Patent application title: Nucleic Acid and Amino Acid Sequences of Infectious Salmon Anaemia Virus and Their Use as Vaccines
Inventors:
Steven Griffiths (Monkton, CA)
Rachael Jame Ritchie (Fredericton, CA)
Joel Heppell (Chelsea, CA)
Assignees:
OTTAWA HEALTH RESEARCH INSTITUTE
NOVARTIS AG
IPC8 Class: AC07K14155FI
USPC Class:
530350
Class name: Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof proteins, i.e., more than 100 amino acid residues
Publication date: 2012-03-22
Patent application number: 20120071630
Abstract:
The present invention provides the use of nucleic acid sequences and/or
amino acid sequences in the preparation of a vaccine for the protection
of fish against infectious salmon anaemia virus. Specifically, such
vaccines contain at least one nucleic acid sequence which is derived from
ISAV or synthetically prepared analogues thereof, or substantially
homologous sequences. These nucleic acid sequences are transcripted and
translated into peptide sequences which are incorporated into a
vaccination strategy to induce and immune response to the surface
antigens of ISAV and therefore ISAV itself. Therefore both the use of a
vaccine against ISAV, and the incorporation of peptide sequences is
herein described.Claims:
1-13. (canceled)
14. A composition comprising a polypeptide having an amino acid sequence that is at least 70% homologous to SEQ ID NO: 10, wherein the polypeptide binds to an antibody obtained from an animal infected with ISAV and wherein the antibody binds to a protein having SEQ ID NO: 10.
15. The composition of claim 14, wherein the polypeptide has an amino acid sequence that is at least 80% homologous to SEQ ID NO: 10.
16. The composition of claim 14, wherein the polypeptide has an amino acid sequence that is at least 90% homologous to SEQ ID NO: 10.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser. No. 12/624,918, filed Nov. 24, 2009, now U.S. Pat. No. 7,998,484, which is a Continuation of U.S. application Ser. No. 11/525,695, filed Sep. 22, 2006, now Abandoned, which is a Continuation of U.S. application Ser. No. 10/734,782, filed Apr. 2, 2004, now U.S. Pat. No. 7,128,917, which is a Divisional of U.S. application Ser. No. 10/049,086, filed May 30, 2002, now U.S. Pat. No. 6,919,083, which is a National Phase Entry under ยง371 of International Application No. PCT/GB00/02976, filed Aug. 7, 2000, which claims priority to GB Application No. 0006674.6, filed Mar. 21, 2000, GB Application No. 0005848.7, filed Mar. 11, 2000 and GB Application No. 9918588.6, filed Aug. 7, 1999.
[0002] The present invention relates to a fish vaccine. More specifically the invention relates to a vaccine to protect salmon against infectious salmon anaemia virus.
[0003] Infectious salmon anaemia virus (ISAV) causes mortality of farmed Atlantic salmon. Typically aquaculture revenue is reduced by over 30%. Accordingly, there is a need for an effective vaccine against ISAV.
[0004] It is an object of the present invention to provide a vaccine to protect against ISAV.
[0005] According to the present invention there is provided a composition containing at least one nucleic acid sequence and/or at least one amino acid sequence, or a synthetically prepared analogue thereof or a substantially homologous sequence, wherein the composition is derived from or based upon infectious salmon anaemia virus and wherein at least one of said nucleotide and/or amino acid sequences does not cause salmon anaemia and is capable of being used as or to prepare a vaccine to ISAV.
[0006] A substantially homologous nucleic acid sequence is a sequence which can be transcribed and/or translated to provide an amino acid sequence which is substantially homologous to at least a part of an antigen of ISAV.
[0007] Preferably the substantially homologous amino acid is at least 70% homologous with a part of an antigen of ISAV which is capable of inducing an immune response.
[0008] More preferably the substantially homologous amino acid sequence is at least 80% homologous with a part of an antigen of ISAV and can induce an immune response.
[0009] Most preferably the substantially homologous amino acid sequence is at least 90% homologous with a part of an antigen of ISAV and can induce an immune response.
[0010] Suitably the amino acid sequence is chosen from the group comprising Sequences ID numbers 2, 4, 6, 7, 8 or 10 as herein described.
[0011] Alternatively the amino acid sequence may comprise at least one fragment of Sequence ID numbers 2, 4, 6, 7, 8 or 10.
[0012] Alternatively said amino acid sequence may be truncated from an amino acid sequence of Sequences ID numbers 2, 4, 6, 7, 8 or 10 as herein described, which can induce an immune response.
[0013] Preferably the substantially homologous nucleotide sequence is at least 60% homologous with a part of a nucleic acid sequence of an antigen of ISAV and the translation product thereof is capable of inducing an immune response.
[0014] Preferably the substantially homologous nucleotide sequence encodes is at least 70% homologous with a part of a nucleic acid sequence of an antigen of ISAV, the translation product of which is capable of inducing an immune response.
[0015] More preferably the substantially homologous nucleotide sequence encodes at least 80% homologous with a part of a nucleic acid sequence of an antigen of ISAV, the translation product of which is capable of inducing an immune response.
[0016] Most preferably the substantially homologous nucleotide sequence is at least 90% homologous to a part of a nucleic acid sequence of an antigen of ISAV, the translation product of which is capable of inducing an immune response.
[0017] Suitably the nucleotide sequences are chosen from the group comprising Sequence ID numbers 1, 3, 5 or 9 as herein described.
