Download Isolation and characterization of a rhabdovirus from starry flounder

Journal of General Virology (2004), 85, 495–505
DOI 10.1099/vir.0.19459-0
Isolation and characterization of a rhabdovirus from
starry flounder (Platichthys stellatus) collected
from the northern portion of Puget Sound,
Washington, USA
Christina Mork,13 Paul Hershberger,2 Richard Kocan,2 William Batts3
and James Winton3
1
University of Washington Friday Harbour Laboratories, 620 University Road, Friday Harbour,
WA 98250, USA
Correspondence
James Winton
2
[email protected]
School of Aquatic and Fishery Sciences, University of Washington, Box 355100, Seattle,
WA 98195, USA
3
Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115, USA
Received 25 June 2003
Accepted 12 November 2003
The initial characterization of a rhabdovirus isolated from a single, asymptomatic starry flounder
(Platichthys stellatus) collected during a viral survey of marine fishes from the northern portion
of Puget Sound, Washington, USA, is reported. Virions were bullet-shaped and approximately
100 nm long and 50 nm wide, contained a lipid envelope, remained stable for at least 14 days at
temperatures ranging from ”80 to 5 6C and grew optimally at 15 6C in cultures of epithelioma
papulosum cyprini (EPC) cells. The cytopathic effect on EPC cell monolayers was characterized
by raised foci containing rounded masses of cells. Pyknotic and dark-staining nuclei that also
showed signs of karyorrhexis were observed following haematoxylin and eosin, May–Grunwald
Giemsa and acridine orange staining. PAGE of the structural proteins and PCR assays using
primers specific for other known fish rhabdoviruses, including Infectious hematopoietic necrosis
virus, Viral hemorrhagic septicemia virus, Spring viremia of carp virus, and Hirame rhabdovirus,
indicated that the new virus, tentatively termed starry flounder rhabdovirus (SFRV), was previously
undescribed in marine fishes from this region. In addition, sequence analysis of 2678 nt of the
amino portion of the viral polymerase gene indicated that SFRV was genetically distinct from other
members of the family Rhabdoviridae for which sequence data are available. Detection of this
virus during a limited viral survey of wild fishes emphasizes the void of knowledge regarding the
diversity of viruses that naturally infect marine fish species in the North Pacific Ocean.
INTRODUCTION
There is limited knowledge about virus infections of marine
fishes in the Pacific Northwest of the United States. In the
past, fish health investigations have focused primarily on
salmonids and few studies have been conducted on the
many other species native to this area. Fish viruses endemic
to this region of the eastern Pacific Ocean that have been
well characterized include a birnavirus, Infectious pancreatic
necrosis virus (IPNV), and two rhabdoviruses, Viral hemorrhagic septicemia virus (VHSV) and Infectious hematopoietic
necrosis virus (IHNV; Wolf, 1988). Other viruses shown to
be present in marine or anadromous fishes in the region,
3Present address: Department of Microbiology, Boston University, 715
Albany Street, Boston, MA 02118, USA.
The GenBank accession number of the sequence reported in this paper
is AY450644.
0001-9459 G 2004 SGM
but which are less completely characterized, include a
herpesvirus, salmonid herpesvirus 1; a presumed iridovirus,
viral erythrocytic necrosis virus; a toga-like virus, salmon
pancreas disease virus; a putative togavirus, erythrocytic
inclusion body syndrome virus; a putative retrovirus
causing plasmacytoid leukaemia and several strains of
paramyxoviruses and reoviruses (Hetrick & Hedrick, 1993;
Traxler et al., 1998; Wolf, 1988).
Salmonids have been shown to harbour these viruses,
especially in freshwater hatcheries or in marine net pens
where the viruses can spread rapidly, sometimes resulting
in high mortality. However, little is known about the host
range of these viruses among various species of marine fish.
In addition to serving as possible reservoirs of viruses that
can be transmitted to salmonids, some of these marine fish
species may themselves be highly susceptible to infection
(Kocan et al., 1997), leading to natural outbreaks in the
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495
C. Mork and others
marine environment (Meyers & Winton, 1995; Meyers
et al., 1992, 1994, 1999; Hershberger et al., 1999; Takano
et al., 2000; Kocan et al., 2001; Hedrick et al., 2003). For
these reasons it is important to sample a range of marine
fish species to determine the prevalence of these viruses in
the wild, and to assess the susceptibility of various marine
fish species to these infectious agents.
In late March of 2000, a survey of marine fish captured in the
San Juan Archipelago of northern Puget Sound, WA, USA,
was conducted to determine the natural prevalence of these
or other viruses in a variety of marine fish species, including
English sole (Parophrys vetulus) and starry flounder
(Platichthys stellatus). During the course of the survey, a
new rhabdovirus of marine fish was isolated. Detection of
this virus, tentatively termed starry flounder rhabdovirus
(SFRV), during a limited survey, highlights the void of
knowledge regarding viruses that naturally infect marine
fish species in the North Pacific Ocean.
