High permissivity of the fish cell line SSN-1 for piscine nodaviruses.

Seventeen isolates of piscine nodavirus from larvae or juveniles of 13 marine fish species affected with viral nervous necrosis (VNN) were examined for their infectivity to a fish cell line SSN-1. Based on cytopathic effects (CPE) and virus antigen detection by fluorescent antibody technique (FAT) after incubation at 25 degrees C, the infectivity of these virus isolates was divided into 4 groups. Group 1, including 9 virus isolates from 4 species of grouper, 2 species of sea bass, barramundi, rock porgy, and Japanese flounder showed CPE characterized by rounded, granular cells with heavy cytoplasmic vacuoles within 3 d post-incubation (p.i.), and the monolayer partially or completely disintegrated over 3 to 6 d p.i. Scattered FAT-positive cells appeared at 3 h p.i. and spread through the cell sheet with an increasing fluorescence signal over 24 h p.i. Group 2, consisting of 3 virus isolates from striped jack, induced CPE with thin or rounded, granular, refractile cells without conspicuous vacuole formation, and extensive FAT-positive reaction was observed in a time course similar to that of Group 1. Cells inoculated with Group 3 (1 isolate from tiger puffer) developed no distinct CPE but viral infection was evidenced by localized FAT-positive cells. There were no FAT-positive cells in Group 4, which included 4 isolates from Japanese flounder, Pacific cod and Atlantic halibut. However, when incubation was performed at 20 degrees C, the SSN-1 cells inoculated with the Group 3 isolate showed CPE similar to that of Group 1 and extensive FAT-positive reaction. Evidence of virus proliferation at 20 degrees C was also obtained in Group 4 isolates. The virus titers in the infected fish varied from 10(11) to 10(16) tissue culture infectious dose (TCID50) g(-1) of fish. There is a good correlation between these infectivities to the SSN-1 cells and the coat protein gene genotypes of the isolates. The present results indicate that SSN-1 cells are useful for propagating and differentiating genotypic variants of piscine nodavirus.     

Temperature-dependency of Betanodavirus infection in SSN-1 cell line

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.     

The piscine SAF-1 cell line: genetic stability and labeling

Fish cell lines are increasingly important research tools. The SAF-1 cell line, fibroblast-like culture derived from the marine fish gilthead seabream (Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their properties and authentication are essential for deeper understanding of their performance and thus more precise experimental design and applicability. In this study we characterized the SAF-1 cell line in terms of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal RNA regions, and DNA content. For genetic labeling 12 microsatellite loci were used. The results indicate that telomerase has been activated in the course of SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell proliferation, thus supporting a permanent cell line. This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA content values, and the genotypic profile allows SAF-1 authentication at the single individual level. This study increases the value of SAF-1 as an in vitro system, which is now one of the few well-characterized cell lines from a marine fish.     

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.     

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.     

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.     

Cloning and characterization of osteoclast precursors from the raw264.7 cell line

Summary Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed β₃ integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.     

Stable expression of bioactive recombinant pleurocidin in a fish cell line

Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limanda limanda and cloned in a vector under the control of a non-viral promoter (the carp β-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.     

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV)

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.     

Cloning of an osteoblastic cell line involved in the formation of osteoclast-like cells.

Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autoradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteoclast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1 alpha, 25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) increased cAMP production in KS-4 cells, and PTH and interleukin-1 alpha also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of beta-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype and represent a single cell line with the ability to promote osteoclast formation by a contact-requiring process.     

Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses

The structure of recombinant virus-like particles of malabaricus grouper nervous necrosis virus (MGNNV), a fish nodavirus isolated from the grouper Epinephelus malabaricus, was determined by electron cryomicroscopy (cryoEM) and three-dimensional reconstruction at 23-A resolution. The cryoEM structure, sequence comparison, and protein fold recognition analysis indicate that the coat protein of MGNNV has two domains resembling those of tomato bushy stunt virus and Norwalk virus, rather than the expected single-domain coat protein of insect nodaviruses. The analysis implies that residues 83 to 216 fold as a beta-sandwich which forms the inner shell of the T=3 capsid and residues 217 to 308 form the trimeric surface protrusions observed in the cryoEM map. The structural similarities between fish nodaviruses and members of the tombusvirus and calicivirus groups provide significant new data for understanding the evolution of the nodavirus family.     

Identification and functional analysis of salmon annexin 1 induced by a virus infection in a fish cell line

In this study, we investigated changes in protein expression of fish cells induced by infection of infectious pancreatic necrosis virus (IPNV) using two-dimensional electrophoresis and matrix-assisted laser desorption-time of flight proton motive force analysis and identified a novel type of salmon annexin 1 that is induced in fish cells by infection with IPNV. Northern blotting showed that this annexin is overexpressed in IPNV-infected cells compared to control cells, and further analysis revealed that it has a 1,509-bp full-length cDNA sequence with an open reading frame encoding 339 amino acids (GenBank accession no. AY944135). Amino acid sequence analysis revealed that this protein belongs to the annexin 1 subfamily. By applying RNA interference, the mRNA levels of salmon annexin 1 were suppressed and, under these conditions, apoptosis of IPNV-infected cells was significantly increased. While small interfering RNA (siRNA) treatment did not affect the levels of the viral proteins significantly until 10 h postinfection, it reduced the titer of extracellular virus to 25% of that of a scrambled siRNA-treated control. These data provide evidence of an antiapoptotic function for salmon annexin 1 that is important for IPNV growth in cultured cells.     

Cloning of fish enzymes and other fish protein genes

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24 h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC₅₀ of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

Cloning and functional characterization of TCRP1, a novel gene mediating resistance to cisplatin in an oral squamous cell carcinoma cell line

To explore the mechanisms of chemotherapy resistance, we previously established a multi-drug resistant cell line, Tca8113/Pingyangmycin (Tca8113/PYM) and identified differential expression in known genes and ESTs using microarray analysis. From among those ESTs we have now identified a novel gene producing an mRNA of 1834 nucleotides translated into a protein having 235 amino acids. This gene was denominated as tongue cancer resistance-associated protein 1 gene (TCRP1, accession number: EF363480). We further determined its functional characteristics. The results demonstrate that TCRP1 mediates a specific resistance to cisplatin in Tca8113 cells by reducing the cisplatin-induced apoptosis. This suggests that TCRP1 might be a novel molecular target to develop agents to reverse cisplatin-induced chemoresistance.