Estudio de la apoptosis en cultivos celulares infectados con el virus de la arteritis equina

La arteritis viral equina es una enfermedad viral muy importante económicamente en la industria equina cuya prevalencia continúa en aumento, posiblemente debido a la intensificación del transporte de caballos y semen congelado. Es una de las principales causas de aborto, neumoenteritis fulminante en recién nacidos y enfermedad respiratoria alrededor del mundo, donde el macho infectado puede convertirse en portador y transmisor del virus durante el servicio. El virus pertenece a la familia Arteriviridae, es envuelto, posee ARN de simple cadena y contiene numerosas proteínas. Las principales proteínas virales, en cuanto a su abundancia y antigenicidad, son la N (de la nucleocápside) y las proteínas gP5 y M (de membrana). El proceso de apoptosis se encuentra estrictamente controlado a nivel genético y diversos factores pueden alterar el proceso de manera de inducir o inhibir esta respuesta celular. Si bien hay estudios que muestran que este virus induce apoptosis en cultivos celulares identificando distintas enzimas participantes, no se conoce cuál o cuáles proteínas virales estarían involucradas en este proceso. El objetivo general de este trabajo es estudiar la inducción de la apoptosis en cultivos celulares tras la infección con el virus. Se estudiaron diferentes mecanismos moleculares implicados en este proceso mediante el empleo de distintas cepas virales de variada patogenicidad a través de la expresión diferencial de proteínas virales en cultivos. Como primer paso se utilizó el virión completo de las tres cepas virales seleccionadas (Bucyrus, LP-01 y GLD-LP-ARG) y se infectaron 3 líneas celulares (RK-13, Vero y BHK-21), incluyendo controles positivos y negativos. Luego se realizaron construcciones en pCDNA3.1(+) para cada uno de los genes correspondientes a las proteínas M, N y gP5 del VAE y se transfectaron cultivos celulares. Además se evaluó la vía del retículo, representada por la caspasa -12, utilizando distintos inhibidores en células RK-13. Se utilizaron tanto métodos cualitativos como cuantitativos. Nuestros resultados indican que la magnitud de apoptosis observada en los cultivos celulares infectados está directamente relacionada con la patogenicidad y la MOI de la cepa viral del VAE y el tiempo posinoculación. Se observó además que todas las construcciones recombinantes inducen un grado variable de apoptosis, siempre menor a la observada con el virión completo pero mayor respecto a los cultivos que expresan el vector vacío. Además, mediante la utilización de staurosporina, reconocido inductor de apoptosis, observamos un efecto antiapoptótico en las construcciones portadoras de la proteína M, mientras que, por el contrario, se observó un efecto sinérgico en aquellas que contenían los genes de las proteínas gP5 y N del VAE. Por último evidenciamos que la vía del retículo, representada por la caspasa 12, es una de las principales rutas apoptóticas activadas por el VAE. La participación en la muerte celular de los distintos genes virales evaluados, abre nuevos interrogantes que estarían destinados a comprender en mayor profundidad la patogenia de enfermedad, con la esperanza de potenciar y perfeccionar las herramientas de prevención, diagnósticas y terapéuticas disponibles actualmente, mediante la puesta a punto de otras herramientas de detección de la actividad viral en la célula huésped.Equine viral arteritis is a highly important viral disease economically in the equine industry whose prevalence continues to increase, possibly due to the intensification of the transport of horses and frozen semen. It is one of the main causes of abortion, fulminating pneumoenteritis in newborns and respiratory disease around the world, where the infected male can become a carrier and transmitter of virus during the service. The virus belongs to the family Arteriviridae, is enveloped, has simple chain RNA and contains numerous proteins. The main viral proteins, in terms of their abundance and antigenicity, are N (nucleocapsid) and proteins gP5 and M (membrane). The process of apoptosis is strictly controlled at the genetic level and various factors can alter the process in order to induce or inhibit this cellular response. Although there are studies that show that this virus induces apoptosis in cell cultures by identifying different participating enzymes, it is not known which viral proteins or proteins would be involved in this process. The general objective of this work is to study the induction of apoptosis in cell cultures after infection with EAV. Different molecular mechanisms involved in this process were studied through the use of different strains of EAV through the differential expression of viral proteins in cultures. As a first step, the complete virion of the three selected viral strains (Bucyrus, LP01 and GLD-LP-ARG) was used and 3 cell lines were infected (RK-13, Vero and BHK-21), including positive and negative controls. Constructs were then made in pCDNA3.1 (+) for each of the genes corresponding to the M, N and gP5 proteins of the VAE and cell cultures were transfected. In addition, the reticulum pathway, represented by caspase -12, was evaluated using different inhibitors in RK-13 cells. Both qualitative and quantitative methods were used. Our results indicate that the magnitude of apoptosis observed in the infected cell cultures is directly related to the pathogenicity and the MOI of the viral strain of the EAV and the posinoculation time. It was also observed that all the recombinant constructs induce a variable degree of apoptosis, always lower than that observed with the complete virion but greater than the cultures that express the empty vector. Furthermore, through the use of staurosporine, a recognized inducer of apoptosis, we observed an antiapoptotic effect in the M protein carrier constructions, whereas, on the contrary, it was observed a synergistic effect in those that contained the genes of the VAE gP5 and N proteins. Finally, we show that the reticulum pathway, represented by caspase 12, is one of the main apoptotic pathways activated by the VAE. The participation in the cell death of the different viral genes evaluated, opens new questions that would be destined to understand in greater depth the pathogenesis of disease, with the hope of strengthening and perfecting the prevention, diagnostic and therapeutic tools currently available, by putting to the point of other tools for detecting viral activity in the host cell.Fil: Abeyá, María Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentin

