Supplement 1
For sequencing, fragments were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from total RNA extracts [3] of virus infected FgV-ch9 and non-infected wild-type strain PH1 mycelium cultivated in liquid complete medium[2]. The amplicons were blunt-end cloned into the pBlueSkriptSK-(Stratagene) with standard techniques[4]. cDNA was synthesized with primers RNA2 rev (5´- gccattaaagtcccatgac-3´), RNA 3 rev (5´- gccatttaagggttctccttaa-3´) and RNA 5 rev (5´- gccatttaggggtaggcc-3´), respectively, using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, St. Leon-Rot, Germany) according to the manufacturers´ protocol. Two µl of the cDNA were amplified with 5 µl 5X Green GoTaq Reaction Buffer (Promega), 2 µl dNTPs (2 mM), 2 µl MgCl2 (25 mM), 1 µl (10 µM) of forward and reverse primer, respectively, and 1 U GoTaq DNA polymerase in a 25 µl volume in a Biometra T1 thermocycler (Biometra GmbH, Göttingen, Germany) with 4 min initial denaturation at 94 °C, 30 cycles with 30 sec 94 °C, 60 sec 58 °C and 60 sec at 72 °C. The reaction was completedafter 5 min at 72 °C. In detail, the 3´ terminus of segment 2 was amplified with the primers RNA 2 rev and RNA 2 fw 1 (5´- catattgtcacgagcgacg-3´). The duplication was verified by using primers RNA 2 rev and RNA 2 fw/rev (5´- agtcatgggactttaatggc-3´). The 3´ terminus of segment 3 was amplified with the primers RNA 3 rev 1 and RNA 3 coding fw 1 (5´- gcacctgtggatgatgactc-3´). The duplication was verified with the primer pair RNA 3 rev/RNA 3 fw/rev (5´- ttaaggagaacccttaaatggc-3´). The result of the verification of the duplication is given in Fig. S1A.
Absence of segments without duplications was verified by amplification of cDNA synthesized with primer RNA 2 fw 1 and RNA 3 coding fw 1The phenol-chloroform purified cDNA was C-tailed with Terminal Deoxynucleotidyl Transferase (Thermo Fisher Scientific) and amplified with primer polyG (5´-cggaattcccg15-3´) and RNA 2 fw 2 (5´-gttggtgattggacgggg-3´) for segment 2 and RNA 3 coding fw 2 (5´-ggtagaagcggcacgggcccgg-3´) for segment 3. For both segments, a single band was obtained (Fig. S1B).
The 3´ terminus of segment 5 was amplified with primers RNA 5 rev (5´- gccatttaggggtaggcc-3´) and RNA 5 fw (5´- gatttgcttcgagccacaacg-3´). The ORF of segment 5 was amplified with RNA 5 ORF rev (5´-tgttgtgtccggcagtatgt-3´) and RNA 5 ORF fw (5´-acataccagcaattccccgggatgtattcg-3´).
The fragment of ORF 5 in the pBlueSkriptSK-was subcloned SmaI/Hind III into the EcoRV/HindIII digested expression vector pET30a (Novagen, USA). The protein was expressed in BL21-Gold cells (Agilent Technologies, Santa Clara) according to the manufacturers´ instructions. A peptide with the amino acid sequence H-CQW SDE RIP TGP GYD L-Nh2 (amino acid 65 to 74 with an additional C for coupling to KLH) was synthesized in vitro at Eurogentec S.A. (Seraing, Belgium), coupled to keyhole limpet haemocyanin (KLH) carrier and used to produce a specific polyclonal antiserum against the ORF of segment 5 using the Speedy mini program (Eurogentec S.A., Seraing, Belgium). SDS-PAGE and Western-Blotting from total protein extracts of mycelium grown 3 days in liquid CM-medium was performed as described in Schwach et al. [5]. Bound antibodies were detected using alkaline phosphatase-conjugated secondary antibodies and nitro-blue tetrazolium chloride(NBT)/ 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP).
Sequencing and sequence analysis were performed as described [1]. Additional sequence alignments were done manually.Zinc finger motifs were predicted with prosite (
Fig. S1: RT-PCR for the verification of duplications at the 3´termini of segment 2 and 3. (A) Primer RNA 2 rev binds at two regions on the 3´ terminus of segment 2. The PCR product can exclusively be synthesized with primer pair RNA 2 rev/RNA fw/rev,if the duplication is present (lane 2).No PCR product was obtained, when total RNA extracted from virus free Fusarium graminearumwas tested (lane 1). (B) Primer RNA 3 rev binds at two regions on the 3´ terminus of segment 3. The PCR product can exclusively be synthesized with primer pair RNA 3 rev/RNA fw/rev, if the duplication is present (lane 1). No PCR product was obtained, when total RNA extracted from virus free Fusarium graminearumwas tested(lane 2). M= pUC9, digested with HaeIII.Fig. S1C: Verification of the absence of sequences without duplication.cDNA of the 3´ termini of segment 2 and 3, respectively, were C-tailed before they were amplified with a pair of polyG-primer and segment specific primer RNA2 fw 2 and RNA 3 coding fw 3, respectively. Amplification of the 3´ terminus of segment 2 (lane 1) and segment 3(lane 2) showed single bands. Amplicons were separated in 1,5 % agarose in TAE buffer and stained with ethidium bromide. M= GeneRuler™ 50 bp ladder (ThermoFisher Scientific).