[0018] Alternatively, the invention provides for fragments of the sequences described in Sequence ID numbers 1, 3, 5 and 9 as herein described and wherein translation products of said fragments result in the induction of an immune response.
[0019] Additionally, the sequences may comprise a truncated form of the sequences given as 1, 3, 5 and 9.
[0020] The nucleotide sequence may be incorporated in a plasmid.
[0021] The nucleotide sequence may be incorporated in a suitable expression vector.
[0022] A further aspect of the present invention provides for the use of a sequence chosen from the group consisting of Sequence ID numbers 1 to 10, as described in the present invention in the preparation of a vaccine and/or therapeutic medicament for the protection of fish from infection with Infectious Salmon Anaemia virus.
[0023] Typical nucleic acid sequences are ISA2cd (previously referred to as p1.38), ISA1mta (previously referred to as p8.17), ISA3mx (previously referred to as p6.28) and
[0024] ISA4ha.
[0025] Preferably the peptide sequences are transcribed and translated from either one, two or all of the nucleic acid sequences; ISA2cd, ISA1mta, ISA3mx or ISA4ha and are incorporated into a vaccination strategy aimed at inducing an immune response to a surface antigen of ISAV and thus infectious salmon anaemia virus itself.
[0026] The invention provides the use of nucleic acid sequences or peptide sequences as defined herein in the preparation of a vaccine for the protection of fish against ISAV.
[0027] The invention further provides a vaccine to protect fish against ISAV wherein the vaccine includes nucleic acid or peptide sequences as defined herein.
CHARACTERISATION OF THE NOVEL SEQUENCES OF THE INVENTION
[0028] The accompanying figures describe the invention in more detail, wherein;
[0029] FIG. 1 is the nucleotide sequence of ISA2cd,
[0030] FIG. 2 is the amino acid sequence which is obtained from translation of the ISA2cd nucleic acid sequence listed in FIG. 1,
[0031] FIG. 3 is the nucleotide sequence of ISA1mta,
[0032] FIG. 4 is the amino acid sequence which is obtained following transcription of the nucleic acid sequence listed in FIG. 3,
[0033] FIG. 5 is the exact nucleotide sequence of ISA3mx,
[0034] FIG. 6a is the amino acid sequence (M1) which is translated from the unspliced nucleic acid sequence of ISA3mx shown in FIG. 5,
[0035] FIG. 6b is the amino acid sequence (M2) which is translated from the spliced nucleic acid sequence of ISA3mx shown in FIGS. 5, and
[0036] FIG. 6c is the amino acid sequence (M3) which is translated from the unspliced nucleic acid sequence of ISA3mx as shown in FIG. 5.
[0037] FIG. 7 is the nucleotide sequence of ISA4ha (SEQ ID NO: 9).
[0038] FIG. 8 is the amino acid sequence of ISA4ha (SEQ ID NO: 10).
[0039] In addition, information detailing the specific molecular weight (MW) and theoretical isoelectric focusing points (pI) is given at the foot of the respective amino acid sequence listings.
[0040] The nucleotide and amino acid sequences shown in the figures are further represented in the accompanying Patent-In generated sequence listings wherein;
[0041] Sequence ID number 1 is the nucleotide sequence of ISA2cd, as shown on FIG. 1,
[0042] Sequence ID Number 2 is the amino acid sequence of the ISA2cd, as shown in FIG. 2,
[0043] Sequence ID number 3 is the nucleotide sequence of ISA1mta, as shown on FIG. 3,
[0044] Sequence ID number 4 is the amino acid sequence of ISA1mta, as shown on FIG. 4,
[0045] Sequence ID number 5 is the nucleotide sequence of ISA3mx, as shown on FIG. 5,
[0046] Sequence ID number 6 is the predicted amino acid sequence of unspliced product of ISA3mx, as shown in FIG. 6a,
[0047] Sequence ID number 7 is the predicted amino acid sequence of spliced ISA3mx, as shown in FIG. 6b,
[0048] Sequence ID number 8 is the predicted amino acid sequence of spliced ISA3mx, as shown in FIG. 6c,
[0049] Sequence ID number 9 is the nucleotide sequence of ISA4ha, as previously shown in FIGS. 7, and
[0050] Sequence ID number 10 is the amino acid sequence of ISA4ha, as previously shown in FIG. 8.
[0051] The genetic sequences shown for ISA1mta and ISA2cd and the unspliced and spliced genetic sequences for ISA3mx have been derived from cloned cDNA wherein the cDNA clones were derived from infectious salmon anaemia virus (ISAV) genomic material. The cloned material was sequenced from the 5' end and the 3' end insertion sites using overlapping amplicons to produce a contig.
[0052] Veracity of the contig was confirmed by Reverse Transcriptase Polymerase Chain Reaction amplification (RT-PCR) of appropriate sized amplicons from ISAV infected salmon tissue and tissue cultures. Such amplicons were however obtained from uninfected control material, indicating that the genetic material was of ISAV origin.
[0053] The open reading frames (ORFs) were completed by rapid amplification of cDNA ends (RACE) from the incomplete sequence from virus-infected tissue culture. Corrections were made for the in vivo transcribed mRNA that were not apparent from the originally cloned cDNAs.
