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Journal of General Virology (2002), 83, 421–428. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Genomic organization of infectious salmon anaemia virus Sharon C. Clouthier,1 Trent Rector,2 Nathan E. C. Brown2 and Eric D. Anderson2 1 2 Microtek International, Ltd., Saanichton, British Columbia, Canada V8M 1Z8 Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, Maine 04469, USA The RNA genome segment order, nucleotide sequence and the putative encoded proteins were determined for infectious salmon anaemia virus (ISAV). Eight segments of genomic viral RNA between 1n0 and 2n4 kb in length were identified. RNA segments 1–6 each had a predicted single open reading frame encoding the P1, PB1, NP, P2, P3 and HA proteins, respectively. Segment 7 encoded the P4/P5 proteins and segment 8 encoded the P6/P7 proteins. Seven virion proteins with molecular masses between 25 and 72 kDa were found by SDS–PAGE analysis. The 72 and 42 kDa proteins were immunoreactive with ISAV antiserum from Atlantic salmon. The molecular mass of the 72 kDa virion protein suggested that it was the NP protein encoded by segment 3. The amino acid sequence was conserved, sharing 96n6 % identity with the NP protein of a Scottish ISAV isolate. Comparison of the amino acid sequences obtained by N-terminal analyses and cDNA nucleotide translation revealed that the 42 and 47 kDa proteins were the HA and P3 proteins encoded by segments 6 and 5, respectively. In addition, analysis provided evidence for their protein synthesis initiation sites. Like the HA protein, the signal sequence and potential glycosylation sites of P3 suggested that it was a surface glycoprotein. The predicted amino acid sequence shared 83n1, 84n0 and 99n6 % identity to the predicted P3 protein sequences for ISAV isolates from Norway, Scotland and Maine, respectively. These results establish the specificity, migration, number and nucleotide sequence of the eight RNA segments of the ISAV genome. Introduction Infectious salmon anaemia (ISA) is caused by an unclassified virus that is related most closely to members of the family Orthomyxoviridae (Krossøy et al., 1999). The primary tissue tropism is endothelial cells of the vascular system of some salmonid fish species (Hovland et al., 1994). Atlantic salmon (Salmo salar) are particularly susceptible to the virus, with infection resulting in overt signs of disease and mortalities within farm populations ranging from 0 to 100 % after several months. ISA virus (ISAV) particles are pleomorphic, enveloped spheres with a diameter of 90–130 nm (Dannevig et al., 1995 ; Nylund et al., 1995). They are covered evenly with surface Author for correspondence : Eric Anderson. Fax j1 207 581 2801. e-mail eandersn!maine.maine.edu The GenBank accession numbers of the sequences reported in this paper are : ISAV isolate CCBB – segments 1, AF404347 ; 2, AF404346 ; 3, AF404345 ; 4, AF404344 ; 5, AF404343 ; 6, AF404342 ; 7, AF404341 ; and 8, AF404340. ISAV isolate Norway – segments 5, AF429987 ; and 7, AF429990. ISAV isolate Scotland – segment 5, AF429988. ISAV isolate ME/01 – segments 5, AF429986 ; 6, AY059402 ; and 7, AF429989. 0001-8175 # 2002 SGM spikes of 13–15 nm in length and contain filamentous nucleocapsid-structures that are released upon partial virion disruption (Sommer & Mennen, 1996). 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ECB S. C. Clouthier and others Virus and RNA purification. Virus was prepared from ISAVinfected 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 104 000 g using an SW28 rotor and a Beckman L8–70M ultracentrifuge. The virus pellet was resuspended in TNE (10 mM Tris, 100 mM NaCl and 1 mM EDTA, pH 7n5), layered onto a 25, 35 and 45 % discontinuous sucrose gradient and centrifuged for 3 h at 132 000 g. Virus was collected from the interface of the 35 and 45 % sucrose layers and centrifuged for 2 h at 104 000 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. GCAAAGA. RNA (100 ng) isolated from purified ISAV or CHSE-214 cells (control) was mixed with the ISAV primer (20 pmol\µl), incubated at 80 mC for 5 min and then combined with the following in a total of 20 µl : 4 µl 5i first-strand buffer (Gibco BRL), 2 µl 10 mM dNTP mixture (Boehringer Mannheim), 1 µl 0n1 M DTT (Gibco BRL) and 1 µl SuperScript II reverse transcriptase (15 U\µl Gibco BRL). The mixture was incubated at 25 mC for 10 min and then at 42 mC for 1 h. The firststrand 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 : 1n5 µl 10 mM dNTP mixture (Boehringer Mannheim), 1n25 µl ISAV primer (20 pmol\µl), 1 µl random hexamers (25 pmol\µl ; Gibco BRL), 10 µl 10i PCR buffer with Mg#+ (Boehringer Mannheim) and 1 µl Taq polymerase (5 U\µl ; Boehringer Mannheim). After 35 cycles of 94 mC for 30 s, 59 mC for 45 s and 72 mC for 1 min, the PCR products were extended for 10 min at 72 mC. 