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Molecular characterisation of infectious pancreatic necrosis viruses isolated from farmed fish in Finland.

ABSTRACT: Infectious pancreatic necrosis virus (IPNV) has been isolated annually since 1987 from salmonids without clinical signs at coastal fish farms in Finland. In the inland area, viral isolations were rare until 2012, when IPNV was detected at several freshwater fish farms. Between 2013 and 2015, the infection spread and IPNV was continuously isolated from several farms, both inland and on the coast. The aim of this study was to genetically characterise the IPNV isolates collected from Finnish coastal and inland fish farms over the last 15 years, and to detect genetic changes that may have occurred in the virus populations during the study period. The partial VP2 gene sequence from 88 isolates was analysed. In addition, a complete genomic coding sequence was obtained from 11 isolates. Based on the genetic analyses, Finnish IPNV isolates belong to three genogroups: 2, 5 and 6. The genetic properties of the isolates appear to vary between inland farms producing juveniles and food fish farms in the coastal region: the inland farms harboured genogroup 2 isolates, whereas at coastal farms, all three genogroups were detected. Little genetic variation was observed within the Finnish genogroup 2 and 5 isolates, whereas among the genogroup 6 isolates, two subgroups were detected. All isolates studied demonstrated amino acid patterns in the viral VP2 gene previously associated with avirulence. However, increased mortality was detected at some of the farms, indicating that more research is needed to clarify the relationship between the pathogenicity and genetic properties of IPNV isolates from different genogroups.

SUBMITTER: Holopainen R

PROVIDER: S-EPMC5640752 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Molecular characterisation of infectious pancreatic necrosis viruses isolated from farmed fish in Finland.

Holopainen Riikka R Eriksson-Kallio Anna Maria AM Gadd Tuija T

Archives of virology 20170809 11

Infectious pancreatic necrosis virus (IPNV) has been isolated annually since 1987 from salmonids without clinical signs at coastal fish farms in Finland. In the inland area, viral isolations were rare until 2012, when IPNV was detected at several freshwater fish farms. Between 2013 and 2015, the infection spread and IPNV was continuously isolated from several farms, both inland and on the coast. The aim of this study was to genetically characterise the IPNV isolates collected from Finnish coasta ...[more]

PMID: 28795226

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Project description: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.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).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.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.

Project description:Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. KEY POINTS: • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

Project description:We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Dataset Information

Molecular characterisation of infectious pancreatic necrosis viruses isolated from farmed fish in Finland.

Publications

Molecular characterisation of infectious pancreatic necrosis viruses isolated from farmed fish in Finland.

Similar Datasets

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