Jpn. J. Infect. Dis.

, 66, 454-457, 2013

Laboratory and Epidemiology Communications

Phylogenetic Analysis of the Nonstructural and Structural Protein Encoding Region Sequences, Indicating Successive Appearance of Genomically Diverse Sapovirus Strains from Gastroenteritis Patients
Seiya Harada1*, Eisuke Tokuoka1, Naoko Kiyota1, Kazuhiko Katayama2, and Tomoichiro Oka2
1Kumamoto 2Department

Prefectural Institute of Public Health and Environmental Science, Kumamoto 869-0425; and of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan
Communicated by Takaji Wakita (Accepted June 13, 2013)

Sapovirus (SaV) is a pathogen that causes acute gastroenteritis (15). The SaV genome is a positive-sense, single-strand RNA molecule of approximately 7.5 kb, containing two open reading frames (ORFs), and ORF1 is predicted to encode at least six nonstructural (NS) proteins and a structural protein (VP1) in the following order: NS1-NS2-NS3-NS4-NS5-NS6-7-VP1. NS6-7 encodes protease and RNA-dependent RNA polymerase (RdRp) (6). The functions of other NS and ORF2-encoded proteins have not yet been experimentally determined. Based on the VP1 sequences, SaV can be divided into at least five genogroups (GIGV), and recently, nine additional genogroups (GVIGXIV) have been proposed (7). Thus far, GI, GII, GIV, and GV SaV strains have been detected in humans. Human SaVs can be further divided into multiple genotypes (at least GI.1-7, GII.1-7, GIV.1, and GV.1) based on the VP1 sequences (8). We previously identified 139 SaVs from 1,367 stool samples of outpatients with acute gastroenteritis at three pediatric clinics from June 2002 to March 2011 in Kumamoto Prefecture, Japan, using RT-PCR targeting the human SaV RdRp-VP1 junction region (approximately 105 bp) (1,2). All 139 SaVs were then successfully amplified in combinations with several RT-PCR assays for the partial VP1 region and classified into 4 genogroups and 11 genotypes: GI.1, GI.2, GI.3, GI.5, GII.1, GII.2, GII.3, GII.4, GII.7, GIV.1 and GV.1 (1). The predominant genotypes changed drastically during the study period (i.e., emerging GIV strains in 2007 changed to predominant GII.3 strains after 2008) (1). The main objective of this study was to obtain the partial RdRp region sequences of all the 4 genogroups and the 11 genotypes detected in our previous studies (1,2) and evaluate whether these detected SaVs were also diverse in the partial RdRp regions as observed in the VP1 regions. In this study, we used novel primer combinations including four published (Sapp36, or SV-S1, -S2, and -S3) (9,10) and one newly designed (SaV GV-GLPSGM) forward primers targeting the human SaV RdRp region
*Corresponding author: Mailing address: Kumamoto Prefectural Institute of Public Health and Environmental Science, Kumamoto 869-0425, Japan. Tel: 81-964-235771, Fax: 81-964-23-5260, E-mail: harada-s-dz pref.kumamoto.lg.jp
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and a common reverse primer (SaV1245R) targeting the most conserved region of human SaV GI, GII, GIV, and GV genomes corresponding to the RdRp-VP1 junction region (11). The SaVs analyzed in this study were originally identified by RT-PCR screening using the SaV 124F, -1F, -5F, and SaV 1245R primers (1,2). Viral RNA extraction, cDNA synthesis, and quantitative real-time PCR were performed as previously described (2). To amplify the partial RdRp region sequences of human SaV, three PCRs were performed to detect the partial SaV RdRp region using step-by-step sequential detection flow: (i) PCR with primers Sapp36 (5?-GTT GCT GTT GGC ATT AAC A-3?) (10) and SaV 1245R (5?-CCC TCC ATY TCA AAC ACT A-3?) (11), (ii) SV-S1 (5?-CCC TGG CAA TAT TGG AGA GAT TTG-3?), SV-S2 (5?-AAG GCC TAT GAA CCA CAT GGG GC-3?), SV-S3 (5?-GAC ATC AAG TTT GAT ACC AAG GA-3?) (9), and SaV 1245R (11), and (iii) the newly designed SaV GV-GLPSGM (5?-GGT CTC CCC TCG GGC ATG-3?) corresponding to nucleotide position 45604577 of the GV NK24 strain (GenBank/ EMBL/DDBJ accession no. AY646856) and SaV 1245R. The expected PCR product sizes were approximately 900 bp for Sapp36 and SaV 1245R, 780, 580, 680 bp for SV-S1, -S2, -S3 and SaV 1245R, respectively, and 660 bp for SaV GV-GLPSGM and SaV 1245R. PCR was performed using a 50-ml reaction volume containing 5 ml of cDNA, 2.5 U of Ex Taq DNA polymerase (Takara Bio, Otsu, Japan) and 20 pmol of each primer. PCR amplification was performed under the C for 3 following conditions: initial denaturation at 949 min, followed by 40 cycles of amplification with denatuC for 30 s, primer annealing at 489 C for ration at 949 C for 1 min, and then a final exten30 s, extension at 729 C for 7 min. The PCR products of expected sion at 729 size were purified and sequenced directly or after cloning as previously described (2,12). The nucleotide sequences corresponding to the partial SaV RdRp region determined in this study have been deposited in GenBank/EMBL/DDBJ under the accession numbers AB812625AB812743. We amplified the partial RdRp sequences of 119 of the 139 SaV strains (85.6z) corresponding to all 4 genogroups and 11 genotypes (GI.1, GI.2, GI.3, GI.5, GII.1, GII.2, GII.3, GII.4, GII.7, GIV.1, and GV.1) (Fig. 1A). Fourteen of the 20 partial RdRp targeting PCR-negative specimens had relatively low viral copy numbers (data not shown), and this may partly explain

