| 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 a-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 alpha-
and beta-tubulin, vimentin, and actin, with the actin organized into
circumferential bundles. Immunofluorescent 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 beta-galactosidase (SA beta-Gal),
revealing four general outcomes. (1) For long-standing fish cell lines
that can be considered immortal, little or no SA beta-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 beta-Gal
even after 40 passages. This suggested that high SA beta-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 beta-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
beta-Gal, but as these were developed into cell lines, SA beta-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 beta-Gal negative cells went onto to form the cell line. Overall
the presence of SA beta-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 a-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.
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