![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Virology, June 2001, p. 5352-5356, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5352-5356.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of the Infectious Salmon Anemia Virus Genomic
Segment That Encodes the Putative Hemagglutinin
Espen
Rimstad,1,*
Siri
Mjaaland,1
Michael
Snow,2
Aase B.
Mikalsen,1 and
Carey
O.
Cunningham2
Department of Pharmacology, Microbiology and
Food Hygiene, The Norwegian School of Veterinary Science, N-0033
Oslo, Norway,1 and FRS Marine
Laboratory, Aberdeen AB11 9DB, United Kingdom2
Received 21 November 2000/Accepted 7 March 2001
 |
ABSTRACT |
The genomic segment encoding the putative hemagglutinin of
infectious salmon anemia virus (ISAV) is described. Expression of the
putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable
region in the hemagglutinin gene.
| |
TEXT |
Infectious salmon anemia virus
(ISAV), an important pathogen for the Atlantic salmon (Salmo
salar) farming industry, shows many properties that closely
resemble those of the Orthomyxoviridae (1, 4, 7,
9). As with the influenza A and B viruses, the genome of ISAV
consists of eight single-stranded RNA segments of negative polarity
with partially complementary 3'- and 5'-end noncoding terminal
sequences (9), and the mRNA synthesis requires 8- to
18-nucleotide 5'-cap structures cleaved from cellular heteronuclear RNAs (11, 13). Transfer of convalescent-phase sera from
surviving fish has been shown to reduce mortality in fish exposed to
ISAV (2). This indicates that humoral immune responses of
the salmon may be protective against ISAV infection.
Molecular cloning.
The ISAV isolates Glesvaer and 390/98,
isolated from ISA epizootics in western Norway in 1990 and in western
Scotland in 1998, respectively, were used in this study. For the
Glesvaer isolate, mRNA from ISAV-infected salmon head kidney cells
(SHK-1 cells) was extracted and used in reverse transcription, adapters
were added, and the cDNA was subsequently ligated into pCRII plasmid vector (Invitrogen, Leek, The Netherlands). Plasmid inserts were excised from gels, 32P labeled, and used as probes in
Northern hybridisation reactions. For the 390/98 isolate, a subtractive
cDNA library was obtained. Total RNA was extracted from both
ISAV-infected and noninfected SHK-1 cells, converted into
double-stranded cDNA, and used for the subtractive hybridization
(PCR-Select cDNA subtraction kit; Clontech Laboratories, Palo Alto,
Calif.). Material resulting from this was PCR amplified and ligated
into the vector pGEM-T Easy (Promega, Madison, Wis.). Insert fragments
were labeled and used as probes in Northern hybridization reactions. To
obtain the 3' and 5' ends of viral RNA from both isolates, rapid
amplification of cDNA ends (RACE) was used (13).
Multiple molecular clones that were tentatively classed as ISAV
specific were sequenced, and alignments of nucleotide and amino acid
sequences were carried out. One molecular clone from the Glesvaer
isolate-infected SHK-1 cells was found to give positive Northern
hybridisation with ISAV-infected SHK-1, with a distinct band of
approximately 1.3 kb (Fig. 1A). Combining
the results of the 5'-end mRNA and 5'-end viral RNA (vRNA) RACE, this
genomic segment of the Glesvaer isolate was found to consist of
1,321 nucleotide residues (Fig. 2a)
(accession number AF220607). A full-length mRNA, excluding the
heterogeneous 5' primer sequence and poly(A) tail, was deduced to be
1,301 nucleotides long, while the similar full-length mRNA of the
Scottish 390/98 isolate was 1,292 nucleotides long (accession number
AJ276859). Alignment of the nucleotide sequences of the Glesvaer and
390/98 isolates showed 96.8% identity. However, the discrepancy
between the nucleotide sequences of the two isolates was unevenly
distributed. Between nucleotide residues 1021 and 1060 (numbers refer
to the Glesvaer isolate), there were 22 residue differences, including
deletions and gaps, yielding 12 of 13 consecutive residues between
residues 339 and 350 in the deduced amino acid sequence nonhomologous
(Fig. 2b and c). To determine whether this localized discrepancy
indicated genetic diversity or was caused by artifacts introduced
through the laboratory procedures, a reverse transcription (RT)-PCR
with subsequent cloning and nucleotide sequencing was performed using RNA extracted from the kidney of a salmon in a natural outbreak of ISA
in Norway (this clone was named Selje A12) and from Scottish ISAV
isolate 1490/98, which is known to differ from 390/98 in the nucleotide
sequences of segments 2 and 8 (8). This revealed identity
between Glesvaer and Selje A12 isolates up to nucleotide 1054. There is
a 6-nucleotide discrepancy (nucleotides 1054 to 1059), including a
3-nucleotide gap, in the Selje A12 sequence between the Glesvaer and
Selje A12 isolates (Fig. 2b and c). The sequence from 1490/98 was
identical to that from Glesvaer, although these isolates are known to
differ in the sequences of segments 2 and 8 (8).