[0054] The ORF from ISA2cd does not have any significant homology at the nucleotide or amino acid sequence with previous submissions to databases accessible by BLAST. However, proteins with similar molecular weights (Mw) and isoelectric points (pI) include 14 viral proteins in the Swiss-Prot database such as Hemagglutinin-Neuraminidase.
[0055] The ORF from ISA1mta is also without any significant homology to previously characterised proteins submitted to the BLAST searchable databases. However it is of interest that it has molecular weight and isoelectric point characteristics (68-69 kDa and pI 8.2) that are nearly identical to one of the most predominant viral proteins identified by two dimensional electrophoresis. The protein appears to be integrally associated with the membranes of the ISAV infected tissue cultures. If the ORF yields such a protein it would be considered valuable in any vaccination strategy to reduce the level of ISAV infection in any salmonoid species.
[0056] Further, in the sequences shown for ISA3mx, the unspliced ORF (the basis for predicted amino acid sequence M1) does not have any significant homology at the nucleotide or amino acid sequence level with the previous submission to databases accessible by BLAST. However, proteins with similar molecular weights and isoelectric focusing points include several viral coat and envelope proteins listed in the Swiss-Prot database. Both the predicted M1 and M2 proteins (obtained from ORF's following splicing of the nucleotide sequence) are predicted to be membrane associated proteins and if the ORFs encoded by ISA3mx yield such proteins it would be considered valuable in any vaccination strategy to reduce the level of ISAV infection in any salomonid species.
[0057] The predicted protein translation of M3 (shown in FIG. 6c and accompanying sequence listing) shows homology to a paromyxovirus fusion protein associated with the cell membrane and thought to be involved in cell adhesion. In view of this exhibited homology, M3 is potentially valuable in any vaccination strategy aimed at reducing the level of ISAV infection in any salmonid species.
[0058] The further sequence relating to ISA4ha nucleotide sequence was obtained by means of the following procedure. The ISA4ha protein was detected by polyclonal antibodies following hybridisation. The protein is found to occur in two alternative forms. These two alternative forms are of different sizes, and can be seen where the proteins are cultured on different cell lines, for example she and chse.
[0059] As these two alternate forms were both detectable by antibody and varied in size depending on how it was grown, the protein is potentially a good candidate for virulence.
[0060] The protein was isolated and sequenced, resulting in a 24 amino acid fragment being produced. When this sequence was submitted, to BLAST searchable databases, it showed similarities to sequences of British and Norwegian strains of ISAV.
[0061] Subsequently, primers were designed based on the amino acid sequence obtained, along with reference to the sequences known for the similar British and Norwegian strains.
[0062] The primers were then subsequently used in polymerase chain reaction to amplify the relevant DNA fragment, which was subsequently sequenced and translated into amino acid coding.
[0063] The open reading frame listings obtained in the present invention, have particular commercial value for the following reasons:
