RESUMEN
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 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 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.
Asunto(s)
Apoptosis/fisiología , Equartevirus/patogenicidad , Células Vero , Replicación Viral/fisiología , Animales , Línea Celular/virología , Chlorocebus aethiops/virología , Cricetinae , HaplorrinosRESUMEN
A novel equine arteritis virus (EAV) was isolated and sequenced from feral donkeys in Chile. Phylogenetic analysis indicates that the new virus and South African asinine strains diverged at least 100 years from equine EAV strains. The results indicate that asinine strains belonged to a different EAV genotype.
Asunto(s)
Infecciones por Arterivirus/veterinaria , Equartevirus/aislamiento & purificación , Equidae , Animales , Infecciones por Arterivirus/virología , Chile , Equartevirus/clasificación , Equartevirus/genética , Masculino , Filogenia , Reacción en Cadena de la Polimerasa/veterinaria , Proteínas Virales/análisisRESUMEN
P34 is an antimicrobial peptide produced by Bacillus sp. P34, isolated from the intestinal contents of a fish from the Amazon basin. This peptide showed antibacterial properties against Gram-positive and Gram-negative bacteria and was characterized as a bacteriocin like substance. It was demonstrated that the peptide P34 exhibited antiviral activity against feline herpesvirus type 1 in vitro. The aim of this work was to evaluate P34 for its antiviral properties in vitro, using RK 13 cells, against the equine arteritis virus, since it has no specific treatment and a variable proportion of stallions may become persistently infected. The results obtained show that P34 exerts antiviral and virucidal activities against equine arteritis virus, probably in the viral envelope. The antiviral assays performed showed that P34 reduces significantly the viral titers of treated cell cultures. The mechanism of action of P34 seems to be time/temperature-dependent. This peptide tends to be a promising antiviral compound for the prevention and treatment of arteriviral infections since it has a high therapeutic index. However, more detailed studies must be performed to address the exact step of viral infection where P34 acts, in order to use this peptide as an antiviral drug in vivo in the future.(AU)
P34 é um peptídeo antimicrobiano produzido pelo Bacillus sp. P34, isolado do conteúdo intestinal de um peixe na Bacia Amazônica. Esse peptídeo demonstrou propriedades antibacterianas contra bactérias Gram-positivas e Gram-negativas e foi caracterizado como uma substância do tipo bacteriocina. Foi demonstrado que o peptídeo P34 exibiu atividade antiviral contra o herpesvírus felino tipo 1 in vitro. O objetivo deste trabalho foi avaliar o P34 in vitro, em cultivo de células RK 13, contra o vírus da arterite equina, uma vez que não há tratamento específico e uma variável proporção de garanhões pode permanecer persistentemente infectada. Os resultados obtidos mostram que o peptídeo exerce atividade antiviral e virucida contra o vírus da arterite equina, agindo provavelmente no envelope viral. Os ensaios antivirais realizados demonstraram que o P34 reduz significativamente os títulos do vírus nas células infectadas e tratadas. O mecanismo de ação do P34 parece ser tempo/temperatura dependente. Esse peptídeo tende a ser um antiviral promissor para o tratamento e a prevenção das infecções por arterivírus, tendo em vista que ele possui um alto índice terapêutico. Contudo, estudos mais detalhados devem ser realizados para precisar a etapa exata da infecção viral em que o P34 age, para que ele possa ser usado como antiviral in vivo no futuro.(AU)
Asunto(s)
Equartevirus , Péptidos/farmacología , Bacillus , Antiinfecciosos/análisis , Antivirales/uso terapéuticoRESUMEN
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.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Células Epiteliales/fisiología , Células Epiteliales/virología , Equartevirus/crecimiento & desarrollo , Animales , Caspasa 3/análisis , Caspasa 8/análisis , Caspasa 9/análisis , Línea Celular , ConejosRESUMEN
A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Infecciones por Arterivirus/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Equartevirus/inmunología , Enfermedades de los Caballos/diagnóstico , Proteínas del Envoltorio Viral/inmunología , Animales , Infecciones por Arterivirus/diagnóstico , Infecciones por Arterivirus/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Equartevirus/aislamiento & purificación , Enfermedades de los Caballos/virología , Caballos , Péptidos/síntesis química , Péptidos/inmunologíaRESUMEN
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.
Asunto(s)
Antígenos Virales/metabolismo , Apoptosis , Equartevirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Factores de Virulencia/metabolismo , Animales , Antígenos Virales/genética , Baculoviridae/genética , Equartevirus/patogenicidad , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Proteínas del Envoltorio Viral/genética , Factores de Virulencia/genéticaRESUMEN
This study investigated the occurrence of equine arteritis virus antibodies using neutralization test in 1,400 horses from Campinas and macro metropolitan paulista mesoregions located at Sao Paulo State, between 2007 and 2008. Eighty (5.7%) samples showed antibodies to virus (titers from 4 to 4,096). Among the 42 cities, 15 (35.7%) presented at least one seropositive animal to equine arteritis virus. Of the 238 farms analyzed 41 showed at least one seropositive animal. The occurrence was higher in sport horses like jumping horses, Quarter horses and was similar for female and male. The rate of seropositive was higher in animals older than 24 months of age. These results suggest the virus circulation among horse population in studied farms, and the need for continuing epidemiological surveillance studies.
