ABSTRACT
Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.
Subject(s)
Animals , Salmonidae/virology , Infectious pancreatic necrosis virus/isolation & purification , Birnaviridae Infections/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/standards , Fish Diseases/diagnosis , RNA, Viral/genetics , Observer Variation , Chile , Sensitivity and Specificity , Infectious pancreatic necrosis virus/genetics , Birnaviridae Infections/virology , Aquaculture , False Negative Reactions , False Positive Reactions , Fish Diseases/virology , LaboratoriesABSTRACT
The infectious pancreatic necrosis (IPNV) is the causative agent of an acute illness well characterized in salmonids worldwide. Clinical signs and mortality rates are dependent on several factors such as the viral dose, the age of the fish, the water temperature, among others. An experimental study was conducted to measure the effect of temperature on the gene expression profile of IFN-1(α), STAT-1 and Mx-1 in rainbow trout fry, exposed to IPNV. Fry (n=198) were exposed at 8, 12 and 16°C, and samples were taken for 21 days to determine the virus titer and gene expression. In the first 11 days the greatest viral titer was recorded at 8°C compared with the values obtained at 12 and 16°C. At 8°C, there was a significant increase on day 4 of mRNA Mx-1 (t-test, p<0.05), time in which the viral titer began to decrease. Furthermore, as the viral titer increased, STAT-1 and Mx-1 (r=0.91) and (r=0.96) increased, respectively. The animals were able to recover from day 4 from some of the symptoms of IPN. Clinical disease was developed only in fish exposed to 12°C and all died between days 6 and 14, despite the highly significant increase shown in the average expression level of Mx-1, compared with the values recorded at 8°C and 16°C (Tukey, p<0.0001). Additionally, the expression profiles of IFN-1(α) and STAT-1 decreased completely (~0.016) and (~0.020 times) on day 7. The highest expression level of IFN-1(α), occurred at 16°C (Tukey, p<0.0005). Fry exposed at 16°C were normal during the experiment. IFN-1(α) possibly generated a protector effect from day 2 when they showed a significant expression increase compared with the results at 8°C and 12°C (t-student, p<0.0001); however, STAT-1 was not significantly affected by temperature, although the highest average expression value was recorded at 16°C. Our research supports the expression of relevant anti-viral response genes as IFN-1(α), STAT-1 and Mx-1 are physiologically modulated by the water temperature, directly influencing the development of the IPN disease in rainbow trout.
El virus de la necrosis pancreática infecciosa (IPNV) es el agente etiológico de una enfermedad aguda bien caracterizada en salmónidos alrededor del mundo. Los signos clínicos y la tasa de mortalidad dependen de varios factores tales como la dosis viral, la edad del pez y la temperatura del agua, entre otros. Un estudio experimental se llevó a cabo para medir el efecto de la temperatura sobre el perfil de expresión génica de IFN-1(α), STAT-1 y Mx-1 en alevines de trucha arcoíris expuestos con IPNV. Los alevines (n=198) fueron expuestos a 8, 12 y 16°C, y se tomaron muestras durante 21 días para determinar el título viral y la expresión génica. En los primeros 11 días el mayor titulo viral se registró a 8ºC en comparación con 12 y 16. A 8°C, existió un incremento significativo en el día 4 del ARNm de Mx-1 (t-test, p<0.05), momento en que el título viral empezó a disminuir. Además conforme el título viral aumentaba, también STAT-1 y Mx-1 aumentaron (r=0.91) y (r=0.96) respectivamente. Los animales fueron capaces de recuperarse desde el día 4 de algunos de los síntomas de IPN. La enfermedad clínica se desarrolló únicamente en peces expuestos a 12°C y todos murieron entre el día 6 y 14, a pesar del incremento altamente significativo mostrado en el nivel promedio de expresión de Mx-1 a 12°C, comparados con los valores registrados a 8 y 16°C (Tukey, p<0.0001). Además los perfiles de expresión de IFN-1(α) y STAT-1 decrecieron el día 7 completamente (~0.016) y (~0.020) veces, respectivamente. El nivel de expresión promedio más alto de IFN-1(α) se registró a 16°C (Tukey, p<0.0005). Los alevines expuestos a 16°C se mostraron normales durante el experimento. IFN-1(α) posiblemente generó un efecto protector desde el día 2 cuando mostró un aumento significativo en comparación con los resultados a 8 y 12°C (t-student, p<0.0001); sin embargo, STAT-1 no fue afectado de manera significativa por la temperatura, aunque el más alto valor de expresión promedio se registró a 16°C. Nuestra investigación confirma que la expresión de genes relevantes de respuesta antiviral como IFN-1(α), STAT-1 y Mx-1 son fisiológicamente modulados por la temperatura del agua, influyendo directamente en el desarrollo de la enfermedad de IPN en trucha arcoíris.
