Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

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.

Effect of temperature on the expression of IFN-1 (α), STAT-1 and Mx-1 genes in Oncorhynchus mykiss (Salmoniformes: Salmonidae) exposed with the virus of the infectious pancreatic necrosis (IPNV) / Efecto de la temperatura sobre la expresión de genes IFN-1(α), STAT-1 y Mx-1 en alevines de trucha arcoíris Oncorhynchus mykiss (Salmoniformes: Salmonidae) expuestos al el virus de la necrosis pancreática infecciosa (IPNV)

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.

Relationship between apoptosis and the BH2 domain sequence of the VP5 peptide of infectious pancreatic necrosis virus / Relación entre apoptosis y secuencia del dominio BH2 del peptido VP5 del virus de la necrosis pancreatica infecciosa

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.

[Evaluation of infectious hematopoietic necrosis, infectious pancreatic necrosis and viral hemorrhagic septicemia viruses in Iranian and imported rainbow trout eggs: a cross sectional study]

Infectious hematopoietic necrosis [IHN], viral hemorrhagic septicemia [VHS] and infectious pancreatic necrosis [IPN] as important viral diseases of salmonids, especially rainbow trout can be led to mass mortality among cultured fish. The aim of the present study was to show if IHN, IPN and VHS viruses can be detected in Iranian and imported rainbow trout eyed eggs and to compare their abundances among the hatcheries of 3 regions [Farsan, Koohrang and Lordegan] in Chaharmahal va Bakhtyari Province. In each area, three hatcheries were selected, 20 eyed eggs were randomly sampled from each farm. The samples were transferred into the sterile tubes and identified by reverse transcriptase -PCR. Meanwhile, physicochemical factors were recorded for each fish. While total infection rate in eggs was 23.3%, the level of infection for IPN, IHN and VHS was 12.5%, 10% and 0.83%, respectively. In this respect, maximum and minimum frequency were observed in Farsan [19.15%] and Koohrang [0.83%]. Comparison of the infected of eggs, based on their origin, showed that the infection rates in Iranian and imported eggs were 20% and 3.33%, respectively. The results revealed that rainbow trout eggs should be considered as major source for transmission of aquatic viruses. Hence, molecular identification of above mentioned viruses in rainbow trout eggs should be done

Recombination and Expression of VP1 Gene of Infectious Pancreatic Necrosis Virus DRT Strain in a Baculovirus,Hyphantria cunea Nuclear Polyhedrosis Virus

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the peal-pol clone (Lee et al 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind 75 phage promoter and the 6x His region of the pQE-30 expression vector, and it was called pQEVP1. Again, the 6xHis-tagged VP1 DNA fragment in the pOEVPl was cleaved with EcoRl and transferred into the VP1 site of the pBacVPl, resulting pBacHis-VPl recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (Lacz-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay, The recombinant virus was named VP1-HcNPV-1. The 6xHis-tagged VP1 protein produced by the pQEVPl was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was 2.0x10(5) pfu/ml at 7 days postinfection.

Caracteristicas etiologicas e clinicas das pancreatites cronicas em Sao Paulo / Etiologic and clinic characteristics of the chronics pancreatitis in Sao Paulo

Foram investigados os fatores etiologicos e as caracteristicas das pancreatites cronicas em 545 portadores desta afeccao. Comprovou-se que o etilismo cronico constitui o fator etiologico preponderante: 509/545 (93,4 por cento). O consumo alcoolico desses pacientes foi intenso (358,6 +-282,0 gramas de etanol diario), precoce (19,8 +-6,5 anos), regular e prolongado (19,8 +- 8,8 anos), sendo o aguardente de cana a bebida mais consumida (99,2 por cento)...

Differentiation of infectious pancreatic necrosis virus isolates by polymerase chain reaction.

Infectious pancreatic necrosis virus (IPNV) from lake trout of Cornwall lake (LT-IPNV), from trout of Jasper (Ja-IPNV) and Arctic char of Northwest Territories (AC-IPNV) are the three isolates recorded from western Canada. Two segments (nt 453-674 and nt 1197-1503) from VP2 coding region of RNA of the isolates were amplified by polymerase chain reaction (PCR) for differentiation. Primers for cDNA synthesis and amplification by PCR were chosen from VP2 coding region of RNA of Ja-IPNV. The segment of 453-674 could be amplified in all the three isolates at annealing temperature from 50 to 60 degrees C. However the segment nt 1197-1503 could be amplified only from Jasper and not from AC or LT-INV at primer annealing temperature ranging from 50 degrees-60 degrees C. PCR product could be obtained from the latter two isolates only at Primer annealing temperature of 45 degrees C. This difference in annealing temperature in PCR amplification between the isolates could be used for differentiating the isolates at genome level.