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Genetic diversity and intergenogroup recombination events of sapoviruses detected from feces of pigs in Japan
Infection, Genetics and Evolution 55 (2017) 209–217
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Research paper
Genetic diversity and intergenogroup recombination events of sapoviruses detected from feces of pigs in Japan
MARK
Moegi Kurodaa,1, Tsuneyuki Masudaa,1, Mika Itob, Yuki Naoic, Yen Hai Doand, Kei Hagad,e, Shinobu Tsuchiakac, Mai Kishimotoc, Kaori Sanoc, Tsutomu Omatsuc, Yukie Katayamac, Mami Obac, Hiroshi Aokif, Toru Ichimarug, Fujiko Sunagah, Itsuro Mukonob, Hiroshi Yamasatoa, Junsuke Shiraic, Kazuhiko Katayamad,e, Tetsuya Mizutanic, Tomoichiro Okad,⁎, Makoto Nagaic,i,⁎⁎ a
Kurayoshi Livestock Hygiene Service Center, Kurayoshi, Tottori 683-0017, Japan Ishikawa Nanbu Livestock Hygiene Service Center, Kanazawa, Ishikawa 920–3101, Japan Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan d Department of Virology II, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan e Laboratory of Viral Infection I, Kitasato Institute for Life Sciences, Graduate School of Infection Control Sciences, Minato, Tokyo 108-8641, Japan f Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan g Department of Health and Medical Sciences, Ishikawa Prefectural Nursing University, Kahoku, Ishikawa 929-1210, Japan h Laboratory of Infectious Diseases, Azabu University, Sagamihara, Kanagawa 252-5201, Japan i Department of Bioproduction Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Genogroup Japan Pig Recombination Sapovirus
Sapoviruses (SaV) are enteric viruses infecting humans and animals. SaVs are highly diverse and are divided into multiple genogroups based on structural protein (VP1) sequences. SaVs detected from pigs belong to eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI), but little is known about the SaV genogroup distribution in the Japanese pig population. In the present study, 26 nearly complete genome (> 6000 nucleotide: nt) and three partial sequences (2429 nt, 4364 nt, and 4419 nt in length, including the entire VP1 coding region) of SaV were obtained from one diarrheic and 15 non-diarrheic porcine feces in Japan via a metagenomics approach. Phylogenetic analysis of the complete VP1 amino acid sequence (aa) revealed that 29 porcine SaVs were classified into seven genogroups; GIII (11 strains), GV (1 strain), GVI (3 strains), GVII (6 strains), GVIII (1 strain), GX (3 strains), and GXI (4 strains). This manuscript presents the first nearly complete genome sequences of GX and GXI, and demonstrates novel intergenogroup recombination events.
1. Introduction Sapoviruses (SaVs) are etiological agents of gastroenteritis in humans and animals and they have non-enveloped positive-sense, singlestranded RNA genome of approximately 7.1–7.7 kb in length with the typical morphology, the Star-of-David structure, as determined by electron microscopy (Oka et al., 2015; Saif et al., 1980). The SaV genome contains two overlapping open reading frames (ORFs) that encode the nonstructural proteins NS1-NS2-NS3 (putative NTPase)NS4-NS5 (genome linked viral protein: VPg)-NS6 (Protease: Pro)-NS7 (RNA dependent RNA polymerase: RdRp) followed by the capsid protein, VP1, and the minor structural protein, VP2 (Oka et al., 2015).
Porcine SaV was first identified by electron microscopy in the United States of America in 1980 (Saif et al., 1980). SaVs have since been identified mostly using reverse transcriptase (RT)-PCR. The metagenomics approach, which does not require sequence-specific primers for PCR amplification, has recently allowed the detection of SaVs. SaVs have been reported from diarrheic and asymptomatic pigs (Cunha et al., 2010; das Merces Hernandez et al., 2014; Di Bartolo et al., 2014; Dufkova et al., 2013; Jeong et al., 2007; Keum et al., 2009; Kim et al., 2006; Liu et al., 2012; Liu et al., 2014; Martella et al., 2008; Martínez et al., 2006; Mauroy et al., 2008; Mijovski et al., 2010; Reuter et al., 2010; Scheuer et al., 2013; Valente et al., 2016; Wang et al., 2006; Zhang et al., 2014; Zhang et al., 2008). Cell culture-adapted and wild-
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Corresponding author. Correspondence to: M. Nagai, Department of Bioproduction Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. E-mail addresses: [email protected] (T. Oka), [email protected], [email protected] (M. Nagai). 1 M. Kuroda and T. Masuda contribute equally. ⁎⁎
http://dx.doi.org/10.1016/j.meegid.2017.09.013 Received 4 April 2017; Received in revised form 11 September 2017; Accepted 13 September 2017 Available online 18 September 2017 1567-1348/ © 2017 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 55 (2017) 209–217 Porcine astrovirus, Picobirnavirus, Posavirus Porcine astrovirus, Porcine enterovirus, Sapelovirus, Porcine rotavirus C Porcine astrovirus, Picobirnavirus Porcine astrovirus, Porcine enterovirus, Porcine picornavirus Japan Porcine astrovirus, Porcine enterovirus, Porcine picornavirus Japan Porcine astrovirus, Posavirus, Techovirus, Picobirnavirus Porcine astrovirus, Sapelovirus, Posavirus, Picobirnavirus Porcine enterovirus, Porcine kobuvirus Teschovirus, Porcine enterovirus, Posavirus, Porcine rotavirus A Teschovirus, Porcine enterovirus, Picobirnavirus, Porcine kobuvirus, Porcine rotavirus A, Porcine rotavirus C Porcine enterovirus, Porcine kobuvirus, Porcine rotavirus C Porcine astrovirus, Sapelovirus, Porcine enterovirus, Porcine rotavirus A, Porcine rotavirus C Porcine astrovirus, Sapelovirus, Porcine rotavirus B, Porcine rotavirus C Porcine astrovirus, Picobirnavirus Porcine enterovirus, Picobirnavirus Porcine astrovirus, Porcine enterovirus, Porcine rotavirus B GV GVII GVII, GXI GIII, GVII GVI GIII, GVII (2), GVIII GIII, GVII GIII GIII, GVI, GX, GXI GVI
GIII (2) GIII GX GIII, GX
2.1. Fecal samples, RNA extraction, and deep sequencing
Without Without Without Without
diarrhea diarrhea diarrhea diarrhea
2 1 1 2
In total, 105 fecal samples collected from 2 to 120-day-old pigs from 12 farms in Tottori Prefecture and Ishikawa Prefecture of Japan in 2015–2016 were used in this study. Samples were collected from pigs with diarrhea (six single samples and 17 pooled samples) or those without diarrhea (70 single samples and 12 pooled samples). Although pooled samples were impossible for recombination analysis, we accepted those samples for search for new genogroups only. Samples were diluted 1:9 (w/v) in sterile phosphate-buffered saline and centrifuged at 10,000 × g for 10 min. The supernatants were collected and stored at − 80 °C until required. Viral RNA was extracted from the fecal sample supernatants, using TRIzol® LS Reagent (Life Technologies, Carlsbad, CA, USA), and was treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) as described previously (Nagai et al., 2015). Deep sequencing was conducted using the MiSeq Reagent Kit v2 (300cycles) (Illumina, San Diego, CA, USA) on a MiSeq bench-top sequencer (Illumina). FASTQ-formatted sequence data files were generated using the MiSeq Reporter (Illumina). Trimmed paired-end sequence reads were assembled into contigs by de novo assembly with the strictest parameter setting (mismatch cost, 2; insertion cost, 3; deletion cost, 3; length function, 0.9; and similarity function, 0.9) in the CLC Genomics Workbench 7.5.5 (CLC bio, Aarhus, Denmark). The nucleotide sequences of SaV strains obtained in this study were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers LC215874–LC215902.
