Microtek International, Ltd., Saanichton, British Columbia, Canada V8M 1Z81
Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, Maine 04469, USA2
Author for correspondence: Eric Anderson. Fax +1 207 581 2801. e-mail eandersn{at}maine.maine.edu
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Abstract |
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Introduction |
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The virus haemagglutinates a variety of fish cells but not erythrocytes from mammals and birds (Falk et al., 1997 ). The virion contains an acetylesterase receptor-destroying activity that does not affect influenza A or C virus haemagglutination, suggesting that the receptors are different for the viruses. Recent evidence shows that ISAV is similar to orthomyxoviruses in that it binds to sialic acid residues on host cell surfaces and undergoes fusion with the cell in acidic endosomes (Eliassen et al., 2000
). The haemagglutinating and acetylesterase activities seem to be carried out by two different proteins in ISAV (Rimstad et al., 2001
).
The ISAV genome is composed of eight segments of single-stranded, negative-polarity RNA. The genes encoding the putative PB1, NP, PA, HA and NS proteins have been described. However, definitive correlation of each gene to their corresponding genomic segment and to their encoded protein product has not been made.
Analysis of the nucleotide sequences encoding the putative NS and PB1 proteins shows that a minimum of two distinct genomic strains of ISAV exist: the North American strain and the European strain (Inglis et al., 2000 ). However, these gene sequences cannot be used to differentiate between the various European strains. Instead, the highly polymorphic region in the putative HA protein is used to identify and separate closely related species (Krossøy et al., 2001
). Further resolution of the ISAV genome and its organization is an important step toward understanding the relationship between the individual virus isolates. Towards this goal, we describe here the genome structure of ISAV isolate CCBB. The antigenic variation of ISAV has not been defined clearly and it is not known whether a cross-neutralizing fish immune response can be elicited. To address this question, we have identified ISAV proteins that are immunoreactive in Atlantic salmon. Together, these results are discussed with respect to their impact on rational vaccine design.
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Methods |
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Virus and RNA purification.
Virus was prepared from ISAV-infected CHSE-214 cell monolayers. Following complete cell lysis, the cell culture supernatant was filtered, dialysed against polyethylene glycol (PEG 8000; Sigma) and centrifuged for 2 h at 104000 g using an SW28 rotor and a Beckman L870M ultracentrifuge. The virus pellet was resuspended in TNE (10 mM Tris, 100 mM NaCl and 1 mM EDTA, pH 7·5), layered onto a 25, 35 and 45% discontinuous sucrose gradient and centrifuged for 3 h at 132000 g. Virus was collected from the interface of the 35 and 45% sucrose layers and centrifuged for 2 h at 104000 g. Viral RNA (vRNA) was then isolated from the pelleted virus using TRIzol reagent (Gibco BRL), as described by the manufacturer, and used for the construction of cDNA libraries.
cDNA library construction.
Two different strategies were employed to clone the ISAV genome. For the first approach, first-strand cDNA was synthesized from ISAV vRNA by reverse transcription with an ISAV-specific primer, 5' AAGCAGTGGTAACAACGCAGAGTAGCAAAGA. RNA (100 ng) isolated from purified ISAV or CHSE-214 cells (control) was mixed with the ISAV primer (20 pmol/µl), incubated at 80 °C for 5 min and then combined with the following in a total of 20 µl: 4 µl 5x first-strand buffer (Gibco BRL), 2 µl 10 mM dNTP mixture (Boehringer Mannheim), 1 µl 0·1 M DTT (Gibco BRL) and 1 µl SuperScript II reverse transcriptase (15 U/µl Gibco BRL). The mixture was incubated at 25 °C for 10 min and then at 42 °C for 1 h. The first-strand ISAV cDNA products synthesized by reverse transcription were amplified by PCR using the ISAV primer and random hexamers. To the first-strand reaction, the following components were added in a total of 100 µl: 1·5 µl 10 mM dNTP mixture (Boehringer Mannheim), 1·25 µl ISAV primer (20 pmol/µl), 1 µl random hexamers (25 pmol/µl; Gibco BRL), 10 µl 10x PCR buffer with Mg2+ (Boehringer Mannheim) and 1 µl Taq polymerase (5 U/µl; Boehringer Mannheim). After 35 cycles of 94 °C for 30 s, 59 °C for 45 s and 72 °C for 1 min, the PCR products were extended for 10 min at 72 °C. The amplified cDNA products were then separated by agarose gel electrophoresis, purified from the gel and cloned into the pGEM-T vector (Promega), according to the manufacturers instructions.
