Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus.

The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly.

Protective and immunogenic effects of Escherichia coli-expressed infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in rainbow trout.

Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae which causes significant losses in the aquaculture industry. To develop a recombinant vaccine for IPNV, a cDNA construct of IPNV VP2-VP3 fusion gene was prepared and cloned into an Escherichia coli (E. coli) expression vector (pET-26b) to obtain recombinant protein products. A study was conducted to determine the antibody responses and protective capacity of this recombinant vaccine expressing VP2-VP3 fusion protein. Subsequently, juvenile rainbow trout were inoculated by injecting purified recombinant IPNV VP2-VP3 proteins, followed by challenge with virulent IPNV in rainbow trout. Our results demonstrate that recombinant E. coli derived VP2-VP3 fusion protein induced a strong and significantly (P < 0.05) higher IgM antibody response in serum samples compared to control groups. Following intraperitoneal challenge, the relative percent survival (RPS) rate of survivors was 83% for the vaccinated group. Statistical analysis of IgM levels indicated that immunogenicity of recombinant VP2-VP3 protein, combined with adjuvant, was much higher than any other groups of rainbow trout challenged with virulent IPNV. This result was confirmed by measuring the viral loads of IPNV in immunized rainbow trout which was drastically reduced, as analyzed by real-time RT-PCR. In summary, we demonstrate that E. coli-expressed IPNV VP2-VP3 injectable vaccine is highly immunogenic and protective against IPNV infection.

Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

Anterior gradient 2 (AGR2), a member of the protein disulfide isomerase family, has been implicated in various cancers including pancreatic ductal adenocarcinoma (PDAC) and is known to promote cancer progression. However, the prognostic value of AGR2 expression and the interaction with epithelial-mesenchymal transition (EMT) remain unclear. We investigated the clinical significance of AGR2 and EMT markers in PDAC patients by immunohistochemical analyses. Although AGR2 expression was not observed in normal pancreas, all pancreatic precursor neoplastic lesions were positive for AGR2, even at the earliest stages, including pancreatic intraepithelial neoplasia-1A, AGR2 expression was reduced in 27.7% (54/195 cases) of PDAC patients. AGR2 downregulation correlated with EMT markers (vimentin overexpression and reduced membranous E-cadherin expression), high Union for International Cancer Control stage (P<0.0001), high histological cellular grade (P<0.0001), and adverse outcome (P<0.0001). In vitro, targeted silencing of AGR2 in cancer cells using siRNA reduced cell proliferation, colony formation, cell invasiveness, and migration, but did not alter EMT markers. To confer a more aggressive phenotype and induce EMT in PDAC cells, we co-cultured PDAC cell lines with primary-cultured pancreatic stellate cells (PSCs) and found that AGR2 was downregulated in co-cultured PDAC cells compared with PDAC monocultures. Treatment with transforming growth factor beta-1 (TGF-ß), secreted from PSCs, decreased AGR2 expression, whereas inhibition of TGF-ß signaling using recombinant soluble human TGF-ß receptor type II and TGF-ß-neutralizing antibodies restored AGR2 expression. We conclude that AGR2 downregulation is a useful prognostic marker, induced by EMT, and that secreted TGF-ß from PSCs may partially contribute to AGR2 downregulation in PDAC patients. AGR2 downregulation does not induce EMT or a more aggressive phenotype, but is a secondary effect of these processes in advanced PDAC.

Molecular characterization of the VP2 gene of infectious pancreatic necrosis virus (IPNV) isolates from Mexico.

Infectious pancreatic necrosis virus (IPNV) is one of the most important viruses in the Pacific salmon Oncorhynchus spp., Atlantic Salmon Salmo salar, and Rainbow Trout O. mykiss industry. This virus has been shown to produce high mortality among salmonid fry and juveniles, and survivors might become carriers. Since 2000, IPNV has affected Mexican Rainbow Trout culture, resulting in considerable economic losses. In the current study, molecular characterization of the VP2 gene of a number of Mexican IPNV isolates was done and the virus's phylogenetic relationships to IPNV reference strains were investigated. The phylogenetic analysis indicated that Mexican IPNV isolates are closely related to strains from the United States and Canada and that all Mexican IPNV isolates belong to genogroup 1. Furthermore, low genetic diversity was found between the Mexican isolates (identity, 95.8-99.8% nucleotides and 95.8-99.6% amino acids). The result of the analysis of the amino acid residues found at positions 217, 221, and 247 (alanine, threonine, and glutamic acid, respectively) could be associated with virulence, although the expression of virulence factors is more complex and may be influenced by the agent and host factors. The high percentage of identity among the VP2 genes from geographically distant IPNV isolates and the evidence of wide distribution in the country might have been facilitated by carrier trout. This hypothesis is supported by the identification of the amino acid threonine at position 221 in all Mexican isolates, a factor related to the carrier state for IPNV, as reported by other studies.