[1] Darissa O, Willingmann P, Schäfer W, Adam G (2011) A novel double-stranded RNA mycovirus from Fusarium graminearum: nucleic acid sequence and genomic structure. Arch Virol 156: 647-658. doi:10.1007/s00705-010-0904-9
[2] Leach J, Lang BR, Yoder, OC (1982) Methods for selection of mutants and in vitro culture of Cochliobolusheterostrophus. J Gen Microbiol 128:1719–1729. doi:10.1099/00221287-128-8-1719
[3]Rott ME, Jelkmann W (2001) Characterization and detection of several filamentous viruses of cherry: adaption of an alternative cloning method (DOP-PCR) and modification of an RNA extraction protocol. Eur J Plant Pathol 107: 411-420. 10.1023/A:1011264400482
[4] Sambrook J, Russell DW, Janssen K, Argentine J (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A.
[5] Schwach F, Adam G, Heinze C (2004) Expression of a modified nucleocapsid-protein of Tomato spotted wilt virus (TSWV) confers resistance against TSWV and Groundnut ringspot virus (GRSV) by blocking systemic spread. Mol Plant Pathol 5: 309-316. doi: 10.1111/j.1364-3703.2004.00229.x
Supplement 2A
FgVCh9 segment 2
1 GCAAAAAAGA GAATAAAGCC ATTTGTGCAC ATAGGTGCGC GGGGAGAAAC TTTCGAACCG
61 TGGCAAATTT GCACCCTTGT TCTAACATAC ATCATGTCAG GACAATTGAA CATTCGTTCA
121 ACCGCTCATC TTGAGCAACA AGCCGTGGCA CCGGATCAAT ATCGTGCCGG CGCTGTGGCT
181 GGTCTTTGCA GTTACAGTGT CCCAACTCCA AGCATCACAC GCCGTGTGCA CTACAACGCA
241 GGTGTGTCAA CAGTCGAATC TGGCGCAGGC AGCGCTGACA AATTAGAGTC GTTCTACCAC
301 CACCGGATTC CAAGGGATGT GGCGCTGACG TCACTGCGCG CCGGAACAAT TAAGCATGAT
361 GTGGTGACAT TTGATGTGAG TGAGCGGGGA CCGACGCAGC CCCTCACCAA CCAGATTGTT
421 AATGTCACGT ACAAGCAGCA ATACAATTTC ATCGACGTGG GCGTGAGCGC AGCCATAGTC
481 AGAACTGAGC TTCAATTCTT CCCCGTCCGT CCACCTGTTG ATAACACGAA GCTGAACCTA
541 CTTAAGAATG CGCTGACTGT GCATGAAGTG GTGAAGCCTC TCAAACTACA ACTTCCCAAG
601 CTGGTGGCGC AATCGCACGC GTCGCCAGGC CTCCCGTTGT ACAGTGATTT CGAGCCACTG
661 CGGCGCGCGA TCGCGAACCA GGCCATAAAT GATGATTATG GCATGAGATT GTCACACATG
721 GCACGCATTG TGCTACACTC GTTCACTGCA CAAGCAGCCG GTTTATCACA CGCGGCTTCA
781 GATGTCGTGT CGACAGTTGA TGTTAGGACG ATGTTTGTGG CTGAAACGTA CCAGATGAGC
841 GCCGCCGAGT TGCAGCAGCG GGCGTTCGTG TATGTGGATG ATGACATTAG TGAAGAATAC
901 TGGGCCTTCC TGGCAATGGC AATGCTGGGC ATAAACACTA ATGTGGGTAA TACGCGCACC
961 GTATACTCAA GGCTAGTGTC GGTGTCAGAG TTGCGCGGCG GTCAGCAGCT ATACTACGTG
1021 ATGAAAAGCG GGGAGAACAC ACCGGCCAAC ATCAGCCAAA ATGCTTGCGC GTCGGTATTG
1081 AACAATCCTG AGAAATGCCT CTCATTCTAT AACGCCTATG CGCAATCATT AGGTTTAGCA
1141 CAAGAGGCGT CATCAGTCCT GCGCCAAGTT GCGGTGATGC CATTCATGTT TACTAAGAAC
1201 GCTACATTAC CGTACAAAGC ACGCGAGAAC CCCAAAGCTG ACGCATACGT GTACTTGCTC
1261 CGCGAGACTG CTGAGGACAT TCGATTGATG TACAATAGCG TTGAGACTCT GGTATATCAA
1321 GCCCCGATTA TCGCGGGCGC TGTTAAGGCA GGTGTGGCAG CGATATCTAT GTCGTACCGT
1381 GTCAAGAACT CGCTAAATGT GCCCTCCATA CTCGCAGCTA TCAAAGAGAT CCTCGCTGAC