1. There is sufficient reason to believe that the nucleotide corresponding amino acid sequences are of ISAV origin. Therefore, their incorporation into nucleic acid vaccines may have an impact on the reduction of mortality of farmed Atlantic salmon caused by ISAV which as previously stated, can typically reduce aquaculture revenues by over 30%. 2. Characterisation of the gene product will lead to the identification of key elements in the pathogenesis of infection and to the design of more accurate diagnostic tests which will also aid in epidemiological studies documenting the dissemination of different strains of the disease.
[0064] The nucleotide sequences ISA1mta, ISA2cd, ISA3mx, ISA4ha and associated derivatives thereof when translated into protein sequences being composed of either identical or equivalent amino acids, should induce a response by the hosts immune system. This principle can be further expanded to use these proteins in diagnostics tests and vaccination procedures.
Sequence CWU
1
1011821DNAInfectious Salmon Anaemia Virus 1caagatggat aacctccgtg
aatgcataaa ccgcaaaaga agactacttg ccttaccaga 60tgttcctgaa acttcggatg
cctttctaag tgatttgaga catctataca tgtgtgttgc 120tttctgtgat caacacaaaa
ccactggaga cgaatcaaga ttcaccaacc tggaattact 180tgaccaagat gaagcactag
gtgcccaaag agcttttgaa gccaaacatg gaataaaagg 240aggttcttta ggagacgttc
ttgaccatga actgaaaaag gtcattgaat ttacttttac 300ttctggaagt ttgtatattg
ccgaacaaag aaaaagaaag actcaagcag actcaataat 360tgtgtgcgtt tcagaaggac
ttaacgactt cagcgtatca cacggagtgc tagacatggg 420acttgtggaa acaggggtga
atgcagtaag agatttctgc acacaaaacg gaataccaat 480gaagataaat caggtaggat
ccacgagaac accaacaccg atcagcacat gcaaaatctc 540tgaacaaata acacgacaga
taaacagtac aattactgaa aggaaaatgg aaacagtact 600ggcagcaatc gcaattaaac
cagaactcaa actaactcag aaaggatgca gaccttgtaa 660agaactagaa gatgaaaata
ttctgtggat ggaccctcaa ttctgtgaaa ttgatgaaag 720ttttccttac agaggagggc
catacgggaa cttcctgcaa gaattgctgc ttacaaccaa 780cgacgtagag accaacggga
aagacagaga agaagtagta aagaagatac tggataacaa 840ggcgttcacc gttgaaagtg
gtgaatgcat aataacactt ccagacaaaa tgacttgttt 900cggagaacag gagaagaaga
gaccagcaac aatagacgaa gtgagaaccg caggagaaag 960gtttgaacag agtgttaaac
cgaaaaccca aagatatgga aggttatcag acaaatggat 1020ggagcttgaa aagtttatct
ttactgcaag caaaacagaa gtggatactt tcctttctgt 1080agggaccgaa agacttgagt
cggttggagt gtgtgtcgga gctttacaca gagcgaccac 1140aaccaggata attagaccta
tgattcaagg agggaaatgt tgggggatga tgttcaaaac 1200aaagtccaaa atgggagaca
cgaggaagga aggatactgt cacgcaatca ttttcggaaa 1260aggggaagat aaatcaggac
aaaacaagat gacaatgatg gggaaaacag tacattggca 1320tctaagagta gttaagtcta
aaggagactg gatggcgcaa caactctgtg caaacaaaag 1380cagaatatgg gaacatgacc
ctgagctagt aacagaagga gtgacagttc taatgacgcc 1440tttttctcag aaaattgcca
ccattagtag atggagggca atgaggttag acagcatgtt 1500tcatgtttct agtgcctggc
atcattcacc tgcgtgtgaa gctgcatcgg caatgctgag 1560aaagtttgtg gagatagtac
atgccatcaa ccagaaaaga gattggggtg ttgtggggag 1620tatggaggac atggtgaagg
aagtggagga aataggggag cacttgcaga cggcatgtga 1680ttttagagtt tacaacatgt
gcaaagcctt gattcagaaa attgcagtca gtacccaatg 1740agtggttatt tacttgtaaa
ttgttgtgtg tttgacgata tgtatttgtc gacgcggccg 1800cggtcgacgc ggccgcgaat t
18212578PRTInfectious Salmon
Anaemia Virus 2Met Asp Asn Leu Arg Glu Cys Ile Asn Arg Lys Arg Arg Leu
Leu Ala1 5 10 15Leu Pro
Asp Val Pro Glu Thr Ser Asp Ala Phe Leu Ser Asp Leu Arg 20
25 30His Leu Tyr Met Cys Val Ala Phe Cys
Asp Gln His Lys Thr Thr Gly 35 40
45Asp Glu Ser Arg Phe Thr Asn Leu Glu Leu Leu Asp Gln Asp Glu Ala 50
55 60Leu Gly Ala Gln Arg Ala Phe Glu Ala
Lys His Gly Ile Lys Gly Gly65 70 75
80Ser Leu Gly Asp Val Leu Asp His Glu Leu Lys Lys Val Ile
Glu Phe 85 90 95Thr Phe
Thr Ser Gly Ser Leu Tyr Ile Ala Glu Gln Arg Lys Arg Lys 100
