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Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan
Accepted Manuscript Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan
Shoko Okitsu, Toshiyuki Hikita, Aksara Thongprachum, Pattara Khamrin, Sayaka Takanashi, Satoshi Hayakawa, Niwat Maneekarn, Hiroshi Ushijima PII: DOI: Reference:
S1567-1348(18)30188-6 doi:10.1016/j.meegid.2018.04.011 MEEGID 3477
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
11 December 2017 1 April 2018 7 April 2018
Please cite this article as: Shoko Okitsu, Toshiyuki Hikita, Aksara Thongprachum, Pattara Khamrin, Sayaka Takanashi, Satoshi Hayakawa, Niwat Maneekarn, Hiroshi Ushijima , Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2017), doi:10.1016/j.meegid.2018.04.011
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Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan
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Shoko Okitsu1,2 , Toshiyuki Hikita3 , Aksara Thongprachum4 , Pattara Khamrin5,6 ,
Division of Microbiology, Department of Pathology and Microbiology, Nihon
2
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University School of Medicine, Tokyo, Japan
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Sayaka Takanashi2 , Satoshi Hayakawa1 , Niwat Maneekarn5,6 and Hiroshi Ushijima1,2
Department of Developmental Medical Sciences, School of International Health,
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Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Hikita Pediatric Clinic, Gunma, Japan
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Faculty of Public Health, Chiang Mai University, Thailand
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Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,
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3
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Thailand
Center of Excellence in Emerging and Re-emerging Diarrheal Viruses, Chiang Mai
*
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University, Chiang Mai, Thailand
Correspondence: Shoko Okitsu
Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi, Tokyo 173-8610, Japan Tel: +81-3-3972-8111 ext: 2263; Fax: +81-3-3972-9560 E-mail: [email protected]
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Abstract This study describes the detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains, which were collected from children with acute gastroenteritis in 2014 in Japan. Among 247 rotaviruses, one G8P[14] (strain 12597)
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and one G3P[3] (strain 12638) rotaviruses were detected. The genotypes of 11 gene
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segments of these two rotavirus strains (RVA/Human-wt/JPN/12597/2014/G8P[14] and RVA/Human-wt/JPN/12638/2014/G3P[3]) were characterized. The genotype
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constellation of strain 12597 was assigned to G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3,
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and this strain possessed a rare T9 genotype of NSP3 gene which has never been reported previously in combination with G8 genotype of VP7 gene. Molecular
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characterization and phylogenetic analysis suggested that the strain 12597 had the consensus G8P[14] backbone that originated from the rotaviruses of animal origins such
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as cows, deer, dogs, and cats. The genotype constellation of strain 12638 was identified as G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6. The VP7 and VP4 genotypes of strain
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12638 was similar to those of the Cat97-like strains, but the VP1, VP2, and VP3 were closely related to those of the AU-1-like strain. Interestingly, the NSP1 to NSP3 genes
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shared highest identities with those of a bat rotavirus (RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3] strain). These findings indicated that the strain 12638 was an intra-genotype reassortant strain among the AU-1-like strains, the Cat97-like strains and the bat strain. Interestingly, the strains 12597 and 12638 possessed the same N2 genotype of NSP2 gene. The results of this study support the possible roles of interspecies transmission and multiple reassortment events for generating the genetic diversity of rotavirus in human. Keywords: G3P[3], G8P[14], interspecies transmission, reassortment, rotavirus
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1. Introduction Group A rotaviruses (RVAs) are the major cause of acute gastroenteritis (AGE) in humans and animals worldwide. RVA is a member of the family Reoviridae, and its genome is composed of 11 double-stranded RNA segments that encode six viral
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structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and six non-structural proteins
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(NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6) (Estes and Greenberg, 2013). The VP7 and VP4 proteins comprise the outer layer of the virion, and the genome sequences of
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two coding genes are used for the classification of G and P genotypes, respectively
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(Estes and Greenberg, 2013). Currently, at least 35G (G1 to G35) and 50P (P[1] to P[50]) have been identified (RCWG; Rojas et al., 2017). In human, five most common
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RVA genotypes, the G1[8], G2P[4], G3P[8], G4P[8], and G9P[8], have been reported worldwide (Banyai et al., 2012). Additionally, G12P[8] is considered as the sixth
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common human RVA genotype (Doro et al., 2014). In addition to the binary classification system, a more complete genotype classification system was proposed and
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recommended by the Rotavirus Classification Working Group (Matthijnssens et al., 2011a; Matthijnssens et al., 2008b). This system gives the genome ordering of
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individual RVA strains as follows; Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx (x is Arabic numbers that
represents each genotype). Based on complete genome analyses
of RVA strains, three genotype constellations, genogroup 1 (Wa-like strains); G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, genogroup 2 (DS-1-like strains); G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and genogroup 3 (AU-1-like strain); G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 have been reported (Doan et al., 2015; Matthijnssens et al., 2008a; Nakagomi and Nakagomi, 1989; Tsugawa et al., 2015). Thus, this classification system provides detail information and increases understanding of the RVA genetic diversities when compared with the binary classification system.
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Several studies have reported the emergence of novel RVAs in humans and animals (Banyai et al., 2012; Doro et al., 2015). Emergence of novel RVA strains could be facilitated through an interspecies transmission and reassortment events of segmental nature of RVA genome between / within human and animal RVA strains (Martella et al.,
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2010).
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The G8P[14] RVA genotype has been reported sporadically in humans worldwide (Banyai et al., 2010; Gautam et al., 2015; Matthijnssens et al., 2009; Medici et al., 2015;
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Mijatovic-Rustempasic et al., 2015; Swiatek et al., 2010; Ward et al., 2016). Most of the
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G8P[14] strains are associated with the DS-1 genotyoe constellation (Heiman et al., 2008; Matthijnssens et al., 2008b). Complete genome analyses of P[14] strains,
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collected from humans and several animal species, revealed that the P[14] human strains may derive originally from sheep or other ungulates, and then introduced to
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humans by interspecies transmission (Matthijnssens et al., 2009). Human G3P[3] RVA strains have been sporadically detected in Thailand, Italy, Israel, the USA, Brazil, and
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China (Dong et al., 2016; Khamrin et al., 2006; Luchs et al., 2012; Matthijnssens et al., 2011b; Tsugawa and Hoshino, 2008), and in animal hosts, such as cats, dogs and rats
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(Ianiro et al., 2017; Matthijnssens et al., 2011b; Papp et al., 2015; Sachsenroder et al., 2014). However, reports on human G3P[3] strains are limited (Matthijnssens et al., 2011b; Tsugawa and Hoshino, 2008; Tsugawa et al., 2015). This study reports two unusual G8P[14] and G3P[3] RVA strains that were identified in the stools of children with AGE in 2014 in Japan. The molecular characterization of the 11 genomic segments of both strains was performed in order to assess the origin of these uncommon RVA strains detected in symptomatic children with diarrhea.
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2. Materials and Methods 2.1 Stool samples A total of 553 stool samples were collected from children with AGE, in Gunma Prefecture, Japan, between July 2014 and June 2015. The samples were stored at -30ºC
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until use.
2.2 Patient medical records
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The G8P[14] RVA strain 12597 was collected from a girl at the aged of 2 years and
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3 months, presenting fever and vomiting. She had abdominal pain and diarrhea twice per day. Her AGE severity (Vesikari) score was 6 points which was classified in a mild
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category (Ruuska and Vesikari, 1990). On the other hand, the G3P[3] RVA strain 12638 was collected from a boy at the aged of 4 years and 7 months, and his diarrheal episodes
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were maximum at 4 times per day for 14 days without vomiting and abdominal pain.
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His Vesikari score was 14 points which was classified in a severe category.
2.3 Viral RNA extraction and reverse transcription
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Stool samples were prepared as 10% (w/v) suspension in distilled water, and centrifuged at 10,600 xg for 10 min. Viral RNA was extracted from the supernatant of 10% suspension by the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The reverse transcription of extracted RNA was performed using the Rever Tra Ace (Toyobo, Osaka, Japan) enzyme and a random primer (Takara, Shiga, Japan) after pretreatment of RNA with 50% dimethylsulfoxide (Thongprachum et al., 2017).
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2.4 RVA screening and genotyping by PCR assay The cDNA was further amplified for the VP7 gene by using sBeg9 and VP7-1’ primers as described previously (Thongprachum et al., 2017). The G genotypes were assigned based on the nucleotide sequence analyses of PCR products. In addition, Con3
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and Con2 primers were used for amplification of VP4 gene (Gentsch et al., 1992), and
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characterized further by nucleotide sequencing for determining P genotypes.