METHODS
Fish sampling. Fifty-eight starry flounder, 25 English sole, 12
butter sole (Isopsetta isolepis) and 36 fish of other species, including
rock sole (Lepidosetta bilineata), C-O sole (Pleuronichthys coenosus),
Dover sole (Microstomus pacificus), sand sole (Psettichthys melanostictus), slender sole (Lyopsetta exilis), great sculpin (Myoxocephalus
polyacanthocephalus), tomcod (Microgadus proximus), shiner perch
(Cymatogaster aggregata), tubesnout (Aulorhynchus flavidus) and
Pacific herring (Clupea pallasi), were captured from waters near San
Juan Island by beach seine or near Orcas Island by otter trawl. Fish
were held at the University of Washington, Friday Harbour
Laboratories, in flow-through seawater tanks at ambient temperature
until necropsies were performed.
Cell culture. Epithelioma papulosum cyprini (EPC; Fijan et al.,
1983) cells were grown in tissue culture flasks using Eagle’s minimum essential medium (MEM) supplemented with 10 % fetal
bovine serum (FBS) and adjusted to pH 7?4 by the addition of
7?5 % sodium bicarbonate (MEM-10-SB). Cells were grown at 25 uC
for the first week and then incubated at 15 uC until used.
Virus isolation. Kidney and spleen tissues from each fish were
removed and diluted 1 : 4 in MEM containing 140 mM Tris and
supplemented with 100 IU penicillin, 100 mg streptomycin, 100 mg
gentamicin sulfate and 2?5 mg amphotericin B ml21 (MEM-AF).
Excised tissue samples were stored at 280 uC until virus assays were
performed. Samples were thawed, homogenized by mortar and
pestle and clarified by low-speed centrifugation for 3–5 min. Serial
10-fold dilutions of the supernatant were made in MEM containing
140 mM Tris and supplemented with 5 % FBS (MEM-5-T), and the
dilutions were inoculated onto EPC monolayers in 24-well plates.
After 30 min incubation at room temperature to allow virus adsorption, 1 ml of an overlay composed of 0?75 % methylcellulose in
MEM-5-T was added to each well and the plates were incubated at
15 uC. Plates were observed regularly for cytopathic effect (CPE) for
7 days, then fixed and stained with a crystal violet and formalin
solution. The initial virus titre in the tissues was reported as plaqueforming units (p.f.u.) (g tissue)21.
Stock viruses. Medium from cell cultures showing CPE was stored
in aliquots at 280 uC for use as stock virus. In addition, reference
strains of IHNV, VHSV, Hirame rhabdovirus (HIRRV), Spring viremia
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of carp virus (SVCV), IPNV and an aquareovirus isolated from
adult chinook salmon (Oncorhynchus tshawytscha) in the Green
River of Washington, USA, were used as controls in various assays.
Plaque assays. Treated tissue culture 24-well plates (Costar) were
seeded with EPC cells in MEM-5-T and incubated at 25 uC for 24 h.
Serial 10-fold dilutions were prepared in MEM-5-T and inoculated
into wells of the 24-well plates from which the medium had been
drained. After 30 min incubation at room temperature to allow
virus adsorption, 1 ml of an overlay composed of 0?75 % methylcellulose in MEM-5-T was added to each well. The cultures were
incubated at 15 uC for 7 days, then fixed and stained with a crystal
violet and formalin solution. Virus titre was reported as p.f.u. (ml
culture fluid)21.
Growth temperature. Medium was decanted from twelve 25 cm2
flasks containing monolayers of EPC cells and each was inoculated
with 0?25 ml stock virus, incubated at room temperature for 30 min
and then rinsed three times with MEM to remove unattached virus.
MEM-10-SB (5 ml) was added to each flask and sets of triplicate
flasks were incubated at 10, 15, 20 or 25 uC. At 1, 2, 3, 4, 5 and
6 days post-inoculation, a 0?2 ml aliquot of culture fluid was
removed from each flask, pooled by temperature and the virus titre
was determined by plaque assay.
Stability to freeze–thaw. To determine the stability of the virus
to repeated freeze–thaw cycles, 1?0 ml aliquots of original stock
virus were held at 280, 220 and 5 uC. After 3, 7 and 14 days of
storage, the frozen aliquots were thawed and the virus titre in the
aliquot stored at each temperature was determined by plaque assay.
The aliquots were then refrozen until the next assay period.