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 manufacturer’s 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 mC for 5 min, placed on ice for 2 min and then combined with the following in a total of 20 µl : 4 µl 5i first-strand buffer (Gibco BRL), 2 µl 10 mM dNTP mixture (Boehringer Mannheim), 1 µl 0n1 M DTT (Gibco BRL) and 1 µl SuperScript II reverse transcriptase (15 U\µl ; Gibco BRL). The mixture was incubated at 25 mC for 10 min and then at 50 mC 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 mC for 30 min and then at 22 mC for 1 h. After spin column purification, the bluntended, double-stranded cDNAs were cloned into dephosphorylated, SmaI-digested pUC18 (Pharmacia), according to the manufacturer’s instructions. For both cDNA libraries, Escherichia coli DH5α 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 mC and stored at k20 mC. 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, 5h AAGCAGTGGTAACAACGCAGAGTA- RT–PCR 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 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. Methods Viruses and cell lines. ISAV strain CCBB was recovered by Micro Technologies from infected Atlantic salmon in Back Bay, New Brunswick, Canada. ISAV isolates from Norway, Scotland (390\98) and Maine (ME\01) and a second Canadian isolate (NB-99) were also obtained from Micro Technologies. ISAV was propagated in a Chinook salmon embryo cell line, CHSE-214 (Lannan et al., 1984). Cell monolayers were infected with ISAV and incubated at 15 mC in minimum essential medium containing Hanks’ salts and supplemented with -glutamine (10 mM) and 5 % foetal bovine serum (MEM-H5 ; Gibco BRL). Table 1. RT–PCR oligonucleotide DNA primers for RNA segments 2, 6 and 8 of ISAV isolate CCBB Segment 2 6 8 ECC Primer name Seg 2-5hF-mRNA Seg 2-3hR-mRNA HA forward HA reverse Class II Seg 8-3hR-mRNA Primer sequence (5h 3h) GAACGCTCTTTAATAACCATG TCAAACATGCTTTTTCTTC AGCAAAGATGGCACGATTC TGCACTTTTCTGTAAACGTACAAC AAGCAGTGGTAACAACGCAGAGTCTATCTACCATG TTATTGTACAGAGTCTTCC Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ISAV genomic organization Table 2. Summary of groups formed from screening ISAV cDNA libraries The cDNA library from approach one had a total of 1364 clones and that from approach two had a total of 768 clones. Number of positive clones in cDNA library Probe 5 : E-6 Segment 2 1-1F2, 5-1F1 2 : C-5, 4 :D-8 5 : E-7 Segment 6 2 : B-10 Segment 8 Origin of probe Approach 1 Approach 2 Approach 2 RT–PCR Approach 1 Approach 2 Approach 2 RT–PCR Approach 2 RT–PCR 0 0 212, 1144 0 0 0 0 6 33 41 6, 43 5, 38 14 0 50 10 amplification was used for second-strand cDNA synthesis ; after 30 cycles of 95 mC for 1 min, 50 mC for 1 min and 72 mC for 2 min, the PCR products were extended for 10 min at 72 mC (see Table 1 for primers). RT–PCR products were gel-purified according to the instructions of the manufacturer (Qiagen). Selection and identification of ISAV clones from the cDNA libraries. The contents of one 96-well plate were transferred onto one Hybond-N+ membrane (Amersham) and then placed on top of an LB agar plate containing 250 µg\ml ampicillin. Clones were grown on the filters at 37 mC overnight and the filters were processed for the times indicated on soaking pads saturated with the following solutions : 0n5 N NaOH for 7 min ; 1 M Tris–HCl, pH 7n4, for 2 min ; 1 M Tris–HCl, pH 7n4, for 2 min ; 0n5 M Tris–HCl, pH 7n4, and 1n5 M NaCl for 4 min. The filters were transferred to a bath of 2i SSC (1i SSC contains 0n15 M NaCl and 0n015 M Na citrate) and 1 % SDS and then soaked in $ 2i SSC. After a brief wash in chloroform, the filters were air-dried and then baked at 80 mC for 2 h. Prehybridization of the filters for 2 h in 6i SSC, 0n5 % SDS, 5i Denhardt’s and 0n1 mg\ml E. coli tRNA (Sigma) was followed by hybridization with a probe labelled with [α-$#P]dCTP (NEN) by nick translation (Amersham). Nick translation was carried out as outlined by the manufacturer. The cDNA libraries were screened initially using cDNA (amplified by RT–PCR and gel purified) for segments 2, 6 or 8 of ISAV isolate CCBB. The remaining segments were identified using probes consisting of gel-purified, restriction enzyme fragments digested from the plasmids of library clones selected randomly. Library clones were grouped based on the probe to which they hybridized (Table 2). Plasmid DNA isolated from representative clones of each group using Qiaprep columns (Qiagen) was sequenced at the University of Maine Core Sequencing Facility, Maine, USA. Only those sequences that matched other orthomyxovirus sequences or that did not match non-viral sequences were analysed further. Northern blot hybridization. Northern blot analysis was used to correlate each representative sequence with a specific ISAV genomic segment. Total RNA was isolated from CHSE-214 cell monolayers or ISAV-infected CHSE-214 cell monolayers using TRIzol reagent. The RNA was separated on a 2 % agarose gel containing formaldehyde and transferred onto a Hybond-N+ membrane in 10i SSC by capillary action, as described by Fourney et al. (1992). The probes used for Northern blot analysis were gel-purified, restriction enzyme-digested fragments from the plasmids of appropriate cDNA library clones. The probes were labelled with [α-$#P]dCTP by nick translation and hybridized to the blots at 42 mC for 18 h in ULTRAhyb (Ambion). The membranes were washed twice for 5 min in 2i SSC and 0n1 % SDS at 42 mC and then twice for 15 min in 0n1i SSC and 0n1 % SDS at 42 mC. 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 RT–PCR 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). SDS–PAGE 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. SDS–PAGE 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 antimouse immunoglobulin G–alkaline phosphatase conjugates (Southern Biotechnology) and visualized with BCIP\NBT (Sigma). N-terminal amino acid sequence analysis. The proteins of purified ISAV were separated by SDS–PAGE, blotted onto PVDF Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ECD S. C. Clouthier and others membranes (Bio-Rad) and stained with 0n1 % 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. Results Genetic analysis The RNA genome segment order, nucleotide sequence and putative encoded proteins were determined for ISAV. To accomplish this, two cDNA libraries were made using genomic RNA from purified virus and screened using known sequences from segments 2, 6 and 8 or randomly chosen cDNA sequences from each library. Eight distinct cDNA hybridization groups were identified. Of these, two groups were found in cDNA library 1 and all but one segment of the ISAV genome in cDNA library 2 (Table 2). Northern blot analysis of RNA from naive or ISAV-infected cells was done using a probe made from each cDNA hybridization group (Fig. 1). The RNA segments were correlated to the ISAV cDNA by consecutively hybridizing a Northern blot of mRNA from ISAV-infected cells or purified virus with cDNA from each hybridization group (Fig. 1). Eight RNA segments were identified ; segments 1 and 2 were both approximately 2400 nucleotides in length. The ISAV RNA segment corresponding to each cDNA clone is summarized in Table 3. The genome segments are numbered with respect to their mobility in agarose gels, from the slowest to the fastest, and comprise a genome of 14 500 nucleotides. The length of each gene, the corresponding encoded polypeptide(s) and the predicted molecular masses of the translated proteins are summarized in Table 3. Only a partial sequence from segment 1 was obtained. The cDNA sequences of segments 1–6 were predicted to encode one ORF. Segments 7 and 8 were each predicted to encode two proteins. 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 82n2–84n5 % 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 ECE Fig. 1. Northern blot analysis of cellular RNA with eight ISAV-specific probes. Purified cellular RNA was separated on a 2 % agarose gel and transferred to a Hybond-N+ membrane. Lanes 1–7 and 10, cellular RNA from CHSE-214 cells infected with ISAV isolate CCBB ; 8, cellular RNA from ISAV isolate ME-01 ; 9, cellular RNA from CHSE-214 cells infected with ISAV isolate NB-99 ; 11, cellular RNA from naive CHSE-214 cells. The RNA blot was consecutively hybridized with radioactively labelled DNA probes specific for one of the ISAV RNA segments. The results recorded by autoradiography after the addition of each single probe to the same RNA blot are shown in lanes 1–11. The probes are identified by segment : lanes 1, segment 3 ; 2, segment 4 ; 3, segment 6 ; 4, segment 1 ; 5, segment 5 ; 6, segment 7 ; 7, segment 8 ; 8–11, segment 2. Molecular mass standards (kb) are indicate on the left. The RNA segments are labelled on the right. the NP protein of ISAV isolate CCBB was highly conserved, sharing 96n6 % 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 76n4, 76n0 and 99n7 % similar to the corresponding sequence of