Evolution of an Extended Pathogenicity Motif in VP2 of Infectious Pancreatic Necrosis Virus Isolates from Farmed Rainbow Trout in Turkey.

Infectious pancreatic necrosis virus (IPNV) causes economic losses with a highly variable mortality rate worldwide, especially in rainbow trout. The virus has a double-stranded bi-partite RNA genome designated segment A and B. New complete genome sequences of nine rainbow trout isolates from Turkey were determined and subjected to phylogenetic analysis, identifying all as genotype 5 (serotype Sp). A time-dependent change in the extended pathogenicity motif of VP2 from P217T221A247 (PTA) to PTE P217T221E247 over a period of 10 years was identified. A wider analysis of 99 IPNV sequences from Turkey and Iran revealed the emergence of the motif PTE from 2007 to 2017, inducing significant morbidity in fry by 2013. In fact, displacement of the PTA motif, by the PTE motif in IPNV isolates appeared to be connected to a production peak of rainbow trout in 2013. An additional CAI analysis provided more evidence, indicating that rainbow trout culture in Turkey has an influence on the evolution of IPNV.

Phylogenetic characterization of Polish isolates of infectious pancreatic necrosis virus in salmonid fish.

INTRODUCTION: Infectious pancreatic necrosis virus belongs to the genus Aquabirnavirus and family Birnaviridae. By VP2 gene similarity, aquatic birnavirus is clustered into seven genogroups. The aim of this study was to genetically analyse IPN viruses occurring on Polish fish farms. MATERIALS AND METHODS: Samples from freshwater fish mostly from 2012 to 2013 and from northern Poland were examined for the presence of IPN virus using isolation on cell cultures, real-time RT-PCR and RT-PCR. Fragments of 1,377 and 1,079 bp of the VP2 and VP5 genes, respectively, were sequenced, and the results were assembled into one consensus and analysed by Geneious software. The same VP2 gene region was compared and a phylogenetic tree generated by the neighbour-joining method and MEGA6 software. RESULTS: All tested Polish isolates belonged to genogroup 5, like other European Spajurup isolates. CONCLUSION: Our findings prove that there is only one IPN virus genogroup in Poland. Polish isolates show close relationships with each other. There is a close relationship between Polish isolates and isolates from Turkey, Spain and Iran. Isolate 57 is a separate branch related to isolates from the United States and Taiwan. This points to the likelihood of past virus introduction via import of stock from those countries.

Molecular epidemiological study on Infectious Pancreatic Necrosis Virus isolates from aquafarms in Scotland over three decades.

In order to obtain an insight into genomic changes and associated evolution and adaptation of Infectious Pancreatic Necrosis Virus (IPNV), the complete coding genomes of 57 IPNV isolates collected from Scottish aquafarms from 1982 to 2014 were sequenced and analysed. Phylogenetic analysis of the sequenced IPNV strains showed separate clustering of genogroups I, II, III and V. IPNV isolates with genetic reassortment of segment A/B of genogroup III/II were determined. About 59 % of the IPNV isolates belonged to the persistent type and 32 % to the low-virulent type, and only one highly pathogenic strain (1.79 %) was identified. Codon adaptation index calculations indicated that the IPNV major capsid protein VP2 has adapted to its salmonid host. Under-representation of CpG dinucleotides in the IPNV genome to minimize detection by the innate immunity receptors, and observed positive selection in the virulence determination sites of VP2 embedded in the variable region of the main antigenic region, suggest an immune escape mechanism driving virulence evolution. The prevalence of mostly persistent genotypes, together with the assumption of adaptation and immune escape, indicates that IPNV is evolving with the host.

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Design and validation of a RT-qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers.

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT-qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT-qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID50  mL-1 , 50 pfu mL-1 or 66 RNA copies mL-1 , depending on the standard. All the standard curves showed high reliability (R2  > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.

A Systematic Approach towards Optimizing a Cohabitation Challenge Model for Infectious Pancreatic Necrosis Virus in Atlantic Salmon (Salmo salar L.).