A natural peptide and its variants derived from the processing of infectious pancreatic necrosis virus (IPNV) displaying enhanced antimicrobial activity: a novel alternative for the control of bacterial diseases.

The larger segment of the infectious pancreatic necrosis virus (IPNV) codifies most of the structural and non-structural proteins of the virus in two overlapping open reading frames (ORFs). The longer of the two ORF is expressed as a polyprotein which generates a number of variable length peptides of unknown function during processing. Since an appealing hypothesis would be that these peptides are generated by the virus to act as antimicrobial agents that favor viral infectivity in their fish host, we decided to test this possibility by selecting a master peptide and using it to generate substitution variants that may enhance their antimicrobial potential. A 20-residue master peptide (p20) was selected from the well-described maturation process of the structural viral protein VP2; several variants were then designed and chemically synthesized, ranging in size from 16 to 20 residues. The synthesized peptides were tested for in vitro activity against several prototype bacterial pathogens using standardized laboratory procedures. Chemically synthesized p20 and all its variants displayed broad activity against the tested bacteria and none of them were toxic to eukaryotic cells at least 10× the concentration used against the bacteria. Interestingly, when p20 was tested against the very aggressive bacterial pathogen Piscirickettsia salmonis, a common co-infectant of IPNV in salmonid fish, the specific activity of the novel peptide was significantly higher than that displayed for bactericidal fish farm antibiotics such as oxolinic acid, flumequine and florfenicol, which are commonly used to control Piscirickettsiosis in the field. It is potentially significant that the approach presented in this report provides a novel alternative for generating new and ideally more efficient and friendly safeguards for bacterial prophylaxis.

Molecular characterization of the VP1 gene of a Mexican isolate of infectious pancreatic necrosis virus.

The presence of infectious pancreatic necrosis virus (IPNV) in salmonids predominantly produces a high mortality rate in first-feeding fry. Genomic analysis of the vp2 gene sequence is most commonly used to determine the genetic diversity of IPNV isolates. Recently, information obtained from the vp1 gene allowed for efficient analysis of the genetic diversity of IPNV. In this study, the vp1 gene from a Mexican IPNV isolate was characterized and compared with IPNV isolates from Europe, North America, and Asia. The results indicate that the Mexican isolate is most closely related genetically to the 2310 strain from Spain.

Effect of a major QTL affecting IPN resistance on production traits in Atlantic salmon.

This study investigated the effect of a major QTL for resistance to IPN in salmon on performance and production traits. The traits studied were related to growth, fillet and gutted yields, and fat content. Two different analyses were performed: (1) regression of the phenotypic data of the production traits on the predicted number of resistant IPN-QTL alleles in individuals and (2) a variance component analysis using the (co)variance matrix calculated at the putative location of the QTL. No significant effect of the QTL was detected on any of the traits investigated by either method. The result has important practical implications in that it encourages the use of MAS to reduce the risks and impact of IPN mortality.

Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells.

Aquatic birnavirus induces post-apoptotic necrotic cell death via a newly synthesized protein-dependent pathway. However, the involvement of viral genome-encoded protein(s) in this death process remains unknown. In the present study, we demonstrated that the submajor capsid protein, VP3, up-regulates the pro-apoptotic protein, Bad, in fish and mouse cells. Western blot analysis revealed that VP3 was expressed in CHSE-214 cells at 4 h post-infection (pi), indicating an early role during viral replication. We cloned the VP3 gene and tested its function in fish and mouse cells; VP3 overexpression induced apoptotic cell death by TUNEL assay. In addition, it up-regulated Bad gene expression in zebrafish ZLE cells by threefold at 12 h post-transfection (pt) and in mouse NIH3T3 cells by tenfold at 24 h pt. VP3 up-regulation of Bad expression altered mitochondria function, inducing mitochondrial membrane potential (MMP) loss and activating initiator caspase-9 and effector caspase-3. Furthermore, reduced Bad expression (65% reduction), MMP loss (up to 40%), and enhanced cell viability (up to 60%) upon expression of VP3 antisense RNA in CHSE-214 cells at 24 h post-IPNV infection was observed. Finally, overexpression of the anti-apoptotic gene, zfBcl-xL, reduced VP3-induced apoptotic cell death and caspase-3 activation at 24 h in fish cells. Taken together, these results suggest that aquatic birnavirus VP3 induces apoptosis via up-regulation of Bad expression and mitochondrial disruption, which activates a downstream caspase-3-mediated death pathway that is blocked by zfBcl-xL.