For the second approach, first-strand cDNA was synthesized from ISAV vRNA by reverse transcription with random hexamer primers. RNA (100 ng) isolated from purified ISAV or CHSE-214 cells (control) was mixed with random hexamers (50 ng/µl; Gibco BRL), incubated at 65 °C for 5 min, placed on ice for 2 min and then combined with the following in a total of 20 µl: 4 µl 5x first-strand buffer (Gibco BRL), 2 µl 10 mM dNTP mixture (Boehringer Mannheim), 1 µl 0·1 M DTT (Gibco BRL) and 1 µl SuperScript II reverse transcriptase (15 U/µl; Gibco BRL). The mixture was incubated at 25 °C for 10 min and then at 50 °C for 50 min. The TimeSaver cDNA Synthesis kit (Pharmacia) was then used for second-strand cDNA synthesis. The first-strand reaction was added to the second-strand reaction mixture, incubated at 12 °C for 30 min and then at 22 °C for 1 h. After spin column purification, the blunt-ended, double-stranded cDNAs were cloned into dephosphorylated, SmaI-digested pUC18 (Pharmacia), according to the manufacturers instructions.
For both cDNA libraries, Escherichia coli DH5 cells (Gibco BRL) were transformed with the ligation reactions and the ampicillin-resistant colonies containing either pGEM-T or pUC18 with cloned ISAV cDNA were selected by blue/white screening. White colonies were transferred onto 96-well plates containing 200 µl LB, 250 µg/ml ampicillin and 15% glycerol per well, grown overnight at 37 °C and stored at -20 °C.
RTPCR amplification of segments 2, 6 and 8 from ISAV isolate CCBB.
First-strand cDNAs for segments 2, 6 and 8 were synthesized from ISAV vRNA by reverse transcription using the primers outlined in Table 1 and under the conditions described above. PCR amplification was used for second-strand cDNA synthesis; after 30 cycles of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min, the PCR products were extended for 10 min at 72 °C (see Table 1
for primers). RTPCR products were gel-purified according to the instructions of the manufacturer (Qiagen).
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Northern blot hybridization.
Northern blot analysis was used to correlate each representative sequence with a specific ISAV genomic segment. Total RNA was isolated from CHSE-214 cell monolayers or ISAV-infected CHSE-214 cell monolayers using TRIzol reagent. The RNA was separated on a 2% agarose gel containing formaldehyde and transferred onto a Hybond-N+ membrane in 10x SSC by capillary action, as described by Fourney et al. (1992) . The probes used for Northern blot analysis were gel-purified, restriction enzyme-digested fragments from the plasmids of appropriate cDNA library clones. The probes were labelled with [
-32P]dCTP by nick translation and hybridized to the blots at 42 °C for 18 h in ULTRAhyb (Ambion). The membranes were washed twice for 5 min in 2x SSC and 0·1% SDS at 42 °C and then twice for 15 min in 0·1x SSC and 0·1% SDS at 42 °C. The results were recorded on Kodak X-OMAT AR film.
The probes used in the Northern blot hybridization experiments were derived from four clones constructed using the second approach, one clone constructed using the first approach and RTPCR products of the three known segments (Table 2). A single RNA blot was probed consecutively with each of the eight individual probes. One probe was hybridized to the Northern blot and the results were visualized by autoradiography. The next probe was hybridized to the same Northern blot, the results were visualized and compared with the results from the previous hybridization. By repeating this process with each of the eight probes, we were able to correlate each individual probe and its corresponding nucleotide sequence with a specific RNA segment.
Construction of full-length clones of each ISAV genome segment.
The full-length cDNA sequences for each of the ISAV RNA segments, with the exception of segment 1, was generated by rapid amplification of cDNA ends (RACE) PCR using the RLM-RACE kit (Ambion). The PCR products were cloned into either pCR2.1-TOPO (Invitrogen) or pGEM-T, as directed by the manufacturers instructions, and then sequenced. AssemblyLIGN, version 1.0.9b (Oxford Molecular Group), was used to order the overlapping sequenced DNA fragments for construction of the full-length sequence.
PCR primers were designed from the consensus sequence obtained for each ISAV RNA segment and used to amplify the full-length cDNA sequence for each segment, with the exception of segment 1. The PCR product for each segment was cloned into pGEM-T and DNA from three representative clones was sequenced. The programs contained in MacVector, version 6.5.3 (Oxford Molecular Group), were used to identify open reading frames (ORFs) and regions of local similarity. The nucleotide and predicted amino acid sequence for each ORF were analysed by BLAST searches through the National Center for Biotechnology Information server (Altschul et al., 1990 ; Pearson & Lipman, 1988
) or the Influenza database (Los Alamos National Laboratory). The most likely cleavage sites for signal peptidase in HA and 5:E-7 were determined using SignalP, version 1.1 (Nielsen et al., 1997
).
Generation of anti-ISAV immune sera.
Anti-ISAV antibodies were generated in Atlantic salmon injected with tissue culture supernatant from ISAV-infected CHSE-214 cell monolayers (Opitz et al., 2000 ). Mouse polyclonal and monoclonal antibodies (pAbs and mAbs, respectively) to ISAV were generated by Rob Beecroft (Immuno-Precise Antibodies).