2016.12.2 2016.12.2 2016.12.2 2016.12.2 Tottori Tottori Tottori Tottori
2 months 2 months 2 months 2 months
GIII GIII, GXI (2) 1 3 Without diarrhea Without diarrhea 2016.11.30 2016.12.2 Ishikawa Tottori
16 2 months
2015.7.1 2015.7.2 2015.7.2 2015.7.2 2015.7.15 2015.11.6 2015.11.6 2015.11.5 2016.11.25 2016.11.25 Tottori Tottori Tottori Tottori Tottori Ishikawa Ishikawa Ishikawa Ishikawa Ishikawa
2 months 2 months 2 months 2 months 2 months 54 30 16 11 11
Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Mild diarrhea Without diarrhea Without diarrhea
1 1 2 2 1 4 2 1 4 1
Co-infection with other viruses Health status Collection date Region
Days (months)
type SaVs were shown to induce diarrhea in experimentally infected gnotobiotic piglets (Flynn et al., 1988; Guo et al., 2001; Lu et al., 2015). Therefore, SaVs are currently suggested as one of the etiological agents of gastroenteritis in pigs; however, the role of SaV as the cause of gastroenteritis in pigs has been obscure. Initially, Farkas et al. classified human SaVs into GI-GV using entire VP1 sequences (Farkas et al., 2004). Thereafter, Scheuer et al. classified SaVs including those from humans and animals into 14 genogroups based on the complete VP1 sequences (Scheuer et al., 2013). Thus far, SaVs are divided into fifteen genogroups (Oka et al., 2016). To date, at least eight SaV genogroups: GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI, have been identified from pigs (Scheuer et al., 2013; Oka et al., 2016). In Japan, GIII, GV, GVII, GVIII, and GX genogroups of SaV have been found from finisher pigs and pigs, whose ages were < 5 months (Nakamura et al., 2010; Yin et al., 2006). Complete or nearly complete genome sequences of SaV GIII, GV, GVI, GVII, and GVIII are available in the DDBJ/EMBL/GenBank database, but those of GIX, X, and GXI are not available. In the present study, 26 nearly complete genome sequences and three partial sequences of SaV were obtained from fecal samples of pigs with or without diarrhea in Japan using a metagenomics approach. This study presents the first report of nearly complete genome data for SaV GX and GXI. Genetic analyses revealed high genetic diversity among SaVs in Japanese pig population and possible intergenogroup recombination events. 2. Materials and methods
Sample status
Single Single Single Single Single Pooled Pooled Pooled Single Single
Single Single
Single Single Single Single
HkKa2-1/2015 HgTa2-1/2015 HgTa2-2/2015 MoI2-1/2015 HgOg2-4/2015 Ishi-Im1/2015 Ishi-Im3/2015 Ishi-Kah3/2015 Ishi-Im7/2016 Ishi-Im9/2016
Ishi-Kah6/2016 HgYa1/2016
HgYa2/2016 HgTa1/2016 HgTa2/2016 HgTa3/2016
2.2. Genome analysis
Sample name
Table 1 Summary of samples used in this study and a number of sapovirus contigs identified in each sample.
Number of sapovirus contigs
Sapovirus genogroup
M. Kuroda et al.
Nucleotide (nt) and amino acid (aa) sequences were aligned using ClustalW (Thompson et al., 1997). Phylogenetic trees were constructed using the 29 SaV strains in this study together with available SaV sequences from the DDBJ/EMBL/GenBank database following the maximum-likelihood method with the best-fit evolutionary models in MEGA 5.22 (Tamura et al., 2011) for rtREV + G + I (RdRp), rtREV + G + I + F (VP1 aa), GTR + G + I (VP1 nt), and rtREV + G + F (VP2). The reliability of the phylogenetic tree obtained for each gene 210
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Table 2 Summary of genomic information of sapoviruses obtained from deep sequencing in this study. Strains
Geno group
MoI2-1-1/2015 Ishi-Im1-3/2015 Ishi-Im3-2/2015 Ishi-Kah3/2015 Ishi-Im7-1/2016 Ishi-Kah6/2016 HgYa1-1/2016 HgYa2-1/2016 HgYa2-2/2016 HgTa1/2016 HgTa3-1/2016 HkKa2-1/2015 HgOg2-4/2015 Ishi-Im7-2/2016 Ishi-Im9/2016 HgTa2-1/2015 HgTa2-2-1/2015 MoI2-1-2/2015 Ishi-Im1-2/2015 Ishi-Im1-4/2015 Ishi-Im3-1/2015 Ishi-Im1-1/2015 HgTa2/2016 HgTa3-2/2016 Ishi-Im7-4/2016 HgTa2-2-2/2015 Ishi-Im7-3/2016 HgYa1-2/2016 HgYa1-3/2016
GIII GIII GIII GIII GIII GIII GIII GIII GIII GIII GIII GV GVI GVI GVI GVII GVII GVII GVII GVII GVII GVIII GX GX GX GXI GXI GXI GXI
Reads and sequences obtained from deep sequencing
DDBJ accession no.
Total reads
Sapovurs reads
Sapovirus reads %
Contig length (excluding poly-A)
2,804,452 1,809,416 3,041,700 2,648,440 1,554,736 536,200 1,083,038 1,341,368 1,341,368 1,405,590 1,074,082 1,128,042 399,976 1,554,736 1,759,264 685,940 862,726 2,804,452 1,809,416 1,809,416 3,041,700 1,809,416 750,634 1,074,082 1,554,736 862,726 1,554,736 1,083,038 1,083,038
9884 1404 842 1496 25,179 40,983 923 40,504 4085 14,962 2683 4412 2419 955 757 745 2382 5022 1227 1241 4265 6092 991 690 108 870 5980 338 449
0.35 0.08 0.03 0.06 1.6 7.6 0.09 3.0 0.30 1.1 0.25 0.39 0.60 0.06 0.04 0.11 0.28 0.18 0.07 0.07 0.14 0.34 0.13 0.06 0.01 0.10 0.38 0.03 0.04
6882 7151 6958 6902 7330 7333 6011 7326 7264 7305 7158 7485 7162 6344 7055 7059 7069 6786 7133 7130 7139 7449 7110 6995 2429 7162 7127 4364 4419
LC215874 LC215875 LC215876 LC215877 LC215878 LC215879 LC215880 LC215881 LC215882 LC215883 LC215884 LC215885 LC215886 LC215887 LC215888 LC215889 LC215890 LC215891 LC215892 LC215893 LC215894 LC215895 LC215896 LC215897 LC215900 LC215898 LC215899 LC215901 LC215902
Table 3 Pairwise amino acid sequence identities of putative RdRp (lower left) and VP2 (upper right) between genotype of sapovirus.
