Genetic diversity of astroviruses detected in wild aquatic birds in Hong Kong

Abstract

Wild waterfowl serve as a reservoir of some astroviruses. Fecal samples from wild waterfowl collected at Hong Kong's Marshes were tested using pan-astrovirus reverse transcription-PCR. Positive samples underwent subsequent host identification using DNA barcoding. Based on deduced partial sequences, noteworthy samples from three astrovirus groups (mammalian, avian and unclassified astroviruses) were further analyzed by next-generation sequencing. One sample of Avastrovirus 4 clade, MP22-196, had a nearly complete genome identified. The results of ORF2 phylogenetic analysis and genetic distance analysis indicate that Avastrovirus 4 is classified as a distinct subclade within Avastrovirus. MP22-196 has typical astrovirus genome characteristics. The unique characteristics and potential differences of this genome, compared to other avian astrovirus sequences, involve the identification of a modified sgRNA sequence situated near the ORF2 start codon, which precedes the ORF1b stop codon. Additionally, the 3' UTR of MP22-196 is shorter than other avian astroviruses. This study expands our understanding of the Avastrovirus 4 clade.

Keywords: Aquatic bird; Astrovirus; Avastrovirus.

Fig. 1

Phylogenetic analysis of MP22-196 and representative astrovirus strains of each clade based on A full genome sequences, B ORF1a, C ORF1b, and D ORF2 region using IQ‐TREE by maximum likelihood. The trees are rooted by the Mamastrovirus clade. The branch values are bootstrap supports (%) with 1000 replicates as statistical support. MP22-196 detected from this study are highlighted in red color. Representative astrovirus references (n = 41) of different clades with GenBank accession numbers are shown. According to the ICTV taxonomic classification, Mamastrovirus 1 to 19 are selected as references in the Mamastrovirus clade. The Avastrovirus strains selected include one reference from Avastrovirus 1, six references from Avastrovirus 2, four references from Avastrovirus 3, and one reference from Avastrovirus 5. Two representative unclassified Astrovirus references are also selected. It is worth noting that some references have only partial sequences so they are not included in some phylogenetic trees

Fig. 2

Schematic representation of the near-complete genome organization of MP22-196. The line at the upper indicated the nucleotide (nt) length and the dashed line stated the location of 5' UTR, 3' UTR, and the starting and final nucleotide for ORF. The total nucleotide length (upper rectangles), different conserved domain hits with nucleotide location, accession number (Cdd), and E-value (below rectangles) are shown in each ORF. TEEEY-like motif and ribosomal frame-shift heptameric signal are stated at ORF1a. Putative sgRNA promoter is indicated between the end of ORF1b and the start of ORF2

Fig. 3

A Prediction of putative viral protein associated with the genome (VPg) domain by FoldIndex and indicated TEEEY-like motif (at 681 to 685). B Prediction of transmembrane helices in proteins by TMHMM and stated as the peak with purple color

Fig. 4

A The predicted secondary structure of ribosomal frame-shift at the ORF1a 3′end. The stop codon of the ORF1a is shown represented by a blue rectangle. B The predicted secondary structure of stem-loop 2 motifs (s2m) at 3′UTR

Fig. 5

Alignment of the partial nucleotide sequences between the ORF1b and ORF2 of MP22-196 and other Avastrovirus strains. Prediction of putative sgRNA promoter indicated in the figure. The sequence variation is highlighted in color. The start codon of the ORF2 and the stop codon of the ORF1b are represented by red and blue rectangles, respectively