1441 CCTAGTTCCT GCAGGAGCAC AATGTCAGCG GTGTTGTGCT CATACGGTGG TATCACGGCC
1501 GACTTGGAGT GGCTACTACC ATTCACAGAA GGAGTGGATA TAGCCATGGA TGCATGCGTG
1561 CGCGCCTACA GGGATTGCGG GTGGCTTATC ACGACTTACA ACGACTTGTC GTGCCTGGGT
1621 GCTTATGCTC CAGCATTTAG TACGGGTGTG GATATGCGCA ATGCACAATT CGGTTACGAC
1681 GTAGCGAGCA GACCGTATCT CGAGTCACTG ATCGCGCTGT CGACTCAAGG TTTACCGTGG
1741 ACGTTCGAAA ACGAATCTTT GGATGGACCA ACACTGCAAC GTGCCGCCAG GGACGTGGTG
1801 CACTGGGAGG CCGTAATTCA ATGGGATGCT TACTCACCAG GCGAAGGCGT AAGCGTGGCC
1861 CACATAACGC CAACCCCCGT CTCACCGAAA CAGGCCCGGC CTTTCGAGAC AGAGCCAAAC
1921 GCTCATCTGA GCGAACGGTG GCAAAAATCG AAGCGCACGC TGAGCGGTAA CGGCCTTAAA
1981 GCATTGACGC TGAGTCCACC ATCGGCTGCA GCACGCAGCG AAGCAACAAA AACGGCATCA
2041 GTAGCCGAAA CTCTGCGCAC ACACACGCGC TCAATGTCAG TCAAACCCAA CCCGGCACCC
2101 GGAACAAAGC CGCACAATAT GCCATGGCCA GTACAGGCGG CAGCCTCAGT ACCATCTGGC
2161 GAAGTTAGTG ATTCTGAGAG TACAACCACC CGAGTAACGC GGGCGAATTC AGTTGCATCA
2221 ACAGATGACA CGGCCAGTGC TGCTACGGTA GTTCAGACTG CCCCACCCGG CGTGACAAGC
2281 CCGCCGCGTG GCCGGTCCGT TACACCTATA GGTACGACTG TGGATTTGAG CCGTGAACCA
2341 ACGCCATCGC TGGCGGAACG GCTGAAGTCA CCATTCCGTC ACCAACGTGT TAGATCACGC
2401 AGTGTGAGTA GTAGTCGTGG TCGTGCGACG AGCGACACTA ACATGGGCCA AATGCTGGAC
2461 CGTGGCGCAA GTGAGTCGAG TGAGATAGCA CTAATACGGA AGACGAAGCC AAAGGACGTG
2521 GCTGAGCTAG GCGAACATAT TGTCACGAGC GACGGGCGAA TTGAGCTTAT AACGCAATTG
2581 GCGGGTAAAC GTGACTACAG TGATGTAGTG CGGGACCTAT TACCAGGCGG TGTAGGTACC
2641 CCAGACGTTG GCGATTGGAC GGGGACGAGT AAGAATGGTG AACAACGCAA TATCACAACG
2701 CTCATGAATG TGAACAAGTGATTTGGTTCG GAAATGCTTG TATAATTGTA TTACAGTTAT
2761 ACGAGGCCGG ATAGGCAAGC TAGCAGCAGT TCGGTACTAC CGTGGTGTTG CAAACCGCAT
2821 AAGGTATTAA AGTCATGGGA CTTTAATGGCGATAGGCAAG CTAGCAGCAG TTCGGTACTA
2881 CCGTGGTGTT GCAAACCGCA TAAGGTATTA AAGTCATGGG ACTTTAATGG C
FgV2 segment 2
1 GCAAAAAAGA GAAAAAGGAT CTTGACGCAC ATAGGTGCGC GGGGAGAAAC TTTCAAACCG
61 TGGCAAATTT GCACCCTTGT TCTAACATAC ATCATGTCAG GACAATTGAA CATTCGTTCA
121 ACCGCTCATC TTGAGCAACA AGCCGTGGCA CCGGATCAAT ATCGTGCCGG CGCTGTGGCT
181 GGTCTTTGCA GTTACAGTGT CCCAACTCCA AACATCACAC GCCGTGTGCA CTACAACGCA
241 GGTGTGTCAA CAGTCGAATC TGGCGCAGGC AGCGCCGACA AATTAGAGTC GTTCTACCAC
301 CACCGGATTC CAAGGGATGT GGCGCTGACG TCACTGCGCG CCGGAACAAT TAAGCATGAT
361 GTGGTGACAT TTGATGTGAG TGAGCGGGGA CCGACGCAGC CCCTCACCAA CCAGATTGTC
421 AATGTCACGT ACAAGCAGCA ATACAATTTC ATCGACGTGG GCGTGAGCGC AGCTATAGTC
481 AGAACTGAGC TCCAGTTCTT CCCCGTCCGC CCACCAGTTG ATAACACGAA GCTGAACCTA
541 CTTAAGAATG CGCTGACTGT GCATGAAGTG GTGAAGCCTC TCAAACTACA ACTTCCCAAG
601 CTGGTAGCGC AATCGCACGC GTCGCCAGGC GCACAGTTAT ACAGTGATTT CGAGCCATTG
661 CGGCGCGCGA TCGCGAACCA GGCCATAAAT GATGATTATG GCATGAGGTT GTCACACATG
721 GCACGCATTG TGCTACACTC GTTCACCGCA CAAGCAGCCG GTTTATCACA CTCGGCTTCA
781 GATGTCGTGT CGACAGTTGA TGTCAGGACG ATGTTTGTGG CAGAAACGTA CCAGATGAGC