GI
GI 72.9-77.7 81.5-87.5
GII
GIII
GIV
GV
GVI
GVII
GVIII
GIX
GX
GXI
GXII
GXIII
GXIV
41.5-43.9
26.3-30.9
53.6
41.3-49.7
24.9-28.8
24.3-29.4
43.0-53.5
27.1-29.4
20.9-25.0
24.3-27.3
18.7-23.1
31.0-33.9
17.3-19.0 16.8-18.4
78.6
GII
71.7-74.6
25.0-28.6
33.9-38.2
32.5-39.4
26.4-28.2
23.6-28.2
39.0-45.3
22.4-23.6
24.1-27.2
21.3-23.1
20.9-23.4
41.9-46.2
GIII
56.0-59.1
55.9-57.8
76.7-100 84.7-100
26.4-29.7
24.0-29.7
20.0-25.1
20.6-28.6
24.9-29.1
18.8-29.1
20.6-24.6
18.8-23.4
14.2-20.1
20.6-23.4
21.8-24.0
GIV
71.5-74.4
93.5-93.9
55.9-57.4
46.2-52.1
22.6
23.2-25.4
46.5-48.3
25.4-29.4
22.6
25.4-28.3
18.6-19.2
29.8
21.2-21.8
GV
63.5-68.4
63.4-68.8
52.2-56.1
63.4-69.0
57.6-74.4 62.5-92.4
19.2-28.3
19.8-29.6
43.4-53.5
21.5-29.7
19.8-26.7
21.5-30.1
14.1-18.6
24.6-28.7
15.6-20.7
GVI
36.6-37.7
37.7-38.1
37.4-38.5
38.1-38.3
34.1-35.8
85.8-99.4 93.4-100
49.7-55.6
21.3-25.4
53.3-58.6
50.3-53.3
49.1-55.0
15.8-20.5
18.1-19.2
18.4-20.1
GVII
33.5-36.0
34.6-37.0
34.4-36.8
35.2-37.4
32.7-35.6
62.4-65.5
63.4-100 62.5-100
23.1-26.4
46.2-58.0
49.1-55.6
48.5-59.8
18.1-28.1
19.8-27.1
18.4-20.1
GVIII
60.0-62.2
62.3-63.9
55.1-57.0
62.3-63.5
62.1-67.0
36.5-38.3
34.6-36.9
55.2-95.9 74.0-98.8
24.7-27.1
19.5-22.6
21.8-26.6
18.0-21.7
26.0-34.7
17.9-21.8
50.3-56.7
54.4-58.6
22.0-24.6
19.2-21.5
17.9-19.0
47.0-55.0
19.6-22.8
20.9-22.0
19.0-20.7
75.7-91.7 67.1-98.8
19.4-24.0
21.5-24.3
19.6-21.2
19.0-20.1
7.3-8.4
98.8
99.4 99.6
59.8
GIX
33.9-35.6
37.2-37.4
36.4-37.2
37.2-37.4
33.7-35.8
64.5-65.7
65.5-71.7
36.1-36.3
GX
34.1-35.6
37.0-37.7
35.4-37.2
37.0-37.4
33.9-36.0
63.9-65.3
64.3-72.5
35.5-36.7
92.8-96.8
GXI
34.6-36.6
36.2-37.9
35.4-37.7
36.8-37.6
32.3-35.6
62.3-65.9
63.5-82.9
35.3-37.5
68.5-74.1
-
76.7 94.4 67.9-74.5
85.3
GXII
57.4-59.0
59.1-59.5
57.6-59.2
59.1-59.5
55.3-58.3
37.4-38.0
35.3-36.4
55.8-58.5
35.9-36.2
35.5-36.1
35.9-36.6
GXIII
66.0-68.4
64.8-65.2
53.2-54.8
64.7-64.8
60.2-65.6
36.0-36.8
33.1-34.4
58.5-60.4
34.4
34.2-34.6
34.4-36.2
55.5-55.6
GXIV
49.4-52.3
52.3
48.5-49.5
51.7-52.1
48.6-49.8
35.7-36.1
32.6-33.9
49.4-50.0
33.9
34.1-34.3
35.1-36.2
45.3-45.7
97.7
49.6
14.5 100 100
in length (26 contigs) and contigs of 2429 nt, 4364 nt, and 4419 nt length including the entire VP1 coding sequence with sufficient coverage (more than three coverage of sequence reads) were identified from one pooled sample with diarrhea, and 2 pooled and 13 single samples without diarrhea (Tables 1 and 2). These samples apart from pooled samples also contained two to six contigs that showed similarities with porcine Astrovirus (9 samples), picobirnavirus (5 samples), posavirus (2 samples), porcine enterovirus (9 samples), sapelovirus (3 samples), porcine picornavirus Japan (2 samples), techovirus (2 samples), porcine kobuvirus (2 samples), porcine rotavirus A (3 samples), porcine rotavirus B (2 samples), and porcine rotavirus C (5 samples) (Table 1). Of the 29 strains, based on complete VP1 sequence analysis,
segment was evaluated by running 1000 bootstrap replicates (Felsenstein, 1985). Pairwise sequence identity calculation was performed on the sequences using the CLC Genomics Workbench (CLC bio). Similarity plot analysis was performed using SimPlot software v. 3.5.1 (Lole et al., 1999). 3. Results 3.1. Identification of nearly complete and partial genome sequences of SaVs Using the previously described metagenomics approach and BLAST search, in total, 29 SaV-like contigs that were longer than about 6000 nt 211
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Fig. 1. Phylogenetic analyses based on amino acid sequences of the full VP1 coding region (approximately 500 aa in length) of 29 SaVs detected in this study with reference strains from DDBJ/EMBL/ GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study.
3.2. Genome analyses of coding regions of the SaV GX and GXI genogroups
11, 1, 3, 6, 1, 3, and 4 strains fell within the GIII, GV, GVI, GVII, GVIII, GX, and GXI genogroups, respectively (Table 2). The strain names and information of the SaV-like contigs obtained in this study are summarized in Table 2. Six single samples showed more than two genogroups (Table 1).
In the putative NTPase and VPg region, three conserved aa motifs, GXPGXGKT, PL(N/D)CD, and WDE(F/Y)D in NTPase, and two conserved aa motifs, KGKXX and XDEYXX in VPg were observed in the GX 212
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Fig. 2. Phylogenetic analyses based on amino acid sequences of VP2 coding region (approximately 165 aa in length) of 27 SaVs detected in this study with reference strains from DDBJ/EMBL/GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study.
strains, where this aa motif was WKDL.
strains (GPPGIGKT, PLNCD, WDEYD, KGKNK, and DDEYTE) and GXI strains (GPPGIGKT, PLNCD, WDEFD, KGKNK, and VDEYLD). For ProRdRp and VP1, five conserved aa motifs, GDCG, KDEL, DYSWDST, GLPSG, and YGDD in Pro-RdRp and two conserved aa motifs PPG and GWS in VP1 were also found in both the GX and GXI strains. The conserved aa motif of putative RdRp was WKGL except in the GXI
3.3. Phylogenetic tree analysis and pairwise amino acid sequence comparison We first analyzed the complete VP1 aa sequences of the SaV strains 213
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Fig. 3. Phylogenetic analyses based on complete amino acid sequences of putative RdRp coding region (approximately 500 aa in length) of 28 SaVs detected in this study with reference strains from DDBJ/EMBL/ GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study. Boxes and dot boxes indicate the cluster including possible intergenogroup strains described previously and the clusters including strains, which branched with other genogroup strains.
92.8–96.8% identities (Fig. 3, Supplementary Table 3). The GXI strain HgYa1-2 clustered with the GVII strains (HgTa2-2-1, HgTa2-1, WG247, Ishi-Im3-1, and Ishi-Im1-2) sharing 82.1–82.9% aa sequence identities but was separated from other GXI strains (HgYa1-3, Ishi-Im7-3, and HgTa2-2-2) with 67.1–67.9% identities (Fig. 3, Supplementary Table 3). Six single samples showed more than two genogroups. (Table 1). These strains, e.g. GIII and GVII strains, and those from pooled samples were similar with respect to each other as shown in Figs. 1–3.