A cohabitation challenge model was developed for use in evaluating the efficacy of vaccines developed against infectious pancreatic necrosis virus (IPNV) in Atlantic salmon (Salmo salar L) using a stepwise approach. The study involved identifying a set of input variables that were optimized before inclusion in the model. Input variables identified included the highly virulent Norwegian Sp strain NVI015-TA encoding the T217A221 motif having the ability to cause >90% mortality and a hazard risk ratio of 490.18 (p<0.000) for use as challenge virus. The challenge dose was estimated at 1x10(7) TCID50/mL per fish while the proportion of virus shedders was estimated at 12.5% of the total number of fish per tank. The model was designed based on a three parallel tank system in which the Cox hazard proportional regression model was used to estimate the minimum number of fish required to show significant differences between the vaccinated and control fish in each tank. All input variables were optimized to generate mortality >75% in the unvaccinated fish in order to attain a high discriminatory capacity (DC) between the vaccinated and control fish as a measure of vaccine efficacy. The model shows the importance of using highly susceptible fish to IPNV in the optimization of challenge models by showing that highly susceptible fish had a better DC of differentiating vaccine protected fish from the unvaccinated control fish than the less susceptible fish. Once all input variables were optimized, the model was tested for its reproducibility by generating similar results from three independent cohabitation challenge trials using the same input variables. Overall, data presented here show that the cohabitation challenge model developed in this study is reproducible and that it can reliably be used to evaluate the efficacy of vaccines developed against IPNV in Atlantic salmon. We envision that the approach used here will open new avenues for developing optimal challenge models for use in evaluating the efficacy of different vaccines used in aquaculture.

Phylogenetic analysis of infectious pancreatic necrosis virus in Ireland reveals the spread of a virulent genogroup 5 subtype previously associated with imports.

Infectious pancreatic necrosis is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality and disease-management costs. Significant outbreaks of the disease occurred in farmed Atlantic salmon in Ireland between 2003 and 2007, associated with imported ova and smolts. As the virus was known to occur in the country since the development of aquaculture in the 1980s, this study examined archived samples to determine whether these older isolates were associated with virulent forms. The study showed that two genotypes of IPNV were present in the 1990s, genotype 3 and genotype 5. A more virulent subtype of the virus first appeared in 2003 associated with clinical outbreaks of IPN, and this subtype is now the most prevalent form of IPNV found in the country. The data also indicated that IPNV in Ireland is more closely related to Scottish and continental European isolates than to Norwegian, Chilean and Australasian genogroup 5 isolates.

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.

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.

Isolation and molecular characterization of a novel infectious pancreatic necrosis virus strain in returning Atlantic salmon Salmo salar from the Connecticut River, USA.

After 22 years of negative viral screening results, the viral pathogen infectious pancreatic necrosis virus (IPNV) was isolated from the ovarian fluid of two pooled samples of returning Connecticut River Atlantic salmon Salmo salar during the 2007 spawning season at Richard Cronin National Salmon Station (RCNSS), Hadley, Massachusetts. Cytopathic effect was observed in Chinook salmon embryo (CHSE-214) cells, and IPNV was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis conducted by the U.S. Geological Survey's Western Fisheries Research Center determined that the isolate closely resembled the Canada_3 strain, falling into Genogroup 4 rather than Genogroup 1, which is more common in the United States. This allowed us to speculate that the Atlantic salmon were not infected during their freshwater life stage in the Connecticut River watershed but somewhere on their migratory route or feeding grounds in the Northwest Atlantic. On November 20, 2007, the Connecticut River Atlantic Salmon Commission voted to depopulate the infected stock at RCNSS and the entire suspect egg lots held at White River National Fish Hatchery, Vermont. Approximately one and a half months later, the 121 Connecticut River Atlantic salmon were euthanized and sampled for a follow-up investigation to determine the prevalence of infection. Only one kidney-spleen homogenate (male) was confirmed IPNV positive via cell culture and RT-PCR. A total of 2,983 base pairs from segment A of the RNA genome were sequenced from this fish and determined to be from a new strain (Connecticut-1) of IPNV that closely resembles Canada_2 and Canada_3 in Genogroup 4. The new strain is genetically identical to one of the first ovarian fluid isolates over a shared 130-nucleotide region, possibly indicating original transmission from a single source. The absence of IPNV from the Connecticut River's subsequent four returning Atlantic salmon year-classes may indicate that the aggressive corrective action was prudent.

Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile.

Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.

Detection of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea by RT-LAMP assay.

The viral diseases have been the serious problem in salmonid farming, and rainbow trout is not an exception. In this study, routine surveys were conducted for detecting of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea during 2009-2010. Head kidneys from individual fish were employed for virus detection by using a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) were the target viruses in this study. 53.5% (46/86) were found to be IPNV-positive, while IHNV and VHSV showed RT-LAMP negative during examination for 2 years. Ten IPNV-positive samples were randomly selected for viral isolation and the cells showing CPEs were subjected to RT-LAMP, RT-PCR, and direct sequencing. Phylogenetic analysis showed that the rainbow trout isolate has high similarity homologies with the VR-299 strain, as previously described.

Sequence similarities of the capsid gene of Chilean and European isolates of infectious pancreatic necrosis virus point towards a common origin.