841 GCCGCCGAGT TGCAGCAGCG GGCGTTCGTG TATGTGGATG ATGACATTAG TGAAGAATAC
901 TGGGCCTTCT TGGCGATGGC AATGCTGGGC ATAAACACTA ATGTGGGTAA TACGCGCACC
961 GTATACTCGA GGCTAGTGTC GGTGTCAGAG TTACGCGGCG GTCAACAGCT ATACTACGTG
1021 ATGAAAAGCG GGGAGAACAC ACCGGCCAAT ATCAGCCAAA ATGCTTGCGC GTCGGTGTTG
1081 AACAACCCCG AGAAATGCCT CTCATTCTAT AACGCCTATG CGCAATCATT AGGTTTAGCA
1141 CAAGAGGCGT CATCAGTCCT ACGCCAAGTT GCGGTGATGC CATTCATGTT CACTAAGAAC
1201 GCTACATTAC CGTACAAAGC ACGCGAGAAC CCCAAAGCTG ACGCATACGT GTACTTGCTC
1261 CGTGAGACTG CTGAGGACAT CCGATTGATG TACAACAGCG TTGAGACTCT GGTGTATCAA
1321 GCCCCAATTA TCGCGGGCGC CGTCAAGGCA GGTGTGGCAG CGATATCTAT GTCGTACCGT
1381 GTCAAGAACT CTCTAAATGT GCCCTCCATA CTCGCAGCTA TCAAAGAGAT CCTCGCTGAT
1441 CCTAGTTCCT GCAGGAGCAC AATGTCAGCG GTGTTGTGCT CATACGGTGG TATCACGGCC
1501 GACTTGGAGT GGCTGTTACC ATTCACAGAA GGGGTGGATA TAGCCATGGA TGCATGCGTG
1561 CGCGCTTACA GAGATTGCGG GTGGCTTATC ACCACCTACA ACGACTTGTC GTGCCTGGGT
1621 GCTTATGCTC CAGCATTTAG CACAGGTGTG GATATGCGCA ACGCGCAATT TGGTTACGAC
1681 GTAGCGAGCA GACCGTATCT CGAATCACTG ATCGCGCTGT CGACTCAAGG TTTACCATGG
1741 ACGTTCGAAA ACGAATCTTT GGATGGACCA ACACTGCAAC GTGCTGCTAG GGACGTGGTG
1801 CACTGGGAGG CCGTAATTCA ATGGGATGCT TACTCACCCG GCGAAGGCGT AAGCGTGGCC
1861 CACATAACAC CAACCCCCGT CTCGCCAAAA CAGGCCCGGC CTTTCGAGAC AGAGCCAAAC
1921 GCTCATCTGA GCGAACGGTG GCAAAAATCG AAGCGCACGC TGAGCGGTAA CGGCCTCAAA
1981 GCATTGACGC TGAGTCCACC ATCAGCTGCA GCACGCAGCG AAGCAACGAA AACGGCAACA
2041 GTAGCTGAAA CCCTGCGCAC ACACACGCGC TCGATGTCAG TTAAGCCCAA CCCAGCACCT
2101 GGAACAAAGC CTCACAGTAT GACATGGCCA GTACAGGCGG CAACCTCAGT ACCATCTGGC
2161 GAAGTTAGTG ATTCTGAAAG TACAACCACC CGAGTAACAC GGGCGAATTC AGTTGCATCA
2221 ACAGATGACA CTGCCAGTGC TGCTACGGTA GTTCAGACTG CCCCACCCGG CGTGACAAGC
2281 CCGCCGCGCG GCCGGTCTGT CACACCTATA GGCACGACTG TGGATTTGAG CCGTGAACCA
2341 ACGCCATCGC TGGCGGAACG GCTGAAATCA CCATTCCGTC ACCAACGTGT TAAATCACGC
2401 AGTGTGAGTA GTAGTCGTGG TCGTGCAACG AGCGACACTA ACATGGGCCA AATGCTGGAC
2461 CGTGGCGCAA GTGAGTCGAG TGAGATAGCA CTGATACGGA AGACGAAGCC AAAGGACGTA
2521 GCTGAGCTAG GCGAACATAT TGTCACGAGC GACGGGCGAA TTGAGCTTAT AACGCAATTG
2581 GCGGGTAGAC GTGACTACAG TGATGTAGTG CGGGACCTGT TACCAGGCGG TGTAGGCACC
2641 CCAGACGTTG GTGATTGGAC GGGGACGAGC AAGAATGGTG AACAACGTAA TATCACAACG
2701 CTCATGAATG TGAACAAGTG ATTTGGTTCG AAAATACTTG TGTAATTGTA TTACAGTTAT
2761 ACGAGGCCGG ATAGGCAAGC TAGCAGCAGT TCGGTACTAC CGTGGTGTTG CAAACCGCAT
2821 AAGGTATTAA AGTCATGGGA CTTTAATGGCGTCATGAATG TGAACAAGTG ATTTGGTTCG
2881 AAAATACTTG TGTAATTGTA TTACAGTTAT ACGAGGCCGG ATAGGCAAGC TAGCAGCAGT
2941 TCGGTACTAC CGTGGTGTTG CAAACCGCAT AAGGTATTAA AGTCATGGGA CTTTAATGGC
FgVCh9 segment 3
1 GCAAAAAAGA AAAGAAGGGT AAGAGTACCT CAACCGCACA TAGGTGCGCG GGGAGAAATT
61 TTCAAACCGT GGCAAATTTG CACAAACCAT ATTTGATTTA CAAATATGGC ATCGAACGCA
121 TTGTTCGACA GACGTGGTGG TGGTGAGCAA GGTGTTAGTG GCAACAACGG AAGTTCTGCG