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2.5 Analyses of eleven genome segments of the RVA strains
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The complete sequences of 11 genome segments were amplified and sequenced by using the primer pairs specific for each genome segment as described previously
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(Fujii et al., 2012; Gouvea et al., 1990; Khamrin et al., 2010; Matthijnssens et al., 2008a). In addition, the forward primer (VP4F-1_20 primer) newly designed in this
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study was also used in combination with the reverse VP4-End primer for amplification of VP4 sequences of P[14] and P[3] genotypes. The nucleotide sequences of primers
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used in this study are shown in Supplemental Table 1.
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2.6 DNA sequencing and phylogenetic analysis The PCR products were sequenced by using the BigDye Terminator Cycle Sequencing Kit (Perkin Elmer-Applied Biosystems, Inc., Foster City, CA., USA) on an automated DNA sequencer (ABI 3100; Perkin Elmer-Applied Biosystems, Inc., Foster City, CA., USA). The nucleotide sequences were compared with those of the reference strains deposited in the GenBank database. Multiple sequence alignments were conducted by the Clustal X program and phylogenetic trees were constructed according to the Maximum Likelihood Method based on the Kimura 2-parameter model using MEGA version 7 software (Kimura, 1980; Kumar et al., 2016). The nucleotide
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sequences of the G3P[3] and G8P[14] strains were deposited in the GenBank database under the following accession numbers: LC340010 to LC340031. The accession numbers of the studied and reference strains in the phylogenetic trees are shown in
Ethical clearance
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2.7
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Supplemental Table 2.
This study was approved by the ethical committee of the Nihon University School
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of Medicine (No. 22-15).
3. Results
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3.1 RVA detection
Among 553 stool samples, 247 (44.7%) were positive for RVA, of which one RVA
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strain was identified as G8P[14], and another was G3P[3] strain. The G8P[14] (strain 12597) and G3P[3] (strain 12638) were collected on December 1st and 11th , 2014,
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respectively. Among 247 RVA samples, G1P[8] was the most prevalent genotype with the detection rate of 82.6% (204 of 247 samples) followed by G2P[4] RVA at 2.4% (6 of
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247 samples). The G3 and G8 RVAs in combination with other P-types, except for the strains 12638 and 12597, were not detected in this study. 3.2 Full genome sequences of the G8P[14] strain 12597 The complete genome sequences of 11 segments of G8P[14] strain 12597 were determined and analyzed. The VP7 nucleotide sequence of the 12597 RVA strain shared 98.6 and 98.8% identities with those of the RVA/Human-wt/AUS/WAG8.2/2003/G8P[14] and the RVA/Cow-tc/Tokushima 9503/JPN/1995/G8P[11] strains, respectively. The phylogenetic analysis of VP7 gene indicated that the strain 12597 was clustered together and formed a monophyletic
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branch with two human and five animal G8 strains (Fig. 1A). It was interesting to note that the G8 strain 12597 detected in this study was distantly related to and formed a separated branch from the G8 strains reported previously from Japan in 2014 (RVA/Human-wt/JPN/To14-0/2014/G8P[8]) (Kondo et al., 2017) and other human G8
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strains reported recently from Africa and Asia with the nucleotide sequence identities
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ranging from 83.3 to 84.7% (Fig. 1A).
The VP4 of strain 12597 was most closely related to that of the
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RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] strain with the nucleotide sequence
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identity of 96.8%. Furthermore, these two sequences were located within lineage VII with 4 other P[14] strains (RVA/Cow-wt/JPN/Sun9/2000/G8P[14],
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RVA/Human-wt/GTM/2009726790/2009/G8P[14],
RVA/Human-wt/HON/2011825363/2011/G10P[14], and
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RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14] strains) from humans and animals (Fig. 1C). The VP4 of strain 12597 shared nucleotide sequence identities with five other
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P[14]-VII strains, ranging from 94.4 to 95.9%. The VP6 of strain 12597 was classified into the I2 genotype and was most closely
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related to the RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14] strain with the nucleotide sequence identity of 96.1% (Fig. 2A). Phylogenetic analysis revealed that the VP1 of strain 12597 was clustered together with two human strains (RVA/Human-wt/USA/VU12-13-176/2013/G1G6P[8] and RVA/Human-wt/AUS/CK2039/2008/G1P[8]), and a vaccine strain (RVA/Vaccine/USA/Rota-teq-W179-4/1992/G6P1A[8]) with the nucleotide sequence identity of 97.3% (Fig. 2B). The VP2 of strain 12597 shared 96.0% nucleotide sequence identity with the RVA/Cow-tc/ZAF/'O' Agent/1965/G8P[1] and RVA/Rhesus-tc/USA/PTRV/1990/G8P[1] (Fig. 2C). The VP3 of strain 12597 presented
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that the highest nucleotide sequence identity (97.1%) with strain RVA/Cat-tc/JPN/FRV537/2004/G6P[5] (Fig. 2D). For the genes that encode non-structural proteins, the NSP1 gene segment of strain 12597 presented the highest nucleotide identity with
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RVA/Rhesus-tc/USA/PTRV/1990/G1P[11] at 97.0% within the A3 genotype (Fig. 3A).
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The NSP2 gene analysis showed the highest nucleotide sequence identity between the strain 12597 and RVA/Simian-tc/USA/RRV/1975/G3P[3] at 97.6% (Fig. 3B).
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The NSP3 nucleotide sequence of strain 12597 was clustered to the rare T9
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genotype (Fig. 3C). Until now, only three strains, two bovine and one human strains, were reported to possess the T9 genotype (Abe et al., 2011; Ward et al., 2016). The
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NSP3 gene of strain 12597 was most closely related to that of the RVA/Human-wt/USA/2012741499/2012/G24P[14] strain at 97.5%. Two other strains
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(RVA/Cow-wt/JPN/AzuK-1/2006/G21P[29] and RVA/Cow-tc/JPN/Dai-10/2008/G24P[33] strains) with the T9 genotype were detected in
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cows in Japan (Abe et al., 2011); however, nucleotide sequence identities of these strains were lower than to that of the strain 2012741499 detected in a human, in the
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USA, at 95.0% and 94.5%, respectively. The NSP4 gene of strain 12597 was clustered together with the strains isolated in humans and cows (Fig. 3D). A highest nucleotide sequence identity at 98.1% was observed between the NSP4 of strain 12597 and that of RVA/Cow-tc/FRA/RF/1982/G6P[1]. The NSP5 nucleotide sequence of strain 12597 was closely related to those of bovine, canine, rhesus, and human strains with the highest nucleotide sequence identity of 98.3% with the RVA /Dog-wt/GER/88977/2012//G8P[1] (Fig. 3E).
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The nucleotide sequence identities of 11 gene segments of the G8P[14] strain 12597 in relation to the most closely related reference strains are shown in Table 1A.
3.3 Full genome sequences of the G3P[3] strain 12638
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The complete genome sequences of 11 segments of G3P[3] strain 12638 were
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determined and analyzed. The VP7 gene of strain 12638 was most closely related to that of RVA/Human-wt/BRA/R2638/2011/G3P[3] with the nucleotide sequence identity of
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97.7%, and also clustered together with those of other human strain
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(RVA/Human-tc/USA/HCR3A/1984/G3P[3]), and two canine strains (RVA/Dog-tc/USA/CU-1/1980/G3P[3] and RVA/Dog-tc/USA/A79-10/1979/G3P[3])
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(Fig. 1B). Phylogenetically, the strains located in the subcluster G3b were found in humans, and animals such as dogs, horses, cows, simian, goats, rabbits, and cats. The
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VP7 gene of strain 12638 was clustered together with those of several animal and human strains (the RVA/Cat-tc/AUS/Cat97/1984/G3P[3],
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RVA/Human-tc/ISR/Ro1845/1985/G3P[3], RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] strains) in the subcluster 3b2.