and classified them into GIII, GV, GVI, GVII, GVIII, GX, and GXI genogroups, with intragenogroup aa identities ≧83.3% (GIII), ≧60.2% (GV), ≧75.7% (GVI), ≧55.8% (GVII), ≧62.1% (GVIII), ≧81.1% (GX), and ≧68.3% (GXI) (Supplementary Table 1). The aa sequence identities of VP1 between each genogroup were 28.6–58.0% (Supplementary Table 2) and the maximum intergenogroup aa sequence identity of VP1 was observed between the GVI strain Ishi-Im9 and the GIX strain F16-7 (Supplementary Table 2). The VP2 tree was similar to that of VP1 but here, the GIX strains did not form an independent cluster (Fig. 2). The pairwise complete VP2 aa sequence identities between each genogroup were 7.3–59.8% (Table 3). A phylogenetic tree of the putative complete RdRp region showed that several SaV strains were branched differently from those in the VP1 tree (Fig. 3). The GIX strain WG214C branched with the GX strains detected in this study, HgTa2 and HgTa3-2, with
3.4. Similarity plot analysis Phylogenetic tree analyses using the nt sequences of VPg-Pro-RdRp and VP1-VP2 clearly showed that the GXI strain HgYa1-2 fell within 214
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Fig. 4. (A) Phylogenetic analysis of GVII and GXI strains. The phylogenetic analysis was performed using nt sequence of VPg-Pro-RdRp and VP1-VP2 coding region. (B) Similarity plots of the nt sequence of nonstructural protein (VPg-Pro-RdRp), VP1, VP2, and 3′ UTR of GXI strain Ishi-Im7-3 (light blue line), GXI strain HgYa1-3 (blue line), GVII strain HgTa2-1 (red line), GVII strain Ishi-Im1-2 (pink line), and GXI strain HgYa1-2 as query sequences. (C) Genome organization of SaV. (D) Sequence alignment at a putative recombination point of GXI strain HgYa1-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The Simplot graph demonstrated that HgYa1-2 showed similarity with the GXI strains Ishi-Im7-3 and HgYa1-3 in the VP1 and VP2 region, whereas it showed similarity with the GVII strains HgTa2-1 and IshiIm2-1 in the VPg-Pro-RdRp region (Fig. 4). The putative recombination
GVII in the VPg-Pro-RdRp region, whereas it fell within GXI in the VP1VP2 region (Fig. 4), suggesting the possibility of recombination events. The standard similarity plot analysis was conducted using SimPlot program with the GXI strain HgYa1-2 nt sequence as a separate query. 215
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Although the putative complete RdRp coding region exhibits low sequence diversity (33.1–74.6%; excluding recombination strains) compared to the VP1 and VP2 region, the RdRp tree showed results similar to the VP1 and VP2 tree with the exception of recombination strains (Figs. 1, 2, and 3). Therefore, the combination of RdRp and VP1-VP2 analyses would be useful for the genetic characterization for SaVs. Although many partial SaV sequences have been reported, further genome information of SaV is warranted for the accumulation of diverse genetic information. In humans, co-infections with different genogroups of SaVs were reported from foodborne gastroenteritis outbreaks (Iizuka et al., 2010; Nakagawa-Okamoto et al., 2009). Co-infection in pigs with SaV has also been reported (Oka et al., 2016; Reuter et al., 2007). In the present study, seven genogroup SaV strains were found to be prevalent in the Japanese pig population and co-infection with two or more SaVs was observed in 6 out of 13 pigs (Table 1). The presence of two or more SaVs within the same animal at the same time is required for initial recombination. In the current pig rearing system in Japan, several genogroup SaV strains co-mingle and recombination events may occur frequently; these situations might be promoting the genetic diversity and evolution of SaVs in pigs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2017.09.013.
breakpoint was indicated by black narrow (Fig. 4. D). The sequence read depths in the putative recombination breakpoint of HgTa2-2-2, Ishi-Im7-3, HgYa1-2, HgYa1-3, HgTa2-1, and Ishi-Im1-2 were 15, 111, 9, 15, 20, and 29, respectively. 4. Discussion In the present study, SaVs were identified from 13 single and two pooled samples from pigs without diarrhea and one pooled sample from pigs with diarrhea. There are several reports describing SaV co-infections with rotavirus, porcine epidemic diarrhea virus, and kobuvirus (Reuter et al., 2007; Wang et al., 2016). In this study, all samples positive for SaVs contained two or more other viruses including enteritis pathogens such as rotaviruses. These findings suggested that SaVs might not be important causes for gastroenteritis in pigs or that specific genogroups may correlate with pathogenicity. The distribution of SaV infection in pigs has been reported to be more frequent in 0–1-monthold pigs than that in older ones (Barry et al., 2008; Valente et al., 2016). In this study, SaVs were predominantly identified from 2-month-old pigs, which were slightly older than those in the previous studies. In this study, seven of eight previously described genogroups were identified and the most predominant genogroups were GIII (11 strains) and GVII (six strains). GIII SaVs appears to be the most common genogroup in pigs in Canada, South Korea, and the USA (L'Homme et al., 2010; Keum et al., 2009; Nakamura et al., 2010; Wang et al., 2006; Yu et al., 2008). However, the sample number screened in this study is less to actually get a true prevalence of the circulating SaV genogroups, our results also showed that the GIII genogroup may be a predominant genogroup in Japanese pigs consistent with the results from previous reports. We determined 26 sequences > 6011 nt in length, including two GX strains and two GXI strains (Table 2). Thus far, there is only one partial sequence for the entire VP1 of GX (accession No. AB242873) and GXI (accession No. DQ359100) strains, which was reported from Japan and Brazil, respectively (Fig. 1, Supplementary Fig. 1) (Oka et al., 2016). To our knowledge, this is the first report to present near whole genomes including complete putative NTPase-VP2 sequences of the GX and GXI strains. The SaV GX and GXI strains possessed conserved aa motifs in the NTPase, VPg, Pro-RdRp, and VP1 regions of SaV strains. Five aa residues at the putative RdRp-VP1 cleavage sites of GX and GXI strains were YVMEG, which is identical to that in certain GVII strains (e.g. WG247) and the GXI strain WG214C (Oka et al., 2016). We constructed phylogenetic trees based on the aa sequences of putative full VP1, VP2, and RdRp regions. The VP1 region is the most diverse region in the genome of at least the GI-GV strains and correlates with antigenicity; thus, VP1 sequences are widely used for SaV classification (Oka et al., 2015). We show here the first full VP2 aa tree using the available sequences of GI-GXIV. The pairwise aa sequence identities between each genogroup of VP1 and VP2 were 28.6–58.0% and 7.3–59.8%, respectively. Oka et al. proposed that the cutoff values for the pairwise aa sequence identity in the VP1 region the between SaV genogroups were designated as those with < 57% aa sequence identity of VP1 (Oka et al., 2016). However, the minimum VP1 aa sequence identity among GVII was 55.8% (Supplementary Table 1) and was slightly lower than this cut-off value. The phylogenetic tree based on the complete putative RdRp region was essentially similar to that of the VP1 and VP2 trees; however, the GIX strain WG214C, and several GXI strains branched together with the GX and GVII strains, respectively (Fig. 3). Recombination analyses using a long sequence (> 4364 nt) covering VPg to the VP2 end revealed that these strains are intergenogroup recombination strains with a putative recombination point located in the RdRp/VP1 junction region (Fig. 4). The intergenogroup recombination strains identified belonged to genogroups GII and GIV (Hansman et al., 2005). The GV strain California sea lion/9775/USA/ 2010 (Accession No. JN420370) is a suggested intergenogroup recombination (Oka et al., 2015). To our knowledge, this is the first report of intergenogroup recombination of GVII and GXI SaV strains.