181 GGGCGCACCG TTAAGCCAGC ATTCCAGGGG GATGATTTCT TTGGAATAGC TTCTGCGCTG
241 GAGATGAACC CAAACATGGG TGTAAACGAC ATGATGCTTG TTGGTCGGGA TAAGCTAATG
301 TCGGGGAAGC AGCAAGCAAA CCCCGGCCTA GTGGGATGGC AGCACGATAT GCAGCGCAAC
361 CTGGCCACAG ACGCATCAGC CTGGGTCGTG CGCAAGGAAA TACGAGACCT GGCTGGCCTC
421 CTACCTGATA ACCCCAATGA TGACACAGCG AGACTGGAGA CCGTGGTCAC GTTCACATAC
481 CAGGTCGAAC CCGTCAACCA GCGAGCTGAT CGGTCACACC GACGCGATGT GGCTGTGCAA
541 TACAAAGCTC CTTCAGTGGG CGATGGTACA GTCATCCAGT TCGCTAAATC CACCAAAGAG
601 TTGCACTACA AGGCGAGCGC AGCTACAGCG TCGTCGGCCG CTATAGGGTA TGCTCAGCAG
661 GTGAATTCCT ATTCCCGGAC TGGCATCTCC GGTGACATGG GCCTGGCAAT GGAAGTTGGT
721 CGTGGACCGG ATATGCTGGG TGTGTTCACG GTGATGTGGC TGTTGCAACT TGATGTGGCG
781 GGTGGAGCAC GTGTGGAGCT CGGCATAAAC GCGAATGATA TGCGCGTGCT GGTGCCATCA
841 TCGGTGTCAC CCCTGGATGA AGCTGACGCA ATGAAGGCAG TGCCCGGCAT CAGGCTACAT
901 TCAGACGCCC TCGACCCTCT AGCATACCGG CTGCTATACA CTGCAACGCA CCGGTCAGCC
961 CTTGGCGCCG CATCTCCGTG GGTCAACAGA TATCGCTGGC CAACCACACC GCTAACTGTG
1021 TATAGCGGCG CACTAAACCC GGGACCGTAC ATGCAAGCAT TCAATGGTAG GCCTGCAGCT
1081 GCGCAGCTAG CCGGGCAGAT TGTCAACATT GCGAACACGT ATGACCTGCG TGACACGTGC
1141 AGTCGTGCTT ACAGCATTGC ATTCTCTCTT TACGGTGATC GTTGTGTGAA CAGGAATTTC
1201 CGCCTGCACT GCGGTAGGCA GACACTGTTC CATGAAATCA GTAATGGGCA GAGCAAAGCA
1261 CAGGTGCCTC AAATGTGTTG GGCCCTCGGT AGTTACCAGC TGCCTATGGC GGCAAAGTTC
1321 AATGGGTATG CGAACGCAGA GTACATGCGC GACCTGTGTC GTTCTACGAT AACTGCTGGC
1381 TTGCGCATCA GGGACCGCGA CGCTGTCCGA CGGGCCGTTG ACCAAATCGA TGTGCATGCC
1441 CTCAAAACTG TGCAGCAACG TTTTGAGGAA GGACAGCAAC TAACGGATGC ATTGCCAATA
1501 GACTACAGGC ATTATGTTAA GCATGGCATC CGACGCGCCT GGGTCGTTCG AGGCATCTTG
1561 CACAATCTCT CGCTCGGCCG TGCTGTGCCG GCAGCGATCA GCGGCTCAGC AGTTAAACAA
1621 TTACGCATGC CAAGTGAAGC AGAACTGGCG GACACCGACG TGCTGGCGTA TGGGAAGGAG
1681 CGTGCGGAGT CAGAAGCAAA TGCGTGGCTC ATAAATGAAC TGATACGAAA CGGTGGTACT
1741 AGCTTATGCC AGAGTGTAGA TGAGGCCATA CCACGATCTG GCGCAACAGC AGTGTTGCAC
1801 ACCGATTGCC GTTATTACAT GGGCATGGAT CTACAATACA AGATTGTTGG ATTCGGTAAT
1861 GATATGCGCA TAAAGTACAA CAGGCCGCTC AACTTTGTGT ATGCACCTAC ACCCACAATG
1921 GTAACACACC AGCGTGATGA TCAACTGCGT GAACCAAGTG CGCTACCGAT GACTGTGGTA
1981 GACAAGGAGC TAGCAGATGA CAAAGCTGAC GACACTGTTG TGACCCCGGA AGATATACTG
2041 CGTGAGGTAA TGCAGATGCG CGGCACTCGC ACGTCAACGT GGTTCAGGGG GAAACCACCC
2101 ACAAAAGCGC CGCCGGTTCG GTACCACGAT AGTGACGTTT TGACCGTGAC AGCTGCCCGT
2161 GATGATGTAC GCCAATCTGT GGTCCAACTA GCCCAAGAAG GTGGTGGTCC TGGACCCACC
2221 AGCGGTGAAA ATCTGCTGTG TGGTGCTCAT GCGATACAGA TGGCGCTAGA ATTCGCAGGT
2281 CACCCCAACC ACATAACAGT GGAACAGTTG GAGGATGAGC TGCGCACTGC CGTGCTCGAT
2341 GCACCTGTGG ATGATGACTC GGAATTGCGG AGTGCAAACA ATTTCACGGC AGACCAATTG
2401 GGCAGAGTAG CTAATCGGCA CGGGTATAAT CTGACAGTCG TGACAGGACA AGGCAAAGGT
2461 GCCGCACTGG CATACAGCGC AACAGAGCTG GTCGGTGCAC CCCACCTTGT TGTGTTCAAC
2521 TTCGGAGGCA ATCACTGGGA AGGTCCGGTC CGGCAAGGTA AGCGTCATGC AATACGAGTA
2581 GAAGGTCAAG ATGCAGGTAC GCCAGCGGTA GAAGCGGCAC GGGCCCGGTC CCAGCGTGGC
2641 AGCGTGTTTG AGAAGACCGT GAACCGTCGA CGTTAAATCA CAAACAATAA ATAGTTAATT
2701 GGTATTACCG GTTAGTCTAT TGCCAAATTG GCGCCAGTAG CAACACACTC GCACGCTACG
2761 CCACAGTCTG TACAGCACTG CTGCCCAGAA TGTCGCCAAA TTAGGTATTT AAGGAGAACC
2821 CTTAAATGGCACGAACCGTC GACGTTAAAT CACAAACAAT AAATAGTTAA TTGGTATTAC
2881 CGGTTAGTCT ATTGCCAAAT TGGCGCCAGT AGCAACACAC TCGCACGCTA CGCCACAGTC
2941 TGTACAGCAC TGCTGCCCAG AATGTCGCCA AATTAGGTAT TTAAGGAGAA CCCTTAAATG
3001 GC
FgV2 segment 3
1 GCAAAAAAGA AAAGAAGGGT AAGAGTACCT CAACCGCACA TAGGTGCGCG GGGAGAAATT
61 TTCAAACCGT GGCAAATTTG CACAAACCAT ATTTGATTTA CAAGTATGGC ATCAAACCCA
121 TTGTTCGACA GACGTGGTGG TGGTGAGCAA GGTGTGAGTG GCAACAACGG CAGTTCCATG
181 GGGCGCACCG TTAAGCCAGC ATTCCAGGGG GATGATTTCT TTGGAATAGC CTCTGCACTG
241 GAGATGAACC CAAACATGGG TGTAAACGAC ATGATGCTTG TTGGTCGGGA CAAGCTGATG
301 TCGGGGAAGC AGCAGGCAAA CCCCGGCCTA GTAGGATGGC AGCACGATAT GCAGCGCAAC
361 CTAGCTACAG ACGCATCGGC CTGGGTCGTA CGCAAGGAAA TACGAGATCT AGCTGGCCTT
421 CTACCTGACA ACCCCAACGA TGACACAGCG AGACTAGAGA CCGTGGTCAC GTTCACATAC
481 CAGGTCGAAC CTGTTAACCA GCGGGCTGAT CGATCACACC GCCGCGATGT GGCTGTGCAA
541 TACAAAGCCC CCTCGGTAGG CGATGGTACA GTCATCCAGT TCGCTAAATC CACTAAAGAG
601 TTGCACTACA AGGCGAGCGC AGCTACGGTG TCATCCGCCG CTATAGGGTA TGCTCAGCAG
661 GTGAATTCCT ATTCCCGGAT TGGCATCTCT GGTGACATGG GCCTGGCAAT GGAGGTTGGT
721 CGTGGACCGG ATATGCTGGG TGTGTTCACG GTGATGTGGC TGTTGCAACT CGATGTGGCG
781 GGTGGAGCAC GTGTAGAGCT CGGCATAGAC GCGAATGATA TGCGCGTGCT AGTGCCATCA
841 TCAGTGTCAC CCCTGGACGA AGCTGACGCA ATGAAGGCAG TGCCCGGCAT CAGGCTTCAT
901 TCAGACGCCC TCGACCCTCT AGCATACCGG CTGCTGTACA CTGCGACGCA CCGATCAGCC
961 CTTGGCGCCG CATCTCCGTG GGTCAACAGG TATCGCTGGC CAACCACACC GCTAACTGTG
1021 TATAGCGGCG CACTAAACCC GGGACCGTAT ATGCAAGCAT TCAATGGTAG GCCTGCAGCT
1081 GCACAGCTAG CCGGGCAGAT TGTTAACATT GCGAACACGT ATGACCTGCG TGACACGTGC
1141 AGTCGTGCTT ACAGCATCGC ATTCTCTCTT TACGGTGATC GCTGTGTGAA CAGGAATTTC
1201 CGCCTGCACT GCGGTAGGCA GACACTGTTC CATGAAATCA GTAATGGGCA GAGCAAAGCA
1261 CAGGTGCCTC AAATGTGTTG GGCCCTCGGT AGTTACCAGC TGCCCATGGC GGCAAAGTTC
1321 AACGGGTATG CGAACGCAGA GTACATGCGC GACCTGTGCC GCTCTACGAT AACCGCTGGC
1381 TTGCGCATCA GGGACCGTGA CGCTGTCCGA CGGGCCGTTG ACCAAATCGA TGTGCATGCC
1441 CTCAAAACTG TGCAGCAACG CTTTGAGGAA GGGCAGCAAC TAACGGGTGC GCTACCAATA
1501 GACTACAGGC ATTATGTTAA GCATGGCATC CGACGTGCTT GGGTCGTTCG AGGTATCTTG
1561 CACAACCTCT CACTTGGCCG TGCTGTGCCA GCAGCGATCA GCGGCTCAGC AGTCAAACAA
1621 TTACGCATGC CAAGTGAAGC AGAACTAGCG GACACTGACG TGCTGGCGTA TGGGAAAGAG
1681 CGTGCAGAGT CAGAAGCAAA TGCGTGGCTC ATAAATGAGC TGATACGAAA CGGTGGCACT
1741 AGCTTATGCC AGAGTGTAGA TGAGGCCATA CCACGATCTG GCGCAACCGC GGTGCTACAC