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The VP4 gene of strain 12638 was also most closely related to that of the RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] strain with the nucleotide identity of 96.7%. These two strains were also clustered together with a human strain (the Ro1845 strain) and a feline strain (the Cat97 strain) (Fig. 1D). The VP6 gene of strain 12638 was closely related to the VP6 of RVA strains with P[3] or P[9] genotypes, which were canine/feline RVAs and/or human RVAs (Fig. 2A). The VP6 gene segment of strain 12638 showed the highest nucleotide sequence identity of 97.0% with the RVA/Human-tc/ITA/PA260-97/1997/G3P[3]. The VP1 gene of 12638 presented the highest nucleotide sequence identity of 96.9% with the
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RVA/Human-wt/PRY/1256A/2009/G3G4P[9]. The VP2 gene of strain 12638 shared highest nucleotide sequence identity of 96.1% with the RVA/Human-tc/THA/T152/1998/G12P[9]. The VP3 gene of strain 12638 presented the highest nucleotide identity with those of three Paraguayan strains, RVA/Human-wt/
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PRY/1259A/2009/G12P[9], RVA/Human-wt/PRY/1701SR/2009/G1P[9], and
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RVA/Human-st/1257A/2009/G1P[9] with the nucleotide sequence identity of 98.3%. Phylogenetically, the VP1, VP2, and VP3 gene segments of strain 12638 were distinct
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from those of the Cat97-like strains (the Cat97, Ro1845, HCR3A strains) (Fig. 2B, 2C,
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and 2D, respectively), and similar to that of the T152 strain.
Furthermore, the NSP1, NSP2 and NSP3 genes of strain 12638 were most closely
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related to those of a bat RVA strain in South Africa
(RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3] strain) with the nucleotide sequence
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idnetities ranging from 96.6 to 98.5% (Fig. 3A , 3B, and 3C, respectively). Interestingly, the strain 12638 possessed the N2 as the NSP2 genotype, which was the same as the
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strain 12597.
The NSP4 gene of strain 12638 was most closely related to those of two canine
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(RVA/Dog-wt/HUN/135/2012/G3P[3], RVA/Dog-tc/ITA/RV52-96/1996/G3P[3] strains), one feline (RVA/Cat-tc/JPN/xxxx/FRV348/G3P[3] strain), and two human strains (RVA/Human-tc/ITA/PA260-97/1997/G3P[3] and RVA/Human-wt/FRA/R1486/2007/G3P[3] strains) with the nucleotide sequence identities ranging from 97.9% to 98.4% (Fig. 3D). The NSP5 gene of strain 12638 formed the same branch with the H6 genotype of the RVA/Human-wt/CHN/M2-102/2014/G3P[3] strain with the nucleotide identity of 97.6%. It should be noted that the M2-102 strain was reported to carry the simian RRV-like NSP5 gene segment (Dong et al., 2016) (Fig. 3E).
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The nucleotide sequence identities of 11 gene segments of the G3P[3] strain 12638 in relation to the most closely related reference strains are shown in Table 1B.
4. Discussion
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The present study demonstrated that the G1P[8] genotype was the major
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epidemic RVA in this area, during 2014 and 2015, and, two rare genotypes, G8P[14] and G3P[3], were identified.
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Until now, only three strains of RVA were reported to possess the T9 as the
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NSP3 genotype worldwide (Abe et al., 2011: Ward et al., 2016), and the strain 12597 is the forth strain which carries the T9 genotype. The T9 genotype was found initially in
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two cows (RVA/Cow-wt/JPN/AzuK-1/2006/G21P[29] and RVA/Cow-tc/JPN/Dai-10/2008/G24P[33] strains) in Japan (Abe et al., 2011), and then,
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another human strain (RVA/Human-wt/USA/2012741499/2012/G24P[14]) was reported in the USA (Ward et al., 2016). The strain 12597 is the second human strain possessing
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the T9 genotype. Previously very rare G24 or G21 genotypes were reported as G genotype combined with the T9 genotype, however, the G8 in this study were found to
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combine with the T9 genotype. The T9 gene of strain 12597 was more closely related to that of human strain detected in the USA than to those of the bovine strains detected in Japan.
Overall, the genotype constellation of the G8P[14] strain 12597 was G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3 (Table 2). The strain 12597 was most closely related to three human P[14] strains (RVA/Human-wt/ITA/PR1300/2004/G8P[14], RVA/Human-wt/ITA/PR1973/2009/G8P[14], and RVA/Human-wt/USA/2012741499/2012/G24P[14]), and one Roe deer strain (RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14]) by sharing 10 genotypes within the
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complete genome constellation. The PR1300, PR1973, and D110-15 strains had different NSP3 genes, and the 2012741499 strain possessed a VP7 genotype distinct from the strain 12597. It was found that the consensus P[14] strains possessed a G6/8-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-E2-H3 genotype constellation with
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reassortment of some genes (Banyai et al., 2010; Matthijnssens et al., 2009), and the
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genotype constellation presented by the P[14] strains were included within genogroup 2 (DS-1-like) (Heiman et al., 2008; Matthijnssens et al., 2008b). The strain 12597 also
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possessed the consensus P[14] genotype constellation with T9 gene reassortment. The
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G8P[14] strains that displayed the nucleotide sequence identities of 11 gene segments most closely related (96.0 to 98.8%) to the strain12597 were a wide range of host
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species including humans, rhesus, cows, deer, cats, and dogs (Table 1A). The human P[14] RVA was suspected to transmit from sheep or other ungulates as host origins to
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human(Matthijnssens et al., 2009). In Japan, even though two RVA strains possessing a P[14] genotype have been reported previously in cows
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(RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] and RVA/Cow-wt/JPN/Sun9/2000/G8P[14] strains), (Fukai et al., 2004; Masuda et al., 2014),
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it has never been reported previously in human. The strain 12597 reported in this study is the first P[14] RVA strain detected in human in Japan and it is distantly related to the P[14] strains detected in cows. Overall, the genotype constellation of the strain 12638 was
G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6 (Table 3). Complete gene sequence analysis revealed that G3P[3]/[9] stains detected in humans, cats, and dogs could be classified into three genogroups (Matthijnssens et al., 2011b; Papp et al., 2015). The Cat97-like genogroup displays the genotyope constellation of G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6 which includes those detected in feline,
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canine, and human (Ro1845 and HCR3A) G3P[3] RVAs (Tsugawa and Hoshino, 2008). The second genogroup (AU-1-like) comprises the G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 genotype constellation, and some feline/canine RVAs, and the human T152 G12P[9] strain belongs to this genogroup
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(Rahman et al., 2007). The third BA222-05-like genogroup possesses the genotype
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constellation of G3-P[9]-I2-R2-C2-M2-A3-N1/2-T6/3-E2-H3, and the human PAI58 and PAH136 strains (Table 3) as well as other feline strains were classified into this
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genogroup. On the other hand, additional heterogenous G3P[3] strains were also
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detected in horses, monkeys, and bats (Papp et al., 2015). The strain 12638 detected in this study carries the genotype constellation of the Cat-97-like strains for the VP7 and
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VP4 genes, similar to two canine-like human R2638 and PA260-97 strains, and the canine RV198-98 strain which have been reported as the Cat-97-like strains. However,
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the nucleotide sequences of VP1, VP2, and VP3 are found to be closely related to those of human RVA strains T152, AU-1 (AU-1 like genogroup). Previously, the human
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PA260-97 strain was reported to be a reassortant strain between the Cat-97 genogroup and the AU-1 genogroup (Matthijnssens et al., 2011b). Similarity, it is possible that the
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strain12638 detected in this study could be an intra-genotype reassortant strain between the Cat97-like genogroup and the AU-1 like genogroup. Furthermore, the NSP1, NSP2, and NSP3 gene segments of strain 12638 have been shown to be closely related to those of bat RVA (Table 1B). Taken together, the findings suggest that the strain 12638 may derive from multiple and intra-genotype reassortment events between the Cat-97 like strain, the AU-1-like G3P[3] strains, and a bat strain. In this study, two rare RVA strains were identified in two Japanese children and the molecular characterization of these two strains were reported. The sources of infection of these rare strains in the patients are still unknown. These two rare RVAs did
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not cause outbreaks in the study area suggesting that they could not adapt and replicate efficiently in human hosts at the present. The Information of the RVA infection in domestic animals like cats, dogs, rabbits, cows, horses, and sinantropic animals like rats, bats, and birds, are limited. Furthermore, contamination of the viruses in environmental
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water like rivers, ponds might be the sources of viral transmission to humans. The
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epidemiological surveillance is needed to monitor circulating RVA genotypes and
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spread of unusual strains in humans.
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5. Conclusion
In 2014, two unusual G8P[14] and G3P[3] human RVA strains were detected in
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the same area in Japan. Both strains were rare RVAs and have never been reported previously in human in Japan. Furthermore, the complete genome sequence analyses of
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these two strains showed new genotype constellations. Molecular characterization demonstrated that the strains 12597 and 12638 detected in this study possessed the
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backbone of the G8P[14] and G3P[3] genotype constellation, respectively, with some gene reassortments. The NSP3 gene segment of the G8P[14] strain 12597 belongs to an
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uncommon T9 genotype, and it is the second human strain with the T9 genotype reported. Analyses of the nucleotide sequences of VP1 to VP7 and NSP4 of 12638 G3P[3] strain revealed that it was a reassortant strain between the Cat-97 genogroup and AU-1 genogroup, however, the other NSP1 to NSP3 gene segments were closely related to that of the bat RVA. Taken together, the phylogenetic analyses showed that genetic diversity of the strain 12638 could be resulted from interspecies transmission and multiple reassortment events.