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
Northern blot hybridization using the ISAV-specific
clone obtained from the Glesvaer isolate as a probe. Molecular sizes
(in kilobases) are indicated. (A) Lanes: 1 RNA extracted from
ISAV-infected SHK-1 cells 6 days p.i.; 2, RNA extracted from
ISAV-infected SHK-1 cells 7 days p.i.; 3, RNA extracted from pelleted
ISAV; 4, noninfected SHK-1 cells. (B) The target was total RNA
extracted from ISAV Glesvaer-infected SHK-1 cells. Lanes 1, 2, 3, 4, and 5 correspond to 1, 3, 5, 7, and 9 days p.i., respectively.
|
|
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 2.
Complete nucleotide sequence of ISAV Glesvaer HA
genomic segment with translated ORFs (a) and alignments of
nucleotide (b) and amino acid (c) sequences of segment from ISAV
Glesvaer, Selje A12, and 390/98 at the variable region. ·, identical
to base in Glesvaer;  , gaps introduced to maximize alignment.
|
|
Computer analysis of the nucleotide sequence of this genomic
segment found one large open reading frame (ORF) (ORF1) that consisted
of 1,176 and 1,167 residues for the Glesvaer and 390/98 isolates,
respectively. In both isolates, ORF1 started at the AUG codon at
nucleotides 8 to 10. The deduced putative polypeptides from
ORF1 were thus 391 and 388 amino acids long and approximately 42.4 to
42.7 kDa, excluding possible posttranslational modifications.
Two hydrophobic domains of the predicted ORF1-encoded
polypeptide scored as certain (from amino acids 150 to 170 and
350 to 370) using the TopPred 2 programme (16), and thus
represented possible transmembrane regions of the deduced amino acid
sequence. Two other regions were also found to be hydrophobic, from
amino acids 1 to 21 and 122 to 142, although the latter was only weakly hydrophobic. The N-terminal hydrophobic part, amino acids 1 to 21, was
deduced by the SignalP program (10) to be a signal
peptide, with the most probable cleavage site between amino acids 16 and 17, and amino acid sequencing of purified ISAV polypeptides
supports this (S. Griffiths, personal communication). A cell attachment sequence, RGD (arginine-glycine-apartate), which indicates membrane association, was found from amino acids 119 to 121. Two possible glycosylation sites were predicted at amino acids 333 and 349 (Glesvaer).
No significant homologies between the nucleotide or deduced amino acid
sequences of this ISAV genomic segment with other viral sequences was found using the Blast program, which reflects that the
evolutionary distance between ISAV and orthomyxoviruses infecting terrestrial animals is vast.
Viral RNA analysis.
A time course study of the occurrence of
intracellular RNA specific to this ISAV genomic segment in
SHK-1 cells was performed by extracting total RNA from ISAV-infected
SHK-1 cells at days 1, 3, 5, 7, and 9 postinfection (p.i.). The RNA was
subjected to electrophoresis, blotted, fixed to a nylon membrane, and
then used as the target in Northern hybridization as previously
described (9). No hybridization signal was found at day
1 p.i., but from day 3 p.i. and onwards, a band representing
a genomic segment of approximately 1.3 kb with increasing
density was observed. A band of approximately 2.6 kb was also observed
at days 5 and 7 p.i. (Fig. 1b). In ISAV-infected cells, there
would be three different classes of viral RNA: mRNA, vRNA
(genomic RNA), and template RNA for the synthesis of vRNA. The
sizes of vRNA, template RNA, and full-length mRNA would correspond
to the 1.3-kb fragment that showed a strong hybridization signal. This
RNA band appeared much stronger from day 5 p.i., which was
probably the result of the production of full-length genomic
RNA. The RNA band with a size of approximately 2.6 kb, which mainly
appeared at days 5 and 7 p.i., was possibly caused by a binding
between vRNA and template RNA.