1801 ACCGATTGCC GTTATTACAT GGGCATGGAT CTGCAATACA AGATTGTTGG ATTCGGTAAT
1861 GATATGCGCA TAAAGTACAA CAGGCCGCTC AACTTTGTGT ATGCACCTAC ACCCACAATG
1921 GTAACACACC AGCGTGATGA TCAACTGCGT GAACCAAGTG CACTACCAAT GACTGTGGTG
1981 GACAAGGAGC TAGCAGATGA CAAAGCTGAC GACACTGTTG TGACCCCGGA AGATATACTG
2041 CGTGAGGTAA TGCAGATGCG CGGCACTCGC ACGTCAACGT TATTCAGGGG AAAACCACCC
2101 ACAAAAGCAC CGCCTGTCCG GTACCACGAT GGTGACGTTT TGACTGTGAC CGCCGCCCGT
2161 GATGATGTGC GCCAATCTGT GGTCCAACTA GCCCAGGAAG GTGGTGGTCC GGGACCCACC
2221 AGCGGTGAAA ATCTACTGTG TGGTGCTCAT GCGATACAGA TGGCGCTAGA ATTCGCAGGT
2281 CACCCCAACC ACATAACAGT AGAACAGTTG GAGGATGAGC TGCGCACTGC CGTGCTCGAT
2341 GCACCCGTGG ATGATGACTC GGAATTGCGG AGTGCAAACA ATTTCACGGC AGATCAATTG
2401 GGCAGAGCAG CTAATCGACA CGGGTATAAT CTGACAGTCG TGACAGGGCA AGGCAAAGGT
2461 GCCGCAGTGG CATACAGCGC AACGGAGCTG GTCGGTGCAC CCCACCTTGT TGTGTTCAAC
2521 TTCGGAGGCA ATCACTGGGA AGGTCCAGTC CGGCAGGGTA AGCGTCATGC AATACGAGTA
2581 GACGGTCAAG ATGCAGGTAC GCCAGCGGTG GAAGCGGCAC GAGCCCGGTC CCAGCGTGGC
2641 AGCGTGTTTG AGAAGACCGT GAACCGTCGA CGTTAAATCA TAAACAATAA ATAGTTAATT
2701 GGTATTACCG GTTAATCTAT TGCCAAATTG GCGCCAGTAG CAACACACTC GCAAGCTACG
2761 CCACAGTCTG TACAGCACTG CTGCCCAGAA TGTCGCCAAA TTAGGTATTT AAGGAGAACC
2821 CTTAAATGGC ACTCATGAAT GTGAACAAGT GATTTGGTTC GAAAATACTT GTGTAATTGT
2881 ATTACAGTTA TACGAGGCCG GATAGGCAAG CTAGCAGCAG TTCGGTACTA CCGTGGTGTT
2941 GCAAACCGCA TAAGGTATTA AAGTCCCATG ACTTTAATGG CG
FgVCh9 segment 5
1 GCAAAAAAGA GAAAAAGCCA CAAACCGCAC ATAGGTGCGC GGGGAGAAAT TTTCTAACTG
61 CAGCTAATTA GCATAACATA CCAGCAATTC GCCGATATGT ATTCGCACTC AAGTTCAAGT
121 GCACAACGCG CAGCTGTTTG GGCGCAACAA CAGCGAACCG TGGCCTTTAC GGCCCATCAA
181 TCAAACTTTA CCGACGTGGC ACTGGGCACA CCACGCCATA CCATGGTGGC TCAGCCCCGG
241 AATGGTGTGG CATCTAGGGA AAATCATGCT CAGCAGTGGT CTGATGAGCG GATTCCAACT
301 GGGCCAGGGT ATGACCTGGT TAACCGCTTC GAGATGATTG CAGACCGTGA GCTTGAGCGT
361 GCTGGGTTGG CAGAGCCACG CACAACGCAC TATGGTGAGG GCGCTGCTAG TTCGGCAAGT
421 TCGACCACGA GCGCTAGACA TTCGTTATTT AGCGTGCAAC CTCAAAGCAC CACTGGTTGT
481 GCTTCGAGTA CTAACGGAGA GAGCGACGAT GATGGAGGCT ATGACTACGA GAGCGATAGT
541 AGTTATGACG ATGATGCACG CTCGGCAGGC GATGACACCT TGTATAACAT GCGTGCACAC
601 ACCCAGTACC AGCATGAGCT GCACGAAGAG AACCTGCTTG ATGAGGCAGA TGATTACATC
661 CATTCTGCTG CACTCACCCG GTCCACACCC CCGCTCTGGT TAGCCGACAC CTACCCGATA
721 ACTGAGCCAG CTATTGAAGC CCGGTACGAC GTTTATTACG ATCAGTACCA CAGTGTAACC
781 ACCCCAATTT ATCCAGGACC AGGTGCAACA CTGCACGAAG ATTTGGTGCT TGACGCCCGG
841 CCCTTTGTTT GCCATAAACC CCTGACCCAG CCAGCACCGA TCGCGTCTCG TACTGGGTCA
901 CTTGAGTATC AGCTAGAATT GCAAGTCGCT GCTATGGCAG CGGGGGACAC GGATTTGGTC
961 GTTAACATGA TCAAGCTCGA ACGATCAAGC AGCCCCGCAT CACCACAGGA TCGGCAGTCT
1021 AGGCACGGTG AGAACCAAGT TGCTGAAGCA TCTGGTCACA GACACCCTCT GCCGGACCTG
1081 GCATGCCCGA TTTGCTTCGA GCCACAACGT TCGCGACGTT CACTTGCCGC CCACCGGAGC
1141 TCATCGCACC CTAAACAAGT ATGTGGTCAG GCGCACTGCA GCTACACTAC TGACGTGCCG
