Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated ß-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye, Sander vitreus (Mitchill), at cold temperatures.

A cell line, WE-cfin11e, with an epithelial-like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast-like cell line, WE-cfin11f, and compared with WE-cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens-1 (ZO-1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE-cfin11e stained for ZO-1 and only WE-cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE-cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE-cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE-cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.

Use of cell lines and primary cultures to explore the capacity of rainbow trout to be a host for frog virus 3 (FV3).

The capacity of rainbow trout, Oncorhynchus mykiss, to be a host for frog virus 3 (FV3) was evaluated at the cellular level. Cell cultures from this species were tested for their ability to express FV3 major capsid protein (MCP) gene, to develop cytopathic effect (CPE), and to produce FV3. After FV3 addition, MCP transcripts were detected in six of six cell lines and in primary macrophage cultures. CPE developed in all cell culture systems, except primary lymphocytes. For the macrophage cell line, RTS11, and primary macrophages, cell death was by apoptosis because DNA laddering and Annexin staining were detected. By contrast, markers of apoptosis did not accompany CPE in three epithelial cell lines from the gill (RTgill-W1), intestine (RTgut-GC), and liver (RTL-W1) and in two fibroblast cell lines from gonads (RTG-2) and skin (RTHDF). Therefore, FV3 was able to enter and begin replicating in several cell types. Yet, FV3 was produced in only two cell lines, RTG-2 and RTL-W1, and only modestly. Overall, these results suggest that if tissue accessibility were possible, FV3 would have the capacity to induce injury, but the ability to replicate would be limited, likely making rainbow trout a poor host for FV3.

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb.

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1-34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects (CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished.

Walleye Sander vitreus (Mitchill) are relatively resistant to experimental infection with VHSV IVb and extant walleye strains vary in susceptibility.

Compared to fathead minnow, walleye demonstrate low susceptibility to experimental infection with VHSV IVb, regardless of route of exposure or water temperature at time of infection. In triplicate and duplicate groups, walleye were intraperitoneally (i.p.) injected (102 -108  pfu/fish) or waterborne-exposed (w; 1.4 × 107  pfu mL-1 ) with VHSV IVb. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV IVb infection were evident only in fish i.p. injected with 108  pfu at 12 °C. These fish had multifocal necrosis of several tissues including the gill and heart. There was no difference in mortality between walleye infected (w or i.p.) at 12 °C (spring stocking) compared with a declining temperature profile from 18 to 12 °C (fall stocking). There were significant differences (P < 0.05) in mortality between four extant walleye strains following i.p. infection, indicating that the choice of walleye strain for stocking might be an important consideration. Viral antigen was found in both i.p. and w-exposed walleye using immunohistochemistry, mostly within the gill and skin of w-exposed fish and most prominently in dermal fibrocytes. VHSV IVb was detected in multiple tissues from 6 to 21 days post-infection using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

Corexit 9500 inactivates two enveloped viruses of aquatic animals but enhances the infectivity of a nonenveloped fish virus.

The effects of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds, and human health have been examined, but there is a significant lack of study of the effect of this dispersant on aquatic viruses. In this study, the effects of Corexit 9500 on four aquatic viruses of differing structural composition were examined. Corexit 9500 reduced the titer of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titer of frog virus 3 (FV3), a virus with both enveloped and nonenveloped virions, was reduced only at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titer of nonenveloped infectious pancreatic necrosis virus (IPNV) but enhanced the titer of chum salmon reovirus (CSV) by 2 to 4 logs. With the ability to inactivate enveloped viruses and possibly enhance some nonenveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere.

Suitability of invertebrate and vertebrate cells in a portable impedance-based toxicity sensor: temperature mediated impacts on long-term survival.

Using ECIS (electric cell-substrate impedance sensing) to monitor the impedance of vertebrate cell monolayers provides a sensitive measure of toxicity for a wide range of chemical toxicants. One major limitation to using a cell-based sensor for chemical toxicant detection in the field is the difficulty in maintaining cell viability over extended periods of time prior to use. This research was performed to identify cell lines suitable for ECIS-based toxicity sensing under field conditions. A variety of invertebrate and vertebrate cell lines were screened for their abilities to be stored for extended periods of time on an enclosed fluidic biochip with minimal maintenance. Three of the ten cell lines screened exhibited favorable portability characteristics on the biochips. Interestingly, all three cell lines were derived from ectothermic vertebrates, and the storage temperature that allowed long-term cell survival on the enclosed fluidic biochips was also at the lower end of reported body temperature for the organism, suggesting that reduced cellular metabolism may be essential for longterm survival on the biochip. Future work with the ectothermic vertebrate cells will characterize their sensitivity to a wide range of chemical toxicants to determine if they are good candidates for use in a field portable toxicity sensor.

Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss).

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb.

Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line.

A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.

Applications and potential uses of fish gill cell lines: examples with RTgill-W1.

