Infectious Pancreatic Necrosis Virus Serotype Research Articles

Preparation and optimization of rapid and sensitive coagglutination test for detection of infectious pancreatic necrosis virus (IPNV)

The aim of this study is the development of a coagglutination test for rapid diagnosis of infectious pancreatic necrosis (IPN) virus which causes an acute infectious viral disease with high mortality in young salmonid fish. Serotypes Sp, Ab, WB of the IPNV were cultivated in BF-2 cells and purified by linear sucrose density gradient centrifugation. Hyperimmune sera against purified IPNV serotypes were collected from immunized guinea pigs. Coagglutination test conjugates were prepared by using sensitized S. aureus and hyperimmune sera. Cell culture derived salmonid pathogens such as infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic hematopoietic necrosis virus (EHNV) were used to determine the specificity of the test as control. Upward comparison of the preferability for the rapid diagnosis was also carried out with ELISA and RT-PCR. The validation studies of newly developed coagglutination test were carried out in accordance with the Manual of Diagnostic Tests for Aquatic Animals. Consequently, determined detection limit of the test was approximately 105.25 TCID50/mL for the Sp and Ab serotypes and 107.75 TCID50/mL for the WB in laboratory conditions. The results indicated that newly developed coagglutination test was compatible with ELISA and RT-PCR in positive cell culture supernatants. It was concluded that this test would be an economic, rapid and reliable method for the diagnosis of IPN virus in cell culture.

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)-GN438A expresses VP2 gene-encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross-protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV-N438A-ΔNV-EGFP and rIHNV-N438A-ΔNV-VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild-type (wt) IHNV HLJ-09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ-09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV-N438A-ΔNV-VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested‐PCR study

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E-stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin-fixed, paraffin-embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.

Moving molecular diagnostics from laboratory to clinical application: a case study using infectious pancreatic necrosis virus serotype A

Using an RT-PCR method for detection of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon as a model, this study examined the optimization and validation required to provide a method suitable for IPNV detection from fish tissue. IPNV-positive Atlantic salmon kidney samples that had been titred or kidney spiked with IPNV were used. The amount of RNA in the reverse transcription (RT), RT denaturation temperature and incubation time, PCR annealing temperature and number of cycles were optimized. The optimized RT-PCR was able to detect IPNV in Atlantic salmon kidney calculated to have a titre of ten infectious units. Extensive optimization is required to produce a PCR for detection of fish pathogens from methods designed in the laboratory. This study demonstrated some of the many variables that should be optimized before a fully validated assay can be claimed and illustrates the extensive validation required to fulfil requirements of the OIE and is of relevance to laboratories carrying out clinical testing.

Molecular characterization of birnaviruses isolated from wild marine fishes at the Flemish Cap (Newfoundland)

Several isolates of aquatic birnaviruses were recovered from different species of wild fish caught in the Flemish Cap, a Newfoundland fishery close to the Atlantic coast of Canada. The nucleotide sequence of a region of the NS gene was identical among the isolates and was most similar to the Dry Mills and West Buxton reference strains of infectious pancreatic necrosis virus (IPNV). Phylogenetic analysis of the sequence of a region of the VP2 gene demonstrated that the isolates were most closely aligned with the American strains of IPNV serotype A1. Electron microscopy of virus structures clarified and concentrated from cultures of infected chinook salmon embryo (CHSE-214) cells revealed a majority of typical IPNV-like icosahedral particles, as well as a low proportion of type I tubules having a diameter of approximately 55 nm and a variable length of up to 2 microm. The tubules could be propagated in cell cultures, but always in the presence of low proportions of icosahedral particles. Cloning of selected isolates by serial dilution yielded preparations with a high proportion of the tubular structures with a density in CsCl gradients of approximately 1.30 g cm(-3). Polyacrylamide gel electrophoresis revealed the material in the band was composed of the IPNV pVP2 and VP2 proteins.

Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures

An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8 h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10 3 TCID 50/ml, whilst the labelling appears at 24 h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent.

Diversity of Infectious Pancreatic Necrosis Virus Strains Isolated from Fish, Shellfish, and Other Reservoirs in Northwestern Spain

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.

Immunogold Technique Applied to Electron Microscopy of Infectious Pancreatic Necrosis Virus(IPNV).

Immunogold technique in electron microscopy proved to be effective to demonstrate the virusantibody specificity using purified reference strains of infectious pancreatic necrosis virus (IPNV). Although some cross reactions were detected, it was possible to distinguish the major IPNV serotypes (Sp, Ab and WB) according to the different affinity for the antisera against the three reference strains which were binded to protein A-gold particles. Moreover, tubular structures with a diameter of 60 nm were detected presenting a high affinity for the gold particles, which suggests a viral origin.

Demonstration of infectious pancreatic necrosis virus strain VR‐299 in Japanese eel, Anguilla japonica Temminck & Schlegel

Abstract. Six isolates of infectious pancreatic necrosis virus (IPNV) were obtained from trout or eel farms in Taiwan in 1987. By using the electrophoretic analysis of 35S‐methionine‐labelled early viral polypeptide patterns and silver‐stained ds RNA gel patterns, a comparison of these isolates with the three archetypal IPNV serotypes (AB, SP and VR‐299) was made. They were all identical with VR‐299, both in the RNA and early viral polypeptide patterns. From the results obtained, it was apparent that the VR‐299 serotype of IPNV was just as widespread in eel as in trout in Taiwan. This is the first case of VR‐299 isolation from eel.

Inoculation of Infectious Pancreatic Necrosis Virus Serotype Sp did not cause Pancreas Disease in Atlantic Salmon (Salmo salar L.)

Atlantic salmon were selected from a fish farm with no previous record of pancreas disease (PD) or infectious pancreatic necrosis virus (IPNV) infection. Groups of fish were inoculated with either IPNV (strain Sp) from cell culture, organ material from fish with PD or control material as phosphate-buffered saline (PBS). Virological, histological and immunohistochemical examinations were carried out throughout the experiment. None of the fish died or showed clinical symptoms of PD. Histological examination revealed no pathological changes, and immunohistochemical studies were negative. Virus was isolated only sporadically from the group inoculated with organ material, whereas it was isolated consistently from the group inoculated with virus propagated in cell culture, as well as from in-contact control fish after the first week. In a latent carrier test, changes were entirely lacking in the first mentioned group, and were only slight in the last mentioned group. The data suggest that PD is not a transmissible disease, and that IPNV isolated from a PD outbreak does not play any part in the etiology of this disease.

Immunohistochemical Identification of Infectious Pancreatic Necrosis Virus in Paraffin-Embedded Tissues of Atlantic Salmon (Salmo Salar)

Infectious pancreatic necrosis virus serotype Sp was identified by immunohistochemistry in formaldehyde-fixed and paraffin-embedded tissue of Atlantic salmon (Salmo salar). The immunoreaction was present in degenerating and necrotic cells in exocrine pancreatic cells. Cross reactions were observed with rabbit antisera against serotypes Sp, Ab, and VR-299 in neutralization tests and western blotting. Immunohistochemically, only Sp antiserum produced positive immunostaining to Sp antigens, whereas antisera to serotypes Ab and VR-299 were negative.

Fish cell lines: Establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella)

Three new cell lines were established from tissues of the grass carp, Ctenopharyngodon idella. Derived from the fin, snout, and swim bladder of two apparently healthy diploid fry, these cell lines have been designated GCF, GCS-2, and GCSB, respectively. The cells grew at temperatures between 24 degrees and 36 degrees C with optimal growth at 32 degrees C and have been subcultured more than 50 times since their initiation in August 1986. Two of the lines remained diploid or pseudodiploid after 38 passages. The cells were tested for microbial contamination, and plating efficiencies were determined. The three cell lines were sensitive to Rhabdovirus carpio (RVC), infectious hematopoietic necrosis virus (IHNV), golden shiner virus (GSV), chum salmon virus (CSV), and infectious pancreatic necrosis virus serotype VR299 IPNV). They were refractory to channel catfish virus (CCV), channel catfish reovirus (CRV), chinook salmon paramyxovirus (CSP), and an Ab serotype of IPNV.

Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon.