SDSPAGE and Western blot analysis.
Whole cell lysates of naive and ISAV-infected CHSE-214 cells as well as purified ISAV were screened for the presence of immunoreactive antigens with sera from vaccinated and challenged Atlantic salmon. SDSPAGE was carried out as described by Laemmli (1970) . Immunoreactive protein bands were visualized by Western blot analysis using sera from ISAV-injected Atlantic salmon, followed by incubation with mouse anti-salmonid immunoglobulin 5F12 mAb (Immuno-Precise Antibodies). Alternatively, the proteins on the membranes were screened with anti-ISAV mouse pAbs and mAbs. Immunoreactive proteins were detected with goat anti-mouse immunoglobulin Galkaline phosphatase conjugates (Southern Biotechnology) and visualized with BCIP/NBT (Sigma).
N-terminal amino acid sequence analysis.
The proteins of purified ISAV were separated by SDSPAGE, blotted onto PVDF membranes (Bio-Rad) and stained with 0·1% Coomassie blue R-250 in 40% methanol and 1% acetic acid. The stained protein bands were removed from the membrane and subjected to N-terminal amino acid sequence analysis using an Applied Biosystems gas-phase sequencer, model 470A, or a liquid-phase sequencer, model 473, with online phenylthiohydantoin analysis.
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Results |
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Comparison of the cDNA nucleotide and predicted amino acid sequences for the ISAV genome to those listed in the Influenza database and in GenBank showed that RNA segments 1 and 5 of ISAV isolate CCBB were unique. RNA segments 2, 3, 4 and 6 were found to encode the putative proteins PB1, NP, PA and HA, respectively. The predicted sequences of the P6 and P7 proteins encoded on RNA segment 8 were similar to the sequences of the two ORFs on segment 8 from other ISAV isolates.
The protein sequence of the partial ORF encoded on segment 1 was unique. The predicted amino acid sequence of the PB1 protein, encoded by RNA segment 2, was 82·284·5% similar to the amino acid sequences of the PB1 proteins from Norwegian (AJ002475) and Scottish (AF262392) ISAV isolates. The assignment of the NP protein to the ORF encoded on RNA segment 3 was based on nucleotide sequence similarity to the influenza A virus NP protein RNA-binding region (Fig. 2) and to the putative NP protein sequence described by Snow & Cunningham (2001)
. The sequence for the NP protein of ISAV isolate CCBB was highly conserved, sharing 96·6% identity to that reported for the NP protein of the Scottish ISAV isolate (AJ276858). The predicted protein sequence of the P2 protein from RNA segment 4 shared 99% identity to the putative PA protein sequence (AF306548) described by Ritchie et al. (2001)
. The nucleotide sequences for segment 5 of the Scottish (AF429988), Norwegian (AF429987) and Maine (AF429986) isolates of ISAV were 76·4, 76·0 and 99·7% similar to the corresponding sequence of ISAV isolate CCBB. The predicted translation of the ORF encoded by RNA segment 6 shared 84·884·3% similarity to the predicted HA protein sequences for ISAV isolates from Norway (AF302799) and Scotland (AJ276859) and 99·2% similarity to the Maine ISAV isolate (AY059402). The nucleotide sequence for ISAV CCBB segment 7 shared 99·6% identity with a reported ISAV sequence (AX083264). The P4 and P5 proteins encoded on segment 7 shared 99·299·3% similarity to the translations predicted for ORFs 1 and 2 from the reported sequence (AX083264). The nucleotide sequence for segment 8 of the Norwegian (AF429990) and ME/01 (AF429989) isolates of ISAV shared 88·799·9% identity to the corresponding sequence from ISAV isolate CCBB. Our results confirmed that segment 8 encoded two proteins, as reported previously by Mjaaland et al. (1997)
. The amino acid sequence translated from the largest ORF shared 75·697·9% similarity to the sequence reported previously for the Norwegian (AF262382), Scottish (AJ242016) and Canadian (AJ242016) isolates of ISAV.
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Discussion |
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Transcription of influenza virus RNA is primed by capped RNA generated by an endonuclease that cleaves cellular RNA 1015 nucleotides from their 5' ends. The heterogeneous cap sequence is then followed by 1216 conserved nucleotides present in the vRNA (Lamb & Choppin, 1983 ). The 5' ends of mRNAs corresponding to ISAV segments 2, 3, 6 and 8 contain seven to eight conserved nucleotides related closely to the conserved nucleotides in other members of the Orthomyxoviridae (Krossøy et al., 1999
; 2001
; Sandvik et al., 2000
; Rimstad et al., 2001
; Snow & Cunningham, 2001
). In this study, segments 2, 3, 6, 7 and 8 contain at least the CAAAGA portion of the consensus sequence. The mRNA polyadenylation signal for orthomyxoviruses is a stretch of five to seven uridine residues, 1522 nucleotides downstream from the 5' end of the vRNA (Li & Palese, 1994
). ISAV genomic segments 7 and 8 each have a polyadenylation signal of four to five uridine residues located 1314 nucleotides downstream from the 5' end of the vRNA (Sandvik et al., 2000
). In this study, segments 2, 4, 5, 6 and 7 contain similar signals at the 3' ends of their mRNA sequences, indicating that CCBB ISAV contains the conserved orthomyxovirus polyadenylation signal.
