifications in viral populations. The possibility of producing viable viruses lacking antigenic site A may find application in the design of new anti-FMD vaccines.

Evolution of cell recognition by viruses.

Evolution of receptor specificity by viruses has several implications for viral pathogenesis, host range, virus-mediated gene targeting, and viral adaptation after organ transplantation and xenotransplantation, as well as for the emergence of viral diseases. Recent evidence suggests that minimal changes in viral genomes may trigger a shift in receptor usage for virus entry, even into the same cell type. A capacity to exploit alternative entry pathways may reflect the ancient evolutionary origins of viruses and a possible role as agents of horizontal gene transfers among cells.

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.

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.

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.

[Quasispecies and molecular evolution of viruses]. / Cuasiespecies y evolución molecular de virus.

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.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

[Variability and development of viral populations: assessment and implications]. / Variabilité et evolution des populations virales: bilan et implications.

RNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases.

Quasispecies structure and persistence of RNA viruses.

Viral quasispecies are closely related (but nonidentical) mutant and recombinant viral genomes subjected to continuous genetic variation, competition, and selection. Quasispecies structure and dynamics of replicating RNA enable virus populations to persist in their hosts and cause disease. We review mechanisms of viral persistence in cells, organisms, and populations of organisms and suggest that the critical interplay between host and viral influences (including in some cases the quasispecies organization) is the main driving force for long-term survival of viruses in nature.

Multiple virulence determinants of foot-and-mouth disease virus in cell culture.

Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.

An RNA virus can adapt to the multiplicity of infection.

RNA viruses evolve as complex distributions of mutants termed viral quasispecies. For this reason it is relevant to explore those environmental parameters that favour the selective advantage of some viral subpopulations over others. In the present study we provide direct evidence that the relative fitness of two competing viral subpopulations may depend on the multiplicity of infection (m.o.i.). Two closely related subpopulations of foot-and-mouth disease virus (FMDV) of serotype C, which differed in their history of cytolytic passages in BHK-21 cells, were subjected to growth-competition experiments in BHK-21 cells. One of the populations, termed S, was found to have a selective advantage over the other population, termed L, only when the competition passages were carried out at low m.o.i. In contrast, both populations, L and S, coexisted during serial passages carried out at high m.o.i. No differences between S and L were detected in assays of inhibition of infectivity by synthetic peptides, in cell binding-competition experiments, or in virulence for BHK-21 cells. However, FMDV S displayed increased heparin binding compared with L, and L higher virulence for Chinese hamster ovary (CHO) cells than S. These results with FMDV suggest that small differences in the interaction of the virus with the host cell may contribute to an m.o.i.-dependent selective advantage of one viral subpopulation over a closely related subpopulation. Therefore, different viral mutants from quasispecies replicating in vivo may be selected depending on the number of variant viruses relative to the number of susceptible cells.

Serum lipids and apolipoproteins in Spanish children and adolescents: a 5 year follow-up.

This study was designed to assess "tracking" of serum lipids and apolipoproteins in three age groups of Spanish children over a 5 year period. A total of 84 6-year-old, 89 10-year-old and 64 14-year-old children were evaluated in 1989 (with measurement of serum total cholesterol, triglycerides, lipoproteins and apolipoproteins A1 and B), and re-evaluated in 1994. Correlation coefficients between initial and final lipid and apolipoprotein values were as follows: total cholesterol, 0.66; low-density lipoprotein (LDL) cholesterol, 0.65; high-density lipoprotein (HDL) cholesterol, 0.61; triglycerides, 0.61; apolipoprotein A1, 0.60; apolipoprotein B, 0.66. When age groups were analysed separately, children who were 14 years old at the beginning of the study showed higher correlation coefficients, particularly for total cholesterol and LDL cholesterol ( > 0.7 in both cases). More than 70% of children who were in the top quintile of total, LDL or HDL cholesterol as well as apolipoprotein A1 or B in 1989 remained in the top quintile 5 years later.