ISAV isolate CCBB. The predicted translation of the ORF encoded by RNA segment 6 shared 84n8–84n3 % similarity to the predicted HA protein sequences for ISAV isolates from Norway (AF302799) and Scotland (AJ276859) and 99n2 % similarity to the Maine ISAV isolate (AY059402). The nucleotide sequence for ISAV CCBB segment 7 shared 99n6 % identity with a reported ISAV sequence (AX083264). The P4 and P5 proteins encoded on segment 7 shared 99n2–99n3 % 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 88n7–99n9 % 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 75n6–97n9 % similarity to the sequence reported previously for the Norwegian (AF262382), Scottish (AJ242016) and Canadian (AJ242016) isolates of ISAV. ISAV proteins N-terminal amino acid sequence analysis was used to correlate viral proteins to the predicted translation of ORFs Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ISAV genomic organization Table 3. RNA segments of ISAV isolate CCBB, their genes and encoded proteins GenBank accession numbers for the segments are as follows : segments 1, AF404347 ; 2, AF404346 ; 3, AF404345 ; 4, AF404344 ; 5, AF404343 ; 6, AF404342 ; 7, AF404341 ; and 8, AF404340. Segment Clone Segment length (kb)* ORF length (bp) Encoded protein Polypeptide length (aa) Predicted molecular mass (kDa) 1749 2127 1851 1737 1335 1185 771 441 705 552 P1 PB1 NP P2 P3 HA P4 P5 P6 P7 – 709 617 579 445 395 257 147 235 184 – 80n5 68n0 65n3 48n8 43n1 28n6 16n3 26n5 20n3 1 2 3 4 5 6 7 5 :E-6 Segment 2 1-1F2\5-5F1 2 :C-5\4 :D-8 5 :E-7 HA 2 :B-10 2n4 2n4 2n2 1n9 1n6 1n5 1n3 8 Segment 8 1n0 * Based on the average length determined from Northern blot hybridization analyses with two to five replicates per probe. Fig. 2. Amino acid sequence alignment of the RNA-binding domain of the NP protein from influenza A and B viruses with the putative NP protein RNA-binding domain from ISAV, as predicted using the CLUSTAL W system. ISAV NP, amino acids 189–307 (AF404345) ; influenza A virus (InfA) NP, amino acids 90–188 (P15675) ; InfB NP, amino acids 149–249 (P04666). Identical amino acids and amino acid residues with similarity in physical and chemical properties are indicated as (*) and (,), respectively. The NP protein RNA-binding domain from influenza A and B viruses was taken from Kobayashi et al. (1994). encoded by RNA segments of ISAV isolate CCBB. Purified ISAV proteins were separated by SDS–PAGE and visualized by Coomassie blue staining (Fig. 3). Seven putative ISAV proteins with estimated molecular masses of between 25 and 72 kDa were evident (Fig. 3). The four prominent proteins had molecular masses of 72, 47, 42 and 25 kDa. N-terminal amino acid sequence analysis was successful with three of the seven proteins (Table 4). The amino acid sequence at the N terminus of both the 40 and 42 kDa proteins was similar to amino acids 17–41 in the predicted translation of the Norwegian (AF302799), Scottish (AJ276859) and North American ISAV HA genes. The region of overlap did not include the first 16 amino acids of the translation, indicating that these amino acids were not present in the virion HA protein. The amino acid sequence of the HA protein from ISAV isolate CCBB had a signal peptidase cleavage site predicted to be between Ser"' and Arg"(. Thus, after cleavage of the signal sequence, the mature HA protein began at Arg"(. Analysis also showed that the 40 kDa protein was a C-terminally truncated form of the 42 kDa HA protein. The 13 N-terminal amino acids from the 47 kDa protein were unique and identical to amino acids in the predicted translation of ISAV RNA segment 5 (Table 4). The translated sequence of the 47 kDa protein had a signal sequence predicted to be 17 amino acids and the N-terminal amino acid sequence results confirmed that the cleavage site was located between Cys"( and Glu"). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ECF S. C. Clouthier and others and 42 kDa proteins of ISAV (Fig. 4A). Similar analyses were performed with ISAV-specific mouse pAbs and mAbs. Of the six immunoreactive proteins present in the cellular preparation of ISAV and recognized by the mouse polyclonal sera, three were present in the purified ISAV sample (42, 25 and 15 kDa ; Fig. 4B). Only the 42 kDa protein was recognized by the mAb (Fig. 4C). For each serum tested, no reaction was observed with the naive CHSE-214 sample, indicating that the immunoreactive proteins were derived from ISAV. Fig. 3. Analyses of proteins from purified ISAV isolate CCBB. Purified ISAV was resuspended in SDS-sample buffer. The solubilized proteins were separated by SDS–PAGE on a 5 % stacking