Elisa Carrillo

Evolution of foot-and-mouth disease virus.

Foot-and-mouth disease virus evolution is strongly influenced by high mutation rates and a quasispecies dynamics. Mutant swarms are subjected to positive selection, negative selection and random drift of genomes. Adaptation is the result of selective amplification of subpopulations of genomes. The extent of adaptation to a given environment is quantified by a relative fitness value. Fitness values depend on the virus and its physical and biological environment. Generally, infections involving large population passages result in fitness gain and population bottlenecks lead to fitness loss. Very different types of mutations tend to accumulate in the foot-and-mouth disease virus (FMDV) genome depending on the virus population size during replication. Quasispecies dynamics predict higher probability of success of antiviral strategies based on multivalent vaccines and combination therapy, and this has been supported by clinical and veterinary practice. Quasispecies suggest also new antiviral strategies based on virus entry into error catastrophe, and such procedures are under investigation. Studies with FMDV have contributed to the understanding of quasispecies dynamics and some of its biological implications.

Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

Association of bovine DRB3 alleles with immune response to FMDV peptides and protection against viral challenge.

We have analysed the influence of bovine MHC (BoLA) polymorphism on the immune response and degree of protection induced by peptide vaccines against foot-and-mouth disease (FMD) in cattle. The peptides used for animal immunisation were: A (VP1(138-156)), AT (peptide A linked to VP1(21-40)) and ACT (peptide A, linked to VP1(196-209) and VP1(21-40)). Sixteen different DRB3 types were found among the 46 cattle analysed by PCR-RFLP typing. No absolute correlation was observed, for any type, with the serum neutralising titres (SNT) values and the protection induced. However, among the most common haplotypes present, associations were observed between expression of different types with the levels of SNT and/or protection induced by peptides A and ACT. Thus, types DRB3.2*1, 3 and 7 were associated with increased levels of protection. In contrast, types DRB3.2*12 and 18 were associated non-protection, and DRB3.2*12 was also associated with low SNT titres. Overall, the results indicate that the polymorphism in BoLA class II molecules affects both the immune response and protection induced by potential FMD peptide vaccines.

Evidence of the Coevolution of Antigenicity and Host Cell Tropism of Foot-and-Mouth Disease Virus In Vivo

ABSTRACT In this work we analyze the antigenic properties and the stability in cell culture of virus mutants recovered upon challenge of peptide-vaccinated cattle with foot-and-mouth disease virus (FMDV) C3 Arg85. Previously, we showed that a significant proportion of 29 lesions analyzed (41%) contained viruses with single amino acid replacements (R141G, L144P, or L147P) within a major antigenic site located at the G-H loop of VP1, known to participate also in interactions with integrin receptors. Here we document that no replacements at this site were found in viruses from 12 lesions developed in six control animals upon challenge with FMDV C3 Arg85. Sera from unprotected, vaccinated animals exhibited poor neutralization titers against mutants recovered from them. Sequence analyses of the viruses recovered upon 10 serial passages in BHK-21 and FBK-2 cells in the presence of preimmune (nonneutralizing) sera revealed that mutants reverted to the parental sequence, suggesting an effect of the amino acid replacements in the interaction of the viruses with cells. Parallel passages in the presence of subneutralizing concentrations of immune homologous sera resulted in the maintenance of mutations R141G and L147P, while mutation L144P reverted to the C3 Arg85 sequence. Reactivity with a panel of FMDV type C-specific monoclonal antibodies indicated that mutant viruses showed altered antigenicity. These results suggest that the selective pressure exerted by host humoral immune response can play a role in both the selection and stability of antigenic FMDV variants and that such variants can manifest alterations in cell tropism.

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus.

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.

Effect of lysosomotropic agents on the foot-and-mouth disease virus replication

The effect of two lysosomotropic agents, NH4Cl and chloroquine, on the foot-and-mouth disease virus (FMDV) replicative cycle was studied. When the drugs were present throughout the viral replicative cycle, an important inhibition of viral RNA synthesis and virus production was detected. The inhibition of viral RNA synthesis was maximal when the drugs were present from 30 min before virus infection up to 30 min after that. Otherwise, if the agents were added once the viral synthesis has started (150 min p.i.) the effect was not evident. The agents neither exerted direct virucidal effects, nor did they affect viral adsorption. The results indicate that the lysosomotropic agents affect preferentially an early intracellular event during the viral infective cycle.