Urinary albumin excretion in Spanish children. Niño Jesus Group.

We measured urinary albumin excretion in 2,224 school-children (1,168 boys, 1,056 girls) aged 2-18 years, between 1989 and 1990 to establish reference values. We recorded all pathological antecedents and findings from physical examination, including anthropometric parameters and arterial blood pressure. The analytical study included serum total protein, albumin and creatinine. The second-morning urine and the nightly (rest) 10-h urine sample were collected and we determined the concentration of albumin and creatinine. We found a positive statistically significant correlation between the urinary albumin excretion (micrograms/10 h) and age, height, weight and body surface area. We suggest that it would be useful to relate the urinary albumin excretion to body surface area. The mean value for albumin excretion was 3.49 micrograms/ml in boys and 3.63 micrograms/ml in girls. The urinary albumin/creatinine ratio showed a high correlation with the albumin excretion (r = 0.958).

Relations between nutrient intake and serum lipid and apolipoprotein levels.

OBJECTIVE: To study the relation between diet and serum lipid and apolipoprotein levels in a large, homogeneous group of Spanish children. DESIGN: Survey. SETTING: Eleven schools chosen at random in Madrid City and the surrounding area. SUBJECTS AND METHODS: Subjects comprised 1682 children, 2 to 12 years of age. Dietary data were obtained with a 24-hour record performed by the child's main caregiver. For statistical analysis the sample was divided into tertiles on the basis of calorie-adjusted consumption of dietary components. RESULTS: Children in the highest tertile of total fat consumption, compared with children in the lowest tertile, had significantly higher mean serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (Apo A) and apolipoprotein B-100 (Apo B). When compared with children in the lowest tertile, children in the highest tertile of saturated fat consumption had significantly higher mean levels of TC, LDL-C, and Apo B, and lower mean levels of HDL-C and Apo A. Children in the highest tertile of monounsaturated fat consumption, compared with children in the lowest tertile, had significantly higher mean levels of HDL-C and Apo A, and lower mean levels of TC, LDL-C, and Apo B. CONCLUSIONS: Our findings suggest that diet composition strongly influences lipid profile in children and point out the importance of monounsaturated fatty acids as modulators of serum lipid and apolipoprotein levels.

Diet therapy for hypercholesterolemia in children and adolescents. A follow-up.

OBJECTIVE: To determine the effectiveness and compliance of dietary restriction in a group of children and adolescents with hypercholesterolemia. RESEARCH DESIGN: Prospective clinical trial. SETTING: Pediatric hospital in Madrid, Spain. PARTICIPANTS: Four hundred fifty-one children and adolescents of both sexes aged 2 to 18 years diagnosed as having hypercholesterolemia and treated with dietary restriction (American Heart Association Step-One and Step-Two Diets). The follow-up period ranged from 6 months to 2 years and was performed by one pediatrician. RESULTS: Total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B-100 levels as well as the low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio decreased significantly (P < .01) from the first month of dietary restriction, whereas the high-density lipoprotein cholesterol level increased (P < .01). The apolipoprotein A-I level increased significantly (P < .01) only after 6 months of diet therapy. After 12 months of follow-up, 30.7% (58/189) of patients did not follow the diet strictly. In this group, significantly higher levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B-100 were found. CONCLUSIONS: Diet therapy is effective in the treatment of hypercholesterolemia in children and adolescents. Dietary restriction has a beneficial effect not only on total cholesterol and low-density lipoprotein cholesterol levels but also on apolipoprotein A-I and B-100 levels.

Smoking and apolipoproteins in adolescents. The Niño Jesus Group.