To investigate the possibility that several different mRNAs could
be transcribed from the present ISAV genomic segment, a PCR was
carried out using primers corresponding to the 5' and 3' ends of
mRNAs. This choice of primers is based on the assumption that ISAV
mRNA, like influenza virus mRNA, always contains a copy of the
3' end of the genomic segment in the 5' end of mRNAs, and similarly the poly(A) addition signal in the 5' end of the
genomic segment is present in the 5' end of the mRNA. The
results from the mRNA RT-PCR did not reveal any mRNA other than
the 1.3-kb mRNA after amplification from Glesvaer-infected SHK-1
cells (5 or 7 days p.i.).
Expression.
ORF1 was amplified by PCR and cloned into the
pEGFP-N1 vector (Clontech Laboratories). The resulting plasmid was
named ORF1-pEGFP-N1. The nucleotide sequence of the insert and adjacent
sequences in the plasmid were determined to ensure that the insert was
in the correct reading frame. The cloned gene was expressed as a fusion to the N terminus of a red-shifted variant of wild-type enhanced green
fluorescent protein (EGFP). EGFP was chosen as a fusion partner because
it enables gene expression to be observed in living cells as
fluorescence without the need for fixation prior to microscopy. No
autofluorescence was seen in negative controls. The capacity of the
ORF1-pEGFP-N1 plasmid to express proteins in fish cells was tested by
transfection of SHK-1 cells. This cell line originates from Atlantic
salmon and was chosen in order to obtain correct processing of the
expressed ISAV polypeptide. The intracellular location of the
EGFP-ORF1 fusion protein was primarily detected as strong fluorescence
in the cytoplasm, but later, in some cells, also as small, intense
fluorescent points with a perinuclear location (Fig.
3). No fluorescence was observed in the
nucleus.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Micrograph of fusion proteins encoded by
ORF1-pEGFP-N1 plasmid in SHK-1 cells, showing cytoplasmic
localization and dense perinuclear granules of the fusion protein.
|
|
Immune staining and hemadsorption.
Transfected SHK-1 cells
were stained with the ISAV-specific monoclonal antibody (MAb) 3H6F8
(3), and sheep anti-mouse immunoglobulin biotinylated
whole antibody (Amersham Pharmacia, Uppsala, Sweden) was used as the
secondary antibody (Fig. 4). To test for
the specificity of 3H6F8 staining, Alexafluor 594-labeled secondary
antibodies were also used (Molecular Probes, Leiden, The Netherlands),
and stained cells showed evenly distributed red fluorescence with no
staining of the nucleus, and the same cells expressed the green fluorescence of the EGFP (data not shown).
View larger version (92K):
[in this window]
[in a new window]
|
FIG. 4.
Micrograph of ORF1-pEGFP-N1-transfected SHK-1 cells. The
cells were incubated with salmon erythrocytes and stained with MAb
3H6F8. A stained cell with adherent salmon erythrocytes is shown.
|
|
Salmon erythrocytes were found to bind to single cells in the
ORF1-pEGFP-N1-transfected SHK-1 monolayer and were observed as dense
aggregations of erythrocytes on the SHK-1 cell surface, demonstrating
the hemadsorptive properties of the ORF1 polypeptide (Fig. 4).
The same SHK-1 cells that aggregated erythrocytes were also recognized
by the 3H6F8 MAb (Fig. 4). It has previously been found that the 3H6M8
MAb inhibits hemagglutination of ISAV and recognizes a surface protein
of the virus particle (3). Together, these data indicate
that the polypeptide encoded by the ORF1 is the putative ISAV hemagglutinin.
ISAV hemagglutinin.
Hemagglutinin is a high-abundance protein
of influenza virus particles and has been calculated to make up
approximately 40% of the weight of influenza B virus particles
(12). Of the ISAV polypeptides, the most abundant,
as observed in gel electrophoresis, had a size of approximately 43 kDa
(4), which corresponds well to the deduced
polypeptide encoded by ORF1. However, it is less than the
hemagglutinin polypeptide of influenza virus, which, prior to
modification, has a molecular size of approximately 61 kDa. However,
electron microscopic studies have shown that the morphology of the
surface spikes of ISAV appear quite different from those of influenza A
virus (4), indicating structural differences between the
surface proteins. The ISAV surface projections were approximately 10 nm
long, while those of influenza A virus were 14 nm long
(4).