Acknowledgements This work was supported by the Grants from the Ministry of Health, Labour and Welfare of Japan, the Research Project for Improving Food Safety and Animal Health of the Ministry of Agriculture, Forestry and Fisheries of Japan, and JSPS KAKENHI Grant Number 15K07718. References Barry, A.F., Alfieri, A.F., Alfieri, A.A., 2008. High genetic diversity in RdRp gene of Brazilian porcine sapovirus strains. Vet. Microbiol. 131, 185–191. Cunha, J.B., de Mendonça, M.C., Miagostovich, M.P., Leite, J.P., 2010. Genetic diversity of porcine enteric caliciviruses in pigs raised in Rio de Janeiro State, Brazil. Arch. Virol. 155, 1301–1305. Di Bartolo, I., Tofani, S., Angeloni, G., Ponterio, E., Ostanello, F., Ruggeri, F.M., 2014. Detection and characterization of porcine caliciviruses in Italy. Arch. Virol. 159, 2479–2484. Dufkova, L., Scigalkova, I., Moutelikova, R., Malenovska, H., Prodelalova, J., 2013. Genetic diversity of porcine sapoviruses, kobuviruses, and astroviruses in asymptomatic pigs: an emerging new sapovirus GIII genotype. Arch. Virol. 158, 549–558. Farkas, T., Zhong, W.M., Jing, Y., Huang, P.W., Espinosa, S.M., Martinez, N., Morrow, A.L., Ruiz-Palacios, G.M., Pickering, L.K., Jiang, X., 2004. Genetic diversity among sapoviruses. Arch. Virol. 149, 1309–1323. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Flynn, W.T., Saif, L.J., Moorhead, P.D., 1988. Pathogenesis of porcine enteric caliciviruslike virus in four-day-old gnotobiotic pigs. Am. J. Vet. Res. 49, 819–825. Guo, M., Hayes, J., Cho, K.O., Parwani, A.V., Lucas, L.M., Saif, L.J., 2001. Comparative pathogenesis of tissue culture-adapted and wild-type Cowden porcine enteric calicivirus (PEC) in gnotobiotic pigs and induction of diarrhea by intravenous inoculation of wild-type PEC. J. Virol. 75, 9239–9251. Hansman, G.S., Takeda, N., Oka, T., Oseto, M., Hedlund, K.O., Katayama, K., 2005. Intergenogroup recombination in sapoviruses. Emerg. Infect. Dis. 11, 1916–1920. Iizuka, S., Oka, T., Tabara, K., Omura, T., Katayama, K., Takeda, N., Noda, M., 2010. Detection of sapoviruses and noroviruses in an outbreak of gastroenteritis linked genetically to shellfish. J. Med. Virol. 82, 1247–1254. Jeong, C., Park, S.I., Park, S.H., Kim, H.H., Park, S.J., Jeong, J.H., Choy, H.E., Saif, L.J., Kim, S.K., Kang, M.I., Hyun, B.H., Cho, K.O., 2007. Genetic diversity of porcine sapoviruses. Vet. Microbiol. 122, 246–257. Keum, H.O., Moon, H.J., Park, S.J., Kim, H.K., Rho, S.M., Park, B.K., 2009. Porcine noroviruses and sapoviruses on Korean swine farms. Arch. Virol. 154, 1765–1774. Kim, H.J., Cho, H.S., Cho, K.O., Park, N.Y., 2006. Detection and molecular characterization of porcine enteric calicivirus in Korea, genetically related to sapoviruses. J. Vet. Med. B Infect. Dis Vet. Public Health 53, 155–159. L'Homme, Y., Brassard, J., Ouardani, M., Gagné, M.J., 2010. Characterization of novel porcine sapoviruses. Arch. Virol. 155, 839–846. Liu, G.H., Li, R.C., Huang, Z.B., Yang, J., Xiao, C.T., Li, J., Li, M.X., Yan, Y.Q., Yu, X.L., 2012. RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China. Trop. Anim. Health Prod. 44, 1335–1339. Liu, Z.K., Li, J.Y., Pan, H., 2014. Seroprevalence and molecular detection of porcine sapovirus in symptomatic suckling piglets in Guangdong Province, China. Trop. Anim.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
Genetic diversity and intergenogroup recombination events of sapoviruses detected from feces of pigs in Japan
MARK
Moegi Kurodaa,1, Tsuneyuki Masudaa,1, Mika Itob, Yuki Naoic, Yen Hai Doand, Kei Hagad,e, Shinobu Tsuchiakac, Mai Kishimotoc, Kaori Sanoc, Tsutomu Omatsuc, Yukie Katayamac, Mami Obac, Hiroshi Aokif, Toru Ichimarug, Fujiko Sunagah, Itsuro Mukonob, Hiroshi Yamasatoa, Junsuke Shiraic, Kazuhiko Katayamad,e, Tetsuya Mizutanic, Tomoichiro Okad,⁎, Makoto Nagaic,i,⁎⁎ a
Kurayoshi Livestock Hygiene Service Center, Kurayoshi, Tottori 683-0017, Japan Ishikawa Nanbu Livestock Hygiene Service Center, Kanazawa, Ishikawa 920–3101, Japan Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan d Department of Virology II, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan e Laboratory of Viral Infection I, Kitasato Institute for Life Sciences, Graduate School of Infection Control Sciences, Minato, Tokyo 108-8641, Japan f Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan g Department of Health and Medical Sciences, Ishikawa Prefectural Nursing University, Kahoku, Ishikawa 929-1210, Japan h Laboratory of Infectious Diseases, Azabu University, Sagamihara, Kanagawa 252-5201, Japan i Department of Bioproduction Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Genogroup Japan Pig Recombination Sapovirus
Sapoviruses (SaV) are enteric viruses infecting humans and animals. SaVs are highly diverse and are divided into multiple genogroups based on structural protein (VP1) sequences. SaVs detected from pigs belong to eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI), but little is known about the SaV genogroup distribution in the Japanese pig population. In the present study, 26 nearly complete genome (> 6000 nucleotide: nt) and three partial sequences (2429 nt, 4364 nt, and 4419 nt in length, including the entire VP1 coding region) of SaV were obtained from one diarrheic and 15 non-diarrheic porcine feces in Japan via a metagenomics approach. Phylogenetic analysis of the complete VP1 amino acid sequence (aa) revealed that 29 porcine SaVs were classified into seven genogroups; GIII (11 strains), GV (1 strain), GVI (3 strains), GVII (6 strains), GVIII (1 strain), GX (3 strains), and GXI (4 strains). This manuscript presents the first nearly complete genome sequences of GX and GXI, and demonstrates novel intergenogroup recombination events.
1. Introduction Sapoviruses (SaVs) are etiological agents of gastroenteritis in humans and animals and they have non-enveloped positive-sense, singlestranded RNA genome of approximately 7.1–7.7 kb in length with the typical morphology, the Star-of-David structure, as determined by electron microscopy (Oka et al., 2015; Saif et al., 1980). The SaV genome contains two overlapping open reading frames (ORFs) that encode the nonstructural proteins NS1-NS2-NS3 (putative NTPase)NS4-NS5 (genome linked viral protein: VPg)-NS6 (Protease: Pro)-NS7 (RNA dependent RNA polymerase: RdRp) followed by the capsid protein, VP1, and the minor structural protein, VP2 (Oka et al., 2015).
Porcine SaV was first identified by electron microscopy in the United States of America in 1980 (Saif et al., 1980). SaVs have since been identified mostly using reverse transcriptase (RT)-PCR. The metagenomics approach, which does not require sequence-specific primers for PCR amplification, has recently allowed the detection of SaVs. SaVs have been reported from diarrheic and asymptomatic pigs (Cunha et al., 2010; das Merces Hernandez et al., 2014; Di Bartolo et al., 2014; Dufkova et al., 2013; Jeong et al., 2007; Keum et al., 2009; Kim et al., 2006; Liu et al., 2012; Liu et al., 2014; Martella et al., 2008; Martínez et al., 2006; Mauroy et al., 2008; Mijovski et al., 2010; Reuter et al., 2010; Scheuer et al., 2013; Valente et al., 2016; Wang et al., 2006; Zhang et al., 2014; Zhang et al., 2008). Cell culture-adapted and wild-
⁎
Corresponding author. Correspondence to: M. Nagai, Department of Bioproduction Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Research and Education Center for Prevention of Global Infectious Disease of Animal, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. E-mail addresses: [email protected] (T. Oka), [email protected], [email protected] (M. Nagai). 1 M. Kuroda and T. Masuda contribute equally. ⁎⁎
http://dx.doi.org/10.1016/j.meegid.2017.09.013 Received 4 April 2017; Received in revised form 11 September 2017; Accepted 13 September 2017 Available online 18 September 2017 1567-1348/ © 2017 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 55 (2017) 209–217 Porcine astrovirus, Picobirnavirus, Posavirus Porcine astrovirus, Porcine enterovirus, Sapelovirus, Porcine rotavirus C Porcine astrovirus, Picobirnavirus Porcine astrovirus, Porcine enterovirus, Porcine picornavirus Japan Porcine astrovirus, Porcine enterovirus, Porcine picornavirus Japan Porcine astrovirus, Posavirus, Techovirus, Picobirnavirus Porcine astrovirus, Sapelovirus, Posavirus, Picobirnavirus Porcine enterovirus, Porcine kobuvirus Teschovirus, Porcine enterovirus, Posavirus, Porcine rotavirus A Teschovirus, Porcine enterovirus, Picobirnavirus, Porcine kobuvirus, Porcine rotavirus A, Porcine rotavirus C Porcine enterovirus, Porcine kobuvirus, Porcine rotavirus C Porcine astrovirus, Sapelovirus, Porcine enterovirus, Porcine rotavirus A, Porcine rotavirus C Porcine astrovirus, Sapelovirus, Porcine rotavirus B, Porcine rotavirus C Porcine astrovirus, Picobirnavirus Porcine enterovirus, Picobirnavirus Porcine astrovirus, Porcine enterovirus, Porcine rotavirus B GV GVII GVII, GXI GIII, GVII GVI GIII, GVII (2), GVIII GIII, GVII GIII GIII, GVI, GX, GXI GVI
GIII (2) GIII GX GIII, GX
2.1. Fecal samples, RNA extraction, and deep sequencing
Without Without Without Without
diarrhea diarrhea diarrhea diarrhea
2 1 1 2
In total, 105 fecal samples collected from 2 to 120-day-old pigs from 12 farms in Tottori Prefecture and Ishikawa Prefecture of Japan in 2015–2016 were used in this study. Samples were collected from pigs with diarrhea (six single samples and 17 pooled samples) or those without diarrhea (70 single samples and 12 pooled samples). Although pooled samples were impossible for recombination analysis, we accepted those samples for search for new genogroups only. Samples were diluted 1:9 (w/v) in sterile phosphate-buffered saline and centrifuged at 10,000 × g for 10 min. The supernatants were collected and stored at − 80 °C until required. Viral RNA was extracted from the fecal sample supernatants, using TRIzol® LS Reagent (Life Technologies, Carlsbad, CA, USA), and was treated with DNase I (Takara Bio, Shiga, Japan). cDNA libraries for deep sequencing were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) as described previously (Nagai et al., 2015). Deep sequencing was conducted using the MiSeq Reagent Kit v2 (300cycles) (Illumina, San Diego, CA, USA) on a MiSeq bench-top sequencer (Illumina). FASTQ-formatted sequence data files were generated using the MiSeq Reporter (Illumina). Trimmed paired-end sequence reads were assembled into contigs by de novo assembly with the strictest parameter setting (mismatch cost, 2; insertion cost, 3; deletion cost, 3; length function, 0.9; and similarity function, 0.9) in the CLC Genomics Workbench 7.5.5 (CLC bio, Aarhus, Denmark). The nucleotide sequences of SaV strains obtained in this study were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers LC215874–LC215902.