Binding of infectious pancreatic necrosis virus (IPNV) to membrane proteins from different fish cell lines.

The early interactions between infectious pancreatic necrosis virus (IPNV) from Atlantic salmon and susceptible cell lines were studied using a virus overlay protein binding assay (VOPBA). Membrane preparations from four different cell lines, bluegill fry (BF)-2 cells, Chinook salmon embryo (CHSE)-214 cells, salmon head kidney (SHK)-1 cells and Atlantic salmon kidney (ASK) cells were separated by SDS-PAGE, blotted to nitrocellulose membranes and incubated with either a highly virulent IPNV field isolate or different recombinant IPNV strains exhibiting variations in virulence. Binding of virus to the respective membrane fractions was detected using either polyclonal or monoclonal antibodies. Independent of variations in virulence, IPNV bound in a specific manner to an approximately 220-kDa membrane protein from the salmonid cell lines CHSE-214, SHK-1 and ASK, whereas the size of the binding protein from the non-salmonid cell line BF-2 was approximately 190 kDa. Neutralization of IPNV prior to incubation with the blotted membrane fractions inhibited binding to the indicated proteins. Complete deglycosylation of the membrane proteins did not interfere with the binding of IPNV, suggesting that the interaction between virus and cells is not carbohydrate specific. The described binding proteins may represent putative cellular binding sites or receptors for IPNV.

Studies on uptake and intracellular processing of infectious pancreatic necrosis virus by Atlantic cod scavenger endothelial cells.

Previous work in our group has identified the scavenger endothelial cells (SECs) of heart endocardium in cod, Gadus morhua L., as the major site for elimination of both physiological and foreign macromolecular waste from the circulation. The present study was undertaken to establish the role of cod SECs in the clearance of virus. We focused on infectious pancreatic necrosis virus (IPNV) as it is a well-known virus with a broad host range, and causes significant economic losses in the salmon industry. Our results showed that cod SEC cultures infected by the IPNV produce high titres of new virus. Ligand-receptor inhibition experiments suggested that the virus did not enter the cells through any of the major endocytosis receptors of cod SECs. Yet, the infection lowered the capacity of the cells to endocytose ligands via the scavenger receptor. Inhibitors of receptor recycling and vesicle acidification did not affect virus infectivity. The finding that SEC cultures prepared from 25% of the cod produced high titres of IPNV without being infected in the laboratory, suggests that SECs of cod may serve as reservoirs for IPNV in persistently infected cod.

VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA.

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.

Characterization of cleavage sites and protease activity in the polyprotein precursor of Japanese marine aquabirnavirus and expression analysis of generated proteins by a VP4 protease activity in four distinct cell lines.

A polyprotein precursor NH(2)-pVP2-VP4-VP3-COOH is encoded in genomic segment A of members of the family Birnaviridae. By N-terminal sequencing analysis, primary cleavage sites of a marine birnavirus (MABV) polyprotein were identified as Ala(508) downward arrow Ser(509) and Ala(734) downward arrow Ser(735), where the cleavage motif was the same as that of infectious pancreatic necrosis virus (IPNV). However, further VP4 and VP3 cleavages occurred at novel sites. Ser(633) and Lys(674) mutations affected the cleavage activity by site-directed mutagenesis. Additional catalytic residues including Ile(543) and Val(686) were MABV-specific. As shown by electron microscopy, pVP2 and further cleaved VP3s (fcVP3s) could not form virus-like particles (VLPs). This suggests that VP3 is necessary for VLP formation. By Western blot analysis of the VP3 expression, fcVP3s were found in RSBK-2 cells and FHM cells, while VP3 was cleaved less in EPC cells, suggesting that fcVP3s might merely be a degraded form. Alternatively, if fcVP3s play functional roles other than in viral assembly, the further VP3 cleavage is, at least, not restricted in FHM cells. Strangely, VP3 was not completely further cleaved in CHSE-214 cells despite the fact that this cell line has a potential proteolytic factor, implying that complicated factors are associated with the further VP3 cleavage.