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Acknowledgements This work was supported by the Japan Society for the Promotion of Science JSPS Kakenhi (16H05360), and the Public Foundation of the Vaccination Research Center
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(2015-34).
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Table 1. Nucleotide sequence identities of the G8P[14] strain 12597 (A) and the G3P[3] strain 12638 (B) with the most closely related reference strains. (A) M ost Gene
closely
related
Genotype
% Host Identity
Tokushima9503
Cow
VP4
P[14]
Tottori-SG
Cow
VP6
I2
D110-15
Roe deer
R2
CK-20039
Human
96.1
97.3
Vaccine
MA
Rota Teq-WI-79-4 C2
O'Agent
Cow
96.0
VP3
M2
FRV537
Cat
97.0
NSP1
A3
PTRV
Rhesus
97.0
NSP2
N2
RRV
Simian
97.6
T9
2012741499
Human
97.5
E2
RF
Cow
98.1
88977
Dog
98.3
EP T
AC C
NSP4
ED
VP2
NSP3
(B)
96.8
Human
NU
VU12-13-176 VP1
98.8
RI
G8
SC
VP7
PT
strains
NSP5
H3
Gene
Genotype
M ost
closely
related
% Host
strains
Identity
VP7
G3
R2638
Human
97.7
VP4
P[3]
RV198-95
Dog
96.7
VP6
I3
PA260-97
Human
97.0
VP1
R3
1256A
Human
96.9
VP2
C3
T152
Human
96.1
1257A
Human
1701SR
Human
98.3
1259A
Human
PT
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M3
A9
LUS12-14
Bat
NSP2
N2
LUS12-14
Bat
NSP3
T3
LUS12-14
NSP4
E3
Bat
H6
M 2-102
ED EP T AC C
96.6
98.4
Human
MA
NSP5
98.5
Dog
NU
RV52-96 R1486
98.5
SC
NSP1
RI
VP3
Human
97.6
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Table 2. Complete genomic constellation of the G8P[14] strain 12597 and the reference strains VP
V
V
V
V
NS
NS
NS
NS
NS
4
P6
P1
P2
P3
P1
P2
P3
P4
P5
P
I
R
C
M
A
N
P[ G8
14/G8P[14]
14]
RVA/Human-wt/ITA/PR1300/20
P[1 G8
04/G8P[14]
I2 4] P[1
G8 4]
RVA/Human-wt/USA/20127414
P[1
EP T
4]
P[1
G8
/2015/G8P[14]
AC C
RVA/Sheep-tc/ESP/OVR762/200
2
R
C
A3
P[1
G8
2
2
2
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
2/G8P[14]
4]
2
2
2
RVA/Rhesus-tc/USA/PTRV/199
P[1
R
C
M
I2
0/G8P[1]
]
2
2
2
RVA/Human-tc/KEN/B12/1987/
P[1
R
C
M
2
2
2
R
C
M
2
2
2
G8 G8P[1]
I2 ]
RVA/Human-tc/ITA/PA169/1988
P[1 G6
/G6P[14]
I2 4]
genotypes
T9
E2
H3
A3
N2
T6
E2
H3
10
A3
N2
T6
E2
H3
10
A3
N2
T9
E2
H3
10
A3
N2
T6
E2
H3
10
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A1
I2
G8
Shared H
M
I2 4]
N2
E
2
I2
ED
G24
RVA/Roe_deer-wt/SLO/D110-15
2
M
I2
09/G8P[14]
99/2012/G24P[14]
C
MA
RVA/Human-wt/ITA/PR1973/20
R I2
T
RI
RVA/Human-wt/JPN/12597/20
SC
G
NU
Strain
PT
VP7
1
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90/2009/G8P[14]
4]
RVA/Human-wt/HON/20118253
R
C
M
A1
2
2
2
3
R
C
M
I2
P[1 G10
I2
63/2011/G10P[14]
4]
2
2
2
RVA/Human-wt/USA/20128411
P[1
R
C
M
3
2
2
R
C
M
2
2
R
C
G8
I2 4] P[1
G15 13/G15P[14]
I2 4]
RVA/Human-wt/HUN/BP1062/2
P[1 I2
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H2
9
T6
E2
H2
9
N2
T6
E2
H3
9
N2
T6
E2
H3
8
N2
T6
E2
H3
8
N2
T6
H3
8
A3
M
A1
004/G8P[14]
4]
2
2
2
1
RVA/Antelope-wt/ZAF/RC-18/2
P[1
R
C
M
A1
G6 008/G6P[14]
I2
4]
RVA/Human-wt/HUN/BP1879/2
P[1
003/G6P[14]
4]
EP T
RVA/Guanaco-wt/ARG/Chubut/
P[1
G8
1999/G8P[14]
AC C
RVA/Vaccine/USA/RotaTeq-WI7
2
2
2
1
R
C
M
A1
2
2
2
1
R
C
M
I2
ED
G6
I2 4] P[8
G6
E1 A3
5
2
2
R
C
M
I2
2
A3
N2
T6
E2
H3
8
A3
N2
T6
E2
H3
8
N2
T6
E2
H3
8
N2
T6
E2
H3
8
9-4/1992/G6P1A[8]
]
2
2
2
RVA/Human-wt/AUS/CK20039/
P[8
R
C
M
2
2
2
R
C
M
A1
2
2
2
1
R
C
M
G1
2008/G1P[8]
I2 ]
RVA/Cow-tc/ZAF/'O'
P[1 G8
Agent/1965/G8P[1]
I2 ]
RVA/Cow-tc/FRA/RF/1982/G6P
P[1 G6
[1]
I2 ]
A3 2
2
N2
2
MA
G8
T6
SC
RVA/Cow-wt/JPN/Tottori-SG/20
NU
74/2012/G8P[14]
N2
PT
P[1 G8
RI
RVA/Human-wt/GTM /20097267
2
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24P[33]
3]
RVA/Cow-wt/JPN/AzuK-1/2006/
R
C
M
A1
2
2
2
3
R
C
M
A1
I2
P[2 G21
I2
G21P[29]
9]
2
2
2
RVA/Goat-wt/ARG/0040/2011/G
P[8
R
C
M
5
2
2
R
C
M
2
2
R
C
G8 8P[1]
I2 ]
RVA/Human-wt/USA/VU12-13-
G1,
P[8
RVA/Cat-tc/JPN/FRV537/2004/
] P[5 I2
M
]
2
2
2
RVA/Human-wt/JPN/To14-0/201
P[8
R
C
M
G8
I2
]
RVA/Guanaco-wt/ARG/Rio_Neg
P[1
ro/1998/G8P[1]
]
EP T
RVA/Human-tc/USA/DS-1/1976/
P[4
G2
G2P[4]
AC C
RVA/Simian-tc/USA/RRV/1975/
P[3
G3
]
RAV/Dog-wt/GER/88977/2013/
P[1 G8
G8P[1]
2
R
C
M
5
2
2
R
C
M
2
2
2
R
C
M
2
I2
P[14]
8
H3
7
N2
T6
N2
T6
E2
H3
7
N2
T6
E2
H3
7
N2
T2
E2
H2
7
N2
T6
H3
6
3
A1
E1
1
2
A2
N2
T2
E2
H2
6
A9
N2
T3
E3
H6
3
A3
N2
T6
E2
H3
-
-
-
-
-
-
-
-
-
-
3
-
-
R
C
M
2
2
2
-
-
-
I2 4]
RVA/Human-wt/AUS/WAG8.2/2
H3
2
P[1 G8
3
E2
C
]
RVA/Cow-tc/JPN/Sun9/2000/G8
P[1 G8
003/G8P[14]
2
I2
G3P[3]
T9
A1
A2
2
I2 ]
N2
2
I2
ED
G8
8
E1
A3
G6P[5]
4/G8P[8]
H3
1
MA
G6
E2
SC
G6*
NU
176/2013/G1G6P[8]
T9
3
A3
I2
N2
PT
P[3 G24
RI
RVA/Cow-tc/JPN/Dai-10/2008/G
4]
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RVA/Cow-tc/JPN/Tokushima950
P[1 G8
3/xxxx/G8P[11]
-
-
-
-
-
-
-
-
-
1]
Gray color indicates the strains that sharing the genotype with the strain 12597. -: Genotypes are not available in the GenBank database.
