ABSTRACT. Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly prevalent disease that affects salmonid fish, mostly during their fresh water life period. Like many other viruses, IPNV produces highly heterogeneous populations. Therefore, diagnostic methods need to be checked constantly so that no variants of the virus escape detection. The IPNV genome is composed of two double-stranded RNA segments: A and B, polymerase chain reaction (PCR) methods normally use segment A as a target. In order to develop an optimized protocol to diagnose IPNV, we present a real-time RT-PCR (reverse transcription) technique, using primers designed to recognize segment B of the virus. To validate the ubiquity of the primers used, the IPNV isolates tested were sequenced and compared with previously published cladograms, which include a wide spectrum of genogroups. These primers made it possible to detect viral isolates belonging to genogroups 1 and 5, which were obtained from different locations linked to fish farming. As expected, we were able to detect the virus from distant Aquabirnavirus genogroups. Keywords: infectious pancreatic necrosis virus (IPNV), real-time RT-PCR assay, VP1 gene, phylogenetic tree.Detección y cuantificación de cepas chilenas del virus de la necrosis pancreática infecciosa por medio de la técnica de RT-PCR en tiempo real usando el segmento B como objetivo RESUMEN. El virus de la necrosis pancreática infecciosa (IPNV) es el agente causal de una enfermedad altamente prevalente que afecta a peces salmónidos, principalmente durante su período de vida en agua dulce. IPNV, así como muchos otros virus, produce poblaciones altamente heterogéneas. Por lo tanto los métodos de diagnóstico necesitan ser constantemente revisados para evitar que ciertas variantes del virus escapen de la detección. El genoma del IPNV está compuesto por dos segmentos de ARN de doble hebra, A y B, los métodos de PCR (reacción en cadena de la polimerasa) normalmente usan el fragmento A como blanco. Con el propósito de generar un protocolo optimizado para el diagnóstico del IPNV, se presenta una técnica de RT-PCR (transcripción reversa-) en tiempo real, usando partidores diseñados para reconocer el segmento B del virus. Para validar la universalidad de los partidores utilizados, los aislados del IPNV probados fueron secuenciados y comparados con cladogramas previamente publicados, los cuales incluyen un amplio rango de genogrupos. Con estos partidores fue posible detectar aislados virales pertenecientes a los genogrupos 1 y 5, provenientes de distintas localidades relacionadas con el cultivo de peces. Como se esperaba, se logró detectar virus pertenecientes a genogrupos distantes de Aquabirnavirus. Palabras clave: virus de la necrosis pancreática infecciosa (IPNV), ensayo en tiempo real RT-PCR, gen VP1, árbol filogenético.
B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.
The effect of dietary β-1.3-glucan, vitamin E, and β-carotene supplements in juvenile brown shrimp, Farfantepenaeus californiensis, inoculated with white spot syndrome virus (WSSV) was evaluated. Groups of 30 organisms (weighing 1 ± 0.5 g) were cultured in 60 L fiberglass tanks and fed daily with β-1.3- glucan (0.1%), vitamin E (0.01%), and β-carotene (0.01%) for 23 days; the specimens were then inoculated with WSSV. The antioxidant activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) were determined in the hepatopancreas and muscle at 0, 1, 6, 12, 24, and 48 h after inoculation. Shrimp fed with β1.3-glucan, vitamin E, and β-carotene significantly increased SOD activity in the hepatopancreas and muscle at 12 and 24 h post-infection, respectively. Shrimp fed with vitamin E and β-1.3-glucan registered an increment in SOD activity from 12 to 48 h post-infection. Shrimp fed with β-carotene increased SOD activity before infection with WSSV, and shrimp fed with β-1.3-glucan and vitamin E increased CAT activity, also before infection. The CAT activity response in shrimp muscle increased with respect to the control group for all treatments tested from 1 to 6 h after inoculation with WSSV. The highest antioxidant response was registered in shrimp fed with vitamin E. Juvenile shrimp fed with vitamin E and later inoculated with WSSV registered 100% mortality at 72 h, but shrimp fed with β-1.3-glucan and β-carotene showed greater resistance to WSSV, with mortality at 144 h post-infection. This study demonstrated the capacity of juvenile Farfantepenaeus californiensis fed β-1.3-glucan, vitamin E, or β-carotene to increase the antioxidant response before and after viral infection.
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