Integrity and wound healing of rainbow trout intestinal epithelial cell sheets at hypo-, normo-, and hyper-thermic temperatures.

How temperature influences fish physiological systems, such as the intestinal barrier, is important for understanding and alleviating the impact of global warming on fish and aquaculture. Monolayers of the rainbow trout cell line, RTgutGC, with or without linear 500 µm wide gaps (wounds) were the in vitro models used to study the integrity and healing of intestinal epithelial sheets at different temperatures. Cultures at hypothermic (4 °C) or hyperthermic (≥ 26 °C) temperatures were compared to normothermic control cultures (18-22 °C). Monolayers remained intact for at least a week at temperatures from 4 to 28 °C, but had lost their integrity after 3 h at 32 °C as the cells pulled away from one another and from the plastic surface. F-actin appeared as prominent stress fibers in cells at 28 °C and as blobs in cells at 32 °C. At normothermia and at 26 °C, cells migrated as sheets into the gaps and closed (healed) the gaps within 5-6 days. By contrast, wounds took 14 days to heal at 4 °C. At 28 °C some cells migrated into the gap in the first few days but mainly as single cells rather than collectively and wounds never healed. When monolayers with wounds were challenged at 32 °C for 3 h and returned to 18-22 °C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26 °C for 24 h and challenged at 32 °C for 3 h prior to being placed at 18-22 °C, cell shape and actin cytoskeleton were maintained, and wounds were healed over 6 days. Thus, intestinal epithelial cells become thermostabilized for shape, cytoskeleton and migration by a prior heat exposure.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

Xela DS2 and Xela VS2: Two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue.

The skin epithelial layer acts as an important immunological barrier against pathogens and is capable of recognizing and responding to pathogen-associated molecular patterns (PAMPs) in human and mouse models. Although presumed, it is unknown whether amphibian skin epithelial cells exhibit the ability to respond to PAMPs such as viral double-stranded RNA (dsRNA). To address this, two cell lines from the dorsal skin (Xela DS2) and ventral skin (Xela VS2) of the African clawed frog (Xenopus laevis) were established. Xela DS2 and Xela VS2 cells have an epithelial-like morphology, express genes associated with epithelial cells, and lack senescence-associated beta-galactosidase activity. Cells grow optimally in 70% Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 26 °C. Upon treatment with poly(I:C), a synthetic analogue of viral dsRNA and known type I interferon inducer, Xela DS2 and Xela VS2 exhibit marked upregulation of key antiviral and pro-inflammatory transcripts suggesting frog epithelial cells participate in the recognition of extracellular viral dsRNA and production of local inflammatory signals; similar to human and mouse models. Currently, these are the only known Xenopus laevis skin epithelial-like cell lines and will be important for future research in amphibian epithelial cell biology, initial host-pathogen interactions, and rapid screening of the effects of environmental stressors, including contaminants, on frog skin epithelial cells.

Understanding the pathogenesis of Flavobacterium psychrophilum using the rainbow trout monocyte/macrophage-like cell line, RTS11, as an infection model.

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.

Effect of selenomethionine on cell viability and heat shock protein 70 levels in rainbow trout intestinal epithelial cells at hypo-, normo-, and hyper-thermic temperatures.

As global warming and environmental pollution modify aquatic environments, the thermal biology of fish could be affected by interactions between temperature and pollutants, such as selenium (Se). Therefore, selenomethionine (SeMet) was studied for effects on cell viability and on heat shock protein 70 (HSP70) levels in the rainbow trout intestinal epithelial cell, RTgutGC, at hypothermic (4 °C), normothermic (14 and 18 °C) and hyperthermic (26 °C) temperatures. RTgutGC cultures remained viable for at least a week at all temperatures, although energy metabolism as measured with Alamar Blue (resazurin) was appreciably diminished at 4 °C. Over a 7-day incubation, HSP 70 levels in cultures remained steady at 4 °C, declined at 18 °C, and increased slightly at 26 °C. When 125 µM SeMet was present, cultures remained viable and HSP70 levels were neither increased nor decreased relative to control cultures, regardless of the temperature. With 500 and 1000 µM SeMet, cell viability was profoundly impaired after 7 days in cultures at 14, 18 and 26 °C but was unchanged at 4 °C. Overall the results suggest that only hypothermia modulated the response of rainbow trout cells to SeMet.

