Differential Transcriptomic Response of Rainbow Trout to Infection with Two Strains of IPNV.

The IPN virus (IPNV) causes a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into seven genogroups, of which two are present in Chile, genogroups 1 and 5. This study aimed to compare the transcriptomic response of rainbow trout fry challenged with two Chilean isolates of IPNV, RTTX (genogroup 1), and ALKA (genogroup 5). Tissue samples from challenged individuals and controls were taken at 1, 7, and 20 days post-challenge and analyzed by RNA-Seq. The results revealed that infection with RTTX elicited a greater modulation of the trout transcriptome compared to ALKA infection, generating a greater number of highly differentially expressed genes in relation to the control fish. Gene Ontology enrichment indicated that functions related to the inflammatory and immune responses were modulated in fish challenged with both isolates throughout the trial, but with different regulation patterns. On day 1 post challenge, these functions were activated in those challenged with ALKA, but suppressed in RTTX-challenged fish. These results suggest that rainbow trout exhibit a differential transcriptomic response to infection with the two genetically distinct IPNV isolates, especially at early times post-infection.

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.

Inducers of salmon innate immunity: An in vitro and in vivo approach.

Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE-214. We found that the butyrate derivatives 4-phenyl butyrate (PBA) and ß-hydroxy-ß-methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24-72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG-1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL-families had lower basal levels of cathelicidin in gills, than non QTL-families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future.

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.

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.

Infectious pancreatic necrosis virus (IPNV) enumeration through epifluorescence microscopy: technical aspects

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.

Visualization of the infectious pancreatic necrosis virus replication cycle by labeling viral intermediates with a TUNEL assay.

Investigating the capacity of the infectious pancreatic necrosis virus (IPNV) to promote apoptosis with a TUNEL kit, designed to visualize fragmented DNA, a striking labeling pattern was found. In addition to the fluorescence observed in the nucleus of cells with fragmented DNA, the infected cells showed an intense particulate fluorescence in their cytoplasm. The cytoplasmic labeling seems to be concomitant with the timing of virus life cycle because labeled viral proteins coexist with TUNEL cytoplasmic fluorescence at different times during infection. In principle the terminal deoxynucleotidyl transferase (TdT) used in TUNEL assays should not be able to label substrates other than DNA, however, our results are consistent with the idea that viral structures are being labeled. We propose that the TdT enzyme label the 3'-hydroxyl ends of viral RNAs during its polymerization. This kind of reaction could be possible because it has been reported that TdT is an enzyme that can use rNTPs as substrates and the 3'-hydroxyl priming end can be provided by a ribonucleotide as well. Thus, allowing the visualization of virus RNA-containing intermediates during IPNV replication.

Visualization of infectious pancreatic necrosis virus (IPNV) particles labeled with fluorescent probes.

Infectious pancreatic necrosis virus (IPNV) particles were labeled with SYBR Green I or a monoclonal antibody and FITC-conjugated secondary antibody and examined in a fluorescence microscope. Labeled viral particles were visualized in a narrow range of pixels. Comparing IPNV particles with fluorescent phage T4 virions, the former, as expected, were seen smaller in size. The method allows the rapid and accurate counting of viral particles both on filters and bound to the cell surface. In addition, IPNV particles can be specifically enumerated in the presence of other virions and the ratio between physical particles and virus infectivity can be easily calculated as well.

Necrosis of infectious pancreatic necrosis virus (IPNV) infected cells rarely is preceded by apoptosis.

Infectious pancreatic necrosis virus (IPNV)-infected cells were labeled with Annexin V and propidium iodide in order to determine the proportion of cells, which developed necrosis and/or apoptosis during the time course of infection. Contrasting with earlier reports, we found that at any time during IPNV multiplication cycle, the percentage of apoptotic cells never exceeded the 12% of the whole population of the infected cells. In addition, the percentage of necrotic cells increased continuously until reaching the 75% of the infected cells at 15 h post infection. Apoptotic cells were also identified by in situ terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling (TUNEL). Our results are in accordance with the idea that apoptosis rarely precedes necrosis.

Rapid simultaneous detection and quantitation of infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) is a pathogen of great concern in the salmon industry as well as in the environment. Taking advantage of the early immunofluorescent visualization of viral proteins in infected cells, a titration method was developed. At 16 h p.i., fluorescent foci were visualized with a monoclonal antibody against VP3-structural protein of the virus. The counting of each fluorescent cell allows the quantitation of infection foci; titres expressed in fluorescent foci/ml were equivalent to plaque forming units (PFU)/ml. With slight modifications, the same method used to detect the virus in field samples, can be applied to estimate virus contents. Some of the samples used during the assays were obtained from routine screening procedures. The titres recorded from positive samples correlated well with the clinical condition of the fish. With this method, rapid diagnosis and quantitation may simultaneously be performed with the same tissue extract.