Inhibition of virion-associated IPNV RNA polymerase, VP1, by radiolabeled nucleotide analogs.

Infectious pancreatic necrosis virus (IPNV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The structural protein VP1 has been postulated as the RNA-dependent RNA polymerase (RdRp), but its transcriptional activity has not been unequivocally identified from viral particles. Here, we assayed partially purified IPNV in an in vitro RNA synthesis system. To test the RdRp, dialdehyde-nucleotide analogs were used to covalently inhibit the polymerase-associated activity. Our results showed that dialdehyde-nucleotide analogs completely abrogated IPNV in vitro RNA synthesis. The protein involved in this process was identified as viral VP1, since: (a) after incubation of IPNV with [alpha-(32)P]2',3'-dialdehyde-UTP, labeled VP1 protein was identified and (b) VP1 was unable to bind [alpha-(32)P]GTP when particles were preincubated with 2',3'-dialdehyde-ATP. Thus, within viral particles, inhibition of the transcriptional activity is a result of the binding of 2',3'-dialdehyde-nucleotide analogs to the RdRp, VP1.

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line.

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.

Genome assembly and particle maturation of the birnavirus infectious pancreatic necrosis virus.

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.

Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis.

VP1, the putative virion-associated RNA-dependent RNA polymerase (RdRp) of infectious pancreatic necrosis virus (IPNV) can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis [Virology 208 (1995) 19]. The role of a template or other virion polypeptides in the reaction is unknown. To shed light on this question, his-tagged recombinant VP(1) (rVP1) was expressed both in Escherichia coli and insect cells and used in the guanylylation reaction. Unlike other viral VPg polypeptides, the purified rVP1 alone could guanylylate itself in vitro in a template-independent manner. Chemical and enzymatic cleavage in combination with site-directed mutagenesis mapped the site of guanylylation to serine 163. The purified rVP1 functioned as a primer as well as an RdRp in vitro, producing labeled dsRNA in the presence of [alpha(32)P] NTP and synthetically produced viral ss + RNA as a template. Only a single cycle of replication was observed and labeled VPg could be recovered from the dsRNA by RNase V(1) digestion. Denaturation of the dsRNA yielded genome-length labeled ssRNA, indicating that RNA synthesis was not initiated by 3'-end snap-back self-priming. Mutating serine 163 to alanine of rVP1 abolished both its self-guanylylating and polymerizing activity.

Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2.

Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline beta elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.

The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.

Expression of infectious pancreatic necrosis virus polyprotein and VP1 in insect cells and the detection of the polyprotein in purified virus.

In order to study the molecular biology of infectious pancreatic necrosis virus (IPNV) replication, six different recombinant baculoviruses were constructed. The following four recombinants contained genome segment A-specific sequences; (i) AcPP contained the complete polyprotein coding region and Spodoptera frugiperda (Sf) cells infected by these recombinants synthesized the 106-kDa polyprotein (NH2-preVP2-NS protease-VP3-COOH), which was only partially processed by the protease to yield preVP2 and VP3 and unprocessed polyprotein; (ii) AcPP(S) and AcPP(Ss) represented 3' truncated sequences of the segment A cDNA where the VP3 coding region and that coding for 30 and 98 carboxy terminal amino acids of NS in the two constructs, respectively, were deleted. AcPP(S) demonstrated partial, and that of AcPP(Ss), complete loss of proteolytic activity, demonstrating that the carboxy one-third of the 29-kDa NS protease is necessary for the formation of the active enzyme; and (iii) AcPP(B/B) contained all but the first 180 nt of the pVP2 gene, the complete NS coding region, and the amino end of VP3. Analysis of cells coinfected with AcPP(Ss) and AcPP(B/B) showed either that the protease did not work in trans or that the alteration of the structure of the substrate prevented cleavage. Recombinant baculoviruses AcVP1VL and AcVP1ETL contained IPNV genome segment B cDNA encoding the 94-kDa VP1 which is the viral RNA-dependent RNA polymerase. AcVP1VL contained the whole segment B cDNA, whereas in AcVP1ETL, the 5' non-coding sequences were deleted resulting in the production of large amounts of VP1 when Sf cells were infected with this recombinant. The use of recombinants AcPP and AcVP1ETL as well as monoclonal antibodies and VP1-specific sera allowed the unambiguous identification of the high molecular weight minor polypeptides present in purified IPNV demonstrating the presence of both VP1 and the polyprotein in purified virus preparations.