Responses of rainbow trout intestinal epithelial cells to different kinds of nutritional deprivation.

In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz's basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.

The characterization of γH2AX and p53 as biomarkers of genotoxic stress in a rainbow trout (Oncorhynchus mykiss) brain cell line.

Rainbow trout cell cultures were exposed to three genotoxicants and examined for effects on γH2AX and p53 levels by western blotting and on cell viability using the indicator dyes Alamar Blue (AB) for energy metabolism and 5'-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Bleomycin induced γH2AX and p53 in a dose- and time-dependent manner and had little cytotoxic effect. However, induction was first seen at 0.3 µM for γH2AX but not until 16.5 µM for p53. Methyl methanesulfonate (MMS) increased H2AX phosphorylation but diminished p53 levels as the dose was increased from 908 µM up to 2724 µM. Over this dose range cell viability was progressively lost. 4-nitroquinoline N-oxide (NQO) induced both γH2AX and p53, beginning at 62.5 nM, which was also the concentration at which cell viability began to decline. As the NQO concentration increased further, elevated γH2AX was detected at up to 2.0 µM, while p53 was elevated up to 1.0 µM. Therefore, H2AX phosphorylation was superior to p53 levels as a marker of DNA damage caused by genotoxicants that act by introducing double-stranded DNA breaks (bleomycin), alkyl groups (MMS), and quinoline adducts (NQO).

Tapasin's protein interactions in the rainbow trout peptide-loading complex.

Major histocompatibility complex (MHC) class I receptors play a key role in the immune system by presenting non-self peptides to T cell lymphocytes. In humans, the assembly of the MHC class I with a peptide is mediated by machinery in the endoplasmic reticulum referred as the peptide loading complex (PLC). Although, the identity of the PLC has been widely explored in humans, this complex has not been characterized in fish. Co-immunoprecipitation and mass spectrometry analysis revealed that the protein-protein interactions which exist in the human PLC are conserved in the monocyte/macrophage rainbow trout cell line (RTS11), in particular the interaction of tapasin with the transporter associated with antigen processing (TAP), MHC class I and ERp57. Importantly, a 20 kDa tapasin version that contains an intact C and N terminal domains was found to associate with ERp57 and form a 75 kDa heterodimer. These results suggest a possible novel alternative spliced version of tapasin may regulate the formation of the peptide-loading complex in teleosts.

Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout.

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18-22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.

Evaluating the toxic potential of benzothiazoles with the rainbow trout cell lines, RTgill-W1 and RTL-W1.

Benzothiazole (BTHs) are environmental contaminants of emerging concern for which little toxicological information is available. Therefore the toxic potential of twelve BTHs was evaluated with two rainbow trout epithelial cell lines, RTgill-W1 and RTL-W1. The BTHs were benzothiazole (BTH), 3,3'-diethylthia dicarbocyanine iodide (DTDC), C.I. sulphur orange 1 (SO), 2-mercaptobenzothiazole (2MBTH), zinc 2-mercaptobenzothiazole (ZnMBTH), sodium 2-mercaptobenzothiazole (NaMBTH), 2-hydroxy-benzothiazole (OHBTH), 2- aminobenzothiazole (2ABTH), C.I. vat yellow 2 (VY), N,N-dicyclohexyl-2-benzothiazolsulfene amide (NNA), 2,2'-dithiobis (benzothiazole) (DBTH) and 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid (MBTHS). All BTHs, except for NNA, DBTH, and MBTHS, caused both cytotoxicity and a transitory elevation in reactive oxygen species (ROS) levels. Yet, neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS imbalance did not contribute to cell death. Cell death was not blocked by Necrostatin-1 nor accompanied by DNA laddering, suggesting that neither necroptosis nor apoptosis took place. The comet assay revealed DNA strand breaks after exposures to 2ABTH and OHBTH for 1 day and to BTH for 12 days. In RTL-W1, cytochrome P4501A was induced noticeably by 2ABTH, OHBTH, and MBTHS and weakly by NaMBTH, ZnMBTH, SO, VY, and NNA, suggesting that these BTHs have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, several toxic actions were initiated in vitro by some but not all BTHs, warranting further study of these BTHs in vivo.

