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.     

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.     

Attachment and entry of infectious pancreatic necrosis virus (IPNV) into CHSE-214 cells

Summary Infectious pancreatic necrosis virus (IPNV) attaches to CHSE-214 cells through two types of cell components: specific and non-specific ones. Competition experiments with inactivated IPNV showed that IPNV requires specific components to productively infect cells. Just a low amount of adsorbed IPNV enters the cell. After 20 minutes, part of the adsorbed IPNV was internalized into acid compartments. Also, the viruses adsorbed on the cell surface require similar periods of time to escape from the neutralization of antibodies.     

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.     

A reliable RT-PCR-ELISA method for the detection of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout

A new method, termed RT-PCR-ELISA, was evaluated for ease of use, reliability and sensitivity when detecting infectious pancreatic necrosis virus (IPNV) present in trout kidney tissue. The method had comparable sensitivity to existing PCR assays and could successfully detect 1.5 x 10(4) pfu IPNV in artificially contaminated trout kidney samples. The technique was easily established in a new laboratory and required no specialised equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. The RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.     

Infectious pancreatic necrosis virus (IPNV) does not requireacid compartments for entry into cells

Summary.  The early events in the infection of unenveloped viruses are still rather unknown and puzzling. However, as in the case of enveloped viruses, the acid pH of endosomes can be important to trigger the virus entry into the cytosol. To test if the infectious pancreatic necrosis virus (IPNV), an unenveloped virus, requires acid endosomal pH to infect cells, we assayed the effect of Bafilomycin A1 on IPNV replication. Concentrations of the antibiotic which inhibited the endosomal acidification of the host cells were unable to affect IPNV replication in CHSE-214 cells; therefore, the acid pH of endosomes seems not to be a mandatory condition for the entry of IPNV into cells. Received March 7, 1997 Accepted July 10, 1997     

Further analysis on the structural proteins of infectious pancreatic necrosis virus

The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.     

Isolation and characterization of infectious pancreatic necrosis virus from pike(Esox lucius)

Summary A nonenveloped icosahedral virus measuring 60 (±5) nm in diameter has been isolated from pike fry(Esox lucius). The isolate is stable to heat, lipid solvent and acid. Iododeoxyuridine has no effect on its replication. Serologically, the virus is related to the major European strains of infectious pancreatic necrosis virus (IPN) Sp and Ab.The isolate is neutralized by normal trout serum and is not pathogenic for trout fry or for young pike. A line of CHSE-214 cells persistently infected with the isolate was established and has been passed 30 times.With 1 Figure     

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins

Summary.  The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm. Received October 25, 1999/Accepted October 26, 1999     

Oligonucleotide fingerprints of the RNAs from infectious pancreatic necrosis virus

Summary T₁-RNase oligonucleotide fingerprints indicated that the two species of RNA from infectious pancreatic necrosis virus were unrelated. Both RNAs had a GC content of 54 per cent.With 3 FiguresSupported by grants from Environment Canada, Fisheries and Marine Service, Ottawa and the National Research Council, Ottawa.     

The haemagglutinating properties of infectious pancreatic necrosis virus

Summary The haemagglutinating properties of infectious pancreatic necrosis virus have been investigated. Erythrocytes from a wide range of animals were used in these tests but only cells derived from mice (Balb/c and Manchester strain) gave reproducible haemagglutination.The tests were carried out at various temperatures but haemagglutination only occurred at 4° and 22° C. The haemagglutination was pH dependent with an optimum being between pH 5.75 and 6.0.Variable results were obtained at the same pH values with erythrocytes derived from rats (Sprague Dawley) but the reaction in this case appeared to be related to individual batches of erythrocytes.With 1 Figure     

Modulation of genes related to the recruitment of immune cells in the digestive tract of trout experimentally infected with infectious pancreatic necrosis virus (IPNV) or orally vaccinated

There are still many details of how intestinal immunity is regulated that remain unsolved in teleost. Although leukocytes are present all along the digestive tract, most immunological studies have focused on the posterior segments and the importance of each gut segment in terms of immunity has barely been addressed. In the current work, we have studied the regulation of several immune genes along five segments of the rainbow trout (Oncorhynchus mykiss) digestive tract, comparing the effects observed in response to an infectious pancreatic necrosis virus (IPNV) infection to those elicited by oral vaccination with a plasmid coding for viral VP2. We have focused on the regulation of several mucosal chemokines, chemokine receptors, the major histocompatibility complex II (MHC-II) and tumor necrosis factor α (TNF-α). Furthermore, the recruitment of IgM⁺ cells and CD3⁺ cells was evaluated along the different segments in response to IPNV by immunohistochemical techniques. Our results provide evidences that there is a differential regulation of these immune genes in response to both stimuli along the gut segments. Along with this chemokine and chemokine receptor induction, IPNV provoked a mobilization of IgM⁺ and IgT⁺ cells to the foregut and pyloric caeca region, and CD3⁺ cells to the pyloric caeca and midgut/hindgut regions. Our results will contribute to a better understanding of how mucosal immunity is orchestrated in the different gut segments of teleost.     