Chloroform sensitivity. Presence of a lipid-containing envelope
was determined by mixing 0?5 ml cell culture fluid containing virus
with an equal volume of chloroform. The mixture was shaken for
10 min, then centrifuged at 200 g for 5 min to separate the aqueous
phase from the chloroform. Control viruses included VHSV as
an enveloped (positive) control and an aquareovirus as a nonenveloped (negative) control. Titres of infectious virus in the
aqueous phases of treated and untreated preparations were determined by plaque assay.
Electron microscopy. Monolayer cultures of EPC cells were inoculated at a relatively high m.o.i. and the cultures were incubated at
15 uC. At 2, 3 and 7 days post-inoculation, control and virusinfected cultures were fixed for 24 h using 4 % glutaraldehyde in
0?1 M cacodylate buffer (pH 7?4). Cells were dislodged into the
medium that was then centrifuged at 1000 g for 10 min, and 0?1 M
sodium cacodylate was added to the pellet. Samples were recentrifuged for 5 min at 1000 g and the pellet was placed in 2 ml cold
1 % OsO4 for 1?5 h. Following three 10 min rinses in 1 ml 0?1 M
sodium cacodylate, 2 ml 1 % aqueous uranyl acetate was added. The
uranyl acetate was removed after 1 h and the samples were dehydrated in a graded ethanol series, then embedded in Spurr’s epoxy
resin. Thin sections were cut using a diamond knife in an ultramicrotome and the sections were placed on grids. The grids were
examined using an electron microscope and sections were photographed at various magnifications.
Staining of infected cells. Monolayers of EPC cells were grown
on sterile cover glasses in 6-well plates. Cells were infected with
0?1 ml 1023 and 1024 dilutions of stock virus to provide approximately 10–100 p.f.u. per cover glass. After 2–3 days incubation at
15 uC, sets of cover glasses were transferred to Petri dishes and fixed
in either MEM containing 10 % formalin or Carnoy’s fixative. After
storage at 5 uC overnight, the fixative was removed and replaced
with MEM. Formalin-fixed cover glasses were stained with either
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Journal of General Virology 85
New rhabdovirus from starry flounder
May–Grunwald Giemsa or with haematoxylin and eosin by standard
methods (Rovozzo & Burke, 1973), then mounted onto slides using
Permount and stored at 25 uC to dry. The stained cover glasses were
examined by light microscopy and photographed. Acridine orange
staining was performed by standard methods (Rovozzo & Burke,
1973) using cover glasses fixed in Carnoy’s fixative. Cover glasses
were kept moist with McIlvaine’s buffer while mounted on the slide
and immediately examined using a fluorescence microscope fitted
with a mercury lamp and appropriate filter blocks.
Analysis of structural proteins. Virus grown in 150 cm2 flasks of
EPC cells was harvested and the culture fluid clarified by centrifugation at 4 uC for 20 min at 1000 g. The supernatant was placed into
ultracentrifuge tubes containing 0?2 ml glycerol, then centrifuged at
82 700 g at 4 uC for 1 h in an SW28 rotor (Beckman). The pellet was
resuspended and layered on top of a step gradient composed of 50,
35 and 20 % sucrose in 0?01 M Tris buffer (pH 7?4). The gradient
was centrifuged at 82 700 g at 4 uC for 1?5 h in an SW28 rotor. The
virus band was then removed with a syringe, diluted in 0?01 M Tris
and the virions were pelleted by centrifugation at 115 000 g at 4 uC
for 1 h in an SW 50.1 rotor (Beckman). SDS-PAGE was used to
analyse the structural proteins of the new virus. The pellet of purified virus was resuspended in sample buffer, heated to 95 uC for
2 min and stored at 220 uC until used. A 10 % SDS-PAGE gel was
prepared and run using standard methods. The gel was stained with
Coomassie blue (Sigma). For comparison, three other fish rhabdoviruses, IHNV, SVCV and VHSV, were purified as indicated and
included in some of the gels.
PCR. RT-PCR assays were used to determine if the new virus was
similar to one of several other fish rhabdoviruses for which genomic
information was available. Nine sets of PCR primers for IHNV (1
set), VHSV (1 set), HIRRV (3 sets) and SVCV (4 sets) were synthesized from published sequences. Culture fluids containing the new
virus or the corresponding virus controls were heated at 95 uC for
2 min to release viral RNA, then cooled on ice, a procedure used
routinely in our laboratory to release nucleic acids from poikilotherm viruses grown in cell culture (Huang et al., 1996), but which
is not recommended for extraction of nucleic acids from tissue
samples. Our standard RT-PCR consisted of 50 pmol each primer
pair in a separate single-tube (50 ml reaction) containing 5 ml viral
RNA, 10 mM Tris/HCl (pH 8?3), 50 mM KCl, 2?5 mM MgCl2,
2?5 units AmpliTaq (Applied Biosystems), 4?5 units reverse transcriptase (Promega), 10 units RNasin ribonuclease inhibitor (Promega)
and 200 mM dNTP. Reverse transcription and annealing temperatures were varied during this study to allow primers to bind to
homologous regions of SFRV genes that may not have been identical. Four reverse transcription and annealing temperatures were
tested in separate experiments: 45, 40, 35 and 30 uC. After a 30 min
reverse transcription reaction and initial denaturation at 95 uC for
2 min, 35 cycles of amplification were performed using the following
conditions: 95 uC for 30 s, 45–30 uC (same as reverse transcription
temperature) for 30 s, 72 uC for 1 min. For final extension, tubes
were incubated at 72 uC for 7 min. The PCR products were then
held at 4 uC until loaded for electrophoresis on a 1?5 % agarose gel
and visualized by ethidium bromide staining.