105 110Thr Gln Ala Asp Ser Ile Ile Val Cys
Val Ser Glu Gly Leu Asn Asp 115 120
125Phe Ser Val Ser His Gly Val Leu Asp Met Gly Leu Val Glu Thr Gly
130 135 140Val Asn Ala Val Arg Asp Phe
Cys Thr Gln Asn Gly Ile Pro Met Lys145 150
155 160Ile Asn Gln Val Gly Ser Thr Arg Thr Pro Thr Pro
Ile Ser Thr Cys 165 170
175Lys Ile Ser Glu Gln Ile Thr Arg Gln Ile Asn Ser Thr Ile Thr Glu
180 185 190Arg Lys Met Glu Thr Val
Leu Ala Ala Ile Ala Ile Lys Pro Glu Leu 195 200
205Lys Leu Thr Gln Lys Gly Cys Arg Pro Cys Lys Glu Leu Glu
Asp Glu 210 215 220Asn Ile Leu Trp Met
Asp Pro Gln Phe Cys Glu Ile Asp Glu Ser Phe225 230
235 240Pro Tyr Arg Gly Gly Pro Tyr Gly Asn Phe
Leu Gln Glu Leu Leu Leu 245 250
255Thr Thr Asn Asp Val Glu Thr Asn Gly Lys Asp Arg Glu Glu Val Val
260 265 270Lys Lys Ile Leu Asp
Asn Lys Ala Phe Thr Val Glu Ser Gly Glu Cys 275
280 285Ile Ile Thr Leu Pro Asp Lys Met Thr Cys Phe Gly
Glu Gln Glu Lys 290 295 300Lys Arg Pro
Ala Thr Ile Asp Glu Val Arg Thr Ala Gly Glu Arg Phe305
310 315 320Glu Gln Ser Val Lys Pro Lys
Thr Gln Arg Tyr Gly Arg Leu Ser Asp 325
330 335Lys Trp Met Glu Leu Glu Lys Phe Ile Phe Thr Ala
Ser Lys Thr Glu 340 345 350Val
Asp Thr Phe Leu Ser Val Gly Thr Glu Arg Leu Glu Ser Val Gly 355
360 365Val Cys Val Gly Ala Leu His Arg Ala
Thr Thr Thr Arg Ile Ile Arg 370 375
380Pro Met Ile Gln Gly Gly Lys Cys Trp Gly Met Met Phe Lys Thr Lys385
390 395 400Ser Lys Met Gly
Asp Thr Arg Lys Glu Gly Tyr Cys His Ala Ile Ile 405
410 415Phe Gly Lys Gly Glu Asp Lys Ser Gly Gln
Asn Lys Met Thr Met Met 420 425
430Gly Lys Thr Val His Trp His Leu Arg Val Val Lys Ser Lys Gly Asp
435 440 445Trp Met Ala Gln Gln Leu Cys
Ala Asn Lys Ser Arg Ile Trp Glu His 450 455
460Asp Pro Glu Leu Val Thr Glu Gly Val Thr Val Leu Met Thr Pro
Phe465 470 475 480Ser Gln
Lys Ile Ala Thr Ile Ser Arg Trp Arg Ala Met Arg Leu Asp
485 490 495Ser Met Phe His Val Ser Ser
Ala Trp His His Ser Pro Ala Cys Glu 500 505
510Ala Ala Ser Ala Met Leu Arg Lys Phe Val Glu Ile Val His
Ala Ile 515 520 525Asn Gln Lys Arg
Asp Trp Gly Val Val Gly Ser Met Glu Asp Met Val 530
535 540Lys Glu Val Glu Glu Ile Gly Glu His Leu Gln Thr
Ala Cys Asp Phe545 550 555
560Arg Val Tyr Asn Met Cys Lys Ala Leu Ile Gln Lys Ile Ala Val Ser
565 570 575Thr
Gln32018DNAInfectious Salmon Anaemia Virus 3gcaaagatyg ctcaaatccc
aaaaataata cagaaaacgt ataagagatg gccgataaag 60gtatgactta ttcttttgat
gtcagagaca acaccttggt tgtaagaaga tctaccgcta 120ctaaaagtgg cattaagatc
tcctacagag aggatcgagg aacatcactt ctccaaaagg 180cattcgccgg gacagaagat
gaattctggg tggagttaga tcaagatgtc tacgttgaca 240aaaagattag aaaattcctg
gaagaagaga aaatgaagga catgagcaca agagtgtctg 300gagcagtggc agcagcaatt
gaaagatcag ttgaatttga caatttctca aaagaagcag 360cagctaacat tgaaatggct
ggtgtagatg atgaagaagc tggaggaagt ggtctggtag 420acaacagaag gaagaacaaa
ggggtctcaa acatggccta caatctgtct ctattcatag 480ggatggtgtt tcctgctctc
actactttct tcagtgctat cctatcagaa ggtgaaatga 540gcatctggca aaatggacaa
gcaatcatca gaattctggc actggcagat gaagacggaa 600agagacaaac aagaacagga
ggacagaggg tggacatggc tgatgtaacc aagctgaacg 660tagtcacggc taacgggaaa
gtcaagcaag ttgaagtaaa cttgaacgat ctcaaagcag 720cattcaggca gagtagacct
aaaagatcgg actacagaaa agggcaaggt tccaaggcta 780cagaatcaag catctccaac
caatgtatgg cactgattat gaaatctgtg ctgtcagcag 840accaactttt tgctccggga
gtgaagatga tgaggacgaa cggtttcaat gcgtcgtaca 900caacactggc agaaggggca
aacattccga gcaagtacct aagacacatg aggaactgcg 960gaggagtagc tctggacctg
atgggaatga agaggatcaa aaactcacct gaaggagcca 1020agtctaagat cttttccatc
atccagaaga aagtaagagg aagatgtcgc acagaggagc 1080aacgcctcct gactagcgca
ctgaaaatca gcgacggtga aaacaagttc cagagaatca 1140tggacactct atgtacaagc