Modification of foot-and-mouth disease virus after serial passages in the presence of antiviral polyclonal sera

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, this virus has a high rate of mutation. It has been proposed that selection exerted by the hosts antibodies could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant populations (Nr), after serial passages of cloned FMDV A24 Cruzeiro strain on secondary monolayers of bovine fetal kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages under selective pressure, the virus population showed the following characteristics: (1) increased resistance to neutralization by APS; (2) altered electrophoretic mobility of structural viral proteins (VP1); (3) remarkable plaque size reduction, (4) a pronounced thermosensitivity (ts); and (5) decreased pathogenicity for mice, in both uncloned and cloned small plaque size populations. This indicates that FMDV populations under antibody pressure in vitro, have acquired, in addition to expected characteristics of natural FMDV variants (resistance to neutralization and altered viral structural proteins), phenotypic markers which correspond to attenuated, less virulent variants.

Early steps in FMDV replication: further analysis on the effects of chloroquine

We have previously demonstrated that chloroquine and NH4Cl, two well-known lysosomotropic drugs inhibit foot-and-mouth disease virus (FMDV) replication. This fact points to the relevance of an acidic environment during FMDV penetration. In the present report, we show that chloroquine prevents the cell-mediated disruption of 140 S virions into 12 S particles. This dissociation, which resembles that caused by low pH in vitro, might be an initial uncoating step. Furthermore, we demonstrated that a decrease in the environmental pH counteracts the effect of chloroquine indicating that viral disruption is a low-pH cell-mediated process. The fact that it still occurs at low temperature (20 degrees) and shortly after viral adsorption suggests not only that prelysosomal vesicles represent the putative site for uncoating but also cause the virion to uncoat.

Molecular Characterization and Phylogenetic Analysis of Newcastle Disease Virus Isolates from Healthy Wild Birds

Abstract Wild waterfowl is considered a natural reservoir of potentially infectious agents and a source of pathogenic viruses like avian paramyxoviruses type 1 (APMV 1). In 1997, commercial poultry in Argentina had reached the status of being free from virulent Newcastle disease virus (NDV) infections. Vaccination and biosecurity measures are actively performed to maintain this preferential sanitary condition. However, the risk of reintroduction of pathogenic viruses is always present. In this context, we conducted a study to describe the status of wild healthy birds in a geographic region relevant for the poultry industry. The presence of anti-NDV antibodies was determined in different species in all areas sampled suggesting previous contact with NDV. Seven ND viruses were isolated and characterized as apathogenic strains by biological and molecular methods. The phylogenetic analysis revealed that the majority of the Argentinian isolates form a subgroup related to viruses of genotype II. The results presented here highlight the importance of maintaining strict biosecurity measures and vaccination programs in poultry industries in order to preserve the virulent NDV-free status for commercial flocks in the country.

Biological and molecular characterization of a pigeon paramyxovirus type-1 isolate found in Argentina.

In this report, we describe the biological and molecular characterization of a paramyxovirus type-1 (PPMV-1) isolate found in wild pigeons in an urban habitat in Buenos Aires, Argentina. Of the nine pigeons captured, three were moribund, and the other six showed diarrhea, ataxia, tremor, torticolis, and wing paralysis. The intracerebral pathogenicity index was 1.29, and the amino acid (aa) sequence at the fusion protein cleavage site was 112GRQ KRF117. These characteristics correspond to a virulent Newcastle disease virus isolate. Nevertheless, it was not possible to reproduce the disease in chickens experimentally although the chickens exhibited seroconversion after inoculation. On the other hand, pigeons inoculated with the isolate became sick. These results provide further evidence about the unusual pathogenicity of PPMV-1 for chickens and show once more the need for more biological determinations in these cases to arrive at a final conclusion.