Astroviruses are non-enveloped, single-stranded, positive-sense RNA viruses. The polyadenylated genomes of these viruses range in size from 6.1 to over 7.7 kb and are arranged in three open reading frames (ORFs 1a, 1b, and 2), as well as a short 5′ untranslated region (UTR) and a 3′UTR [3]. ORF1a and ORF1b encode the non-structural proteins, including several transmembrane (TM) helical motifs, a serine protease, a nuclear localization signal (NLS), and an RNA-dependent RNA polymerase (RdRp). ORF2 encodes the capsid protein that is required for virion formation [6, 11].

Avian astroviruses are members of the genus Avastrovirus, one of the two genera in the family Astroviridae. As of 2011, the International Committee on Taxonomy of Viruses (ICTV) recognized three species of the genus Avastrovirus. These three species are 1) Avastrovirus 1, including turkey astrovirus 1 (TAstV-1); 2) Avastrovirus 2, including avian nephritis virus (ANV) 1 and 2; and 3) Avastrovirus 3, including turkey astrovirus 2 (TAstV-2) and duck astrovirus 1 (DAstV-1) [4, 15]. Within the genus Avastrovirus, there are also a number of unassigned species [9, 12,13,14,15, 18, 19, 21]. Here, we report the complete genomic sequence of a novel avastrovirus of goose-origin (GAstV).

The FLX strain of GAstV was identified in the liver of a 15-day-old goose with enteritis, which was collected in June 2014 from a commercial goose farm in Hunan province, China. RNA was extracted from the liver sample as described previously [12]. The presence of the astrovirus was demonstrated by amplification of a 720 nt ORF1b–2 sequence using a reverse transcription (RT)-PCR assay with primers Picof and Picor. The astrovirus genome was amplified using RT-PCR with new primers based on conserved regions of chicken, turkey and duck astroviruses [9, 10, 12,13,14], as well as specific sequences from the original 720 nt ORF1b–2 FLX RT-PCR. The 5′ and 3′ ends of the genome were derived using 5′ and 3′ rapid amplification of cDNA ends (RACE) strategies. The initial genome sequence was verified by determination of overlapping DNA fragments amplified with additional primers. The primers applied to amplify the genome sequence are shown in Supplementary Table S1. The complete genomic sequence of FLX has been deposited in GenBank under accession number KY271027.

ORFs in the genome were predicted with DNAMAN 5.2.2 (Lynnon). Sequence similarity searches were conducted by BLASTP in GenBank [17]. Pairwise comparisons were performed using CLUSTALW [16]. Genetic distances were computed by the p-dist method of MEGA6 [20], using parameters as described previously [4]. Neighbor-joining trees were constructed by MEGA6, using Jones-Taylor-Thornton matrix-based model and 1000 bootstrap replications [20]. TM domains in ORF1a were predicted using TMHMM [1].

The GAstV FLX genome was 7299 nt in length with three overlapping ORFs (ORF1a, 3283 nt; ORF1b, 1545 nt; and ORF2, 2124 nt) and had a 5′UTR of 22 nt and a 3′UTR of 307 nt (Supplementary Fig. S1). The three ORFs were in different reading frames, as seen in DAstV-1 and duck astrovirus 3 (DAstV-3) [3, 13]. As expected, the overlap region between ORF1a and ORF1b in FLX contained a ribosomal frameshift signal [6, 11], consisting of a heptameric AAAAAAC sequence (nt 3295–3301) and a downstream stem-loop structure (nt 3308–3332). Similar to most avastroviruses [3, 8,9,10, 12,13,14], the conserved CCGAA motif was found at the 5′ end of the genome as well as the 29-nt space between the ORF1b stop codon and the ORF2 start codon. A stem-loop II-like motif (s2m) [7] was detected in the 3′UTR of the genome. The ORF1a and ORF1b amino acid sequences of FLX contained characteristic motifs conserved in other astroviruses [3, 5, 6, 8,9,10,11,12,13,14, 18]. These included motifs typical of: serine proteases (685GNSG688), a NLS (residues 784–815), and a RdRp (264DWTRYD269, 325GNPSG329, 375YGDD378, and 403FGMWVK408). Five TM domains were detected at positions 216–238, 371–388, 401–423, 433–455, and 468–490 of ORF1a.

Amino acid sequences deduced from the three ORFs of FLX by BLAST searches displayed low identity with other astroviruses (ORF1a: <48%; ORF1b: <66%; ORF2: <51%). Low identity at the nucleotide (genome: 51–59%) and amino acid (ORF1a: 27–54%; ORF1b: 52–66%; ORF2: 19–50%) level was also found between FLX and representative members of recognized and unassigned species in the genus Avastrovirus, by pairwise comparisons (Table 1). These analyses suggest that FLX is a novel member of the genus Avastrovirus.

Table 1 The percentage identity between genomic RNA sequences, the amino acids sequences of the three ORFs, and the genetic distances between the ORF2 regions of GAstV FLX and representative members of the genus Avastrovirus
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The avastrovirus species are currently defined based on genetic analysis of the complete capsid amino acid sequences. The species demarcation criteria are that mean genetic distances range from 0.576–0.742 between, and 0.204–0.284 within, species, respectively [4]. To classify FLX, we conducted a genetic analysis of the FLX capsid sequence and those of representative members of the classified avastrovirus species as well as unassigned avastroviruses (Table 1). FLX shared a genetic distance of 0.574–0.719 with three official species, suggesting that it could be classified as a member of a novel species in the genus Avastrovirus. FLX also shared high levels of genetic distance (0.605–0.784) with DAstV-3 [13], duck astrovirus 4 (DAstV-4) [14], chicken astrovirus B (CAstV-B) [9, 19], and northern pintail astrovirus (NpAstV) MPJ1332 [2], indicating FLX represents an additional species to the four unassigned avastroviruses. FLX showed relatively low levels of genetic distance (0.468–0.518) with the duck astrovirus 2 (DAstV-2) [12] and the chicken astrovirus A (CAstV-A) [18, 19], indicating that they are closely related, yet distinct from one another.

To gain further insight into the evolutionary relationship of FLX with other avastroviruses where corresponding sequences are available, we performed phylogenetic analyses based on the amino acid sequence of full-length ORF2. Avastroviruses formed three major groups (groups 1, 2, and 3), consistent with those deduced from analyses of the partial ORF1b sequence and the 5′ region of ORF2 [2]. Group 1 contained most avastroviruses (except ANV) previously identified from poultry, which was divided into eight clades as described previously (12–14, 19, 21). FLX formed a distinct clade within group 1, and was most closely related to the DAstV-2 clade (Fig. 1). Phylogenetic analyses of the amino acid sequences of ORF1a and ORF1b demonstrated that FLX was highly divergent from other avastroviruses (Supplementary Fig. S2).

Fig. 1
figure 1

Phylogenetic analysis of avastroviruses based on the amino acid sequence of full-length ORF2. Numbers on the branches indicate bootstrap percentages obtained using 1000 replicates (only values of 70% and above are shown). A human astrovirus (HAstV) isolate was included as an outgroup. GenBank accession numbers of the sequences are indicated in parentheses. FpAstV, feral pigeon astrovirus; GfAstV, guinea fowl astrovirus; PhAstV, pond heron astrovirus; WpAstV, wood pigeon astrovirus. The virus determined in this study is highlighted in bold

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Taken together, our data on the full-length genomic sequence suggest that FLX can be identified as a member of a novel species in the genus Avastrovirus. Based on the proposal of Chu et al. (2012) [2], the virus belongs to a novel clade in avastrovirus group 1. The present work contributes to our understanding of the molecular epidemiology and ecology of avastroviruses in domestic geese.