Sequencing of a region of the viral polymerase gene.
Relatively conserved primer sites were identified in the rhabdovirus
polymerase (L) gene by comparing published sequences of SVCV,
IHNV, VHSV, Vesicular stomatitis New Jersey virus (VSNJV), Rabies
virus (RABV) and Mokola virus (MOKV). Three degenerate primers,
homologous to positions 1592–1611, 1938–1919 and 2157–2141 of
the L gene of Vesicular stomatitis Indiana virus (VSIV; GenBank
accession no. K02378), were synthesized and used to amplify a
region of the SFRV polymerase gene in a semi-nested PCR reaction
using RT and annealing temperatures of 40 and 30 uC, respectively.
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The first round of PCR used degenerate sense primer 59-AARGTCAAGGCGATGGARYT-39 and antisense primer 59-TGATTGTCCCCCTGNGC-39 in standard RT-PCR reaction mixes with SFRV
RNA extracts to amplify a 577 bp product. A semi-nested PCR was
conducted on the first round PCR product by using the same sense
primer, but with reverse primer 59-TCAAAAGTCTCGTGAACTCT39 to amplify a 348 bp region. For both amplifications, DNA was
analysed on a 1?5 % agarose gel and visualized by ethidium bromide
staining. The visible PCR product from the second round was
purified with a StrataPrep kit (Stratagene) and labelled for automated sequencing by BigDye terminator cycle sequencing (Applied
Biosystems) on an Applied Biosystems 310 genetic analyser. A
243 nt region of authentic SFRV sequence was obtained, consisting
of a portion of an intact ORF. This authentic SFRV sequence, when
compared with other known rhabdovirus sequences, allowed the
creation of primers for obtaining additional sequence information.
Because the sequence was located near the 59 end of the L gene
mRNA, a 59 RACE kit (Invitrogen) was used with three gene-specific
primers (GSP) to gain additional sequence information. Primer
GSP1 was 59-AAAGATCAAYTCDCGGGAWG-39, GSP2 was 59AAYTCDCGGGAWGGTTSTG-39 and GSP3 was 59-GAGAGMTCKAYGAATTAGA-39. Additional primers were used to sequence toward
the 59 end of the mRNA of the L protein gene. Sequence information was also obtained later by using conserved primers toward the
39 end of the L mRNA combined with known SFRV primer sequences.
Phylogenetic analysis. To perform a phylogenetic comparison of
SFRV with other fish rhabdoviruses, the polymerase gene sequence
of SFRV and selected L gene sequences from GenBank were truncated to align with the 456 aa of the amino terminus of the polymerase protein. Sequences were aligned with CLUSTAL W followed by
analysis with PAUP 4.0. Both neighbour-joining and parsimony
analyses were performed using the homologous region of the
polymerase sequence of Human parainfluenza virus 1 (HPIV-1), a
member of the family Paramyxoviridae, as the outgroup. Phylogenetic trees were generated containing 1000 bootstrapped samples
and values shown as percentages were transferred to the nodes of
the branches.
RESULTS
Virus isolation
Tissues were collected from a total of 131 marine fish
representing 13 species. Of the 58 starry flounder surveyed,
only one fish tested positive for a virus using the plaque
assay isolation procedure. None of the other fish sampled
produced evidence of a viral infection. After 7 days incubation, plaques appeared in a well containing 0?1 ml of the
tissue homogenate (prepared at 1 : 40) from one starry
flounder, indicating that the original tissue contained a
moderate titre (~104 p.f.u. g21). The initial plaques were
approximately 0?5 mm in diameter and appeared similar
to plaques produced by IHNV or VHSV. Reinoculation of
25 cm2 flasks of fresh EPC cells and incubation at 15 uC
resulted in a rapid progression of CPE (Fig. 1b) with
subsequent virus titres in the culture fluid that exceeded
107 p.f.u. ml21. Culture fluid containing the virus was
passed through a nitrocellulose filter having a pore size of
0?22 mm. The virus titre was 6?06106 p.f.u. ml21 before
filtration and 1?56105 p.f.u. ml21 after filtration, confirming the presence of a filterable agent and indicating the
virus was somewhat smaller than 220 nm.
