Cell Cultures from Walleye (Sander vitreus) for Use in Cell Biology and Virology

Abstract

This thesis has developed from the walleye, Sander vitreus (Mitchill), cell lines (WE) and used them to study viral hemorrhagic septicemia virus (VHSV IVb), which is the causative agent for viral hemorrhagic septicaemia (VHS), and to contribute to the comparative cellular biology and physiology of fish. The outcomes have been organized into six chapters whose major discoveries are abstracted below. A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a caudal fin and used to study the intersection of thermobiology of walleye with the thermal requirements for replication of VHSV IVb. WE-cfin11f proliferated from 10 °C to 32 °C and endured as a monolayer for at least a week at 1 °C to 34 °C. WE-cfin11f adopted an epithelial-shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 °C 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. A cell line, WE-cfin11e, with an epithelial-like morphology was developed from a caudal fin, characterized as distinct from the fibroblast-like cell line, WE-cfin11f, and compared with WE-cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscope were used to localize the intermediate filament protein, vimentin, the tight junction protein, zona occludin (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 fibers 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 low temperature can modulate viral tropism. A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen 6 cells had a spindle 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-spleen 6 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 cell line from the walleye caudal fin, WE-cfin11f. 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. A cell line, WEBA, has been developed from the bulbus arteriosus (BA). WEBA produced collagen I, and when held at confluency for days or weeks, spontaneously formed capillary-like tubes. WEBA cells bound fluorescently-labeled Ulex europaeus lectin agglutinin I (UEA-1), took up acetylated low density lipoprotein (Ac-LDL), stained for von Willebrand factor (vWF), and produced nitric oxide (NO). The cytoskeleton consisted at least of α- and β- tubulin, vimentin, and actin, with the actin organized into circumferential bundles. Immunofluoresecent staining revealed at least two tight junction proteins, zona occludens -1 (ZO-1) and claudin 3. Together these results suggest that WEBA is an endothelial cell line. Relatively high doses of 2,3, 7,8-tetrachlorodibenzodioxin (TCDD) induced cytochrome P4501A (CYP1A) protein and 7-ethoxyresorufin o-deethylase (EROD) activity in WEBA. As one of the first fish endothelial and BA cell lines, WEBA should be useful in many disciplines in which the teleost cardiovascular system is a focus. Cell lines and primary cultures from several teleost tissues and species were stained for senescence-associated β-galactosidase (SA β-Gal), revealing four general outcomes. (1) For long-standing fish cell lines that can be considered immortal, little or no SA β-Gal staining was observed, regardless of the culture conditions. (2) For a new walleye cell line from the bulbous arteriosus (WEBA), most cells stained for SA β-Gal even after 40 passages. This suggested that high SA β-Gal activity was a unique property of WEBA, perhaps reflecting their endothelial character, rather than cellular senescence. (3) For cell lines developed from the walleye caudal fin and from somatic cells in rainbow trout coelomic fluid, no SA β-Gal staining was observed in the earliest cultures to over 70 passages later. This suggested that cells from these anatomical sites do not undergo senescence in vitro. (4) By contrast, for cell lines developed from the walleye brain and from somatic cells in rainbow trout milt, most cells in the early stage cultures stained for SA β-Gal, but as these were developed into cell lines, SA β-Gal negative cells became dominant. This suggested that if cellular senescence occurred in vitro, this happened early in these cultures and subsequently a few SA β-Gal negative cells went onto to form the cell line. Overall the presence of SA β-Gal positive cells in cultures could be interpreted in several ways, whereas their absence predicted that in these cultures cells would proliferate indefinitely. Twenty three cell lines from seven fish species were examined immunocytochemically with the monoclonal antibody (mAb), 6-11B-1, for acetylated α-tubulin, revealing universal staining of some structures, such as midbodies, but more restricted staining for others, such as primary cilia. The midbody and mitotic spindle stained strongly in all the cell lines. As well as being from two salmonid species, Chinook salmon (Onchorhynchus tshawytscha) and rainbow trout (O. mykiss), the cell lines were from Pacific herring (Clupea pallasii), haddock (Melanogrammus aeglefinus), walleye (Sander vitreus), fathead minnow (Pimephales promelas), and zebrafish (Danio rerio). Strong cytoplasmic staining of microtubule networks was observed in only a few cell lines. These were from walleye brain (astroglial-like), spleen (epithelia-like), skin (fibroblastic) and liver (fibroblastic). For the same species, cell lines with an endothelial-like shape from the bulbous arteriosus and epithelial-like shape from the caudal fin and eye retina had no cytoplasmic staining. Primary cilia stained intensely in seven of eight walleye cell lines and in cell lines from fathead minnow (EPC), zebrafish (ZSSJ), and Pacific herring (PHL). By contrast, few or no primary cilia were observed in cultures of the other twelve cell lines, one from haddock and eleven from salmonids, and in cultures of these cell lines a portion of the cells had weak staining in either the nucleus or cytoplasm. Overall this is the first demonstration of primary cilia in fish cell lines and of cells from a particular taxonomic group, in this case the salmonids, being unable to maintain primary cilia in vitro. In the future, these fish cell lines could be used to study the formation and function of primary cilia, but as well, primary cilia could be a useful marker for characterizing new fish cell lines.