2016.12.2 2016.12.2 2016.12.2 2016.12.2 Tottori Tottori Tottori Tottori
2 months 2 months 2 months 2 months
GIII GIII, GXI (2) 1 3 Without diarrhea Without diarrhea 2016.11.30 2016.12.2 Ishikawa Tottori
16 2 months
2015.7.1 2015.7.2 2015.7.2 2015.7.2 2015.7.15 2015.11.6 2015.11.6 2015.11.5 2016.11.25 2016.11.25 Tottori Tottori Tottori Tottori Tottori Ishikawa Ishikawa Ishikawa Ishikawa Ishikawa
2 months 2 months 2 months 2 months 2 months 54 30 16 11 11
Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Without diarrhea Mild diarrhea Without diarrhea Without diarrhea
1 1 2 2 1 4 2 1 4 1
Co-infection with other viruses Health status Collection date Region
Days (months)
type SaVs were shown to induce diarrhea in experimentally infected gnotobiotic piglets (Flynn et al., 1988; Guo et al., 2001; Lu et al., 2015). Therefore, SaVs are currently suggested as one of the etiological agents of gastroenteritis in pigs; however, the role of SaV as the cause of gastroenteritis in pigs has been obscure. Initially, Farkas et al. classified human SaVs into GI-GV using entire VP1 sequences (Farkas et al., 2004). Thereafter, Scheuer et al. classified SaVs including those from humans and animals into 14 genogroups based on the complete VP1 sequences (Scheuer et al., 2013). Thus far, SaVs are divided into fifteen genogroups (Oka et al., 2016). To date, at least eight SaV genogroups: GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI, have been identified from pigs (Scheuer et al., 2013; Oka et al., 2016). In Japan, GIII, GV, GVII, GVIII, and GX genogroups of SaV have been found from finisher pigs and pigs, whose ages were < 5 months (Nakamura et al., 2010; Yin et al., 2006). Complete or nearly complete genome sequences of SaV GIII, GV, GVI, GVII, and GVIII are available in the DDBJ/EMBL/GenBank database, but those of GIX, X, and GXI are not available. In the present study, 26 nearly complete genome sequences and three partial sequences of SaV were obtained from fecal samples of pigs with or without diarrhea in Japan using a metagenomics approach. This study presents the first report of nearly complete genome data for SaV GX and GXI. Genetic analyses revealed high genetic diversity among SaVs in Japanese pig population and possible intergenogroup recombination events. 2. Materials and methods
Sample status
Single Single Single Single Single Pooled Pooled Pooled Single Single
Single Single
Single Single Single Single
HkKa2-1/2015 HgTa2-1/2015 HgTa2-2/2015 MoI2-1/2015 HgOg2-4/2015 Ishi-Im1/2015 Ishi-Im3/2015 Ishi-Kah3/2015 Ishi-Im7/2016 Ishi-Im9/2016
Ishi-Kah6/2016 HgYa1/2016
HgYa2/2016 HgTa1/2016 HgTa2/2016 HgTa3/2016
2.2. Genome analysis
Sample name
Table 1 Summary of samples used in this study and a number of sapovirus contigs identified in each sample.
Number of sapovirus contigs
Sapovirus genogroup
M. Kuroda et al.
Nucleotide (nt) and amino acid (aa) sequences were aligned using ClustalW (Thompson et al., 1997). Phylogenetic trees were constructed using the 29 SaV strains in this study together with available SaV sequences from the DDBJ/EMBL/GenBank database following the maximum-likelihood method with the best-fit evolutionary models in MEGA 5.22 (Tamura et al., 2011) for rtREV + G + I (RdRp), rtREV + G + I + F (VP1 aa), GTR + G + I (VP1 nt), and rtREV + G + F (VP2). The reliability of the phylogenetic tree obtained for each gene 210
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Table 2 Summary of genomic information of sapoviruses obtained from deep sequencing in this study. Strains
Geno group
MoI2-1-1/2015 Ishi-Im1-3/2015 Ishi-Im3-2/2015 Ishi-Kah3/2015 Ishi-Im7-1/2016 Ishi-Kah6/2016 HgYa1-1/2016 HgYa2-1/2016 HgYa2-2/2016 HgTa1/2016 HgTa3-1/2016 HkKa2-1/2015 HgOg2-4/2015 Ishi-Im7-2/2016 Ishi-Im9/2016 HgTa2-1/2015 HgTa2-2-1/2015 MoI2-1-2/2015 Ishi-Im1-2/2015 Ishi-Im1-4/2015 Ishi-Im3-1/2015 Ishi-Im1-1/2015 HgTa2/2016 HgTa3-2/2016 Ishi-Im7-4/2016 HgTa2-2-2/2015 Ishi-Im7-3/2016 HgYa1-2/2016 HgYa1-3/2016
GIII GIII GIII GIII GIII GIII GIII GIII GIII GIII GIII GV GVI GVI GVI GVII GVII GVII GVII GVII GVII GVIII GX GX GX GXI GXI GXI GXI
Reads and sequences obtained from deep sequencing
DDBJ accession no.
Total reads
Sapovurs reads
Sapovirus reads %
Contig length (excluding poly-A)
2,804,452 1,809,416 3,041,700 2,648,440 1,554,736 536,200 1,083,038 1,341,368 1,341,368 1,405,590 1,074,082 1,128,042 399,976 1,554,736 1,759,264 685,940 862,726 2,804,452 1,809,416 1,809,416 3,041,700 1,809,416 750,634 1,074,082 1,554,736 862,726 1,554,736 1,083,038 1,083,038
9884 1404 842 1496 25,179 40,983 923 40,504 4085 14,962 2683 4412 2419 955 757 745 2382 5022 1227 1241 4265 6092 991 690 108 870 5980 338 449
0.35 0.08 0.03 0.06 1.6 7.6 0.09 3.0 0.30 1.1 0.25 0.39 0.60 0.06 0.04 0.11 0.28 0.18 0.07 0.07 0.14 0.34 0.13 0.06 0.01 0.10 0.38 0.03 0.04
6882 7151 6958 6902 7330 7333 6011 7326 7264 7305 7158 7485 7162 6344 7055 7059 7069 6786 7133 7130 7139 7449 7110 6995 2429 7162 7127 4364 4419
LC215874 LC215875 LC215876 LC215877 LC215878 LC215879 LC215880 LC215881 LC215882 LC215883 LC215884 LC215885 LC215886 LC215887 LC215888 LC215889 LC215890 LC215891 LC215892 LC215893 LC215894 LC215895 LC215896 LC215897 LC215900 LC215898 LC215899 LC215901 LC215902
Table 3 Pairwise amino acid sequence identities of putative RdRp (lower left) and VP2 (upper right) between genotype of sapovirus.