The receptor-destroying enzyme activity of ISAV has been suggested to
be an acetylesterase and not neuraminidase (4). A similar
activity is found in influenza C virus, for which receptor binding and
cleavage activities are combined in one molecule (6). In
order to test for eventual acetylesterase activity of the expressed ORF1 polypeptide 4, methylumbelliferyl acetate was added to
both transfected and control SHK-1 cultures, and the turnover of
substrate by acetylesterase activity was measured (HTS 7000 Bio Assay
reader; Perkin Elmer Applied Biosystems, Foster City, Calif.). No
difference in acetylesterase activity was observed between transfected
and control cells, indicating that the ORF1 polypeptide did not
have acetylesterase activity.
The influenza virus hemagglutinin proteins, in contrast to several
other influenza virus proteins, never enter the nucleus during the
replication cycle. Similarly, the ORF1 polypeptide was not
found in the nucleus. The influence of the ORF1 polypeptide fusion partner, EGFP, on intracellular localization is unknown; however, it follows the localization signal of its fusion partner (5), and without a fusion partner, EGFP is distributed
throughout the cell cytoplasm and nucleus. It has been found earlier
that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid
environment of endosomes (1), indicating that surface proteins of ISAV have activities similar to those of influenza virus
hemagglutinin. The influenza virus hemagglutinin polypeptide has an N-terminal signal peptide that is cleaved by a signal peptidase, and it has two further hydrophobic regions, of which the one at the C
terminus crosses the viral envelope. The other hydrophobic region of
influenza virus hemagglutinin is an essential component of the
hemagglutinin fusion process. The putative ISAV hemagglutinin also has
two hydrophobic regions that scored as probable membrane-crossing regions besides the signal peptide, and the locations of these corresponded well with those in influenza virus hemagglutinin. If the
putative ISAV hemagglutinin has the same orientation as influenza virus
hemagglutinin, it would thus have a cytoplasmatic C-terminal tail of 20 to 22 amino acids (from 391 to approximately 370), and influenza virus
A (H3) has a cytoplasmatic tail of 10 amino acids (15).
The ISAV hemagglutinin hydrophobic domain from residues 350 to 370 could cross the viral envelope, and the ectodomain would consist of 334 amino acid residues. The two predicted N-linked glycosylation sites on
ISAV hemagglutinin would thus be in the ectodomain, compared to between
three and nine (depending on the isolate) N-linked oligosaccharide
chains per subunit added to the ectodomain of influenza virus A
hemagglutinin (14).
The region in the ISAV genomic segment that showed sequence
discrepancy between isolates is of particular interest. This region encodes a hydrophilic part of the ORF1 protein just upstream of the
hydrophobic domain predicted to cross the viral envelope. Nucleotide
sequencing of the Selje A12 sample revealed closer nucleotide sequence
homology to the Glesvaer isolate than to the 390/98 isolate in the
variable region. This homology may reflect closer genetic and
epidemiological relationship, as 390/98 originated from a different
geographical area. The Selje A12 sample was amplified from RNA obtained
from diseased fish and has thus never been cultivated in SHK-1 cells.
The similarity between Glesvaer and Selje A12 indicated that there had
been no selection for genetic variation caused by adaptation of ISAV to
SHK-1 cells. The variable region of this segment may prove useful in
further epidemiological and phylogenetic analyses.
In conclusion, we have described the cloning and nucleotide sequencing
of the ISAV genomic segment that encodes the putative hemagglutinin. The putative ISAV hemagglutinin differs remarkably in
size from those of influenza viruses, but otherwise conforms to many of
the properties of orthomyxoviral hemagglutinins.
| |
ACKNOWLEDGMENTS |
This work was supported by grant 110562/122 from the Norwegian
Research Council.
The skillful technical assistance of Ann Teig and Julie Inglis is appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Norwegian School
of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway. Phone: 47 2296 4766. Fax: 47 2296 4818. E-mail:
espen.rimstad{at}veths.no.
| |
REFERENCES |
| 1.
|
Eliassen, T. M.,
M. K. Froystad,
B. H. Dannevig,
M. Jankowska,
A. Brech,
K. Falk,
K. Romoren, and T. Gjoen.
2000.
Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step.
J. Virol.
74:218-227[Abstract/Free Full Text].
|
| 2.
|
Falk, K., and B. H. Dannevig.
1995.
Demonstration of a protective immune-response in infectious salmon anemia (ISA)-infected atlantic salmon salmo-salar.
Dis. Aquat. Org.
21:1-5.
|
| 3.
|
Falk, K.,
E. Namork, and B. H. Dannevig.
1998.
Characterization and applications of a monoclonal antibody against infectious salmon anaemia virus.
Dis. Aquat. Org.
34:77-85[Medline].
|
| 4.
|
Falk, K.,
E. Namork,
E. Rimstad,
S. Mjaaland, and B. H. Dannevig.
1997.
Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.).
J. Virol.
71:9016-9023[Abstract].
|
| 5.
|
Flach, J.,
M. Bossie,
J. Vogel,
A. Corbett,
T. Jinks,
D. A. Willins, and P. A. Silver.
1994.
A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.
Mol. Cell. Biol.
14:8399-8407[Abstract/Free Full Text].
|
| 6.
|
Herrler, G.,
I. Durkop,
H. Becht, and H.-D. Klenk.
1988.
The glycoprotein of influenza C virus is the haemagglutinin, esterase and fusion factor.
J Gen. Virol.
69:839-846[Abstract/Free Full Text].
|
| 7.
|
Hovland, T.,
A. Nylund,
K. Watanabe, and C. Endresen.
1994.
Observation of infectious salmon anemia virus in Atlantic salmon, Salmo-salar L.
J. Fish Dis.
17:291-296.
|
| 8.
|
Inglis, J. A.,
J. Bruce, and C. O. Cunningham.
2000.
Nucleotide sequence variation in isolates of infectious salmon anaemia virus (ISAV) from Atlantic salmon Salmo salar in Scotland and Norway.
Dis. Aquat. Org.
43:71-76[Medline].
|
| 9.
|
Mjaaland, S.,
E. Rimstad,
K. Falk, and B. H. Dannevig.
1997.
Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost.
J. Virol.
71:7681-7686[Abstract].
|
| 10.
|
Nielsen, H.,
J. Engelbrecht,
S. Brunak, and G. von Heijne.
1997.
Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.
Protein Eng.
10:1-6[Abstract/Free Full Text].
|
| 11.
|
Plotch, S. J.,
M. Bouloy,
I. Ulmanen, and R. M. Krug.
1981.
A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.
Cell
23:847-858[CrossRef][Medline].
|
| 12.
|
Ruigrok, R. W. H.
2000.
Structure of influenza A, B and C viruses, p. 29-42.
In
K. G. Nicholson, R. G. Webster, and A. J. Hay (ed.), Textbook of influenza. Blackwell Science Ltd., Oxford, U.K.
|
| 13.
|
Sandvik, T.,
E. Rimstad, and S. Mjaaland.
2000.
The viral RNA 3'- and 5'-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses.
Arch. Virol.
145:1659-1669[CrossRef][Medline].
|
| 14.
|
Schulze, I. T.
1997.
Effects of glycosylation on the properties and functions of influenza virus hemagglutinin.
J. Infect. Dis.
176(Suppl. 1):S24-S28.
|
| 15.
|
Verhoeyen, M.,
R. Fang,
W. M. Jou,
R. Devos,
D. Huylebroeck,
E. Saman, and W. Fiers.
1980.
Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75.
Nature
286:771-776[CrossRef][Medline].
|
| 16.
|
von Heijne, G.
1992.
Membrane protein structure prediction: hydrophobicity analysis and the positive-inside rule.
J. Mol. Biol.
225:487-494[CrossRef][Medline].
|
Journal of Virology, June 2001, p. 5352-5356, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5352-5356.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Aspehaug, V., Mikalsen, A. B., Snow, M., Biering, E., Villoing, S.
(2005). Characterization of the Infectious Salmon Anemia Virus Fusion Protein. J. Virol.
79: 12544-12553
[Abstract]
[Full Text]
-
Hellebo, A., Vilas, U., Falk, K., Vlasak, R.
(2004). Infectious Salmon Anemia Virus Specifically Binds to and Hydrolyzes 4-O-Acetylated Sialic Acids. J. Virol.
78: 3055-3062
[Abstract]
[Full Text]
-
Falk, K., Aspehaug, V., Vlasak, R., Endresen, C.
(2004). Identification and Characterization of Viral Structural Proteins of Infectious Salmon Anemia Virus. J. Virol.
78: 3063-3071
[Abstract]
[Full Text]
-
Clouthier, S. C., Rector, T., Brown, N. E. C., Anderson, E. D.
(2002). Genomic organization of infectious salmon anaemia virus. J. Gen. Virol.
83: 421-428
[Abstract]
[Full Text]
-
Kibenge, F. S. B., Kibenge, M. J. T., McKenna, P. K., Stothard, P., Marshall, R., Cusack, R. R., McGeachy, S.
(2001). Antigenic variation among isolates of infectious salmon anaemia virus correlates with genetic variation of the viral haemagglutinin gene. J. Gen. Virol.
82: 2869-2879
[Abstract]
[Full Text]
Top
Abstract
Text