O presente estudo investigou a ocorrência de anticorpos contra o vírus da arterite equina, utilizando a técnica de soroneutralização em 1.400 equinos criados nas mesorregiões macro metropolitana paulista e Campinas do estado de São Paulo (SP), entre os anos de 2007 e 2008. Do total das amostras, 80 (5,7%) apresentaram anticorpos para o vírus (títulos entre 4 e 4096). Dentre os 42 municípios amostrados, 15 (35,7%) apresentaram pelo menos um animal sororeagente. Foram analisadas 238 propriedades das quais 41 apresentaram ao menos um animal sororeagente. A ocorrência de animais reagentes foi maior em cavalos que eram utilizados para práticas esportivas, particularmente das raças utilizadas para salto e Quarto de Milha e foi semelhante entre machos e fêmeas. A soropositividade também foi maior em animais com idade superior a 24 meses. Os resultados obtidos sugerem a circulação do vírus da arterite equina nos criatórios amostrados e reforçam a necessidade de estudos continuados de vigilância epidemiológica.
RESUMEN
This study investigated the occurrence of equine arteritis virus antibodies using neutralization test in 1,400 horses from Campinas and macro metropolitan paulista mesoregions located at Sao Paulo State, between 2007 and 2008. Eighty (5.7%) samples showed antibodies to virus (titers from 4 to 4,096). Among the 42 cities, 15 (35.7%) presented at least one seropositive animal to equine arteritis virus. Of the 238 farms analyzed 41 showed at least one seropositive animal. The occurrence was higher in sport horses like jumping horses, Quarter horses and was similar for female and male. The rate of seropositive was higher in animals older than 24 months of age. These results suggest the virus circulation among horse population in studied farms, and the need for continuing epidemiological surveillance studies.
O presente estudo investigou a ocorrência de anticorpos contra o vírus da arterite equina, utilizando a técnica de soroneutralização em 1.400 equinos criados nas mesorregiões macro metropolitana paulista e Campinas do estado de São Paulo (SP), entre os anos de 2007 e 2008. Do total das amostras, 80 (5,7%) apresentaram anticorpos para o vírus (títulos entre 4 e 4096). Dentre os 42 municípios amostrados, 15 (35,7%) apresentaram pelo menos um animal sororeagente. Foram analisadas 238 propriedades das quais 41 apresentaram ao menos um animal sororeagente. A ocorrência de animais reagentes foi maior em cavalos que eram utilizados para práticas esportivas, particularmente das raças utilizadas para salto e Quarto de Milha e foi semelhante entre machos e fêmeas. A soropositividade também foi maior em animais com idade superior a 24 meses. Os resultados obtidos sugerem a circulação do vírus da arterite equina nos criatórios amostrados e reforçam a necessidade de estudos continuados de vigilância epidemiológica.
RESUMEN
Equine viral arteritis is an infectious viral disease of horses that causes serious economic losses due to the presentation of abortions, respiratory disease and loss of performance. After the initial infection males can become persistently infected carriers scattering infection through semen, a situation that brings indirect economic losses by restrictions on international trade of horses and semen from breeding and from countries at risk of infection with the virus. Outbreaks of infection have been reported in several American countries with which Colombia has active links of import and export of horses and semen. This article focuses on creating awareness of the key points of infection and on examples of how infection has been distributed in countries that have developed horse breeding, such as Argentina, a country aware of the origin and distribution of the infection. This study will help us understand how to assess, prevent and/ or control the entry of such a devastating infection for the equine industry.
La arteritis viral equina es una enfermedad viral infectocontagiosa de los equinos que puede llegar a causar graves pérdidas económicas debidas a la presentación de abortos, enfermedad respiratoria y pérdida del desempeño; luego de la primoinfección los machos pueden resultar infectados persistentemente convirtiéndose en portadores los cuales diseminan la infección a través del semen; hecho que acarrea pérdidas económicas indirectas por las restricciones en el comercio internacional tanto de ejemplares como de semen procedente de criaderos y países con riesgo de infección con el virus. Se han reportado brotes de infección en diversos países de América con los cuales Colombia tiene activos vínculos de importación y exportación de equinos y semen. El presente artículo trata de dar a conocer los puntos clave de la infección y ejemplos de cómo se ha distribuido la infección en países con sistemas de explotación de equinos tales como Argentina, en el cual se conoce el origen de la infección y su distribución; cuyo estudio, nos ayudará a entender cómo debemos evaluar, prevenir y/o controlar la entrada de una infección devastadora para la industria equina nacional.
A arterites viral eqüina é uma doença infecto-contagiosa dos eqüinos que pode chegar a causar graves perdas económicas devidas à apresentação de abortos, enfermidade respiratória e perda do desempenho; logo da primoinfecção, os machos podem resultar infectados persistentemente, convertendo-se em portadores e disseminam a infecção a través do sémen; fato que acarreia perdas económicas indirectas pelas restrições no comercio internacional de reprodutores e sémen procedente de criatórios e países com risco de infecção com o vírus. Tem-se reportado surtos de infecção em diversos países da América, com os quais Colômbia tem activos vínculos de importação e exportação de equinos e sémen. O presente artigo trata de dar a conhecer os pontos chave da infecção e exemplos de como tem-se distribuído a infecção em países com sistemas de exploração de eqüinos como Argentina, na qual se conhece a origem da infecção e sua distribuição; para o qual o estudo, nos ajudará a entender como devemos avaliar, prevenir e/ou controlar a entrada da infecção devastadora para a industria eqüina nacional.
RESUMEN
Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.
La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.