GI
GI 72.9-77.7 81.5-87.5
GII
GIII
GIV
GV
GVI
GVII
GVIII
GIX
GX
GXI
GXII
GXIII
GXIV
41.5-43.9
26.3-30.9
53.6
41.3-49.7
24.9-28.8
24.3-29.4
43.0-53.5
27.1-29.4
20.9-25.0
24.3-27.3
18.7-23.1
31.0-33.9
17.3-19.0 16.8-18.4
78.6
GII
71.7-74.6
25.0-28.6
33.9-38.2
32.5-39.4
26.4-28.2
23.6-28.2
39.0-45.3
22.4-23.6
24.1-27.2
21.3-23.1
20.9-23.4
41.9-46.2
GIII
56.0-59.1
55.9-57.8
76.7-100 84.7-100
26.4-29.7
24.0-29.7
20.0-25.1
20.6-28.6
24.9-29.1
18.8-29.1
20.6-24.6
18.8-23.4
14.2-20.1
20.6-23.4
21.8-24.0
GIV
71.5-74.4
93.5-93.9
55.9-57.4
46.2-52.1
22.6
23.2-25.4
46.5-48.3
25.4-29.4
22.6
25.4-28.3
18.6-19.2
29.8
21.2-21.8
GV
63.5-68.4
63.4-68.8
52.2-56.1
63.4-69.0
57.6-74.4 62.5-92.4
19.2-28.3
19.8-29.6
43.4-53.5
21.5-29.7
19.8-26.7
21.5-30.1
14.1-18.6
24.6-28.7
15.6-20.7
GVI
36.6-37.7
37.7-38.1
37.4-38.5
38.1-38.3
34.1-35.8
85.8-99.4 93.4-100
49.7-55.6
21.3-25.4
53.3-58.6
50.3-53.3
49.1-55.0
15.8-20.5
18.1-19.2
18.4-20.1
GVII
33.5-36.0
34.6-37.0
34.4-36.8
35.2-37.4
32.7-35.6
62.4-65.5
63.4-100 62.5-100
23.1-26.4
46.2-58.0
49.1-55.6
48.5-59.8
18.1-28.1
19.8-27.1
18.4-20.1
GVIII
60.0-62.2
62.3-63.9
55.1-57.0
62.3-63.5
62.1-67.0
36.5-38.3
34.6-36.9
55.2-95.9 74.0-98.8
24.7-27.1
19.5-22.6
21.8-26.6
18.0-21.7
26.0-34.7
17.9-21.8
50.3-56.7
54.4-58.6
22.0-24.6
19.2-21.5
17.9-19.0
47.0-55.0
19.6-22.8
20.9-22.0
19.0-20.7
75.7-91.7 67.1-98.8
19.4-24.0
21.5-24.3
19.6-21.2
19.0-20.1
7.3-8.4
98.8
99.4 99.6
59.8
GIX
33.9-35.6
37.2-37.4
36.4-37.2
37.2-37.4
33.7-35.8
64.5-65.7
65.5-71.7
36.1-36.3
GX
34.1-35.6
37.0-37.7
35.4-37.2
37.0-37.4
33.9-36.0
63.9-65.3
64.3-72.5
35.5-36.7
92.8-96.8
GXI
34.6-36.6
36.2-37.9
35.4-37.7
36.8-37.6
32.3-35.6
62.3-65.9
63.5-82.9
35.3-37.5
68.5-74.1
-
76.7 94.4 67.9-74.5
85.3
GXII
57.4-59.0
59.1-59.5
57.6-59.2
59.1-59.5
55.3-58.3
37.4-38.0
35.3-36.4
55.8-58.5
35.9-36.2
35.5-36.1
35.9-36.6
GXIII
66.0-68.4
64.8-65.2
53.2-54.8
64.7-64.8
60.2-65.6
36.0-36.8
33.1-34.4
58.5-60.4
34.4
34.2-34.6
34.4-36.2
55.5-55.6
GXIV
49.4-52.3
52.3
48.5-49.5
51.7-52.1
48.6-49.8
35.7-36.1
32.6-33.9
49.4-50.0
33.9
34.1-34.3
35.1-36.2
45.3-45.7
97.7
49.6
14.5 100 100
in length (26 contigs) and contigs of 2429 nt, 4364 nt, and 4419 nt length including the entire VP1 coding sequence with sufficient coverage (more than three coverage of sequence reads) were identified from one pooled sample with diarrhea, and 2 pooled and 13 single samples without diarrhea (Tables 1 and 2). These samples apart from pooled samples also contained two to six contigs that showed similarities with porcine Astrovirus (9 samples), picobirnavirus (5 samples), posavirus (2 samples), porcine enterovirus (9 samples), sapelovirus (3 samples), porcine picornavirus Japan (2 samples), techovirus (2 samples), porcine kobuvirus (2 samples), porcine rotavirus A (3 samples), porcine rotavirus B (2 samples), and porcine rotavirus C (5 samples) (Table 1). Of the 29 strains, based on complete VP1 sequence analysis,
segment was evaluated by running 1000 bootstrap replicates (Felsenstein, 1985). Pairwise sequence identity calculation was performed on the sequences using the CLC Genomics Workbench (CLC bio). Similarity plot analysis was performed using SimPlot software v. 3.5.1 (Lole et al., 1999). 3. Results 3.1. Identification of nearly complete and partial genome sequences of SaVs Using the previously described metagenomics approach and BLAST search, in total, 29 SaV-like contigs that were longer than about 6000 nt 211
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Fig. 1. Phylogenetic analyses based on amino acid sequences of the full VP1 coding region (approximately 500 aa in length) of 29 SaVs detected in this study with reference strains from DDBJ/EMBL/ GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study.
3.2. Genome analyses of coding regions of the SaV GX and GXI genogroups
11, 1, 3, 6, 1, 3, and 4 strains fell within the GIII, GV, GVI, GVII, GVIII, GX, and GXI genogroups, respectively (Table 2). The strain names and information of the SaV-like contigs obtained in this study are summarized in Table 2. Six single samples showed more than two genogroups (Table 1).
In the putative NTPase and VPg region, three conserved aa motifs, GXPGXGKT, PL(N/D)CD, and WDE(F/Y)D in NTPase, and two conserved aa motifs, KGKXX and XDEYXX in VPg were observed in the GX 212
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Fig. 2. Phylogenetic analyses based on amino acid sequences of VP2 coding region (approximately 165 aa in length) of 27 SaVs detected in this study with reference strains from DDBJ/EMBL/GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study.
strains, where this aa motif was WKDL.
strains (GPPGIGKT, PLNCD, WDEYD, KGKNK, and DDEYTE) and GXI strains (GPPGIGKT, PLNCD, WDEFD, KGKNK, and VDEYLD). For ProRdRp and VP1, five conserved aa motifs, GDCG, KDEL, DYSWDST, GLPSG, and YGDD in Pro-RdRp and two conserved aa motifs PPG and GWS in VP1 were also found in both the GX and GXI strains. The conserved aa motif of putative RdRp was WKGL except in the GXI
3.3. Phylogenetic tree analysis and pairwise amino acid sequence comparison We first analyzed the complete VP1 aa sequences of the SaV strains 213
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Fig. 3. Phylogenetic analyses based on complete amino acid sequences of putative RdRp coding region (approximately 500 aa in length) of 28 SaVs detected in this study with reference strains from DDBJ/EMBL/ GenBank database. Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates) above 70 are shown. The scale bar indicates nucleotide substitutions per site. The genogroups are indicated at the right-hand side. ● indicates SaV strains detected in this study. Boxes and dot boxes indicate the cluster including possible intergenogroup strains described previously and the clusters including strains, which branched with other genogroup strains.
