La Escuela de Salud Pública de México: innovación educativa y tecnológica en el nuevo milenio / School of Public Health of Mexico: Educational and technological innovation in the new millennium

Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.

This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants.

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryptophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3'-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.

Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.

Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed.

Quantification of endogenous viral polymerase, 3D(pol), in preparations of Mengo and encephalomyocarditis viruses.

Measurement of an antigenic response to the aphthovirus infection-associated antigen (VIA), the viral RNA polymerase 3D(pol), is frequently used as a discriminating assay for the extent of viral replication in animals. In practice, animals seropositive for VIA are assumed to have been exposed to live virus, although in fact it is suspected that endogenous 3D(pol) in commercial inactivated vaccines may occasionally stimulate analogous responses and result in false-positive tests for virus exposure. Cardiovirus infections in mice produce similar anti-VIA antibodies, and in view of recently developed attenuated Mengo vaccines and live Mengo vectors, these VIA responses are also under investigation as potential correlates of vaccine efficacy. We have purified recombinant Mengo 3D(pol), developed monoclonal antibodies to the protein, and used these reagents in highly sensitive Western blot assays to quantify the levels of endogenous 3D(pol) in Mengo and encephalomyocarditis virus (EMCV) preparations. The presence of 3D(pol) was detected at all stages of standard vaccine purification procedures, including materials purified by CsCl. Clarified suspensions of Mengo- or encephalomyocarditis virus-infected HeLa cells were found to contain very high quantities of 3D(pol), averaging approximately 1.2-1.5 micrograms of protein/micrograms of virus. Pelleting through 30% sucrose or purification by CsCl removed much of this material, but even these samples retained approximately 0.2-0.4 ng of 3D(pol)/micrograms virus. These ratios represent approximately 1 3D(pol) molecule/20 virus particles in the most highly purified materials and probably indicate that 3D(pol) is a contaminant on the particle surface rather than an intrinsically packaged molecule. In clarified cell lysates, which are commonly used as vaccine inocula, the protein to virus ratio was approximately 210:1, a level that could represent serious contamination problems for future VIA detection if such inocula are used without further purification.

Genetic analysis of mengovirus protein 2A: its function in polyprotein processing and virus reproduction.

To examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is replaced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro junction was highly efficient. Although the resulting L-P1 precursor was cleaved at the L-VP4 junction, further processing of the P1 precursor was abrogated. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro construct did not result in viable virus. Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was reduced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged. The results also confirm that the processing cascade of L-P1-2A occurs sequentially and is probably regulated by subsequent conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.

Mengo virus 3C proteinase: recombinant expression, intergenus substrate cleavage and localization in vivo.

Mengo virus 3C proteinase was cloned and expressed to high levels in a bacterial vector system. The protein was solubilized from inclusion bodies then purified to homogeneity (> 95%) by ion exchange chromatography. The recombinant enzyme was proteolytically active in cell-free processing assays with a Mengo capsid precursor substrate, L-P1-2A, correctly and proficiently cleaving it into L, 1AB, 1C, 1D and 2A protein products. Further analyses with synthetic peptide substrates encompassing the Mengo or rhinovirus-14 2C/3A cleavage sequences, showed the Mengo 3C could recognize and process specific glutamine-glycine sites within these peptides. The reactivity with the rhinovirus peptide was unexpected, because cross-reactivity between a picornavirus 3C enzyme and a protein substrate from different genus of this family has otherwise never been observed. In reciprocal reactions, a rhinovirus-14 3C preparation was unable to cleave the Mengo-derived synthetic peptide substrate. The recombinant Mengo 3C reactions were also characterized with regard to substrate Km, optimum pH and temperature. The protein was additionally used to raise monoclonal antibodies (mAbs) in mice, which in turn localized natural 3C, 3ABC, 3CD and P3 in immunoblots, immunoprecipitations and indirect immunofluorescence assays of Mengo-infected HeLa cells. The monoclonals showed cross-reactivity with 3C and 3C-containing precursors from encephalomyocarditis virus (EMCV), but did not react with 3C proteins from rhinovirus-14 or poliovirus-1M.

Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase.

The cDNA coding sequence of the RNA-dependent RNA polymerase (3Dpol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3Dpol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3Dpol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3Dpol from poliovirus type 1 and 3 or rhinovirus type 14 and 16.

Inhibition of mengovirus RNA-dependent RNA polymerase by an isatinisothiosemicarbazone and a piperidine-thiocarbonyl-hydrazone derivative in a cell-free system.

The inhibitory action of two antiviral compounds used at the maximum tolerated dosis in a cellular system led to a complete suppression of the infectious mengovirus yield and to a 100% plaque reduction. The products of the RNA polymerase reaction catalyzed by the microsomal-mitochondrial fraction of mengovirus-infected FL cells were analyzed by linear sucrose gradient centrifugation and polyacrylamid gel electrophoresis. The results showed that the inhibitors cause a general reduction of the synthesis of single-stranded viral RNA. The influence on double-stranded viral RNA was more obvious in the case of the isatinisothiosemicarbazone derivative.

The isolation of Mengo virus stable non-capsid polypeptides from infected L cells and preliminary characterization of an RNA polymerase activity associated with polypeptide E.

Isolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS. Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.

Effects of pyrimidine derivatives on RNA-dependent RNA polymerase of mengovirus-infected Fogh and Lund (FL) cells.

Two newly synthesized pyrimidine derivatives were found to possess antiviral activity against Mengovirus in Fogh and Lund (FL) cells and in a cell-free system. The inhibitory effect on RNA-dependent RNA polymerase of Mengovirus-infected FL cells was assayed using 14C-UTP as precursor. Addition of 50 or 100 muM of the inhibitors in a cell-free system of crude enzyme and nucleoside triphosphate medium for 60 min incubation at 37 degrees C resulted in about 40 to 60% lower counting rates for drug-treated reaction mixtures. The analysis of the polymerase synthesis product (virus RNA extracted from the cell-free reaction mixture and deproteinized by the phenol-SDS method) was carried out by means of agarose-acrylamide gel electrophoresis. The main finding was a reduction of single-stranded Mengovirus RNA (RNase-sensitive and LiCl-precipitable). The rates of synthesis of the replicative intermediate (LiCl-precipitable) and the replicative form of RNA (LiCl-soluble) were not significantly influenced.

Alteration in tRNA methyltransferase activity in mengovirus infection: host range specificity.

The tRNA methyltransferase activity in mengovirus-infected L cells, HeLa cells, and Maden Derby bovine kidney cells has been examined during the course of infection. The first two cell lines yield a productive infection, but have different kinetics of inhibition of host RNA synthesis, whereas the bovine kidney cells are a restrictive host. In infected L cells the enzymes show altered capacity and base specificity throughout the infection. In infected HeLa cells and in infected bovine kidney cells less marked changes were seen. No inhibitors or activators of the enzymes were detected in any of the infected cell lines. Labeling experiments in infected cells indicated that in infected L cells synthesis of RNA was inhibited to a greater degree than was methylation of RNA. The consequence of this would be a hypermethylation of RNA. The methylated derivatives synthesized in infected L cells showed changes in relative proportions. Infection of HeLa cells and bovine kidney cells did not show such marked effects on methylation of RNA.