ttcctgattg accctccaag aactaccaaa tgcttcattc 1200cacctatttc cagtctcatg
atgtacatcc aagaaggcaa ctctgtactg gcaatggatt 1260tcatgaaaaa cggagaggac
gcctgcaaga tctgcagaga agccaaactg aaagtggggg 1320taaacagtac gttcacaatg
tcagtagcta gaacatgcgt tgcagtgtca atggttgcaa 1380cagctttttg ttctgcagat
atcatcgaga atgcagtgcc tggttccgaa aggtacagat 1440ccaacatcaa ggctaacaca
accaaaccaa aaaaggactc cacttacaca attcaaggac 1500ttagattgtc taacgtgagg
tatgaagcaa gacctgaaac atcacaaagc aacacagaca 1560gaagttggca agtgaacgtg
actgacagct tcggaggact tgctgtgttc aaccaagggg 1620caattagaga aatgctagga
gacggaacat cagagacaac tagtgtgaac gtcagagccc 1680tggtgaagag aattctgaaa
tcagcttcag agaggagtgc aagagctgta aagacattta 1740tggtgggaga acaagggaaa
tcagctattg ttatctctgg tgtgggactg ttctctattg 1800actttgaagg ggtagaggaa
gcggaaagga taactgacat gacacctgaa attgagtttg 1860acgaggacga cgaggaagag
gaagacattg acatttagag tgacaattat gtaactttct 1920aattacccta tattgtttga
atatataatg aaactattgt gtgttaaagg ttgtgggttt 1980gattattaaa tttaaattga
aacggtattg acgatatt 20184616PRTInfectious
Salmon Anaemia Virus 4Met Ala Asp Lys Gly Met Thr Tyr Ser Phe Asp Val Arg
Asp Asn Thr1 5 10 15Leu
Val Val Arg Arg Ser Thr Ala Thr Lys Ser Gly Ile Lys Ile Ser 20
25 30Tyr Arg Glu Asp Arg Gly Thr Ser
Leu Leu Gln Lys Ala Phe Ala Gly 35 40
45Thr Glu Asp Glu Phe Trp Val Glu Leu Asp Gln Asp Val Tyr Val Asp
50 55 60Lys Lys Ile Arg Lys Phe Leu Glu
Glu Glu Lys Met Lys Asp Met Ser65 70 75
80Thr Arg Val Ser Gly Ala Val Ala Ala Ala Ile Glu Arg
Ser Val Glu 85 90 95Phe
Asp Asn Phe Ser Lys Glu Ala Ala Ala Asn Ile Glu Met Ala Gly
100 105 110Val Asp Asp Glu Glu Ala Gly
Gly Ser Gly Leu Val Asp Asn Arg Arg 115 120
125Lys Asn Lys Gly Val Ser Asn Met Ala Tyr Asn Leu Ser Leu Phe
Ile 130 135 140Gly Met Val Phe Pro Ala
Leu Thr Thr Phe Phe Ser Ala Ile Leu Ser145 150
155 160Glu Gly Glu Met Ser Ile Trp Gln Asn Gly Gln
Ala Ile Ile Arg Ile 165 170
175Leu Ala Leu Ala Asp Glu Asp Gly Lys Arg Gln Thr Arg Thr Gly Gly
180 185 190Gln Arg Val Asp Met Ala
Asp Val Thr Lys Leu Asn Val Val Thr Ala 195 200
205Asn Gly Lys Val Lys Gln Val Glu Val Asn Leu Asn Asp Leu
Lys Ala 210 215 220Ala Phe Arg Gln Ser
Arg Pro Lys Arg Ser Asp Tyr Arg Lys Gly Gln225 230
235 240Gly Ser Lys Ala Thr Glu Ser Ser Ile Ser
Asn Gln Cys Met Ala Leu 245 250
255Ile Met Lys Ser Val Leu Ser Ala Asp Gln Leu Phe Ala Pro Gly Val
260 265 270Lys Met Met Arg Thr
Asn Gly Phe Asn Ala Ser Tyr Thr Thr Leu Ala 275
280 285Glu Gly Ala Asn Ile Pro Ser Lys Tyr Leu Arg His
Met Arg Asn Cys 290 295 300Gly Gly Val
Ala Leu Asp Leu Met Gly Met Lys Arg Ile Lys Asn Ser305
310 315 320Pro Glu Gly Ala Lys Ser Lys
Ile Phe Ser Ile Ile Gln Lys Lys Val 325
330 335Arg Gly Arg Cys Arg Thr Glu Glu Gln Arg Leu Leu
Thr Ser Ala Leu 340 345 350Lys
Ile Ser Asp Gly Glu Asn Lys Phe Gln Arg Ile Met Asp Thr Leu 355
360 365Cys Thr Ser Phe Leu Ile Asp Pro Pro
Arg Thr Thr Lys Cys Phe Ile 370 375
380Pro Pro Ile Ser Ser Leu Met Met Tyr Ile Gln Glu Gly Asn Ser Val385
390 395 400Leu Ala Met Asp
Phe Met Lys Asn Gly Glu Asp Ala Cys Lys Ile Cys 405
410 415Arg Glu Ala Lys Leu Lys Val Gly Val Asn
Ser Thr Phe Thr Met Ser 420 425
430Val Ala Arg Thr Cys Val Ala Val Ser Met Val Ala Thr Ala Phe Cys
435 440 445Ser Ala Asp Ile Ile Glu Asn
Ala Val Pro Gly Ser Glu Arg Tyr Arg 450 455
460Ser Asn Ile Lys Ala Asn Thr Thr Lys Pro Lys Lys Asp Ser Thr
Tyr465 470 475 480Thr Ile
Gln Gly Leu Arg Leu Ser Asn Val Arg Tyr Glu Ala Arg Pro
485 490 495Glu Thr Ser Gln Ser Asn Thr
Asp Arg Ser Trp Gln Val Asn Val Thr 500 505
510Asp Ser Phe Gly Gly Leu Ala Val Phe Asn Gln Gly Ala Ile
Arg Glu 515 520 525Met Leu Gly Asp
Gly Thr Ser Glu Thr Thr Ser Val Asn Val Arg Ala 530