Use of the rainbow trout cell lines, RTgill-W1 and RTL-W1 to evaluate the toxic potential of benzotriazoles.

Epithelial cell lines, RTgill-W1 and RTL-W1 from respectively gill and liver of rainbow trout, Onchorhynchus mykiss (Walbaum), were used to evaluate the toxic potential of six benzotriazoles (BTRs) and tolytriazole (TT), which is a commercial mixture of 4-methyl-1H-benzotriazole (4MBTR) and 5-methyl-1H-benzotriazole (5MBTR). The other BTRs were 1H-benzotriazole (1H-BTR), 5-chlorobenzotriazole (5CBTR), 1-hydroxybenzotriazole (1OHBTR) and 5,6-dimethyl-1H-benzotriazole monohydrate (DM). Except for DM, all BTRs were cytotoxic at concentrations above 15mg/L and transitorily elevated reactive oxygen species (ROS) levels. Neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS were not the major cause of the cell death. Cell death was not blocked by Necrostatin nor accompanied by DNA laddering, suggesting that the cell death mechanism was neither necroptosis nor apoptosis. As judged by the comet assay, DNA strand breaks were detected with three BTRs: 4MBTR, 5MBTR and 5CBTR. In RTL-W1, the BTRs weakly induced cytochrome P4501A, suggesting that they have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, the toxic potential of BTRs appears to be limited to only high concentrations, which are higher than have been measured in the environment to date.

The p53 inhibitor, pifithrin-α, disrupts microtubule organization, arrests growth, and induces polyploidy in the rainbow trout gill cell line, RTgill-W1.

Pifithrin-α (PFT-α) blocks p53-dependent transcription and is an example of the many drugs being developed to target the p53 pathway in humans that could be released into the environment with potential impacts on aquatic animals if they were to become successful pharmaceuticals. In order to understand how p53 drugs might act on fish, the effects of PFT-α on rainbow trout gill epithelial cell line, RTgill-W1, were studied. PFT-α was not cytotoxic to RTgill-W1 in cultures with or without fetal bovine serum (FBS), but at 5.25µg/ml, PFT-α completely arrested proliferation. When FBS was present, PFT-α increased the number of polyploid cells over 12days. Those results suggest that like in mammals, p53 appears to regulate ploidy in fish. However, several effects were seen that have not been observed with mammalian cells. PFT-α caused a transient rise in the mitotic index and a disruption in cytoskeletal microtubules. These results suggest that in fish cells PFT-α affects microtubules either directly through an off-target action on tubulin or indirectly through an on-target action on p53-regulated transcription.

Demonstration of primary cilia and acetylated α-tubulin in fish endothelial, epithelial and fibroblast cell lines.

Primary cilia (PC) were demonstrated for the first time in fish endothelial, epithelial and fibroblast cell lines through immunofluorescence staining with the monoclonal antibody, 6-11B-1, against acetylated α-tubulin. The study was carried out with eight recently developed cell lines from the walleye, Sander vitreus (Mitchill). These were three fibroblast-like cell lines, WE-cfin11f, WE-skin11f and WE-liver3 from, respectively, the caudal fin, skin and liver, and three epithelial-like cell lines, WE-cfin11e, WE-spleen6 and WErpe from, respectively, the caudal fin, spleen and retina. Also, endothelial-like WEBA from the bulbus arteriosus and glial-like WE-brain5 from the brain were used. Immunocytochemistry revealed strong staining for acetylated α-tubulin in mitotic spindles and midbodies for all cell lines, and in PC for all cell lines except WE-skin11f. Staining of cytoplasmic microtubules (fibrils) was absent in three cell lines, including WEBA, but present in the others, especially WE-skin11f, which might have obscured PC detection in these cells. Tubacin, an inhibitor of histone deacetylase 6, induced cytoplasmic fibrils in WEBA and the intensity of their staining in WE-cfin11f. These results suggest that the cell lines might differ in their deacetylase activities, making them useful for studying this tubulin modification in teleosts, as well as for studying PC.

Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA.

A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle.