The structure of infectious pancreatic necrosis virus RNA

The RNA from infectious pancreatic necrosis virus has been purified and had a sedimentation velocity of 14S on sucrose gradients, a buoyant density of 1-60 g/ml in CS2SO4 and pyrimidine to purine ratios near unity. The RNA had the appearance of a linear double stranded molecule with an average length of 0-92 mum and a standard deviation of 0-07 mum when observed under the electron microscope using the Kleinschmidt protein film technique. This would correspond to a mol. wt. of 2-4 +/- 0-2 X 10(6). The RNase A resistance of IPN virus RNA exhibited a marked salt dependence; it was 92% resistant in 0-1 M-NaCl, but only 9% resistant, or less, in 0-*1 M-NaCl. The RNA was resistant to denaturation by boilding at NaCl concentrations of 0-04 M or higher, but did denature at lower concentrations. Polyacrylamide gel electrophoresis of the RNA indicated that two RNA species were present and the standard deviation of lengths in the electron microscope indicated that they could not differ by more than 4 X 10(5) in mol. wt.     

Characterization of a membrane protein (VP001L) from infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of megalocytivirus, Iridoviridae. A novel membrane protein corresponding to the first open reading frame (ORF001L) of ISKNV genome was identified. This 378-residue protein, termed the VP001L protein, has a high content of hydrophobic sequences and contains 10–11 putative transmembrane domains, indicating it may be a membrane protein. The VP001L mRNA start site was extended 433 bp upstream of the start codon and the temporal analysis showed that the VP001L gene was first transcribed at 8 h post-infection (h.p.i.). VP001L protein was detected on the plasma membrane of ISKNV infected cells by immunofluresence. In order to further investigate different transmembrane domains’ influence on subcellular localization of VP001L, series of truncated or deleted mutants were constructed with GFP at the C terminus. The transfection results indicated that the second putative transmembrane domain played a determinative role in VP001L’s membrane localization and the translocation of the first and third transmembrane domains depended on their interactions with the second one. Therefore, this novel VP001L protein is considered to serve as a model for analyzing the topology and roles of different hydrophobic regions in multi-transmembrane proteins.     

Method for the concentration of infectious pancreatic necrosis virus from hatchery water

A method was developed for concentrating infectious pancreatic necrosis virus from hatchery water using positively charged 1-MDS filters. The method consists of passing large volumes (Ca. 1001) of hatchery water through 1-MDS microporous filter followed by the elution of the adsorbed virus using a high pH buffer. The virus adsorbed efficiently to 1-MDS filters when the pH of the water was 5.5 and was eluted optimally with 3% beef extract solution (pH 10). This procedure permitted the processing of 100 1 of hatchery water which resulted in a 300-fold reduction in the volume of water and greater than 90% recovery of the seeded virus.     

Sequence analysis of infectious pancreatic necrosis virus isolated from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly contagious disease that affects many species of fish and shellfish. This virus causes economically significant diseases of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in Iran, which is often associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry samples were collected during an outbreak of IPNV in three different fish farms in north and west provinces of Iran in 2012; and we investigated the full genome sequence of Iranian IPNV and compared it with previously identified IPNV sequences. The sequences of different structural and nonstructural-protein genes were compared to those of other aquatic birnaviruses sequenced to date. Our results show that the Iranian isolate falls within genogroup 5, serotype A2 strain SP, having 99 % identity with the strain 1146 from Spain. These results suggest that the Iranian isolate may have originated from Europe.     

Genomic and phenotypic divergence among three serotypes of aquatic birnaviruses (infectious pancreatic necrosis virus)

The existence of three serotypes (VR299, Ab, Sp) of infectious pancreatic necrosis virus (IPNV) has been confirmed by the kinetics of their neutralization in the presence of excess antibody. Other isolates from fish and molluscs that have been reported to represent new serotypes were found to be identical to one of the established three serotypes. Variants of the North American serotype (VR299) were found to differ in their sensitivity to antiserum, but rate values from reciprocal neutralizations showed that they did not belong to a new serotype. Comparison of the size of the RNA species, and the size and composition of the proteins in the virion showed that each serotype was unique. Phenotypic markers (host range, plaque size, plaque morphology, and inherent temperature sensitivity) were also unique for each serotype. Although all of these phenotypic markers could be modified by mutation, they were useful for the initial rapid characterization of isolates. The implications from this study are that IPNV has a wide host range (infecting many species of fish as well as molluscs) and that the three serotypes are all closely related (they cross-reacted immunologically), but the molecular weights of their RNAs and the protein composition of their virions showed divergence.