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. 2018 Aug 7;8(1):11837.
doi: 10.1038/s41598-018-30217-y.

Next-generation amplicon sequencing identifies genetically diverse human astroviruses, including recombinant strains, in environmental waters

Akihiko Hata  1   2 , Masaaki Kitajima  3 , Eiji Haramoto  4 , Suntae Lee  5 , Masaru Ihara  5 , Charles P Gerba  6 , Hiroaki Tanaka  5
Affiliations

Next-generation amplicon sequencing identifies genetically diverse human astroviruses, including recombinant strains, in environmental waters

Akihiko Hata et al. Sci Rep. .

Abstract

Human astroviruses are associated with gastroenteritis and known to contaminate water environments. Three different genetic clades of astroviruses are known to infect humans and each clade consists of diverse strains. This study aimed to determine the occurrence and genetic diversity of astrovirus strains in water samples in different geographical locations, i.e., influent and effluent wastewater samples (n = 24 each) in Arizona, U.S., and groundwater (n = 37) and river water (n = 14) samples collected in the Kathmandu Valley, Nepal, using next-generation amplicon sequencing. Astrovirus strains including rare types (types 6 and 7 classical human astroviruses), emerging type (type 5 VA-astroviruses), and putative recombinants were identified. Feline astrovirus strains were collaterally identified and recombination between human and feline astroviruses was suggested. Classical- and VA-astroviruses seemed to be prevalent during cooler months, while MLB-astroviruses were identified only during warmer months. This study demonstrated the effectiveness of next-generation amplicon sequencing for identification and characterization of genetically diverse astrovirus strains in environmental water.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Simplot (left) and bootscan (right) analysis of CAstV OTUs, including FAstV-like OTUs, identified from U.S. samples. OTUs suggested to be recombinants by the phylogenetic analysis (Fig. S1) were subjected to the analysis here. Representative OTUs resulted in 100% bootstrap values among 100 replicates with two different reference strains at the 5′ and 3′ end in the bootscan analysis are shown here. Results obtained from other OTUs are shown in Fig. S2 in the Supplementary Information. Names of OTUs used as a query sequence in each analysis are shown on upper side of the figures by the following manner, “Origin of the sample (- “type of wastewater”)” - “year or month of sample collection”- “OTU number”. A” and “B” indicate places of WWTP and “in” and “out” indicate influent and treated effluent, respectively. GenBank accession numbers of referential strains are as follows, CAstV type 1: L23513; -2: L13745; -3: AF141381; -4: DQ344027; -5: DQ028633; -6: GQ495608; -7: AF248738; -8: AF260508; and FAstV: KM017742. Window size and step size for each analysis were set as 100 and 20 nt, respectively. Nucleotide position shown on x-axis corresponds to CAstV type 1 strain Oxford (GenBank accession number: L23513). Arrows and lines indicate locations of ORF1b and ORF2.
Figure 2
Figure 2
Simplot (left) and bootscan (right) analysis of MLB-AstV OTUs identified from Nepal samples. OTUs suggested to be recombinants by the phylogenetic analysis (Fig. S1) were subjected to the analysis here. Representative OTUs resulted in 100% bootstrap values among 100 replicates with two different reference strains at the 5′ and 3′ end in the bootscan analysis are shown here. Results obtained from other OTUs are shown in Fig. S2 in the Supplementary Information. Names of OTUs used as a query sequence in each analysis are shown on upper side of the figures by the following manner, “Origin of the sample (- “type of wastewater”)” - “year or month of sample collection”- “OTU number”. GenBank accession numbers of referential strains are as follows, MLB-AstV type 1: FJ222451;-2: JF742759; and-3: JX857870. Window size and step size for each analysis were set as 100 and 20 nt, respectively. Nucleotide position shown on x-axis corresponds to MLB-AstV type1 (GenBank accession number: FJ222451). Arrows and lines indicate locations of ORF1b and ORF2.
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