Patogenicidad viral de cepas del virus de arteritis equina en la activación de las vías intrínseca y extrínseca de apoptosis

La apoptosis se considera un componente vital de varios procesos fisiológicos normales de las células. Se ha encontrado que un gran número de virus modulan el proceso de muerte celular programada o apoptosis de las células que infectan. Las dos vías mejor caracterizadas y estudiadas son la vía intrínseca y la vía extrínseca de apoptosis celular. Dentro de los virus que han encontrado una ventaja en la inducción de este mecanismo, se destacan los de genoma ARN (Archambault & St-Laurent, 2000; Koyama, 1995; Lin et al., 2002; Maestre et al., 2011; St-Louis et al., 2007; Sur et al., 1998; Thomson, 2001). El virus de arteritis equina (VAE) es un virus ARN de importancia en medicina veterinaria equina en el cual se ha asociado la infección in vitro por este virus con la inducción del proceso de muerte celular (Archambault & St-Laurent, 2000). El siguiente trabajo busca analizar los cambios morfológicos observados in vitro y correlacionarlos con cambios moleculares ocurridos en las células infectadas durante el proceso de muerte celular programada o apoptosis.Eje: Virología MolecularLaboratorio de Virologí

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

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

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

First Study of Different Insect Cells to Triatoma Virus Infection

The use of viruses for biological control is a new option to be considered. The family Dicistroviridae, which affects only invertebrates, is one of the families that have been proposed for this purpose. The Triatoma virus (TrV), a member of this family, affects triatomine transmitters of Chagas disease, which is endemic in Latin America but also expanding its worldwide distribution. To this end, we attempted virus replication in Diptera, Aedes albopictus (clone C6/36) and Lepidoptera Spodoptera frugiperda (SF9, SF21) and High Five (H5) cell lines. The methodologies used were transfection process, direct inoculation (purified virus), and inoculation of purified virus with trypsin. Results were confirmed by SDS-PAGE, Western blotting, RT-PCR, electron microscopy, and immunofluorescence. According to the results obtained, further analysis of susceptibility/infection of H5 cells to TrV required to be studied.Centro de Estudios Parasitológicos y de VectoresFacultad de Ciencias Veterinaria

Clinical and molecular aspects of veterinary coronaviruses

Coronaviruses are a large group of RNA viruses that infect a wide range of animal species. The replication strategy of coronaviruses involves recombination and mutation events that lead to the possibility of cross-species transmission. The high plasticity of the viral receptor due to a continuous modification of the host species habitat may be the cause of cross-species transmission that can turn into a threat to other species including the human population. The successive emergence of highly pathogenic coronaviruses such as the Severe Acute Respiratory Syndrome (SARS) in 2003, the Middle East Respiratory Syndrome Coronavirus in 2012, and the recent SARS-CoV-2 has incentivized a number of studies on the molecular basis of the coronavirus and its pathogenesis. The high degree of interrelatedness between humans and wild and domestic animals and the modification of animal habitats by human urbanization, has favored new viral spreads. Hence, knowledge on the main clinical signs of coronavirus infection in the different hosts and the distinctive molecular characteristics of each coronavirus is essential to prevent the emergence of new coronavirus diseases. The coronavirus infections routinely studied in veterinary medicine must be properly recognized and diagnosed not only to prevent animal disease but also to promote public health.Laboratorio de Virologí

Caracterización de cepas de campo de <i>Herpesvirus suino 1</i> aisladas en Argentina

Se caracterizaron cepas argentinas de herpesvirus suino tipo 1 (HVS-1) mediante corte con las enzimas de restricción BamHI, BstEII y XhoI. La presencia del tipo genómico II había sido reportada en la Argentina una sola vez y, hasta el momento, no se han informado nuevos casos. Este estudio reveló una homogeneidad de los sitios BamHI, acorde con el número y el tamaño de los fragmentos. La presencia del fragmento BamHI #7 en los aislamientos argentinos analizados sugiere que éstos pertenecen al tipo salvaje (wild-type). Además, se caracterizó el principal dominio inmunogénico de la glicoproteína E de todas las cepas argentinas y se lo comparó con los de las cepas de referencia y con las secuencias disponibles en la base de datos GenBank. Los porcentajes de similitud obtenidos oscilaron entre 99 y 100%.The genomic characterization of Suid herpesvirus 1 (SHV-1) isolates from Argentina was accomplished by restriction pattern analysis using the BamHI, BstEII and XhoI enzymes. Type II genome has been described only once in Argentina. This study revealed considerable homogeneity of BamHI endonuclease sites in all the strains analyzed, according to the number and size of the fragments. No deletion of BamHI fragment #7 among the Argentinean isolates suggests that these strains are wild-type. In addition, the main antigenic domain of glycoprotein E of all the Argentinean strains, as well as the reference strains and sequences available in the GenBank, were characterized. The similarity percent oscillated between 99 and 100%.Facultad de Ciencias Veterinaria

Caracterización de cepas de campo de <i>Herpesvirus suino 1</i> aisladas en Argentina

Se caracterizaron cepas argentinas de herpesvirus suino tipo 1 (HVS-1) mediante corte con las enzimas de restricción BamHI, BstEII y XhoI. La presencia del tipo genómico II había sido reportada en la Argentina una sola vez y, hasta el momento, no se han informado nuevos casos. Este estudio reveló una homogeneidad de los sitios BamHI, acorde con el número y el tamaño de los fragmentos. La presencia del fragmento BamHI #7 en los aislamientos argentinos analizados sugiere que éstos pertenecen al tipo salvaje (wild-type). Además, se caracterizó el principal dominio inmunogénico de la glicoproteína E de todas las cepas argentinas y se lo comparó con los de las cepas de referencia y con las secuencias disponibles en la base de datos GenBank. Los porcentajes de similitud obtenidos oscilaron entre 99 y 100%.The genomic characterization of Suid herpesvirus 1 (SHV-1) isolates from Argentina was accomplished by restriction pattern analysis using the BamHI, BstEII and XhoI enzymes. Type II genome has been described only once in Argentina. This study revealed considerable homogeneity of BamHI endonuclease sites in all the strains analyzed, according to the number and size of the fragments. No deletion of BamHI fragment #7 among the Argentinean isolates suggests that these strains are wild-type. In addition, the main antigenic domain of glycoprotein E of all the Argentinean strains, as well as the reference strains and sequences available in the GenBank, were characterized. The similarity percent oscillated between 99 and 100%.Facultad de Ciencias Veterinaria

Extended phylogeny of the equine arteritis virus sequences including South American sequences

Objective: To perform genetic analysis of the ORF5 of equine arteritis virus (EAV) may provide new insights into the genetic evolution and origin of the Argentinean EAV sequences. Methods: A total of 76 sequences were analyzed by neighbor joining (NJ), maximum parsimony and maximum likelihood algorithms. The analysis of the selective pressures was performed using the Tajima's test. Results: The trees showed similar topologies. Two clades were identified: the first clade was formed by strains isolated mainly from a donkey, whereas the second clade presented four large groups from different geographic regions exclusively from Equus caballus. In this clade, we identified a group formed by South African and another one by South American and European sequences. In the latter, the monophyletic group was formed by seven Argentinean sequences. In the NJ tree, we identified a group formed by six Argentinean sequences. The Tajima's test showed a D value of 1.73663, indicating that the sequences analyzed follow a neutral evolution model. Conclusion: We concluded that the Argentinean sequences have a paraphyletic origin and that the fixation of point mutation might follow the neutral model evolution; however, we identified purifying pressures that may be involved in the differentiation of the EAV sequences.Facultad de Ciencias Veterinaria