Gills are unique structures involved in respiration and osmoregulation in piscinids as well as in many aquatic invertebrates. The availability of the trout-derived gill cell line, RTgill-W1, is beginning to make impacts in fish health and toxicology. These cells are available from the American Type Culture Collection as ATCC CRL 2523. The cells have an epithelioid morphology and form tight monolayer sheets that can be used for testing epithelial resistance. The cells can be grown in regular tissue culture surfaces or in transwell membranes in direct contact with water on their apical surfaces. The ability of RTgill-W1 to withstand hypo- and hyper-osmotic conditions and their optimal growth capacity at room temperature, make these cells ideal sentinel models for in vitro aquatic toxicology as well as model systems to study fish gill function and gill diseases. RTgill-W1 support growth of paramyxoviruses and orthomyxoviruses like salmon anemia virus. RTgill-W1 also support growth of Neoparamoeba pemaquidensis, the causative agent of amoebic gill disease. The cells have been used to understand mechanisms of toxicity, ranking the potencies of toxicants, and evaluating the toxicity of environmental samples. These cells are also valuable for high throughput toxicogenomic and toxicoproteomic studies which are easier to achieve with cell lines than with whole organisms. RTgill-W1 cell line could become a valuable complement to whole animal studies and in some cases as gill replacements in aquatic toxicology.

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells.

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.

Induction of homotypic aggregation in the rainbow trout macrophage-like cell line, RTS11.

The rainbow trout monocyte/macrophage-like cell line, RTS11, has been used to study homotypic aggregation (HA), which is a well-studied feature of leucocytes in mammals but less understood in fish. HA is the aggregation of cells of the same cell type. RTS11 underwent HA in response to polyinosinic:cytidylic acid (poly IC), polyadenylic acid (poly A), lipopolysaccharide (LPS), zymosan, and phorbol 12-myristate 13-acetate (PMA). Poly IC was the best inducer of HA and did so in a dose- and time-dependent manner. The induction of RTS11 aggregation by poly IC required divalent cations but was not blocked by either an inhibitor of lymphocyte function-associated molecule-1 (LFA-1) or the tripeptide integrin adhesion recognition sequence, RGD. Poly IC-induced HA was inhibited by colchicine and latrunculin B, which act on microtubules and microfilaments, respectively, implying the necessity for an intact cytoskeleton. HA induction by poly IC did not occur at 4 degrees C and was blocked by the transcriptional and translational inhibitors, actinomycin D and cycloheximide, respectively, suggesting the requirement for de novo protein synthesis. Poly IC-induced RTS11 aggregation was blocked by two inhibitors of dsRNA-dependent protein kinase (PKR). This is the first indication that PKR could have a role in the HA of leucocytes.

Development of a continuous cell line, PBLE, from an American eel peripheral blood leukocyte preparation.

A continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitz's L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18 degrees C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36 degrees C, and proliferated at temperatures from 5 degrees C to at least 30 degrees C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.

Cell adhesion characteristics of a monocytic cell line derived from rainbow trout, Oncorhynchus mykiss.

In experiments investigating the adhesive properties of the rainbow trout splenic monocyte-like cell line RTS11 it was found that the cells bound with low affinity to plates coated with bovine serum albumin (BSA) but that phorbol ester-induced activation/differentiation greatly increased adhesion to BSA. Similarly, pre-exposure to 500 microM MnCl(2) at time of plating, increased RTS11 adhesion to BSA coated plates, in agreement with the reported ability of divalent cations such as Mn(2+) to activate integrins. Integrins are a diverse family of heterodimeric cell surface glycoproteins that have been shown to mediate cell-cell and cell-extracellular matrix adhesion. Transcripts of the beta(2)-integrin CD18 were detected by PCR in RTS11 but not in RTG-2 cells, a fibroblastic lineage derived from rainbow trout gonads. These results suggest that differentiated RTS11 express molecules related to members of the beta(2)-integrin subfamily such as the macrophage lineage marker Mac-1 (CD11b/CD18) and/or p150,95 (CD11c/CD18) and possibly as well alpha(4)beta(1) of the beta(1)-integrin subfamily.

Gliotoxin-induced cytotoxicity in three salmonid cell lines: cell death by apoptosis and necrosis.

Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 microM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 microM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation.

Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA.

The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.

In vitro and in vivo comparisons of fish-specific CYP1A induction relative potency factors for selected polycyclic aromatic hydrocarbons.

Induction of cytochrome P450 (CYP1A), as measured by liver ethoxyresorufin-O-deethylase (EROD) activity in juvenile rainbow trout (Oncorhynchus mykiss), was used to derive relative potency factors (RPFs) for several polycyclic aromatic hydrocarbons (PAHs), chosen for their induction potency in a rainbow trout liver cell line (RTL-W1). Potency for causing induction was estimated as the median effective concentration (EC50) from exposure-response curves. With the exception of phenanthrene, all PAHs tested induced EROD activity in juvenile trout, ranked as: benzo[k]fluoranthene>benzo[b]fluoranthene>benzo[b]fluorene>beta-napthoflavone>retene (7-isopropyl-1-methylphenanthrene). When induction potency was expressed relative to benzo[k]fluoranthene, RPFs ranged from 0.02 to 1, and the rank order in vivo was identical to the rank order with RTL-W1-derived values. The additivity of PAHs in mixtures in RTL-W1 cells was compared to whole-fish results from a previous study. EROD induction showed additive interactions for PAHs with exposure-response curves of similar slopes. This study demonstrates that assays of CYP1A induction using rainbow trout liver cells in culture would be a convenient substitute for assays with whole fish as part of testing programs for risk assessment of PAHs.

Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss.

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were >or=10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities.