Supplementary Table S1Primers used for amplification of the genomesequence of GAstV FLX
Primera / Sequence (5′→3′) / Positionb / Size of PCR
product (bp)
Picfc / GCGGAGTGCTGYYCCCCAAG / –
Picrc / AGTTGTTGGTTTTGTGGTAGAGGAG / – / 720
5′f / ATAAGAATGCGGCCGCA27 / – / 367
367r / GCGAAAGACTTTTGCTTGC / 367–349
434r / TCACAAGGCGGGTTTTTTCC / 434–415
101f / GGCGTYTTYHASTTCCARGG / 101–120 / 402d
415f / GGAAAAAACCCGCCTTGTGA / 415–434 / 1048
1462r / CGACCAGCCAATAAAAGTGA / 1462–1443
1379f / GCWTTYTWTTGGARTGADGGRHY / 1379–1401 / 561
1939r / AAGTGGTTTTACCATCTGTAGCTC / 1939–1916
1674f / CAGDCAAGAGGNRTHATACCATC / 1674–1695 / 684
2357r / GCATCAGCCAGAAATCATCATA / 2357–2336
2060f / TTTRAYACMMARTTTGGRAAYAGTGG / 2060–2085 / 3111
5170r / TGTCTTGACAAGGAATCATCAGG / 5170–5148
5367f / GCGACTGCAGAAAGCCCTGATAC / 5367–5389 / 1933
3′r1 / CTACACAGTCGGGT21 / –
1f / CCGAAAGCGTTGGTGAGAGGC / 1–21 / 938
938r / TCTTCCGGGGATTAAAAGCTGA / 938–917
841f / CGTGCCAGTTCCTACCTACG / 841–860 / 1255
2095r / GTAAGGGGCACCACTATTGC / 2095–2076
1781f / ACTGCAGGACATGTTGTTAGAGG / 1781–1803 / 967
2747r / TGTGCAGTAGTGCCTGTTGTATG / 2747–2725
2653f / GATTGACCGCAAGATTGAAGAAG / 2653–2675 / 1343
3995r / TGTGCAGCATGGTCTTCTGTT / 3995–3975
3915f / AGTTTTTACACGTATTGGAGCAT / 3915–3937 / 1353
5267r / CCATAGACCATATTGGCTCGCTC / 5267–5245
5132f / TCGTGTCCTTGGAAATCCTGATG / 5132–5154 / 1350
6481r / GTGAACCCTTCACAAGATGTAAATG / 6481–6457
6370f / TTTTCCAGAGCACACACAC / 6370–6388 / 930
3′r2 / GACTCGAGTCGACATCGAT17 / –
af: forward primer; r: reverse primer. Roman: primers applied to determine the initial genome sequence.Italics: primers applied to confirm the genome sequence.
bPositions in the FLX genome.
cPrimers originally designed to amplify the 5′UTR–VP0 sequence of picornaviruses. The reverse primer was hybridized to the sequences from nt 4944 to 4968 and nt 5639 to 5663 of the FLX genome, resulting in a fragment of approximately 720 nucleotides.
dProductamplified by using a thermal asymmetric interlaced (tail)-PCR [4] with primers 101f, 1462r, 1939r and 2357r.

Supplementary Fig.S1 Genomic organization of GAstVFLX. The translation start sites of ORF1a and ORF2 are indicated by black triangles. The numbers represent the nucleotide positions in the genome.*, ribosomal frameshifting signal;TM, transmembranedemain; PRO, serine protease; NLS, nuclear localization signal (NLS); RdRp, RNA-dependent RNA polymerase;s2m,stem-loop II-like motif.

Supplementary Fig.S2Neighbour-Joining trees based on alignments of the full-length ORF1a (A) and ORF1b (B) amino acid sequences of astroviruses. The avastrovirus clades are in agreement with previous reports (1–3, 5,6) and are shown on the right side of the trees. The GenBank accession numbers of theastrovirus sequences are indicated in parentheses. The virus determined in this study is highlighted in bold. The mamastroviruses used for this comparison included astrovirus ML1 (AstV ML1), astrovirus ML2 (AstV ML2), astrovirus VA1 (AstV VA1), feline astrovirus 2 (FAstV-2), human astrovirus 1 (HAstV-1), mink astrovirus (MAstV), ovine astrovirus (OAstV), and porcine astrovirus 3 (PAstV-3).

References

  1. Liao Q, Liu N, Wang X, Wang F, Zhang D (2015) Genetic characterization of a novel astrovirus in Pekin ducks. Infect Genet Evol 32:60–67
  2. Liu N, Wang F, Zhang D (2014) Complete sequence of a novel duck astrovirus. Arch Virol 159:2823–2827
  3. Liu N, Wang F, Shi J, Zheng L, Wang X, Zhang D (2014) Molecular characterization of a duck hepatitis virus 3-like astrovirus. Vet Microbiol 170:39–47
  4. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics25:674–681
  5. Smyth VJ, Todd D, Trudgett J, Lee A, Welsh MD (2012) Capsid protein sequence diversity of chicken astrovirus. Avian Pathol 41:151–159
  6. Todd D, Smyth VJ, Ball NW, Donnelly BM, Wylie M, Knowles NJ, Adair BM (2009) Identification of chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses. Avian Pathol 38:21–30