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ED
MA
NU
SC
RI
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*: The VU12-13-176 strain was reported to possess mixed genotype of VP
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Table 3. Complete genomic constellation of the G3P[3]strain 12638 and the reference strains VP
V
V
V
V
NSP
NS
NS
NS
NS
4
P6
P1
P2
P3
1
P2
P3
P4
P5
P
I
R
C
M
A
N
P[3 G3
/2014/G3P[3]
]
RVA/Dog-wt/HUN/135/2012
P[3 G3
/G3P[3]
RVA/Dog-tc/ITA/RV198-95/
P[3
ED
EP T
AC C
R
C
M
A9
3
3
C
M
3
3
3
R
C
M
3
2
3
R
C
M
3
2
3
R
C
M
I3
P[3
I3
]
RVA/Dog-tc/USA/CU-1/198
3
R
]
G3
996/G3P[3]
3
MA
]
RVA/Dog-tc/ITA/RV52-96/1
3
I3
7/1997/G3P[3]
G3
M
3
P[3 G3
1995/G3P[3]
C
I3 ]
RVA/Human-tc/ITA/PA260-9
R I3
P[3
G3
I3
2/G3P[3]
]
3
2
3
RVA/Dog-tc/AUS/K9/1981/
P[3
R
C
M
G3
I3
G3P[3]
]
3
2
3
RVA/Dog-tc/USA/A79-10/19
P[3
R
C
M
3
2
3
R
C
M
3
2
3
G3 79/G3P[3]
I3 ]
RVA/Cat-tc/AUS/Cat97/1984
P[3 G3
/G3P[3]
I3 ]
T
RI
RVA/Human-wt/JPN/12638
N2
SC
G
E
Shared H genotypes
T3
E3
H6
A15
N2
T3
E3
H6
10
A15
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
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Strain
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VP7
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1985/G3P[3]
]
RVA/Human-tc/USA/HCR3
R
C
M
3
2
3
R
C
M
I3
P[3 G3
I3
A/1984/G3P[3]
]
3
2
3
RVA/Horse-wt/ARG/E3198/
P[3
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
G3
I3 ] P[3
G3 2/2014/G3P[3]
I3 ]
RVA/Bat-tc/CHN/M SLH14/2
P[3 G3
I8 3
RVA/Simian-tc/USA/RRV/19
P[3
R
P[3
94/G3P[3]
]
P[9
EP T
RVA/Human-tc/CHN/L621/2
G3
006/G3P[9]
AC C
P[9
G3
/2011/G3P[9]
H6
10
A9
N2
T3
E3
H6
10
A9
N3
E3
H6
10
T3
E3
H6
10
2
3
3
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
I3
T3
E3
H6
9
A9
N2
T3
E3
H6
9
A15
N3
T3
E3
H6
9
A3
N3
T3
E3
H6
8
A3
N3
T3
E3
H6
8
N3
T3
E3
H6
8(7)
A9
N2
T3
E2
H3
7
A3
N3
T3
E3
H3
7
3
3
R
C
M
A3,
3
3
3
A1*
R
C
M
2
2
3
R
C
M
P[9
2009/G3G4P[9]
G4*
]
I3
RVA/Bat-wt/ZM B/LUS12-14
P[3 G3
/2012/G3P[3]
I3 ]
RVA/Human-tc/JPN/AU-1/1
P[9 G3
I3 ]
3
3
N3
N3
3
G3,
3
T3
A9
]
RVA/Human-wt/PRY/1256A/
982/G3P[9]
M
I3
]
RVA/Human-wt/CHN/E2451
C
I3
ED
G3
3
I2 ]
RVA/Cat-tc/JPN/FRV348/19
3
MA
]
G3
E3
A9
012/G3P[3]
75/G3P[3]
T3
SC
RVA/Human-wt/CHN/M 2-10
N2
NU
2008/G3P[3]
A9
PT
P[3 G3
RI
RVA/Human-tc/ISR/Ro1845/
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998/G12P[9]
]
RVA/Human-wt/PRY/1259A/
R
C
M
3
3
3
R
C
M
I3
P[9 G12
I3
2009/G12P[9]
]
3
3
3
RVA/Human-wt/PRY/1701S
P[9
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
G1
I3 ] P[9
G1 2009/G1P[9]
I3 ]
RVA/Cat-tc/AUS/Cat2/1984/
P[9 G3
I3 3
RVA/Rhesus-tc/USA/TUCH/
P[2
R
P[9
5/G3P[9]
]
P[9
EP T
RVA/Human-wt/ITA/PAI58/1
G3
996/G3P[9]
AC C
P[9
G3
6/1996/G3P[9]
]
RVA/Human-wt/FRA/R1486/
P[3 G3
2007/G3P[3]
H6
7
A3
N3
T3
E3
H6
6
A3
N3
E3
H6
6
T3
E3
H6
6
3
3
3
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
I2
T3
N3
A3
N1
T6
E3
H3
5
A9
N1
T1
E1
H1
5
A3
N1
T3
E2
H3
2
A3
N2
T6
E2
H3
2
A3
N1
T6
E2
H3
2
2
2
2
I3
-
-
-
-
-
-
E3
-
I8
-
-
-
-
-
-
E3
-
-
-
-
-
-
-
-
-
-
]
RVA/Human-wt/THA/CM H2
P[3 G3
22/2001/G3P[3]
]
RVA/Human-wt/BRA/R2638
P[3 G3
/2011/G3P[3]
M
I2
]
RVA/Human-wt/ITA/PAH13
C
I2
ED
G3
3
I9 4]
RVA/Cat-wt/ITA/BA222/200
2
MA
]
G3
E3
A3
G3P[9]
2002/G3P[24]
T3
SC
RVA/Human-wt/PRY/1257A/
N3
NU
R/2009/G1P[9]
A12
PT
P[9 G12
RI
RVA/Human-tc/THA/T152/1
]
Gray color indicates the strain that sharing the genotypes with the strain 12638.
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-: Genotypes are not available in the GenBank database.
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EP T
ED
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NU
SC
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*: The 1256A strain was reported to possess mixed genotypes of VP7 and NSP1.
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Figure legend. Fig 1. Phylogenetic trees of the viral structural protein VP7 and VP4 genes of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap
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values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of
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nucleotide substitutions per site. The strains 12597 and 12638 are indicated by the black dots. (A): VP7-G8 (841 nucleotides); (B): VP7-G3 (841 nucleotides); (C): VP4-P[14]
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(1975 nucleotides); 1D; VP4-P[3] (2290 nucleotides). Within 1B, arbitrary subclusters
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(b1 and B2) were indicated.
Fig 2. Phylogenetic trees of the vial structural protein VP 6, VP1, VP2, and VP3 genes
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of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap
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values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of nucleotide substitutions per site. (A): VP6 (1154 nucleotides); (B): VP1 (3208
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nucleotide); (C): VP2 (2631 nucleotides); (D): VP3 (2507 nucleotides). Fig.3. Phylogenetic trees of the non-structural protein NSP1, NSP2, NSP3, NSP4 and
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NSP5 genes of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of nucleotide substitutions per site. The strains 12597 and 12638 are indicated by the black dots. (A): NSP1 (1477 nucleotides); (B): NSP2 (953 nucleotides); (C): NSP3 (942 nucleotides); (D): NSP4 (527 nucleotides); (E): NSP5 (599 nucleotides).
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AC C
EP T
ED
MA
NU
SC
RI
PT
Fig . 1A
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AC C
EP T
ED
MA
NU
SC
RI
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Fig . 1B
AC C
EP T
ED
MA
NU
SC
RI
PT
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Fig . 1 (C) VP4-P[14]
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AC C
EP T
ED
MA
NU
SC
RI
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Fig . 1 (D) VP4-P[3]
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Fig . 2
AC C
EP T
ED
MA
NU
SC
RI
PT
(A) VP6
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Fig . 2
AC C
EP T
ED
MA
NU
SC
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(B) VP1
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Fig . 2
AC C
EP T
ED
MA
NU
SC
RI
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(C) VP2
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Fig . 2
AC C
EP T
ED
MA
NU
SC
RI
PT
(D) VP3
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Fig . 3
AC C
EP T
ED
MA
NU
SC
RI
PT
(A) NSP1
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Fig . 3
AC C
EP T
ED
MA
NU
SC
RI
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(B) NSP2
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Fig .3
AC C
EP T
ED
MA
NU
SC
RI
PT
(C) NSP3
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Fig .3
AC C
EP T
ED
MA
NU
SC
RI
PT
(D) NSP4
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Fig . 3
AC C
EP T
ED
MA
NU
SC
RI
PT
(E) NSP5
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Highlights Detection of two rare rotavirus strains, G8P[14] and G3P[3], in Japanese children.