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C. Mork and others
Fig. 1. Cytopathic effects produced by the
rhabdovirus isolated from starry flounder. (a)
Uninfected EPC cells. (b) EPC cells infected
for 3 days. (c) EPC cells grown on cover
glasses and stained with haematoxylin and
eosin. (d) Infected EPC cells stained with
haematoxylin and eosin.
Staining
Infected EPC monolayers stained by haematoxylin and
eosin revealed foci containing rounded masses of cells that
were raised from the monolayer. Infected cells contained
pyknotic and dark-staining nuclei that also showed signs
of karyorrhexis (Fig. 1d). Cell monolayers stained with
acridine orange and May–Grunwald Giemsa also revealed
evidence of these changes.
shown). No plaques appeared when the virus isolated from
starry flounder was mixed with chloroform before inoculation of EPC cells, indicating the virus possessed a lipidcontaining envelope. The enveloped control virus (VHSV)
was also inactivated, while the non-enveloped aquareovirus,
used as a negative control, showed no loss of infectious titre
following treatment with chloroform.
Electron microscopy
Growth temperature and physical stability
Using cultures of EPC cells incubated at selected temperatures, the optimal growth temperature was determined to be
15 uC (Fig. 2). The virus also grew well at 10 uC. The virus
replicated poorly at 20 uC and there was no evidence of virus
growth at 25 uC. Aliquots of tissue homogenate stored at 5,
220 or 280 uC showed little loss in titre over the 14-day
period that included three freeze–thaw cycles (data not
Thin sections of infected EPC cells observed by electron
microscopy revealed bullet-shaped virions budding from
cell surfaces or around the outer membrane (Fig. 3). No
virus particles were observed within the cytoplasm of the
cells. The virion shape was typical of members of the
Rhabdoviridae; however, the particles were somewhat
shorter than the sizes reported for IHNV or VHSV, the
two fish rhabdoviruses known to be endemic in the Pacific
Northwest. The virus particles averaged approximately
100 nm in length and 50 nm in width.
Analysis of structural proteins
Purification of the virus from starry flounder produced an
opalescent band in sucrose gradients that yielded a visible
pellet of purified virus. Following SDS-PAGE analysis, the
molecular masses of the viral structural proteins were
estimated by comparison with the relative mobilities of
proteins of known molecular mass (Fig. 4). The molecular
masses of the structural proteins of SFRV were more similar
to those of the fish vesiculovirus-like viruses, as shown by
Nishizawa et al. (1991) and Bjorklund et al. (1994), rather
than those of the novirhabdoviruses.
Fig. 2. Effect of temperature on the replication of the rhabdovirus isolated from starry flounder. Diamonds, 10 6C; squares,
15 6C; circles, 20 6C; triangles, 25 6C.
498
Rhabdoviruses typically possess five structural proteins
(Walker et al., 2000). Of the proteins clearly resolved on the
gels, the large 150–190 kDa polymerase (L) was not evident.
Among the four proteins observed, the 57 kDa protein was
assumed to be the glycoprotein (G), the 46 kDa protein
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New rhabdovirus from starry flounder
Fig. 3. Electron micrographs of the rhabdovirus isolated from starry flounder. (a) EPC cells infected for 48 h and showing
bullet-shaped virions at the surface of an infected cell. Bar, 200 nm. (b and c) Higher magnification showing the bullet-shaped
virions at the surface of an infected cell. Bars, 100 nm.
the nucleoprotein (N), the 36 kDa protein the phosphoprotein (P) and the 24 kDa protein the matrix protein (M).
The apparent relative concentration of the four proteins
was in good agreement with Walker et al. (2000) in that N
and M appeared to be in greatest abundance, while fewer
copies of the P were observed.
PCR
Control virus templates produced RT-PCR products of
the expected size, while no products were obtained using
the unknown virus template. Reduction of the annealing
temperature to lower the stringency of the reaction did not
alter the results, indicating the new virus was not an isolate
of IHNV, SVCV, HIRRV or VHSV.
Sequence analysis of SFRV polymerase gene
The SFRV polymerase gene sequence obtained was 2678 nt
long and had a single large ORF starting with an AUG codon
in strong initiation context at nt 11–13. The first 13 nt of
the gene were identical between SFRV and SVCV, while 12
of the 13 nt were identical to SSTV and 903/87. The SFRV
ORF encoded an 889 aa portion of the 59 end of the putative polymerase protein and this region was most homologous to that of species and tentative species of the genus
Vesiculovirus, having 53 % amino acid identity to VSIV and
53 % amino acid identity to SVCV. The next closest isolates,
45–46 % identical to SFRV, were Bovine ephemeral fever
virus (BEFV), Flanders virus (FLAV) and the European
lake trout isolate (903/87). Less related (38 % identity) was
RABV, the type species of the genus Lyssavirus. Identities
of 21 % or lower were obtained between SFRV and representatives of the genera Novirhabdovirus, Cytorhabdovirus
and Nucleorhabdovirus, and for HPIV-1, a member of the
family Paramyxoviridae.