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological 10 changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal 15 marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and −9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.Facultad de Ciencias Veterinaria

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological 10 changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal 15 marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and −9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.Facultad de Ciencias Veterinaria

Expression of M Protein from LP02/C Equine Arteritis Virus Inhibits Growth of <i>Escherichia Coli</i> M15-pQE30 System

Our objective was to obtain Equine Arteritis Virus M protein in prokaryotic system to test it as immunogen. LP02/C Equine Arteritis Virus cDNA was used as template to obtain and clone this protein. Equine Arteritis M protein was cloned in the expression vector pQE30 and the recombinant plasmid pQE30/M was transformed in Escherichia Coli M15 cells. The OD600 values of the IPTG-induced M15-pQE30/M culture showed an inhibition of the kinetics growth compared with the non-induced M15-pQE30/M and positive M15-pQE40/DHFR cultures. Several factors such as growth temperature, IPTG concentration and different inductors were analyzed but any of them showed an improvement in protein expression. Instead of E. coli M15strain, a new strain (E. coli BL21) was used and transformed with the pQE30/M. This resolved in part the growth inhibition observed in E. coli M15 cells, but no the recovery yield of the protein. So, as all gene products that affect cells kinetics growth are considered to be toxic, we argue that the lower yields in M protein recovery could be attributed to an associated toxicity of EAV-M protein from LP02/C strain in this expression system.Facultad de Ciencias Veterinarias (FCV

Equine arteritis virus cytopathic effect: caspase-dependent cell death as the major consequence observed

Equine arteritis virus infection in horse populations could be confirmed by the OIE recommended Virus Neutralization (VN) test and by the gold standard Virus Isolation (VI). These two techniques involve the observation of the cytopathic effect (CPE) of EAV. The characteristic CPE in EAV infections is the cellular lysis. The presence/absence of this CPE in cells in the VI/VN respectively, indicate the positivity of each test. CPE refers to morphological and molecular changes that where evidence in infect cells after viral infections. Most viral infections eventually result in the death of the host cell by different cellular mechanisms. The causes of death include cell lysis by alterations to the cell’s surface membrane and by various modes of programmed cell death such as necrosis, apoptosis, autophagy and others. EAV CPE is always refers as cellular lysis but the mechanism involve in this lytical effect has never specifically determined. So, our objective is to extend the concept of the cytopathic effect of EAV infections. To study the effect of different cell death mechanism in EAV CPE we used different inhibitors. Consequently, we concluded that the most important mechanism of cell death in EAV infections is caspase-dependent cell death.Facultad de Ciencias Veterinarias (FCV

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.Instituto de Genética VeterinariaFacultad de Ciencias Veterinaria

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.Fil: Metz, German Ernesto. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Serena, Maria Soledad. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Abeyá, María Mercedes. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dulbecco, Andrea Belén. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Massone, Adriana. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Diaz, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; ArgentinaFil: Echeverria, Maria Gabriela. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; Argentin

Expression of M Protein from LP02/C Equine Arteritis Virus Inhibits Growth of <i>Escherichia Coli</i> M15-pQE30 System

Our objective was to obtain Equine Arteritis Virus M protein in prokaryotic system to test it as immunogen. LP02/C Equine Arteritis Virus cDNA was used as template to obtain and clone this protein. Equine Arteritis M protein was cloned in the expression vector pQE30 and the recombinant plasmid pQE30/M was transformed in Escherichia Coli M15 cells. The OD600 values of the IPTG-induced M15-pQE30/M culture showed an inhibition of the kinetics growth compared with the non-induced M15-pQE30/M and positive M15-pQE40/DHFR cultures. Several factors such as growth temperature, IPTG concentration and different inductors were analyzed but any of them showed an improvement in protein expression. Instead of E. coli M15strain, a new strain (E. coli BL21) was used and transformed with the pQE30/M. This resolved in part the growth inhibition observed in E. coli M15 cells, but no the recovery yield of the protein. So, as all gene products that affect cells kinetics growth are considered to be toxic, we argue that the lower yields in M protein recovery could be attributed to an associated toxicity of EAV-M protein from LP02/C strain in this expression system.Facultad de Ciencias Veterinarias (FCV

Arterivirus

Dentro del orden Nidovirales, familia Arteriviridae se agrupan varias especies virales siendolas más representativas en medicina veterinaria el virus del Síndrome Respiratorio yReproductivo Porcino (PRRSV, de sus siglas en inglés) y el virus de la Arteritis Equina (VAE),siendo este último la especie representativa del género. Una característica importante quepresentan todas las especies dentro de esta familia viral es que presentan patogenicidadespecie específica.Los virus incluidos en la familia Arteriviridae poseen envoltura, la que los hace sensible asolventes lipídicos y detergentes y una nucleocápside de simetría icosaédrica. Su genoma estáconstituido por ARN simple cadena policistrónico de polaridad positiva (ARNsc +) con untamaño de entre 12-17 Kb dependiendo la especie viral.Fil: Metz, German Ernesto. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Abeyá, María Mercedes. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentin

Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with equine arteritis virus

The modulation of the expression of caspases by viruses influences the cell survival of different cell types. Equine arteritis virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation.Fil: Metz, German Ernesto. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galindo, Inmaculada. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria; EspañaFil: Abeyá, María Mercedes. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echeverria, Maria Gabriela. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Covadonga. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria; Españ