Subject(s)
Animals , Temperature , Trout/abnormalities , Infectious pancreatic necrosis virusABSTRACT
Objective. To determine whether the level of apoptosis induced by infectious pancreatic necrosis virus (IPNV) is related to the amino acid sequence of the BH2 domain of the VP5 protein and the level of infectivity. Materials and methods. Three IPNV strains were used, the VP2 protein gene was amplified for genotyping and the VP5 sequence was also obtained. The infectivity of the strains was calculated using the viral titer obtained at 12, 24, 36 and 45 hpi in CHSE-214 cells. The percentage of apoptosis in infected cells was visualized by TUNEL assay and immunohistochemistry (caspase 3 detection). Results. The V70/06 and V33/98 strains corresponded to genotype Sp, while V112/06 to VR-299; the amino acid analysis of the V70/06 strain allows its classification as middle virulent strain and V33/98 and V112/06 strains as low virulent ones; infection with the V112/06 strain produced a lower viral titer (p<0.05). The VP5 gene of the 3 strains showed four homologous domains to Bcl-2, however, the BH2 domain was truncated in V70/06 and V33/98 (12 kDa), being complete (15kDa) in V112/06, which also showed the Trp155 residue, equivalent to Trp188 considered as a critical factor for the function of Bcl-2. The average apoptosis was below 12%, showing no differences between strains (p>0.05). Conclusions. The results showed that the differences in the BH2 sequence of the VP5 protein, infectivity and the VP2 sequence are not associated with the modulation of apoptosis.
Objetivo. Determinar si el nivel de apoptosis inducido por cepas del virus de la necrosis pancreática infecciosa (IPNV) tiene relación con la secuencia aminoacídica del dominio BH2 de la proteína VP5 y el nivel de infectividad. Materiales y métodos. Se utilizaron tres cepas de IPNV; el gen de la proteína VP2 fue amplificado para genotipificación y se obtuvo la secuencia de VP5. La infectividad de las cepas se calculó mediante el título viral obtenido a 12, 24, 36 y 45 hpi en células CHSE-214. Los porcentajes de apoptosis en células infectadas se visualizaron mediante ensayo TUNEL e inmuno-histoquímica (detección de caspasa 3). Resultados. Las cepas V70/06 y V33/98 correspondieron a genotipo Sp, mientras que V112/06 a VR-299; el análisis aminoacídico relacionó a V70/06 como cepa de mediana virulencia y a V33/98 y V112/06 de baja virulencia; la infección con V112/06 produjo menor título viral (p<0.05). El gen VP5 de las 3 cepas presentó los cuatro dominios homólogos a Bcl-2; sin embargo, el dominio BH2 fue truncado en V70/06 y V33/98 (12 kDa); siendo completo (15kDa) en V112/06, que además, presentó el residuo Trp155, equivalente a Trp188 considerado factor crítico para la función de Bcl-2. El promedio de apoptosis fue inferior a 12%, no se observaron diferencias entre cepas (p>0.05). Conclusiones. Los resultados mostraron que las diferencias en la secuencia de BH2 de la proteína VP5, la infectividad y en la secuencia de la proteína VP2 no están asociadas con la modulación de apoptosis.
Subject(s)
Apoptosis , Infectious pancreatic necrosis virus , VirusesABSTRACT
A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.