Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.     

Isolation of different types of birnavirus from ayu Plecoglossus altivelis and amago salmon Oncorhynchus rhodurus cultured in the same geographic area

A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.     

Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

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.     

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.     

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.     

Solar UV radiation does not inactivate marine birnavirus in coastal seawater

We examined the inactivation kinetics of marine birnavirus (MABV) in a coastal sea, in seawater samples collected from 50 cm depth. MABV was added to both natural and autoclaved seawater at a concentration of 6 x 10(6.43) TCID50 (50% tissue culture infectious dose) ml(-1), put in dialysis tubes and incubated at the original depth. The inactivation of MABV by solar UV radiation was examined using light and dark tubes. The infectivity titer of MABV was measured by the TCID50 method using CHSE-214 cells. Virus infectivity in natural seawater decreased quickly and was below the detection limit by 270 min in both light and dark conditions; however, virus infectivity was maintained in the autoclaved seawater until 420 min. These results suggest that the loss of virus infectivity is not caused by sunlight UV radiation.     

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.     

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.     

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.     

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.     

Distribution of marine birnavirus (MABV) in marine organisms from Okinawa,Japan, and a unique sequence variation of the VP2/NS region

Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25°C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus

We have identified a region related to the protease domain of bacterial and organelle ATP-dependent Lon proteases in virus protein 4 (VP4) of infectious bursal disease virus strain P2 (IBDVP2), a two-segmented double-stranded RNA virus. Unlike canonical Lons, IBDVP2 VP4 possesses a proteinase activity though it lacks an ATPase domain. Ser652 and Lys692 of IBDVP2 VP4 are conserved across the Lon/VP4 family and are essential for catalysis. Lys692 has the properties of a general base, increasing the nucleophilicity of Ser652; a similar catalytic dyad may function in the other Lons. VP4 can cleave in trans and is responsible for the interdomain proteolytic autoprocessing of the pVP2- VP4-VP3 polyprotein encoded by RNA segment A. VP2, which is later derived from pVP2, and VP3 are major capsid proteins of birnaviruses. Results of the characterization of a range of the IBDVP2 VP4 mutants in cell cultures implicate VP4 in trans-activation of the synthesis of VP1, putative RNA-dependent RNA polymerase encoded by RNA segment B, and in cleavage rate-dependent control of process(es) crucial for the generation of the infectious virus progeny.     

Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework.

The polyomavirus proteins VP1, VP2, and VP3 move from their cytoplasmic site of synthesis into the nucleus, where virus assembly occurs. To identify cellular or viral components which might control this process, we determined the distribution of VP1, VP2, and VP3 in a soluble fraction, a cytoplasmic cytoskeleton fraction, and a nuclear framework fraction of infected cells. All three proteins were detected in a detergent-extractable form immediately after their synthesis in polyomavirus-infected cells. Approximately 50, 25, and 40% of pulse-labeled VP1, VP2, and VP3, respectively, associated with the skeletal framework of the nucleus within 10 min after their synthesis. The remaining portion of each labeled protein failed to accumulate on the nuclear framework during a 40-min chase and was degraded. When expressed separately by recombinant vaccinia viruses, VP1 and VP2, but not VP3, accumulated on the nuclear framework. This association was not dependent on other polyomavirus proteins or viral DNA. The amount of total VP1 and VP2 which was bound to the nuclear framework approximated 45 and 20%, respectively. Indirect immunofluorescence demonstrated an exclusive nuclear localization of VP1 in situ. In coinfection experiments, a greater percentage of total VP2 and VP3 was bound to the nuclear framework of cells which cosynthesized VP1. These results indicate that although VP1 and VP2 can bind independently to the insoluble nuclear framework, the association of VP3 with this nuclear structure is promoted by the presence of VP1.     

The maturation process of pVP2 requires assembly of infectious bursal disease virus capsids

Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly.     

Cell Culture Evaluation of the Semliki Forest Virus Expression System As a Novel Approach for Antigen Delivery and Expression in Fish

Heterologous gene expression by Semliki Forest virus (SFV) expression vectors was investigated in fish cell culture. Experiments performed using an infectious strain of SFV, replication-defective SFV particles, and recombinant SFV RNA constructs encoding the Escherchia coli LacZ or firefly luciferase reporter genes indicated that levels of SFV-mediated expression in fish cells were dependent on cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9 at 25°C and 20°C. As the temperature was lowered to 15°C or below, levels of reporter gene expression were reduced up to 1000-fold, indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems to function in fish cells was further investigated by analyzing the expression of the protective VP2 antigen of infectious pancreatic necrosis virus (IPNV) from the various constructs, including a novel DNA-based SFV plasmid. The VP2 protein produced in CHSE-214 and F95/9 cells transfected or infected with the recombinant SFV-IPNV VP2 constructs appeared to be synthesized in an antigenically correct form, as evidenced by the ability to react with several conformation-dependent IPNV-specific monoclonal antibodies. Whether the temperature-restricted replication and expression displayed by SFV-based constructs in fish cell culture also occurs in vivo remains to be determined. Received January 29; accepted June 29, 1999.     

Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.