535 540Leu Val Lys Arg Ile Leu Lys Ser Ala Ser Glu Arg
Ser Ala Arg Ala545 550 555
560Val Lys Thr Phe Met Val Gly Glu Gln Gly Lys Ser Ala Ile Val Ile
565 570 575Ser Gly Val Gly Leu
Phe Ser Ile Asp Phe Glu Gly Val Glu Glu Ala 580
585 590Glu Arg Ile Thr Asp Met Thr Pro Glu Ile Glu Phe
Asp Glu Asp Asp 595 600 605Glu Glu
Glu Glu Asp Ile Asp Ile 610 61551050DNAInfectious
Salmon Anaemia Virus 5atgtctggat ttaacctcga ggtaatggtg ccggaacaag
gaggaaaagt ggtcttcagc 60cttactgaaa cggggtcatg tgtctcgttt tacggagatg
atgaaccagg tgaagggtcc 120tgcgaacttg cctctgaaaa catggatttt ccaagttgtc
ctctggggaa tggagatgac 180ttctgtctgt cgctggcgct aagcacaatg agatggtctg
ggatgaccaa gagaaacaac 240ttcatggaca gattcattgg aagttttgtt cactgtacac
cagtgatgat ctggtcgtat 300ggaaatttgt ccaagaaaag ccatcacaaa atggtttgcc
acacttgccc agacgagtac 360aagttcagtg acaaggacga gatgcaggga tactatgagg
gatgtctaga ggcttctact 420gacattttcc ttgatgaact tgctactgtt gttacaggtg
gcttctttcc tgtcggactc 480aaaggttcct ggggaggatg gtacctcaag tacgtcaggt
atgctggacc tcttgcggga 540tcaagtggat tcattgtcaa tcaacgattc tacgacagag
cccaaaacaa gactggatcc 600agggttgtat ccatggttga aatggacgga gacggcttat
cgttcatcta cgagaagcct 660agcgtctacc atagtgatgg gtgcactggg tcagcagcga
ggttctggaa acgggatcac 720aatgagagag ctggagttga gcttagggct ggacttcact
tcagaatgtg attggttgaa 780aacttgttat gtaaacaaga attttgtgtt tttgtcagaa
aaagaaattg ctgtaaacat 840ggaagttgaa aaattcattt gtaatgagaa ctaaagatgt
ctttgtgttc aaattttaac 900taatgacaat atatgaaata tgtcgtacat ggtgttgatg
ataattttta aaacgaaaag 960gagaattttt actaaaataa aaaaaaaata aaaaaaaaaa
aaaagaaaaa aaaaaaaaaa 1020aaaaaaagtc gacatcgata cgcgtggtca
10506256PRTInfectious Salmon Anaemia Virus 6Met Ser
Gly Phe Asn Leu Glu Val Met Val Pro Glu Gln Gly Gly Lys1 5
10 15Val Val Phe Ser Leu Thr Glu Thr
Gly Ser Cys Val Ser Phe Tyr Gly 20 25
30Asp Asp Glu Pro Gly Glu Gly Ser Cys Glu Leu Ala Ser Glu Asn
Met 35 40 45Asp Phe Pro Ser Cys
Pro Leu Gly Asn Gly Asp Asp Phe Cys Leu Ser 50 55
60Leu Ala Leu Ser Thr Met Arg Trp Ser Gly Met Thr Lys Arg
Asn Asn65 70 75 80Phe
Met Asp Arg Phe Ile Gly Ser Phe Val His Cys Thr Pro Val Met
85 90 95Ile Trp Ser Tyr Gly Asn Leu
Ser Lys Lys Ser His His Lys Met Val 100 105
110Cys His Thr Cys Pro Asp Glu Tyr Lys Phe Ser Asp Lys Asp
Glu Met 115 120 125Gln Gly Tyr Tyr
Glu Gly Cys Leu Glu Ala Ser Thr Asp Ile Phe Leu 130
135 140Asp Glu Leu Ala Thr Val Val Thr Gly Gly Phe Phe
Pro Val Gly Leu145 150 155
160Lys Gly Ser Trp Gly Gly Trp Tyr Leu Lys Tyr Val Arg Tyr Ala Gly
165 170 175Pro Leu Ala Gly Ser
Ser Gly Phe Ile Val Asn Gln Arg Phe Tyr Asp 180
185 190Arg Ala Gln Asn Lys Thr Gly Ser Arg Val Val Ser
Met Val Glu Met 195 200 205Asp Gly
Asp Gly Leu Ser Phe Ile Tyr Glu Lys Pro Ser Val Tyr His 210
215 220Ser Asp Gly Cys Thr Gly Ser Ala Ala Arg Phe
Trp Lys Arg Asp His225 230 235
240Asn Glu Arg Ala Gly Val Glu Leu Arg Ala Gly Leu His Phe Arg Met
245 250 2557140PRTInfectious
Salmon Anaemia Virus 7Met Ser Gly Phe Asn Leu Glu Val Met Val Pro Glu Gln
Gly Gly Lys1 5 10 15Val
Val Phe Ser Leu Thr Glu Thr Gly Ser Cys Val Ser Phe Tyr Gly 20
25 30Asp Asp Glu Pro Gly Gly Phe Phe
Pro Val Gly Leu Lys Gly Ser Trp 35 40
45Gly Gly Ser Tyr Leu Lys Tyr Val Arg Tyr Ala Gly Pro Leu Ala Gly
50 55 60Ser Ser Gly Phe Ile Val Asn Gln
Arg Phe Tyr Asp Arg Ala Gln Asn65 70 75
80Lys Thr Gly Ser Arg Val Val Ser Met Val Glu Met Asp
Gly Asp Gly 85 90 95Leu
Ser Phe Ile Tyr Glu Lys Pro Ser Val Tyr His Ser Asp Gly Cys
100 105 110Thr Gly Ser Ala Ala Arg Phe
Trp Lys Arg Asp His Asn Glu Arg Ala 115 120
125Gly Val Glu Leu Arg Ala Gly Leu His Phe Arg Met 130
135 1408146PRTInfectious Salmon Anaemia Virus
