Evidence for the Detection of the Infectious Pancreatic Necrosis Virus Polyprotein and the 17-kDa Polypeptide in Infected Cells and of the NS Protease in Purified Virus

doi:10.1006/viro.1994.1572

Virology

Volume 204, Issue 2, 1 November 1994, Pages 580-589

https://doi.org/10.1006/viro.1994.1572 Get rights and content

Abstract

The larger genome segment A of infectious pancreatic necrosis virus (IPNV) contains two open reading frames (ORFs): (i) the large ORF encodes a 106-kDa polyprotein (PP) (NH2-pVP2-NS-VP3-COOH) which is cotranslationally cleaved by the NS protease to generate the major capsid polypeptides VP2 and VP3; (ii) the second small ORF, which overlaps the 5′ end of the PP ORF but in a different reading frame, encodes a 17-kDa arginine-rich polypeptide. Hitherto, neither the PP nor the 17-kDa polypeptide have been identified in infected cells, and the NS (nonstructural) polypeptide was thought not to be part of the virion. The smaller genome segment B of IPNV encodes a 94-kDa minor polypeptide VP1. Using recombinant baculoviruses expressing VP1 and PP as markers, the PP could be unambiguously identified in Western blots of infected fish-cell lysates and in purified IPNV. Anti-17-kDa and anti-NS serum was produced by injecting rabbits with bacterially expressed fusion proteins containing these polypeptides. Labeled 17-kDa polypeptide was immune-precipitated from infected cell lysates using the anti-17-kDa serum, whereas the NS and NS, (a truncated form of NS) polypeptides were identified in infected cells by immune precipitation and Western blotting using the anti-NS serum. Western blots of purified virus revealed two forms of truncated NS: (i) the NS, found in infected cells and (ii) a smaller polypeptide NS_ta. The identity of the virion NSt/NSta was also demonstrated by peptide mapping.

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Cited by (75)

Molecular docking of phyto-compounds against VP1 and VP2 proteins of IPNV (Infectious Pancreatic Necrosis Virus) for deriving possible therapeutic agents
2023, Materials Today: Proceedings
Citation Excerpt :
An additional small ORF of segment-A genome partially overlaps the amino terminal end of the polyprotein [8]. This tiny ORF encodes a 17-kDa arginine-rich minor polypeptide (VP5) with uncertain function [11]. The sequencing of a marine birnavirus VP5 gene revealed that VP5 is a basic protein which may participate in RNA binding.
Infectious Pancreatic Necrosis Virus (IPNV) is able to produce severe and congenital infections in fingerlings and fry salmonid fishes with high mortality up to 90%. Infected fishes that overcome IPN remain as asymptomatic carriers. Carrier fish may be the most important source for ensuring the presence of virus as these fish shed the virus through their body fluids. There are currently no therapeutic medications available to treat IPNV in asymptomatic fish. Here, we have focused upon screening of potential components derived from medicinal plants using molecular docking. 130 phyto-compounds from different medicinal plants were analyzed by molecular docking against VP1 and VP2 proteins of IPNV. Molecular docking revealed that out of 130 compounds analyzed, isovitexin exclusively forms a stable complex with VP1. Likewise, isoorientin forms a stable complex exclusively with VP2. It was observed that the phyto-compounds baicalin, and luteoloside form less stable complexes with these viral proteins. These phyto-components, isovitexin and isoorientin could be a potential candidate for eliminating IPNV from asymptomatic brood stock of fishes.
Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss)
2022, Fish and Shellfish Immunology
Citation Excerpt :
The first genomic segment, named A, contains two open reading frames. The first ORF, overlaps with the second ORF and codes for a non-structural protein called VP5 (17 kDa), which is detected only in infected cells [11]. The second ORF codes for a 106 kDa polyprotein (NH2-pV2-VP4-VP3-COOH) which, during the synthesis of viral proteins is co-translationally proteolyzed by the viral protease VP4 to produce the pVP2 precursor (62 kDa), VP4 (29 kDa) and VP3 (31 kDa).
In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1⁺ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-β1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.
An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)
2022, Fish and Shellfish Immunology
Infectious pancreatic necrosis virus (IPNV), belonging to the genus Aquabirnavirus within the family Birnaviridae, causes huge economic loss to the global salmonid industry every year. Recently, outbreaks of disease caused by genogroup I IPNV were found in many rainbow trout (Oncorhynchus mykiss) farms worldwide. An inactivated vaccine was prepared using a genogroup I IPNV isolate with an optimized procedure as incubation with β-propanolactone (BPL) at the final concentration of 0.5% at room temperature for 48 h. The inactivated vaccine was used to immunize rainbow trout, and the protection efficiency was evaluated by viral loads determination, immune-related genes quantification, and neutralizing antibody tests. The viral loads in immunized rainbow trout were significantly decreased and the strongest antiviral effect was observed on 30 days post-immunization (d.p.i). The expression of innate immune-related genes IFN-1, and Mx-1 genes were significantly up-regulated on 3, 7, and 15 d.p.i (p < 0.05), and adaptive immune-related genes CD4, CD8, and IgM genes were significantly up-regulated on 15 and 30 d.p.i (p < 0.05). Neutralizing antibodies were firstly detected on 30 d.p.i and the highest titer was observed on 45 d.p.i, which began to decrease on 60 d.p.i, but was still significantly higher than that in negative control fish. The results indicated that the vaccine prepared in this study could stimulate the non-specific and specific immune response and provide significant immune protection to the vaccinated rainbow trout.
In vitro reassortment between Infectious Pancreatic Necrosis Virus (IPNV) strains: The mechanisms involved and its effect on virulence
2017, Virology
Citation Excerpt :
IPNV is a non-enveloped icosahedral virus, with a bi-segmented ds RNA (Lightner and Post, 1969; Cohen et al., 1973; Dobos, 1977). Segment A contains two partially overlapping open reading frames (ORFs): the smaller one is located at the 5′ end and encodes the VP5 protein (Duncan et al., 1987; Magyar and Dobos, 1994; Dobos, 1995a), and the larger encodes a polyprotein, which is processed by the protease activity of VP4, yielding VP2, VP3 and VP4. VP2 is the major structural protein and is related to virulence (Heppell et al., 1995; Blake et al., 2001; Santi et al., 2004).
Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses.
Aquatic viruses induce host cell death pathways and its application
2016, Virus Research
Citation Excerpt :
Segment A contains a large open reading frame (ORF) encoding a 107-kDa polyprotein that is processed into the major structural proteins VP2 and VP3 by the viral protease VP4 (Duncan et al., 1987; Macdonald and Dobos, 1981; Petit et al., 2000). A second ORF, overlapping the N-terminal region of VP2, encodes the small non-structural protein VP5 (Håvarstein et al., 1990; Magyar and Dobos, 1994). Replication of IPNV takes place in the cytoplasm of susceptible cells, and within 16–20 h post infection (p.i.) that the infection manifests itself by a dramatic cytopathic effect (Dorson, 1982).
Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell’s death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases.
Birnavirus VP4 Processing Endopeptidase
2013, Handbook of Proteolytic Enzymes

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Regular ArticleEvidence for the Detection of the Infectious Pancreatic Necrosis Virus Polyprotein and the 17-kDa Polypeptide in Infected Cells and of the NS Protease in Purified Virus

Abstract

Regular Article
Evidence for the Detection of the Infectious Pancreatic Necrosis Virus Polyprotein and the 17-kDa Polypeptide in Infected Cells and of the NS Protease in Purified Virus