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Within the polymerase gene of members of the Mononegavirales, a consistent pattern of conserved sequence domains
and subdomains has been described (Kamer & Argos, 1984;
Poch et al., 1990). These include major domains I–VI and
more highly conserved subdomains A–D within domain
III. Rhabdoviruses, in contrast to paramyxoviruses, do not
contain a variable hinge region between conserved domains
II and III. Our partial sequence of SFRV L protein contained the first three conserved domains: I, II and III.
Pairwise alignments of the SFRV L protein in these regions
with other rhabdoviruses and with HPIV-1, a paramyxovirus, showed levels of identity that generally conformed to
this previously described pattern (Table 1). Domains II and
III were the most conserved of the major domains, while
domain I was the least conserved, showing little identity
above the overall level seen throughout the region of the
L portion for which sequence was determined. Within
domain III, subdomain IIIA was the most conserved,
showing 100 % aa identity between SFRV and VSIV, SVCV
and FLAV (Fig. 5). One conserved stretch of 35 aa, encompassing domain IIIA, was identical between SFRV and SVCV
from nucleotide position 1808 to 1912 of the putative SFRV
polymerase gene. Virus isolate 903/87 from European lake
trout had the highest identity to SFRV in domain I.
Unfortunately, the sequence available for that virus is
limited and could not be compared for the other domains.
Phylogenetic analysis
The deduced amino acid sequence of the amino-terminal
portion of the putative L gene of SFRV was used to infer
phylogenetic relationships with other members of Rhabdoviridae using HPIV-1, a member of the Paramyxoviridae,
as an outgroup. All sequences were truncated to approximately 460 aa at the amino end of the protein to allow
inclusion of the fish rhabdovirus 903/87, for which only a
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C. Mork and others
1
2
kDa
Table 1. Percentage amino acid identity of three conserved
domains and four conserved subdomains within the polymerase gene of selected viruses compared to SFRV
Virus
Domain and subdomain
I
57
46
36
II
III
SFRV
100 100 100
VSIV
51 77 74
SVCV
51 71 75
903/87D 54 NA NA
FLAV
44 68 58
BEFV
45 69 63
RABV
37 55 50
IHNV
14 28 21
SYNV
12 35 27
NCMV
21 36 25
HPIV-1 23 17 19
Overall*
IIIA IIIB IIIC IIID
100
100
100
100
88
88
100
90
90
100
92
85
NA
NA
NA
NA
100
85
85
23
38
38
15
65
85
77
35
46
42
31
80
80
80
70
50
60
60
77
62
69
15
15
23
23
100
53
53
45 (partial)
45
46
38
19
20
21
17
NA,
24
Not available for sequence comparison.
*Overall refers to the percentage amino acid identity compared to
the 889 aa sequence of the SFRV polymerase.
DFor isolate 903/87 only 462 aa were available for comparison.
It was anticipated that this survey might identify additional
species of marine fish that could serve as hosts for the
viruses known to be endemic in the region, especially,
IHNV or VHSV. Surprisingly, none of these viruses was
detected in the tissues of the fish examined during the
survey. Also unexpected was the isolation of an apparently
new rhabdovirus from the tissues of one starry flounder.
DISCUSSION
Electron microscopy and the physical characteristics of
the new isolate confirmed that it was a member of the
Rhabdoviridae. Only two rhabdoviruses are known to be
endemic among freshwater or marine fish species on the
west coast of North America, IHNV and VHSV, while
several other fish rhabdoviruses have been isolated from
freshwater and marine species in Europe and Asia (Table 2).
Results from biological, physical, chemical and molecular
assays convincingly demonstrated that the new virus was
not a strain of either IHNV or VHSV. These assays also
showed that SFRV was not related to three other fish
rhabdoviruses known to occur in the Pacific Ocean for
which limited sequence data are available: SVCV, reported
from penaeid shrimp (Penaeus stylirostris and Penaeus
vannamei) cultured in the Hawaiian Islands (Johnson
et al., 1999); HIRRV, reported from Japanese flounder
(Paralichthys olivaceus) ayu (Plecoglossus altivelis), reared on
the Japanese islands of Hokkaido and Honshu, (Kimura
et al., 1986); and snakehead rhabdovirus (SHRV) isolated
from striped snakehead (Ophicephalus striatus) cultured in
Southeast Asia (Ahne et al., 1988).