8Met Asn Leu Leu Leu Leu Leu Gln Val Ala Ser Phe Leu Ser Asp Ser1
5 10 15Lys Val Pro Gly Glu Asp
Gly Thr Ser Ser Thr Ser Gly Met Leu Asp 20 25
30Leu Leu Arg Asp Gln Val Asp Ser Leu Ser Ile Asn Asp
Ser Thr Thr 35 40 45Glu Pro Lys
Thr Arg Leu Asp Pro Gly Leu Tyr Pro Trp Leu Lys Trp 50
55 60Thr Glu Thr Ala Tyr Arg Ser Ser Thr Arg Ser Leu
Ala Ser Thr Ile65 70 75
80Val Met Gly Ala Leu Gly Gln Gln Arg Gly Ser Gly Asn Gly Ile Thr
85 90 95Met Arg Glu Leu Glu Leu
Ser Leu Gly Leu Asp Phe Thr Ser Glu Cys 100
105 110Asp Trp Leu Lys Thr Cys Tyr Val Asn Lys Asn Phe
Val Phe Leu Ser 115 120 125Glu Lys
Glu Ile Ala Val Asn Met Glu Val Glu Lys Phe Ile Cys Asn 130
135 140Glu Asn14591033DNAInfectious Salmon Anaemia
Virus 9cagtcgtcta tgtcttagaa accatcctga caccacctgg ataggtgact cccgaagcga
60tcaatcaagg gtgaaccaac agtctcttga tctggttaca aacttcaagg gaattctaca
120agccaagaac gggaatggtc tcatgaagca gatgagcgga aggttcccaa gtgattggta
180ccaacctact acaaagtata ggattctata cattggtaca aacgactgca ctgagggccc
240taacgacgtg atcataccga cgtcaatgac actagacaat gtggcaaggg acctgtacct
300gggagcatgt cgaggagatg taagagtgac accaaccttc gtgggagcag ctgagcttgg
360actgattggg agaacagatg ccttaacagg attttctgta aaggtgctga ctttcaacaa
420ccctactatt gtagtagttg gactaaatgg aatgtcagga atctacaagg tctgcattgc
480tgcctcttct ggaaacgtag gcggagtcaa cttggtgaac ggatgcggat acttcagcgc
540tcctctgaga ttcgacaact tcaaaggaca gatctacgtg tcagacacct ttgaagtcag
600aggaacaaag aacaaatgtg tcatacttag atcttctagc aatgctcctt tgtgtacaca
660tatcaaaaga aacattgagt tggatgagta cgttgacaca ccaaacactg ggggcgtata
720tccttctgat gggtttgatt ctcttcacgg ctctgcttcg attagaactt ttttaacaga
780ggcactgaca tgtccaggtg tagattggga cagaattgat gcagcttcat gcgagtatga
840cagttgtcct aaacttgtga aagaatttga ccaaacaggg ctcggaaaca cagatactca
900aataatgaga gagctagaag cacaaaagga gatgattggt aaacttggca gaaacattac
960agacgtaaac aacagagtag atgctattcc accacagctt agcaacatct tcatctctat
1020gggagtggca ggt
103310344PRTInfectious Salmon Anaemia Virus 10Ser Arg Leu Cys Leu Arg Asn
His Pro Asp Thr Thr Trp Ile Gly Asp1 5 10
15Ser Arg Ser Asp Gln Ser Arg Val Asn Gln Gln Ser Leu
Asp Leu Val 20 25 30Thr Asn
Phe Lys Gly Ile Leu Gln Ala Lys Asn Gly Asn Gly Leu Met 35
40 45Lys Gln Met Ser Gly Arg Phe Pro Ser Asp
Trp Tyr Gln Pro Thr Thr 50 55 60Lys
Tyr Arg Ile Leu Tyr Ile Gly Thr Asn Asp Cys Thr Glu Gly Pro65
70 75 80Asn Asp Val Ile Ile Pro
Thr Ser Met Thr Leu Asp Asn Val Ala Arg 85
90 95Asp Leu Tyr Leu Gly Ala Cys Arg Gly Asp Val Arg
Val Thr Pro Thr 100 105 110Phe
Val Gly Ala Ala Glu Leu Gly Leu Ile Gly Arg Thr Asp Ala Leu 115
120 125Thr Gly Phe Ser Val Lys Val Leu Thr
Phe Asn Asn Pro Thr Ile Val 130 135
140Val Val Gly Leu Asn Gly Met Ser Gly Ile Tyr Lys Val Cys Ile Ala145
150 155 160Ala Ser Ser Gly
Asn Val Gly Gly Val Asn Leu Val Asn Gly Cys Gly 165
170 175Tyr Phe Ser Ala Pro Leu Arg Phe Asp Asn
Phe Lys Gly Gln Ile Tyr 180 185
190Val Ser Asp Thr Phe Glu Val Arg Gly Thr Lys Asn Lys Cys Val Ile
195 200 205Leu Arg Ser Ser Ser Asn Ala
Pro Leu Cys Thr His Ile Lys Arg Asn 210 215
220Ile Glu Leu Asp Glu Tyr Val Asp Thr Pro Asn Thr Gly Gly Val
Tyr225 230 235 240Pro Ser
Asp Gly Phe Asp Ser Leu His Gly Ser Ala Ser Ile Arg Thr
245 250 255Phe Leu Thr Glu Ala Leu Thr
Cys Pro Gly Val Asp Trp Asp Arg Ile 260 265
270Asp Ala Ala Ser Cys Glu Tyr Asp Ser Cys Pro Lys Leu Val
Lys Glu 275 280 285Phe Asp Gln Thr
Gly Leu Gly Asn Thr Asp Thr Gln Ile Met Arg Glu 290
295 300Leu Glu Ala Gln Lys Glu Met Ile Gly Lys Leu Gly
Arg Asn Ile Thr305 310 315
320Asp Val Asn Asn Arg Val Asp Ala Ile Pro Pro Gln Leu Ser Asn Ile
325 330 335Phe Ile Ser Met Gly
Val Ala Gly 340
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