1201 GCTGACTTTG CTTTCCATAT GGATGAAGAA CACCCTGTCT TAGGGTGTGA TTCAACAGGC
1261 TGCGATTACA CTACACGCAG CAGCCAACTA CACTGGACTC ATATGGTTGG GCACCTGAGT
1321 TGCACAGTGT GTGGGCTGCT AAGTGCCAAC CTCGCAGCGG CTGCTGATCA CATTCGCGAA
1381 CATGCTGGAA AGTGGTCCTG TTCACTGTGC AACGAGCCCT TCGAGGACGC GTTCAGCCTT
1441 AATGAGCATA ATCAGGATGT GCACTTTACC TGCTGCACTC AATGTGAAGC CATTGTAAGA
1501 GACAAGAACG AACTACTGGA CCACTGCCAT GATGTTCACC CGTACTTACG GTGTCGTGAA
1561 GTAGGCTGCG CGTTCCGCAC ATTTGAACCA CCGGAGCTAG CAGCACACCA TGATTGGCAT
1621 GATCAATACC CATTGTCGTT CTTCGTGGTG AAGTTGCAAT GCCCTGACCT CGGTTGCACT
1681 CATGCGGCAT CAAGTCATGA GATGGCAGCG CTACATTACA ACACTCACTT GCAGAAACCG
1741 AATTACGCGT GTGAAAGTTG TGACGCTTGG TGCCACAGCC ACACTGCCCT AATCAGGCAC
1801 TCCCGGATGC ACAAGCAGGT AGAGCAGAAT CTGCGAGAAC AGCCCGCACT CCAACAAGCA
1861 CCTGCAACGG CGTCTGGCGA TGTGTGCAAA TGTCCACATT GTGCGTTCAC GAGTAAGTCA
1921 AAAGTTGAAT GGCAAGATCA TATCAACATC AATCACCGTT CATCCCAACA TCTAGCATTA
1981 CCTTGTAGCT TATGCGGCAA GGTGTTCACT GGCGTGAATG CGGTCCGGAA ACATCACTGT
2041 CACAAACGTG ATTGGCATGA TAAACCGCAC CGTTGTGGTA AATGTAATGC ACGATTTGGT
2101 CAGTCTCGCG ACCTAGAGCG CCATAAAGCG ACACATGAGG CAGCTAGGCC CCGACACCAA
2161 TGTCAGGTTT GCCACGAAAC ATTCGCGCGG CTGGACCATT TGCAACGCCA CGTTAGACGG
2221 CGGCACTCCC GGATGCACAA GCAGGTAGAG CAGAATCTGC GAGAACAGCC TGCACTCCAA
2281 CAAGCACCTG CAACGGCGTC TGGCGATGTG TACAAATGTC CACATTGTGC GTTCACGAGT
2341 AAGTCAAAAG TTGAATGGCA AGATCATATC AACATCAATC ACCGTTCATC CCAACATCTA
2401 GCATTACCTT GTAGCTTATG CGGCAAGGTG TTCACTGGCG TGAATGCGGT CCGGAAACAT
2461 CACTGTCACA AACGTGATTG GCATGATAAA CCGCACCGTT GTGATAAATG TAATGCACGA
2521 TTTGGTCAGT CTCGCGACCT AGAGCGCCAT AAAGCGACAC ATGAGGCAGA TAGGCCCCGA
2581 CACCAATGTC AGGTTTGCCA CGAAACATTC GCGCGGCTGG ACCATTTGCA ACGCCACGTT
2641 AGACGGCGAC ATACATAATG GCACCACACT GGTACGTTGG CATACTTGTG GTATTACCGC
2701 AGTACTTGCC ACCCTAATTT GGGATGCTGC TAGCATGTAA TGGCACCACA CTGGTACGTT
2761 GGCATACTTG TGGTATTACC GCAGTACTTG CCACCCTAAT TTGGGATTGC TGCTAGCAAT
2821 GCATGAGTAC CAGTAACATA CTGCCGGACA CAACAGCCAA ACACCTGTTT TGGCACCAGG
2881 CAGTATGGTA ATTAATAATT ATTTAGGCTT GGCCTACCCC TAAATGGC
The sequence, which is duplicated, is underlined. The duplications themselves are dotted underlined. Start and stop codons are shown in bold letters. Nucleotides, which connect the duplications or are inserted (T in FgVCh9 segment 5) are shown in boxes.
In segment 3 of FgV2 the underlined sequence is a duplication of the 151 3´ terminal bp of segment 2 of this virus, however with the exception of 4 bp (bold italics) in the extreme end.
The internal duplication in segment 5 of FgV-ch9 is composed of a 5´and 3´ duplicated sequence (underlined), which are both connected by non-homologous nucleotides (shown in boxes).
Supplement 2B
1 MYSHSSSSAQRAAVWAQQQRTVAFTAHQSNFTDVALGTPRHTMVAQP