In late March of 2000, a virus survey was conducted of 131
marine fish collected from the San Juan Archipelago in
the north-western portion of the state of Washington, USA.
While our assays, including PCR and sequence analysis,
confirmed that SFRV was not an isolate of VHSV, IHNV,
SVCV, HIRRV or SHRV, its relatedness to the other fish
Fig. 4. SDS-PAGE of viral structural proteins. Lanes: 1, molecular mass markers; 2, SFRV proteins. The positions and estimates of molecular mass for the structural proteins are indicated.
456 aa sequence was available (Johansson et al., 2001).
Neighbour-joining distance methods revealed that SFRV
was closest to isolate 903/87, branching near BEFV and
FLAV, at the base of the Vesiculovirus and SVCV branches
(Fig. 6). Although the common ancestral node joining
SFRV and 903/87 was supported by a highly significant
bootstrap confidence value of 99 in neighbour-joining
analyses, this was not evident when the same sequences
were analysed with parsimony methods. In parsimony trees
(Fig. 6), SFRV appeared as a polytomy, equally separated
from FLAV, BEFV, 903/87 and an ancestral node leading
to SVCV, VSIV and VSNJV.
500
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Journal of General Virology 85
New rhabdovirus from starry flounder
Fig. 5. Amino acid sequence alignment of the conserved domain III of selected rhabdovirus polymerase genes. Subdomains
IIIa, IIIb, IIIc and IIId are shown in bold. A dot represents an amino acid identical to that of SFRV. Dashes were added to aid
CLUSTAL W alignment of the sequences.
Fig. 6. Phylogenetic trees showing the relationship of SFRV to fish rhabdoviruses for which partial polymerase gene
sequences were available and to other selected rhabdoviruses representing established genera of the Rhabdoviridae. The
neighbour-joining distance tree is on the left and the parsimony tree is on the right. HPIV-1 was used as the outgroup.
Phylogenetic trees were generated using 1000 bootstrapped samples and values greater than 70 % were transferred to the
nodes of the branches. SYNV, Sonchus yellow net virus; RYSV, Rice yellow stunt virus; NCMV, Northern cereal mosaic virus.
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501
C. Mork and others
Table 2. Rhabdoviruses isolated from fish
Virus
Abbreviation
I. Members and probable members of the genus Novirhabdovirus
Viral hemorrhagic septicemia virus (Egtved virus)
VHSV
Brown trout rhabdovirus (VHSV)
Cod ulcus syndrome rhabdovirus (VHSV)
Carpione brown trout rhabdovirus (VHSV)
583
Infectious hematopoietic necrosis virus
IHNV
Oregon sockeye virus (IHNV)
OSV
Sacramento River chinook virus (IHNV)
SRCV
Hirame rhabdovirus
HIRRV
Snakehead rhabdovirus
SHRV
Eel viruses B12 and C26
EEV-B12, EEV-C26
Rio Grande perch rhabdovirus
II. Vesiculovirus-like viruses from fishes
Spring viremia of carp virus (Rhabdovirus carpio)
SVCV
Swim bladder inflammation virus (SVCV)
SBI
Pike fry rhabdovirus
PFRV
Grass carp rhabdovirus (PFRV)
GCV
Eel rhabdovirus (Rhabdovirus anguilla)
Eel virus American
EVA
Eel virus European X
EVEX
Eel virus C30, B44 and D13
C30, B44, D13
Perch rhabdovirus
Pike-perch rhabdovirus
Pike rhabdovirus
DK5533
European lake trout rhabdovirus
903/87
Swedish sea trout rhabdovirus
SSTV
Ulcerative disease rhabdovirus
UDRV
III. Uncharacterized fish rhabdoviruses
Rhabdovirus salmonis
rhabdoviruses listed in Table 2 could be ruled out based
upon the results of various biological or physical tests.
First, the structural proteins separated by SDS-PAGE did
not produce patterns that were typical of members of the
Novirhabdovirus genus of the Rhabdoviridae. This eliminated, in addition to IHNV, VHSV, HIRRV and SHRV,
the two eel rhabdovirus isolates, B12 and C26 (Castric
et al., 1984) and the rhabdovirus from Rio Grande perch
(Malsberger & Lautenslager, 1980).