The G8P[14] strain 12597 possessed a rare T9 NSP3 genotype.
The G3P[3] strain 12638 was an intra-genotype reassortant strain.
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NU
SC
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Shoko Okitsu, Toshiyuki Hikita, Aksara Thongprachum, Pattara Khamrin, Sayaka Takanashi, Satoshi Hayakawa, Niwat Maneekarn, Hiroshi Ushijima PII: DOI: Reference:
S1567-1348(18)30188-6 doi:10.1016/j.meegid.2018.04.011 MEEGID 3477
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
11 December 2017 1 April 2018 7 April 2018
Please cite this article as: Shoko Okitsu, Toshiyuki Hikita, Aksara Thongprachum, Pattara Khamrin, Sayaka Takanashi, Satoshi Hayakawa, Niwat Maneekarn, Hiroshi Ushijima , Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2017), doi:10.1016/j.meegid.2018.04.011
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Detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains collected from children with acute gastroenteritis in Japan
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Shoko Okitsu1,2 , Toshiyuki Hikita3 , Aksara Thongprachum4 , Pattara Khamrin5,6 ,
Division of Microbiology, Department of Pathology and Microbiology, Nihon
2
NU
University School of Medicine, Tokyo, Japan
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1
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Sayaka Takanashi2 , Satoshi Hayakawa1 , Niwat Maneekarn5,6 and Hiroshi Ushijima1,2
Department of Developmental Medical Sciences, School of International Health,
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Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Hikita Pediatric Clinic, Gunma, Japan
4
Faculty of Public Health, Chiang Mai University, Thailand
5
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,
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3
6
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Thailand
Center of Excellence in Emerging and Re-emerging Diarrheal Viruses, Chiang Mai
*
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University, Chiang Mai, Thailand
Correspondence: Shoko Okitsu
Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi, Tokyo 173-8610, Japan Tel: +81-3-3972-8111 ext: 2263; Fax: +81-3-3972-9560 E-mail: [email protected]
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Abstract This study describes the detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains, which were collected from children with acute gastroenteritis in 2014 in Japan. Among 247 rotaviruses, one G8P[14] (strain 12597)
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and one G3P[3] (strain 12638) rotaviruses were detected. The genotypes of 11 gene
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segments of these two rotavirus strains (RVA/Human-wt/JPN/12597/2014/G8P[14] and RVA/Human-wt/JPN/12638/2014/G3P[3]) were characterized. The genotype
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constellation of strain 12597 was assigned to G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3,
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and this strain possessed a rare T9 genotype of NSP3 gene which has never been reported previously in combination with G8 genotype of VP7 gene. Molecular
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characterization and phylogenetic analysis suggested that the strain 12597 had the consensus G8P[14] backbone that originated from the rotaviruses of animal origins such
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as cows, deer, dogs, and cats. The genotype constellation of strain 12638 was identified as G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6. The VP7 and VP4 genotypes of strain
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12638 was similar to those of the Cat97-like strains, but the VP1, VP2, and VP3 were closely related to those of the AU-1-like strain. Interestingly, the NSP1 to NSP3 genes
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shared highest identities with those of a bat rotavirus (RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3] strain). These findings indicated that the strain 12638 was an intra-genotype reassortant strain among the AU-1-like strains, the Cat97-like strains and the bat strain. Interestingly, the strains 12597 and 12638 possessed the same N2 genotype of NSP2 gene. The results of this study support the possible roles of interspecies transmission and multiple reassortment events for generating the genetic diversity of rotavirus in human. Keywords: G3P[3], G8P[14], interspecies transmission, reassortment, rotavirus
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1. Introduction Group A rotaviruses (RVAs) are the major cause of acute gastroenteritis (AGE) in humans and animals worldwide. RVA is a member of the family Reoviridae, and its genome is composed of 11 double-stranded RNA segments that encode six viral
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structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and six non-structural proteins
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(NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6) (Estes and Greenberg, 2013). The VP7 and VP4 proteins comprise the outer layer of the virion, and the genome sequences of
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two coding genes are used for the classification of G and P genotypes, respectively
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(Estes and Greenberg, 2013). Currently, at least 35G (G1 to G35) and 50P (P[1] to P[50]) have been identified (RCWG; Rojas et al., 2017). In human, five most common
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RVA genotypes, the G1[8], G2P[4], G3P[8], G4P[8], and G9P[8], have been reported worldwide (Banyai et al., 2012). Additionally, G12P[8] is considered as the sixth
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common human RVA genotype (Doro et al., 2014). In addition to the binary classification system, a more complete genotype classification system was proposed and
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recommended by the Rotavirus Classification Working Group (Matthijnssens et al., 2011a; Matthijnssens et al., 2008b). This system gives the genome ordering of
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individual RVA strains as follows; Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx (x is Arabic numbers that
represents each genotype). Based on complete genome analyses
of RVA strains, three genotype constellations, genogroup 1 (Wa-like strains); G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, genogroup 2 (DS-1-like strains); G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and genogroup 3 (AU-1-like strain); G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 have been reported (Doan et al., 2015; Matthijnssens et al., 2008a; Nakagomi and Nakagomi, 1989; Tsugawa et al., 2015). Thus, this classification system provides detail information and increases understanding of the RVA genetic diversities when compared with the binary classification system.
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Several studies have reported the emergence of novel RVAs in humans and animals (Banyai et al., 2012; Doro et al., 2015). Emergence of novel RVA strains could be facilitated through an interspecies transmission and reassortment events of segmental nature of RVA genome between / within human and animal RVA strains (Martella et al.,
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2010).
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The G8P[14] RVA genotype has been reported sporadically in humans worldwide (Banyai et al., 2010; Gautam et al., 2015; Matthijnssens et al., 2009; Medici et al., 2015;
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Mijatovic-Rustempasic et al., 2015; Swiatek et al., 2010; Ward et al., 2016). Most of the
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G8P[14] strains are associated with the DS-1 genotyoe constellation (Heiman et al., 2008; Matthijnssens et al., 2008b). Complete genome analyses of P[14] strains,
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collected from humans and several animal species, revealed that the P[14] human strains may derive originally from sheep or other ungulates, and then introduced to
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humans by interspecies transmission (Matthijnssens et al., 2009). Human G3P[3] RVA strains have been sporadically detected in Thailand, Italy, Israel, the USA, Brazil, and
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China (Dong et al., 2016; Khamrin et al., 2006; Luchs et al., 2012; Matthijnssens et al., 2011b; Tsugawa and Hoshino, 2008), and in animal hosts, such as cats, dogs and rats
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(Ianiro et al., 2017; Matthijnssens et al., 2011b; Papp et al., 2015; Sachsenroder et al., 2014). However, reports on human G3P[3] strains are limited (Matthijnssens et al., 2011b; Tsugawa and Hoshino, 2008; Tsugawa et al., 2015). This study reports two unusual G8P[14] and G3P[3] RVA strains that were identified in the stools of children with AGE in 2014 in Japan. The molecular characterization of the 11 genomic segments of both strains was performed in order to assess the origin of these uncommon RVA strains detected in symptomatic children with diarrhea.
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2. Materials and Methods 2.1 Stool samples A total of 553 stool samples were collected from children with AGE, in Gunma Prefecture, Japan, between July 2014 and June 2015. The samples were stored at -30ºC
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until use.
2.2 Patient medical records
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The G8P[14] RVA strain 12597 was collected from a girl at the aged of 2 years and
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3 months, presenting fever and vomiting. She had abdominal pain and diarrhea twice per day. Her AGE severity (Vesikari) score was 6 points which was classified in a mild
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category (Ruuska and Vesikari, 1990). On the other hand, the G3P[3] RVA strain 12638 was collected from a boy at the aged of 4 years and 7 months, and his diarrheal episodes
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were maximum at 4 times per day for 14 days without vomiting and abdominal pain.
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His Vesikari score was 14 points which was classified in a severe category.
2.3 Viral RNA extraction and reverse transcription
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Stool samples were prepared as 10% (w/v) suspension in distilled water, and centrifuged at 10,600 xg for 10 min. Viral RNA was extracted from the supernatant of 10% suspension by the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The reverse transcription of extracted RNA was performed using the Rever Tra Ace (Toyobo, Osaka, Japan) enzyme and a random primer (Takara, Shiga, Japan) after pretreatment of RNA with 50% dimethylsulfoxide (Thongprachum et al., 2017).