Virus particles isolated from hatchery reared fish with infectious pancreatic necrosis (IPN) were neutralized by homologous immune sera but not by immune sera raised against IPN virus serotype 1, 2, and 3. This virus isolate, called the N1 strain, was detected in one year old Atlantic salmon (Salmo salar) during an outbreak with histopathological lesions of IPN and slightly increased mortality. The polypeptide pattern of N1 virus differed markedly from that of the three classical IPN virus serotypes. Double stranded RNA isolated from the N1 virus particles, co-migrated during agarose gel electrophoresis with nucleic acid isolated from the IPN virus Jasper and Ab strains. Nucleic acid hybridizations using low stringency washing conditions and a synthetic DNA oligonucleotide probe (representing the 3' end of the A segment of the Jasper strain) gave positive results with the IPN virus Jasper, Ab, Sp, and N1 strains. The results presented in this paper show that the N1 isolate differs immunologically and biochemically from the IPN virus serotypes 1, 2, and 3 and may represent a new serotype of IPNV.

Isolation of infectious pancreatic necrosis virus (serotype Ab) from diverse species of estuarine fish

Two significant fish kills occurred in the Pamlico River estuary (North Carolina, USA), one in December 1981 and January 1982, and the other in June 1982. The first involved only the southern flounder(Paralichthys lethostigma). Histopathologic examination of morbid and moribund flounder revealed extensive sloughing and necrosis of the mucosa of the pyloric caeca and intestine, and inflammation of the submucosa of the pyloric caeca. Brain and internal organ homogenates from morbid and moribund flounder were assayed on CHSE-214 cells, and a virus was isolated. Virus titers ranged from≤8.4 · 102 to 6.3 · 107 TCID50 per gram of tissue. Cross-plaque neutralization assays indicated that the southern flounder virus was infectious pancreatic necrosis virus serotype Ab. Immersion challenge showed the isolate is only slightly virulent for fry of brook trout(Salvelinus fontinalis). The second fish kill involved the southern flounder and six other species: hogchoker(Trinectes maculatus), Atlantic silverside(Menidia menidia), spot(Leiostomus xanthurus), Atlantic croaker(Micropogon undulatus), silver perch(Bairdiella chrysura), and striped mullet(Mugil cephalus). Virus was isolated from southern founder, hogchoker, Atlantic silverside, and spot. Neutralization assays indicated that the four isolates were nearly identical; however, the diversity of species affected suggests that the virus might not have been the specific cause of mortality.

Rapid detection of infectious pancreatic necrosis virus (IPNV) by the enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the presence of infectious pancreatic necrosis virus (IPNV) antigen in cell cultures and in fish. Virus antigen could be detected in infected cell cultures before visible cytopathic effect (c.p.e.) was evident and cell cultures showing complete viral c.p.e. produced intense colour reactions. Virus antigen was detected in infected fry during and immediately after an epizootic, but although IPNV-carrier fish could be detected by the ELISA technique, the sensitivity of detection was not as great as that of isolation of the infectious virus in cell culture. The major IPNV serotypes, Sp, Ab and Vr, crossreacted at only a low level and it was shown that the ELISA technique could be used to serotype IPNV strains rapidly. None of 10 other fish pathogenic viruses reacted with plates sensitized for IPNV detection. The time taken to perform the technique was reduced to 1 h 35 min at room temperature and this still allowed the results to be readily assessed visually as antigen-positive or antigen-negative.

Isolation and characterization attempts of three viruses from European EEL, Anguilla anguilla: Preliminary results

A virological survey of the elver population along the French Atlantic coast led to the isolation of three viruses, B 6 and B 12 in 1977, and C 30 in 1978. B 6 and B 12 were isolated from the same lot of elver. B 6 was demonstrated to be an infectious pancreatic necrosis virus serotype Sp. B 12 and C 30 are two rhabdoviruses differing on their cell susceptibility and growth properties. The first one multiplied in ⪡epithelioma papulosum Carpio⪢ cells at 10 and 14°C, but not at 20°C. C 30 replicated in the same cells at temperature ranging from 10 to 25°C. B 12 and C 30 were both similar in their morphological properties to the other eel and salmonid rhabdoviruses known at present, but were serologically different from them and from the virus of spring viraemia of carp. However, C 30 was shown to be antigenically related to two other eel rhabdoviruses, EVEX and EVA. B 6 was experimentally demonstrated to induce severe mortalities in a group of rainbow trout fry infected per bath at 10°C, but was not pathogenic for elvers and young eel. B 12, C 30 and EVEX failed to produce any significant mortalities in various groups of rainbow trout fry and eel under definite experimental conditions.