Among the vesiculovirus-like fish rhabdoviruses, growth
temperature, results from the PCR assays and sequence
data indicated that the SFRV was not an isolate of SVCV
nor, presumably, of the closely related pike fry rhabdovirus
found in European freshwater fishes (Rowley et al., 2001;
Stone et al., 2003). Also, the inability of SFRV to replicate
at temperatures of 25 uC or greater indicated the virus was
not similar to the ulcerative disease rhabdovirus (UDRV;
Frerichs et al., 1986), a vesiculovirus-like agent recovered
from snakehead fish in Southeast Asia (Kasornchandra
et al., 1992). Similarly, the eel rhabdoviruses, EVA, EVEX,
C30, B44 and D13 from Japan (Sano, 1976; Sano et al.,
1977) or Europe (Ahne et al., 1987; Castric et al., 1984) have
temperature optima greater than 25 uC and are serologically
502
Reference
Jensen (1963)
de Kinkelin & Le Berre (1977)
Jensen et al. (1979)
Bovo et al. (1995)
Amend et al. (1969)
Wingfield et al. (1969)
Wingfield & Chan (1970)
Kimura et al. (1986)
Ahne et al. (1988)
Castric et al. (1984)
Malsberger & Lautenslager (1980)
Fijan et al. (1971)
Bachmann & Ahne (1973)
de Kinkelin et al. (1973)
Ahne (1975)
Hill et al. (1980)
Sano (1976)
Sano et al. (1977)
Castric et al. (1984)
Dorson et al. (1984)
Nougayrede et al. (1992)
Jorgensen et al. (1993)
Koski et al. (1992)
Johansson et al. (2002)
Frerichs et al. (1986)
Osadchaya & Nakonechnaya (1981)
related to each other (Castric et al., 1984; Hill et al., 1980),
having been grouped together under the name Rhabdovirus
anguilla by Hill et al. (1980). Finally, the rhabdoviruses
of perch (Perca fluviatilis; Dorson et al., 1984), pike-perch
(Stizostedion lucioperca; Nougayrede et al., 1992), pike (Esox
lucius; Jorgensen et al., 1993) and lake (brown) trout (Salmo
trutta lacustris; Koski et al., 1992) in Europe do not replicate
well in the EPC cell line in which the virus from starry
flounder grew to high titre, have been shown to be closely
related by serology (Bjorklund et al., 1994; Dannevig et al.,
2001; Johansson et al., 2001; Jorgensen et al., 1993;
Nougayrede et al., 1992), and at least one of which
(903/87) was genetically distinguishable from SFRV. In
summary, it appears that SFRV, while related to other
vesiculovirus-like isolates from fish, should be considered
a novel virus. The final placement of SFRV and the other
fish vesiculovirus-like viruses within the appropriate genus
(or genera) of the family Rhabdoviridae will have to await
further resolution.
Compared with other rhabdoviruses of fish, the virus from
starry flounder had a relatively low temperature optimum.
In addition to indicating the virus was unlike many of the
previously described fish rhabdoviruses, the low temperature
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Journal of General Virology 85
New rhabdovirus from starry flounder
at which this agent replicates is similar to the mean temperature of Puget Sound and further suggests the agent is
probably well adapted to a marine host in this region and
not an introduced pathogen. Thus the starry flounder may
represent one of the normal hosts for this virus in nature;
however, the prevalence of the agent may be low. For
example, soon after the initial report of VHSV in North
America in 1989, more than 6000 samples were obtained
from both marine and freshwater fish species collected from
waters of Western Washington State (Winton et al., 1991).
This survey included 543 marine fish of 12 species from
northern Puget Sound and the Straits of Juan de Fuca. The
samples were examined for viruses using standard cell
culture assays and no virus of any type, including the SFRV,
was isolated from any fish examined (Amos et al., 1998).
The initial discovery of this virus in wild fish has several
important implications. Many of the known fish viruses
were first isolated from fish reared in aquaculture facilities
and their later discovery among wild fish was considered,
by some, as evidence that fish culture practices were affecting wild stocks. Furthermore, the discovery of a new virus
in cultured stocks has often led to the mandated destruction
of fish thought to be infected with an exotic virus that was
later found to be endemic, but previously undiscovered, in
the region. Should the virus from starry flounder be isolated
in the future from fish cultured in the Puget Sound region,
it will be recognized as an endemic agent.
Two young starry flounder were injected with virus and
observed for 7–14 days. One fish was necropsied after
1 week and the other after 2 weeks with no outward signs
of infection. Plaque assays did not show evidence of virus
infection in either fish; however, fish collected from the
wild may have been previously exposed to the virus with
resulting immunity. Additionally, pathogen-free young
rainbow trout were injected with the virus from starry
flounder and observed for 12 days without visible signs of
disease; however, a more in-depth study is needed using
various species of marine fish.
providing access to the electron microscope and Mechthild Jonas for
operation of the electron microscope. This project was conducted at
the University of Washington, Friday Harbour Laboratories (FHL) as
part of an undergraduate research apprenticeship funded by the
Washington Research Foundation, the Mary Gates Endowment
and the Tools for Transformation Program of the University of
Washington. The assistance of the Director of the FHL, Dr Dennis
Willows, in obtaining support for this research opportunity is
gratefully acknowledged.
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