The relation between smoking and blood lipids and apolipoproteins (A1,B100) were studied in a group of 1024 12- to 18-year-old school children in the Comunidad de Madrid. The percentage of smokers was 19% (17% for girls and 21% for boys). The average consumption of cigarettes per day was 7.83 +/- 5.06 in boys and 6.04 +/- 3.49 in girls (p less than 0.05). As compared with male nonsmokers, male smokers showed a higher mean level of low-density lipoprotein (LDL) cholesterol (112 versus 100 mg/dL, p less than 0.05), a higher LDL cholesterol to HDL-cholesterol ratio (2.27 versus 1.94, p less than 0.001), a higher mean level of apolipoprotein B100 (59 versus 53 mg/dL, p less than 0.05), and a higher apolipoprotein B100 to apolipoprotein A1 ratio (0.45 versus 0.40, p less than 0.01). Female smokers tended to show the same results, although significant differences were only found for LDL cholesterol to HDL cholesterol ratio and apolipoprotein B100 to apolipoprotein A1 ratio (1.8 versus 1.59 and 0.41 versus 0.38 respectively, both p less than 0.05). This work provides new data about the effects of smoking on apolipoproteins in adolescents and emphasizes on the need for preventive programs.

[Variations in the excretion of N-acetyl-glucosaminidase in the first year of life]. / Variación de la excrección de N-acetil-glucosaminidasa en el primer año de vida.

We have studied 122 patients, all younger than 12 months of age, to whom we check the N-acetylglucosaminidase/creatinine (NAG/creatinine) ratio in the first morning urine. The NAG/creatinine values were decreasing with age increase of the patients, being significantly different (p less than 0.001) in the younger 3 months of age comparative with the older than six months of age. The NAG/creatinine values were inversely correlated with age, height and weight. We have made normal values percentils of the NAG/creatinine ratio for each age group.

[The Fuenlabrada study: relationship between anthropometric variables and cardiovascular risk factors]. / Estudio de Fuenlabrada: relación de las variables antropométricas con los factores de riesgo cardiovascular.

Anthropometric variables were studied in 2,153 healthy children, 1,115 males and 1,038 females, aged 0 to 18 years, of the Fuenlabrada population, Madrid. Height was measured by an anthropometer and weight by a weighting scale. Tricipital and subscapular skinfold thickness were measured by Holtain caliper. Body mass index (kg/m2) and ponderal index (kg/m3) were calculated from weight and height. In the total population skinfold thickness had the highest correlation with total body fat. But, when only obese children (greater than 95 p) were analyzed, other variables like body mass index and ponderal index had also high correlation. Different fatness trends were observed between sexes, although females always were the fattest. In our population the correlation of skinfold thickness, body mass index and ponderal index with lipids, blood pressure, glycemia and uric acid were significant. Obese children and adolescents had nearly a twofold increase in relative risk of arterial hypertension, hypertriglyceridemia, hyperuricemia and low C-HDL.

[The Fuenlabrada study: a familial aggregation of ischemic cardiopathy and cardiovascular risk factors]. / Estudio de Fuenlabrada: agregación familiar de la cardiopatía isquémica y de los factores de riesgo cardiovascular.

UNLABELLED: Familial aggregation of coronary heart disease (CHD) and coronary risk factors (CRF) were evaluated by clinical history in adult relatives of children surveyed for CRF. Population was divided into two groups: Group I included 2,153 children without parental history of CHD. Group II included 266 children of 112 families with parental history of early CHD (before 56 years). In 105 cases the patients were the fathers and in 7 cases the mothers. All the patients were admitted to a coronary care unit. Familial aggregation of CHD was 9.7 times more frequent in paternal families of group II (p less than or equal to 0.0001) than in group I; there was no differences in maternal families. Higher prevalence of hyperlipidaemia, blood pressure hypertension, smoking habits, diabetes and obesity in fathers of group II was observed. Children in group II showed increased levels of C-LDL and decreased levels of C-HDL compared with those of group I. The prevalence of CRF was also significantly higher in children of group II, although in the 33% of the children of group II no CRF was detected. CONCLUSION: a substantial proportion, but not all cases, of familial aggregation of CHD could be explained by known CRF.