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2.4 RVA screening and genotyping by PCR assay The cDNA was further amplified for the VP7 gene by using sBeg9 and VP7-1’ primers as described previously (Thongprachum et al., 2017). The G genotypes were assigned based on the nucleotide sequence analyses of PCR products. In addition, Con3
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and Con2 primers were used for amplification of VP4 gene (Gentsch et al., 1992), and
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characterized further by nucleotide sequencing for determining P genotypes.
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2.5 Analyses of eleven genome segments of the RVA strains
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The complete sequences of 11 genome segments were amplified and sequenced by using the primer pairs specific for each genome segment as described previously
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(Fujii et al., 2012; Gouvea et al., 1990; Khamrin et al., 2010; Matthijnssens et al., 2008a). In addition, the forward primer (VP4F-1_20 primer) newly designed in this
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study was also used in combination with the reverse VP4-End primer for amplification of VP4 sequences of P[14] and P[3] genotypes. The nucleotide sequences of primers
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used in this study are shown in Supplemental Table 1.
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2.6 DNA sequencing and phylogenetic analysis The PCR products were sequenced by using the BigDye Terminator Cycle Sequencing Kit (Perkin Elmer-Applied Biosystems, Inc., Foster City, CA., USA) on an automated DNA sequencer (ABI 3100; Perkin Elmer-Applied Biosystems, Inc., Foster City, CA., USA). The nucleotide sequences were compared with those of the reference strains deposited in the GenBank database. Multiple sequence alignments were conducted by the Clustal X program and phylogenetic trees were constructed according to the Maximum Likelihood Method based on the Kimura 2-parameter model using MEGA version 7 software (Kimura, 1980; Kumar et al., 2016). The nucleotide
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sequences of the G3P[3] and G8P[14] strains were deposited in the GenBank database under the following accession numbers: LC340010 to LC340031. The accession numbers of the studied and reference strains in the phylogenetic trees are shown in
Ethical clearance
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2.7
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Supplemental Table 2.
This study was approved by the ethical committee of the Nihon University School
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of Medicine (No. 22-15).
3. Results
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3.1 RVA detection
Among 553 stool samples, 247 (44.7%) were positive for RVA, of which one RVA
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strain was identified as G8P[14], and another was G3P[3] strain. The G8P[14] (strain 12597) and G3P[3] (strain 12638) were collected on December 1st and 11th , 2014,
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respectively. Among 247 RVA samples, G1P[8] was the most prevalent genotype with the detection rate of 82.6% (204 of 247 samples) followed by G2P[4] RVA at 2.4% (6 of
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247 samples). The G3 and G8 RVAs in combination with other P-types, except for the strains 12638 and 12597, were not detected in this study. 3.2 Full genome sequences of the G8P[14] strain 12597 The complete genome sequences of 11 segments of G8P[14] strain 12597 were determined and analyzed. The VP7 nucleotide sequence of the 12597 RVA strain shared 98.6 and 98.8% identities with those of the RVA/Human-wt/AUS/WAG8.2/2003/G8P[14] and the RVA/Cow-tc/Tokushima 9503/JPN/1995/G8P[11] strains, respectively. The phylogenetic analysis of VP7 gene indicated that the strain 12597 was clustered together and formed a monophyletic
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branch with two human and five animal G8 strains (Fig. 1A). It was interesting to note that the G8 strain 12597 detected in this study was distantly related to and formed a separated branch from the G8 strains reported previously from Japan in 2014 (RVA/Human-wt/JPN/To14-0/2014/G8P[8]) (Kondo et al., 2017) and other human G8
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strains reported recently from Africa and Asia with the nucleotide sequence identities
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ranging from 83.3 to 84.7% (Fig. 1A).
The VP4 of strain 12597 was most closely related to that of the
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RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] strain with the nucleotide sequence
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identity of 96.8%. Furthermore, these two sequences were located within lineage VII with 4 other P[14] strains (RVA/Cow-wt/JPN/Sun9/2000/G8P[14],
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RVA/Human-wt/GTM/2009726790/2009/G8P[14],
RVA/Human-wt/HON/2011825363/2011/G10P[14], and
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RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14] strains) from humans and animals (Fig. 1C). The VP4 of strain 12597 shared nucleotide sequence identities with five other
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P[14]-VII strains, ranging from 94.4 to 95.9%. The VP6 of strain 12597 was classified into the I2 genotype and was most closely
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related to the RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14] strain with the nucleotide sequence identity of 96.1% (Fig. 2A). Phylogenetic analysis revealed that the VP1 of strain 12597 was clustered together with two human strains (RVA/Human-wt/USA/VU12-13-176/2013/G1G6P[8] and RVA/Human-wt/AUS/CK2039/2008/G1P[8]), and a vaccine strain (RVA/Vaccine/USA/Rota-teq-W179-4/1992/G6P1A[8]) with the nucleotide sequence identity of 97.3% (Fig. 2B). The VP2 of strain 12597 shared 96.0% nucleotide sequence identity with the RVA/Cow-tc/ZAF/'O' Agent/1965/G8P[1] and RVA/Rhesus-tc/USA/PTRV/1990/G8P[1] (Fig. 2C). The VP3 of strain 12597 presented
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that the highest nucleotide sequence identity (97.1%) with strain RVA/Cat-tc/JPN/FRV537/2004/G6P[5] (Fig. 2D). For the genes that encode non-structural proteins, the NSP1 gene segment of strain 12597 presented the highest nucleotide identity with
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RVA/Rhesus-tc/USA/PTRV/1990/G1P[11] at 97.0% within the A3 genotype (Fig. 3A).
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The NSP2 gene analysis showed the highest nucleotide sequence identity between the strain 12597 and RVA/Simian-tc/USA/RRV/1975/G3P[3] at 97.6% (Fig. 3B).
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The NSP3 nucleotide sequence of strain 12597 was clustered to the rare T9
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genotype (Fig. 3C). Until now, only three strains, two bovine and one human strains, were reported to possess the T9 genotype (Abe et al., 2011; Ward et al., 2016). The
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NSP3 gene of strain 12597 was most closely related to that of the RVA/Human-wt/USA/2012741499/2012/G24P[14] strain at 97.5%. Two other strains
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(RVA/Cow-wt/JPN/AzuK-1/2006/G21P[29] and RVA/Cow-tc/JPN/Dai-10/2008/G24P[33] strains) with the T9 genotype were detected in
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cows in Japan (Abe et al., 2011); however, nucleotide sequence identities of these strains were lower than to that of the strain 2012741499 detected in a human, in the
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USA, at 95.0% and 94.5%, respectively. The NSP4 gene of strain 12597 was clustered together with the strains isolated in humans and cows (Fig. 3D). A highest nucleotide sequence identity at 98.1% was observed between the NSP4 of strain 12597 and that of RVA/Cow-tc/FRA/RF/1982/G6P[1]. The NSP5 nucleotide sequence of strain 12597 was closely related to those of bovine, canine, rhesus, and human strains with the highest nucleotide sequence identity of 98.3% with the RVA /Dog-wt/GER/88977/2012//G8P[1] (Fig. 3E).
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The nucleotide sequence identities of 11 gene segments of the G8P[14] strain 12597 in relation to the most closely related reference strains are shown in Table 1A.
3.3 Full genome sequences of the G3P[3] strain 12638
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The complete genome sequences of 11 segments of G3P[3] strain 12638 were
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determined and analyzed. The VP7 gene of strain 12638 was most closely related to that of RVA/Human-wt/BRA/R2638/2011/G3P[3] with the nucleotide sequence identity of
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97.7%, and also clustered together with those of other human strain
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(RVA/Human-tc/USA/HCR3A/1984/G3P[3]), and two canine strains (RVA/Dog-tc/USA/CU-1/1980/G3P[3] and RVA/Dog-tc/USA/A79-10/1979/G3P[3])
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(Fig. 1B). Phylogenetically, the strains located in the subcluster G3b were found in humans, and animals such as dogs, horses, cows, simian, goats, rabbits, and cats. The
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VP7 gene of strain 12638 was clustered together with those of several animal and human strains (the RVA/Cat-tc/AUS/Cat97/1984/G3P[3],
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RVA/Human-tc/ISR/Ro1845/1985/G3P[3], RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] strains) in the subcluster 3b2.