Delineating cellular interactions between ciliates and fish by co-culturing Tetrahymena thermophila with fish cells.

Although several species of Tetrahymena are often described as histophagous and opportunistic pathogens of fish, little is known about ciliate/fish cell interactions, but one approach for studying these is in vitro with cell lines. In this study, T. thermophila, B1975 (wild type) and NP1 (temperature sensitive mutant for phagocytosis) were cultured on monolayers of 3 fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and the rabbit kidney epithelial cell line, RK-13. Generally the ciliates flourished, whereas the monolayers died, being completely consumed over several days. The destruction of monolayers required that the ciliates could make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could be phagocytosed. The ciliates internalized into food vacuoles ZEB2J from cell monolayers as well as from cell suspensions. Phagocytosis was essential for monolayer destruction as monolayers remained intact under conditions where phagocytosis was impeded, such as 37°C for NP1 and 4°C for B1975. Monolayers of fish cells supported the proliferation of ciliates. Thus T. thermophila can 'eat' animal cells or be histophagous in vitro, with the potential to be histophagous in vivo.

Induction of rainbow trout MH class I and accessory proteins by viral haemorrhagic septicaemia virus.

Major histocompatibility (MH) class I receptors are glycoproteins which play a critical role during responses to intracellular pathogens by presenting endogenous peptides to cytotoxic T cell lymphocytes (CD8+). To date, little is known about MH class I regulation at the protein level during viral infections in fish. In this study, we characterised the MH class I pathway response to polyinosinic-polycytidylic acid (poly I:C) and upon infection with viral haemorrhagic septicemia virus (VHSV) genotype IVa using the rainbow trout monocyte/macrophage cell line RTS11. A 14-day challenge with VHSV IVa at 14°C demonstrated enhanced expression of the class I heavy chain, ß2 microglobulin (ß2M) and tapasin, while the expression of other accessory molecules ERp57 and calreticulin remained unchanged. However, when infection occurred at 2°C no change in expression levels of any of these molecules was observed. ß2M accumulated in the media of RTS11 over time, however the ß2M concentrations were 2 fold higher in cultures infected with VHSV 14 days post infection. Strikingly, when cells were maintained at 2°C the secretion of ß2M was significantly reduced in both infected and non-infected cultures. These results indicate that VHSV infection alters the kinetics of ß2M release as well as the expression of MH class I and suggests that cellular immunity against VHSV can be compromised at low temperatures which may increase host susceptibility to this virus during the winter.

The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells.

The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.

Expression of tapasin in rainbow trout tissues and cell lines and up regulation in a monocyte/macrophage cell line (RTS11) by a viral mimic and viral infection.

Tapasin is a transmembrane glycoprotein that acts as a bridge between the transporter associated with antigen processing and the MHC class I receptor in mammals. Through the development of antibody against trout tapasin, this report demonstrates the detection of trout tapasin as a N-glycosylated 48 kDa protein. Tissue and cell line distribution revealed that tapasin protein is expressed mainly in immune system organs and in rainbow trout epithelial cell lines from gill (RTgill-W1), liver (RTL-W1), and intestine (RTgutGC). An additional 20 kDa band was observed in tissues and cell lines, and appeared to be most prominent in RTgutGC but was absent in peripheral blood leukocytes. Tapasin 48 kDa protein was most strongly expressed in RTS11 (monocyte/macrophage cell line) and its regulation following dsRNA stimulation was explored. Upon poly I:C treatment and Chum Salmon Reovirus (CSV) infection, tapasin protein expression was upregulated up to 3.5 fold and 3 fold respectively, in parallel with increased expression of the glycosylated MH class I heavy chain, whereas the expression of the 20 kDa form remained unchanged. Overall this work demonstrates the induction of tapasin protein by dsRNA stimulation, which implies its possible conserved regulation during viral infection in teleost cells.

Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three non-host species.

Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), chinook salmon embryonic (CHSE-214) and fathead minnow Epithelioma papulosum cyprinid (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were challenged with heat-inactivated GIV and UV-treated GIV (GIV(UV)). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIV(UV). Relative to GIV, GIV(UV) caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC is a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titer. The three non-host cell lines present a good system for potentially identifying different components of GIV-induced apoptotic pathways in future studies.