92.8–96.8% identities (Fig. 3, Supplementary Table 3). The GXI strain HgYa1-2 clustered with the GVII strains (HgTa2-2-1, HgTa2-1, WG247, Ishi-Im3-1, and Ishi-Im1-2) sharing 82.1–82.9% aa sequence identities but was separated from other GXI strains (HgYa1-3, Ishi-Im7-3, and HgTa2-2-2) with 67.1–67.9% identities (Fig. 3, Supplementary Table 3). Six single samples showed more than two genogroups. (Table 1). These strains, e.g. GIII and GVII strains, and those from pooled samples were similar with respect to each other as shown in Figs. 1–3.
and classified them into GIII, GV, GVI, GVII, GVIII, GX, and GXI genogroups, with intragenogroup aa identities ≧83.3% (GIII), ≧60.2% (GV), ≧75.7% (GVI), ≧55.8% (GVII), ≧62.1% (GVIII), ≧81.1% (GX), and ≧68.3% (GXI) (Supplementary Table 1). The aa sequence identities of VP1 between each genogroup were 28.6–58.0% (Supplementary Table 2) and the maximum intergenogroup aa sequence identity of VP1 was observed between the GVI strain Ishi-Im9 and the GIX strain F16-7 (Supplementary Table 2). The VP2 tree was similar to that of VP1 but here, the GIX strains did not form an independent cluster (Fig. 2). The pairwise complete VP2 aa sequence identities between each genogroup were 7.3–59.8% (Table 3). A phylogenetic tree of the putative complete RdRp region showed that several SaV strains were branched differently from those in the VP1 tree (Fig. 3). The GIX strain WG214C branched with the GX strains detected in this study, HgTa2 and HgTa3-2, with
3.4. Similarity plot analysis Phylogenetic tree analyses using the nt sequences of VPg-Pro-RdRp and VP1-VP2 clearly showed that the GXI strain HgYa1-2 fell within 214
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Fig. 4. (A) Phylogenetic analysis of GVII and GXI strains. The phylogenetic analysis was performed using nt sequence of VPg-Pro-RdRp and VP1-VP2 coding region. (B) Similarity plots of the nt sequence of nonstructural protein (VPg-Pro-RdRp), VP1, VP2, and 3′ UTR of GXI strain Ishi-Im7-3 (light blue line), GXI strain HgYa1-3 (blue line), GVII strain HgTa2-1 (red line), GVII strain Ishi-Im1-2 (pink line), and GXI strain HgYa1-2 as query sequences. (C) Genome organization of SaV. (D) Sequence alignment at a putative recombination point of GXI strain HgYa1-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The Simplot graph demonstrated that HgYa1-2 showed similarity with the GXI strains Ishi-Im7-3 and HgYa1-3 in the VP1 and VP2 region, whereas it showed similarity with the GVII strains HgTa2-1 and IshiIm2-1 in the VPg-Pro-RdRp region (Fig. 4). The putative recombination
GVII in the VPg-Pro-RdRp region, whereas it fell within GXI in the VP1VP2 region (Fig. 4), suggesting the possibility of recombination events. The standard similarity plot analysis was conducted using SimPlot program with the GXI strain HgYa1-2 nt sequence as a separate query. 215
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Although the putative complete RdRp coding region exhibits low sequence diversity (33.1–74.6%; excluding recombination strains) compared to the VP1 and VP2 region, the RdRp tree showed results similar to the VP1 and VP2 tree with the exception of recombination strains (Figs. 1, 2, and 3). Therefore, the combination of RdRp and VP1-VP2 analyses would be useful for the genetic characterization for SaVs. Although many partial SaV sequences have been reported, further genome information of SaV is warranted for the accumulation of diverse genetic information. In humans, co-infections with different genogroups of SaVs were reported from foodborne gastroenteritis outbreaks (Iizuka et al., 2010; Nakagawa-Okamoto et al., 2009). Co-infection in pigs with SaV has also been reported (Oka et al., 2016; Reuter et al., 2007). In the present study, seven genogroup SaV strains were found to be prevalent in the Japanese pig population and co-infection with two or more SaVs was observed in 6 out of 13 pigs (Table 1). The presence of two or more SaVs within the same animal at the same time is required for initial recombination. In the current pig rearing system in Japan, several genogroup SaV strains co-mingle and recombination events may occur frequently; these situations might be promoting the genetic diversity and evolution of SaVs in pigs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2017.09.013.
breakpoint was indicated by black narrow (Fig. 4. D). The sequence read depths in the putative recombination breakpoint of HgTa2-2-2, Ishi-Im7-3, HgYa1-2, HgYa1-3, HgTa2-1, and Ishi-Im1-2 were 15, 111, 9, 15, 20, and 29, respectively. 4. Discussion In the present study, SaVs were identified from 13 single and two pooled samples from pigs without diarrhea and one pooled sample from pigs with diarrhea. There are several reports describing SaV co-infections with rotavirus, porcine epidemic diarrhea virus, and kobuvirus (Reuter et al., 2007; Wang et al., 2016). In this study, all samples positive for SaVs contained two or more other viruses including enteritis pathogens such as rotaviruses. These findings suggested that SaVs might not be important causes for gastroenteritis in pigs or that specific genogroups may correlate with pathogenicity. The distribution of SaV infection in pigs has been reported to be more frequent in 0–1-monthold pigs than that in older ones (Barry et al., 2008; Valente et al., 2016). In this study, SaVs were predominantly identified from 2-month-old pigs, which were slightly older than those in the previous studies. In this study, seven of eight previously described genogroups were identified and the most predominant genogroups were GIII (11 strains) and GVII (six strains). GIII SaVs appears to be the most common genogroup in pigs in Canada, South Korea, and the USA (L'Homme et al., 2010; Keum et al., 2009; Nakamura et al., 2010; Wang et al., 2006; Yu et al., 2008). However, the sample number screened in this study is less to actually get a true prevalence of the circulating SaV genogroups, our results also showed that the GIII genogroup may be a predominant genogroup in Japanese pigs consistent with the results from previous reports. We determined 26 sequences > 6011 nt in length, including two GX strains and two GXI strains (Table 2). Thus far, there is only one partial sequence for the entire VP1 of GX (accession No. AB242873) and GXI (accession No. DQ359100) strains, which was reported from Japan and Brazil, respectively (Fig. 1, Supplementary Fig. 1) (Oka et al., 2016). To our knowledge, this is the first report to present near whole genomes including complete putative NTPase-VP2 sequences of the GX and GXI strains. The SaV GX and GXI strains possessed conserved aa motifs in the NTPase, VPg, Pro-RdRp, and VP1 regions of SaV strains. Five aa residues at the putative RdRp-VP1 cleavage sites of GX and GXI strains were YVMEG, which is identical to that in certain GVII strains (e.g. WG247) and the GXI strain WG214C (Oka et al., 2016). We constructed phylogenetic trees based on the aa sequences of putative full VP1, VP2, and RdRp regions. The VP1 region is the most diverse region in the genome of at least the GI-GV strains and correlates with antigenicity; thus, VP1 sequences are widely used for SaV classification (Oka et al., 2015). We show here the first full VP2 aa tree using the available sequences of GI-GXIV. The pairwise aa sequence identities between each genogroup of VP1 and VP2 were 28.6–58.0% and 7.3–59.8%, respectively. Oka et al. proposed that the cutoff values for the pairwise aa sequence identity in the VP1 region the between SaV genogroups were designated as those with < 57% aa sequence identity of VP1 (Oka et al., 2016). However, the minimum VP1 aa sequence identity among GVII was 55.8% (Supplementary Table 1) and was slightly lower than this cut-off value. The phylogenetic tree based on the complete putative RdRp region was essentially similar to that of the VP1 and VP2 trees; however, the GIX strain WG214C, and several GXI strains branched together with the GX and GVII strains, respectively (Fig. 3). Recombination analyses using a long sequence (> 4364 nt) covering VPg to the VP2 end revealed that these strains are intergenogroup recombination strains with a putative recombination point located in the RdRp/VP1 junction region (Fig. 4). The intergenogroup recombination strains identified belonged to genogroups GII and GIV (Hansman et al., 2005). The GV strain California sea lion/9775/USA/ 2010 (Accession No. JN420370) is a suggested intergenogroup recombination (Oka et al., 2015). To our knowledge, this is the first report of intergenogroup recombination of GVII and GXI SaV strains.
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