ISAV segments 3 and 6 encode the NP (68 kDa) and HA (43 kDa) proteins, respectively. Antibodies in serum from Atlantic salmon infected with ISAV recognize a 72 and a 42 kDa protein. The assignment of the 72 kDa protein to NP is based on size. The putative nucleocapsid protein from the Scottish strain of ISAV shares 96·6% identity to the NP protein from the CCBB isolate. The high sequence conservation suggests that the ISAV NP protein may be a type-specific antigen, like the NP proteins of influenza viruses (Lamb & Krug, 1996 ). A protective humoral immune response to influenza viruses is made to the surface protein HA. The HA-specific antibodies in fish sera indicate that ISAV HA proteins may play a similar role in protecting Atlantic salmon against ISAV. These findings suggest that the ISAV NP and HA proteins will be important antigens for ISA vaccine design and typing of ISAV.
There are three predicted N-linked glycosylation sites in the ISAV HA protein (Kornfeld & Kornfeld, 1985 ): one is unique to the North American isolates of ISAV (positions 155157) and one is conserved among the European and North American isolates of ISAV (positions 333335). Also of interest is the putative N-glycosylation site in the hypervariable region of the HA protein from the Norwegian ISAV isolate Glesvaer/2/90. The carbohydrates on the HA protein may contribute to differences in immunogenicity and the ability of various isolates of ISAV to haemagglutinate the red blood cells of different species of vertebrates.
Segment 5 has a single ORF of 1335 nucleotides encoding 445 amino acids (48·8 kDa). N-terminal amino acid sequence analysis confirms that the 47 kDa protein is encoded by segment 5. The function of the protein remains unknown but P3 is likely to be the 53 kDa protein reported for other ISAV strains (Falk et al., 1997 ; Kibenge et al., 2000
). In this study, antibodies in serum from Atlantic salmon infected with ISAV do not recognize the P3 protein. However, the hydrophobic signal sequence of 17 amino acids and potential glycosylation sites suggest that it is a surface glycoprotein. If this is the case, then, like influenza A and B viruses, ISAV has two virion surface glycoproteins.
RNA segment 7 of ISAV isolate CCBB contains two ORFs. The largest ORF (+1) extends from the ATG codon at nucleotides 4749 to a termination codon at nucleotides 815817 and encodes a 257 amino acid protein (28·6 kDa). This segment contains a second ORF (+2) that could code for 147 amino acids (16·3 kDa). The eighth RNA segment of ISAV isolate CCBB also encodes two ORFs: the first (+1) comprises 703 nucleotides and encodes a 235 amino acid protein (26·5 kDa); the second (+2) is 552 nucleotides and encodes a 184 amino acid protein (20·3 kDa). RNA segments 7 and 8 of influenza A virus encode two proteins each, the membrane proteins M1 and M2 and the non-structural proteins NS1 and NEP, respectively (Lamb, 1989 ). These proteins from influenza A, B and C viruses are encoded by spliced transcripts. If ISAV uses a similar coding strategy, the proteins on segment 7 may also be made via alternative splicing. This is in contrast to the ORF (+2) on segment 8, which begins one nucleotide after the ATG of the second ORF (+1) and makes splicing unlikely.
The ISAV genome comprises eight segments of RNA. There are ten predicted encoded proteins but more may be found. The biological functions of the ISAV proteins remain unknown, with the exception of the HA protein. The NP and HA proteins are immunoreactive and may prove to be important vaccine candidates or type-specific antigens. It may be that the detection of anti-P3 antibodies in the Atlantic salmon serum were masked by the antibody response to the HA protein. It will be interesting to determine if antibodies to the second glycoprotein, P3, can be generated in Atlantic salmon in the absence of the HA protein. The North American and European ISAV isolates are genetically different and may represent distinct strains. Studies to determine if ISAV genetic reassortment occurs and the consequences with respect to pathogenicity are required. Reassortant studies with the various strains of ISAV may also provide insight into the biochemical structure/function of a given gene and its encoded protein. These studies should provide the basis for rational design of vaccines for ISAV.
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Acknowledgments |
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Footnotes |
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References |
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Received 18 October 2001;
accepted 26 October 2001.