The Chilean salmonid industry was developed by importing breeding materials, a practice still in effect due to deficits in the national supply of roe. Importation of breeding materials is often associated with the transmission of pathogens. The objectives of this study were to compare the infectious pancreatic necrosis virus (IPNV) isolates from Chile to those of European origin and to determine the diversity of the Chilean IPNV. The VP2 genes of IPNV from Chilean fish (whose eggs originated from Scotland, Iceland and Norway) were compared to isolates from fish in Norway and Ireland. The results show that the isolates are identical (97-99%) and cluster into one genogroup. Our findings support previous reports of association between the trade-in breeding materials and transmission of pathogens. Furthermore, our results demonstrate the genotypic diversity of Chilean IPNV isolates. These findings have important implications for IPNV disease diagnosis and control in Chile.

Detection and genetic analysis of aquabirnaviruses in subclinically infected aquarium fish.

Aquabirnaviruses (ABVs) cause serious diseases in a variety of fish species used worldwide in aquaculture and have been isolated from a variety of healthy fish and shellfish species. The type species of ABV is Infectious pancreatic necrosis virus (IPNV), which is the causative agent of a highly contagious disease in juvenile salmonid fish. Marine birnaviruses (MABVs) have been isolated from various marine fish and shellfish. In Korea, ABV infection has been identified in several fish and shellfish. The current study presents sequence data from nested polymerase chain reaction products of 3 ABV strains obtained from different species of asymptomatic aquarium fish collected from a private commercial aquarium in Korea. Phylogenetic analysis of these strains, based on the partial nucleotide sequence of the VP2/NS junction, placed them within the genogroup VII (95-99% bootstrap confidence), which also contains MABV. The subclinically infected fish may be a source of MABV infection for other susceptible fish species inside the aquarium and potentially represent a serious challenge for the management of MABV infections. Additionally, the presence of MABV in these subclinically infected aquarium fish imported from other countries indicates that there is a need for the establishment of appropriate quarantine practices.

Molecular differentiation of infectious pancreatic necrosis virus isolates from farmed and wild salmonids in Ireland.

This study investigated the genotypes and sub-groups of infectious pancreatic necrosis virus (IPNV) present in farmed and wild salmonid fish in Ireland. An 1100-bp portion of the VP2 region of segment A from each of 55 IPNV isolates collected over 2003-2007 was amplified by reverse-transcription-polymerase chain reaction and the product directly sequenced. The nucleotide sequences of each isolate were aligned and compared with each other and with the corresponding sequences of a number of reference isolates. All the 55 sequenced isolates belonged to genogroup 5 (Sp serotype) and could be divided into two subgroups. Irish subgroup 1 consisted of isolates from farmed salmon originating from an Irish salmon broodstock. Irish subgroup 2 consisted of isolates from imported farmed stock and all reported clinical outbreaks of IPN were associated with isolates from subgroup 2. Isolates from wild fish were identical to some isolates from subgroup 2, and therefore are believed to have originated from infected farms. These results highlight the importance of import risk analysis for diseases not listed under current legislation.

Detection and genotyping of an infectious pancreatic necrosis virus from asymptomatic rainbow trout (Oncorhynchus mykiss) facilities in Mexico.

BACKGROUND: Infectious pancreatic necrosis virus (IPNV) is a birnavirus that causes a lethal disease in both hatchery-reared juvenile salmonids and other non-salmonid fishes. IPNV has been classified into seven genogroups based on the analysis of the VP2/NS junction region of the viral A RNA segment. METHODS: Ten organisms from two trout-rearing farms were used for viral isolation in RTG-2 cells. Cells were inoculated with samples from spleen, kidneys and pyloric caeca. The viral isolate was initially identified by electron microscopy, and confirmed by a commercial ELISA, RT-PCR and sequencing. A phylogenetic tree was constructed based on the deduced amino acids sequence of VP2. RESULTS: An IPNV genogroup 1, labeled as EdoMex07, was isolated from a pool of renal tissues of five asymptomatic trout. The amino acid sequence analysis of VP2 showed that this IPNV presented the putative VP2 residue (221) already described in asymptomatic trout carriers. CONCLUSIONS: The EdoMex07 IPNV isolate belongs to genogroup 1, and has a VP2 phenotype which has been suggested to be involved in the establishment of the carrier state. This EdoMex07 IPNV is currently used as the standard positive control for detection of IPNV in rainbow trout farms in Mexico.

Genetic analysis of infectious pancreatic necrosis virus from Scotland.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: Extensive optimization is required to produce a PCR for detection of fish pathogens from methods designed in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).