Fig. 1. Phylogenetic tree of SaV based on (A) partial VP1 or (B) partial RdRp nucleotide sequences. SaV strains were labeled with strain number, detected year and month. The reference SaV strains are indicated with GenBank/ EMBL/DDBJ accession number in parenthesis. The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replications. The numbers on each branch indicate the bootstrap values, where the values of 950 or higher were indicated. The scale represents the nucleotide substitutions per site. The 139 partial VP1 sequences were determined in our previous studies and deposited under the accession numbers AB429079AB429159 and AB689798AB689855 (1,2), and the 119 partial RdRp sequences have been determined in this study and deposited under the accession numbers AB812625AB812743.

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Fig. 1. Continued

the negative result (Fig. 1A). As shown in Fig. 1B, the Sapp36 and SaV-1245R or SV-S1, -S2, -S3, and SaV1245R primer sets successfully detected the partial RdRp regions of the SaV GI, GII, and GIV strains, whereas GV strains could only be amplified using the SaV GV-GLPSGM and SaV 1245R primer set. A single
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GI strain was also amplified using this primer combination. On the phylogenetic tree, partial RdRp regions of the 119 SaVs corresponding to the 4 genogroups and 11 genotypes were basically segregated into distinct clusters or branches along with their VP1-based genogroup and genotype except GII.2 and GII.3 strains (Fig. 1B). SaV

GII.2 and GII.3 analyzed in this study formed distinct clusters by the VP1 region (Fig. 1A), whereas they were not segregated well by the RdRp region (Fig. 1B). This is consistent with the previous report that the GII.2 strain (GenBank/EMBL/DDBJ accession no. AY237420) detected from an infant hospitalized with acute gastroenteritis in Thailand in 2000 and the GII.3 strain (AY603425) detected from an infant with gastroenteritis in Japan in 2001 were grouped in the same cluster by the RdRp region (13). These genetically similar SaV GII.2 and GII.3 strains likely spread and persist in different countries and areas. Our results suggest that the total clusters based on the RdRp region sequences may not be identical to those based on VP1 region sequences. In this study, we amplified the partial RdRp region sequence for SaV strains GII.4 and GII.7 for the first time; however, more RdRp sequences corresponding to all VP1-based genogroups and genotypes with a statistically reliable analytical scheme is necessary to establish RdRp sequence-based typing in the future. Three children (Patients A, B, and C) were positive for SaV twice during the study period as previously described (1). SaVs detected from Patient A consisted of strains GI.1 (29/2007/Jan) and GIV.1 (60/2007/Nov) based on partial VP1 sequences (Fig. 1A). These strains also formed the GI.1 and GIV.1 clusters, respectively, based on the partial RdRp sequences (Fig. 1B). Patient B was positive for SaV strain GI.1 (25/2006/Dec) and then strain GII.3 (101/2009/Mar) (Fig. 1A). The SaV strain isolated from the first sample of this patient also clustered into GI.1 based on the partial RdRp sequence (Fig. 1B); however, we could not amplify the partial RdRp region of the second sample (Figs. 1A and B). The low viral level in the second sample (1.0 106 copies/g stool) may partly explain this result, although mismatches in the primer target region could also have been possible. Patient C was positive for SaV strain GII.3 twice within the same month (89/2008/Nov and 90/2008/Nov). The SaV strains from the two stool samples contained sequences with 99.0z (420/ 424) nucleotide identity within the comparable partial RdRp regions and was consistent with that of partial VP1 region (99.0z), as previously described (1). In conclusion, our data demonstrated that genetically diverse SaV strains with both nonstructural and structural protein encoding region sequences appeared in succession from 2002 to 2011 in the same geographic area in Kumamoto Prefecture, Japan. The novel primer combinations described in this study can be used as an additional tool to accumulate human SaV partial RdRp region sequences of strains from multiple genogroups

and genotypes to furthre characterize the human SaV genome.

Acknowledgments We thank Drs. Yasushi Shimada, Shigeru Ikezawa, and Takehiko Ueno for collecting samples. We also thank Kelly A. Scheuer for the linguistic revision of the manuscript. This work was supported in part by a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan. Conflict of interest None to declare. REFERENCES
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