gel and a 12 % resolving gel and visualized by Coomassie blue staining. Lanes 1, 2n5 % ; 2, 5n0 % ; 3, 7n5 % total virus preparation. Molecular mass standards (kDa) are indicated on the left. Arrows on the right point to four prominent protein bands and are labelled with the corresponding molecular masses (kDa). Table 4. N-terminal amino acid sequence analysis of proteins from ISAV isolate CCBB Protein (kDa) 40 42 47 Sequence analysis Similarity analysis RLXLRNHPDTTWIGDSRSDQSRXNQ RLXLRNHPDTTWIGDSRSDQSRXNQ EPXIXENPTXLAI HA HA 5 :E-7 Immunoreactive polypeptides encoded by the RNA segments were identified by Western blot analysis performed on naive and ISAV-infected CHSE-214 cells (Fig. 4). Sera collected from Atlantic salmon injected with ISAV reacted with the 72 Discussion This paper reports the organization of the ISAV genome segments as well as their genes and encoded proteins. The genome size and number of segments correspond to those reported previously for the European strain of ISAV (Mjaaland et al., 1997). Analyses show that each of the eight segments of the ISAV genome encodes one ORF, with the exception of segments 7 and 8. The gene assignment for ISAV and the proteins for which they encode are as follows : RNA segment 1 encodes P1, 2 encodes PB1, 3 encodes NP, 4 encodes P2, 5 encodes P3, 6 encodes HA, 7 encodes P4 and P5, and 8 encodes P6 and P7. This organization differs from that reported for other members of the Orthomyxoviridae but is most similar to influenza A and B viruses. Phylogenetic distance calculations also support a closer relationship of ISAV to influenza viruses than to Thogoto and Dhori viruses (Krossøy et al., 1999). The gene order for influenza A and B viruses and ISAV RNA segment 2 is the same. However, the ISAV gene order does have distinct differences relative to the following influenza A and B viruses, RNA segments and the proteins for which they encode : segments 3 (PA), 4 (HA), 5 (NP) and 6 (NA) (Lamb, 1989). RNA segments 7 and 8 for ISAV as well as influenza A and B viruses are predicted to encode two proteins each. As Fig. 4. Analyses of the immunoreactive antigens of ISAV isolate CCBB. Whole cell lysates were separated by SDS–PAGE, transferred to a nitrocellulose membrane and the immunoreactive antigens were detected using Western blot analysis. (A) Atlantic salmon anti-ISAV sera ; (B) ISAV pAb ; (C) ISAV mAb. Lanes 1, whole cell lysate of CHSE-214 cells ; 2, whole cell lysate of CHSE-214 cells infected with ISAV isolate CCBB ; 3 (B and C only), semi-purified ISAV isolate CCBB. Molecular mass standards (kDa) are indicated on the left. ECG Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 06 May 2017 13:32:35 ISAV genomic organization yet, which of these segments of ISAV encodes the NS and M proteins is unknown. Transcription of influenza virus RNA is primed by capped RNA generated by an endonuclease that cleaves cellular RNA 10–15 nucleotides from their 5h ends. The heterogeneous cap sequence is then followed by 12–16 conserved nucleotides present in the vRNA (Lamb & Choppin, 1983). The 5h 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, 15–22 nucleotides downstream from the 5h 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 13–14 nucleotides downstream from the 5h end of the vRNA (Sandvik et al., 2000). In this study, segments 2, 4, 5, 6 and 7 contain similar signals at the 3h 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 96n6 % 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 HAspecific 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 155–157) and one is conserved among the European and North American isolates of ISAV (positions 333–335). 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 (48n8 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 (j1) extends from the ATG codon at nucleotides 47–49 to a termination codon at nucleotides 815–817 and encodes a 257 amino acid protein (28n6 kDa). This segment contains a second ORF (j2) that could code for 147 amino acids (16n3 kDa). The eighth RNA segment of ISAV isolate CCBB also encodes two ORFs : the first (j1) comprises 703 nucleotides and encodes a 235 amino acid protein (26n5 kDa) ; the second (j2) is 552 nucleotides and encodes a 184 amino acid protein (20n3 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 (j2) on segment 8, which begins one nucleotide after the ATG of the second ORF (j1) 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. The authors wish to thank Micro Technologies, Inc. for providing the isolates of ISAV used in this study. 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