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The VP4 gene of strain 12638 was also most closely related to that of the RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] strain with the nucleotide identity of 96.7%. These two strains were also clustered together with a human strain (the Ro1845 strain) and a feline strain (the Cat97 strain) (Fig. 1D). The VP6 gene of strain 12638 was closely related to the VP6 of RVA strains with P[3] or P[9] genotypes, which were canine/feline RVAs and/or human RVAs (Fig. 2A). The VP6 gene segment of strain 12638 showed the highest nucleotide sequence identity of 97.0% with the RVA/Human-tc/ITA/PA260-97/1997/G3P[3]. The VP1 gene of 12638 presented the highest nucleotide sequence identity of 96.9% with the
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RVA/Human-wt/PRY/1256A/2009/G3G4P[9]. The VP2 gene of strain 12638 shared highest nucleotide sequence identity of 96.1% with the RVA/Human-tc/THA/T152/1998/G12P[9]. The VP3 gene of strain 12638 presented the highest nucleotide identity with those of three Paraguayan strains, RVA/Human-wt/
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PRY/1259A/2009/G12P[9], RVA/Human-wt/PRY/1701SR/2009/G1P[9], and
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RVA/Human-st/1257A/2009/G1P[9] with the nucleotide sequence identity of 98.3%. Phylogenetically, the VP1, VP2, and VP3 gene segments of strain 12638 were distinct
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from those of the Cat97-like strains (the Cat97, Ro1845, HCR3A strains) (Fig. 2B, 2C,
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and 2D, respectively), and similar to that of the T152 strain.
Furthermore, the NSP1, NSP2 and NSP3 genes of strain 12638 were most closely
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related to those of a bat RVA strain in South Africa
(RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3] strain) with the nucleotide sequence
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idnetities ranging from 96.6 to 98.5% (Fig. 3A , 3B, and 3C, respectively). Interestingly, the strain 12638 possessed the N2 as the NSP2 genotype, which was the same as the
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strain 12597.
The NSP4 gene of strain 12638 was most closely related to those of two canine
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(RVA/Dog-wt/HUN/135/2012/G3P[3], RVA/Dog-tc/ITA/RV52-96/1996/G3P[3] strains), one feline (RVA/Cat-tc/JPN/xxxx/FRV348/G3P[3] strain), and two human strains (RVA/Human-tc/ITA/PA260-97/1997/G3P[3] and RVA/Human-wt/FRA/R1486/2007/G3P[3] strains) with the nucleotide sequence identities ranging from 97.9% to 98.4% (Fig. 3D). The NSP5 gene of strain 12638 formed the same branch with the H6 genotype of the RVA/Human-wt/CHN/M2-102/2014/G3P[3] strain with the nucleotide identity of 97.6%. It should be noted that the M2-102 strain was reported to carry the simian RRV-like NSP5 gene segment (Dong et al., 2016) (Fig. 3E).
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The nucleotide sequence identities of 11 gene segments of the G3P[3] strain 12638 in relation to the most closely related reference strains are shown in Table 1B.
4. Discussion
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The present study demonstrated that the G1P[8] genotype was the major
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epidemic RVA in this area, during 2014 and 2015, and, two rare genotypes, G8P[14] and G3P[3], were identified.
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Until now, only three strains of RVA were reported to possess the T9 as the
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NSP3 genotype worldwide (Abe et al., 2011: Ward et al., 2016), and the strain 12597 is the forth strain which carries the T9 genotype. The T9 genotype was found initially in
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two cows (RVA/Cow-wt/JPN/AzuK-1/2006/G21P[29] and RVA/Cow-tc/JPN/Dai-10/2008/G24P[33] strains) in Japan (Abe et al., 2011), and then,
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another human strain (RVA/Human-wt/USA/2012741499/2012/G24P[14]) was reported in the USA (Ward et al., 2016). The strain 12597 is the second human strain possessing
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the T9 genotype. Previously very rare G24 or G21 genotypes were reported as G genotype combined with the T9 genotype, however, the G8 in this study were found to
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combine with the T9 genotype. The T9 gene of strain 12597 was more closely related to that of human strain detected in the USA than to those of the bovine strains detected in Japan.
Overall, the genotype constellation of the G8P[14] strain 12597 was G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3 (Table 2). The strain 12597 was most closely related to three human P[14] strains (RVA/Human-wt/ITA/PR1300/2004/G8P[14], RVA/Human-wt/ITA/PR1973/2009/G8P[14], and RVA/Human-wt/USA/2012741499/2012/G24P[14]), and one Roe deer strain (RVA/Roe_deer-wt/SLO/D110-15/2015/G8P[14]) by sharing 10 genotypes within the
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complete genome constellation. The PR1300, PR1973, and D110-15 strains had different NSP3 genes, and the 2012741499 strain possessed a VP7 genotype distinct from the strain 12597. It was found that the consensus P[14] strains possessed a G6/8-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-E2-H3 genotype constellation with
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reassortment of some genes (Banyai et al., 2010; Matthijnssens et al., 2009), and the
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genotype constellation presented by the P[14] strains were included within genogroup 2 (DS-1-like) (Heiman et al., 2008; Matthijnssens et al., 2008b). The strain 12597 also
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possessed the consensus P[14] genotype constellation with T9 gene reassortment. The
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G8P[14] strains that displayed the nucleotide sequence identities of 11 gene segments most closely related (96.0 to 98.8%) to the strain12597 were a wide range of host
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species including humans, rhesus, cows, deer, cats, and dogs (Table 1A). The human P[14] RVA was suspected to transmit from sheep or other ungulates as host origins to
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human(Matthijnssens et al., 2009). In Japan, even though two RVA strains possessing a P[14] genotype have been reported previously in cows
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(RVA/Cow-wt/JPN/Tottori-SG/2013/G15P[14] and RVA/Cow-wt/JPN/Sun9/2000/G8P[14] strains), (Fukai et al., 2004; Masuda et al., 2014),
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it has never been reported previously in human. The strain 12597 reported in this study is the first P[14] RVA strain detected in human in Japan and it is distantly related to the P[14] strains detected in cows. Overall, the genotype constellation of the strain 12638 was
G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6 (Table 3). Complete gene sequence analysis revealed that G3P[3]/[9] stains detected in humans, cats, and dogs could be classified into three genogroups (Matthijnssens et al., 2011b; Papp et al., 2015). The Cat97-like genogroup displays the genotyope constellation of G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6 which includes those detected in feline,
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canine, and human (Ro1845 and HCR3A) G3P[3] RVAs (Tsugawa and Hoshino, 2008). The second genogroup (AU-1-like) comprises the G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 genotype constellation, and some feline/canine RVAs, and the human T152 G12P[9] strain belongs to this genogroup
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(Rahman et al., 2007). The third BA222-05-like genogroup possesses the genotype
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constellation of G3-P[9]-I2-R2-C2-M2-A3-N1/2-T6/3-E2-H3, and the human PAI58 and PAH136 strains (Table 3) as well as other feline strains were classified into this
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genogroup. On the other hand, additional heterogenous G3P[3] strains were also
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detected in horses, monkeys, and bats (Papp et al., 2015). The strain 12638 detected in this study carries the genotype constellation of the Cat-97-like strains for the VP7 and
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VP4 genes, similar to two canine-like human R2638 and PA260-97 strains, and the canine RV198-98 strain which have been reported as the Cat-97-like strains. However,
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the nucleotide sequences of VP1, VP2, and VP3 are found to be closely related to those of human RVA strains T152, AU-1 (AU-1 like genogroup). Previously, the human
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PA260-97 strain was reported to be a reassortant strain between the Cat-97 genogroup and the AU-1 genogroup (Matthijnssens et al., 2011b). Similarity, it is possible that the
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strain12638 detected in this study could be an intra-genotype reassortant strain between the Cat97-like genogroup and the AU-1 like genogroup. Furthermore, the NSP1, NSP2, and NSP3 gene segments of strain 12638 have been shown to be closely related to those of bat RVA (Table 1B). Taken together, the findings suggest that the strain 12638 may derive from multiple and intra-genotype reassortment events between the Cat-97 like strain, the AU-1-like G3P[3] strains, and a bat strain. In this study, two rare RVA strains were identified in two Japanese children and the molecular characterization of these two strains were reported. The sources of infection of these rare strains in the patients are still unknown. These two rare RVAs did
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not cause outbreaks in the study area suggesting that they could not adapt and replicate efficiently in human hosts at the present. The Information of the RVA infection in domestic animals like cats, dogs, rabbits, cows, horses, and sinantropic animals like rats, bats, and birds, are limited. Furthermore, contamination of the viruses in environmental
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water like rivers, ponds might be the sources of viral transmission to humans. The
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epidemiological surveillance is needed to monitor circulating RVA genotypes and
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spread of unusual strains in humans.
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5. Conclusion
In 2014, two unusual G8P[14] and G3P[3] human RVA strains were detected in
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the same area in Japan. Both strains were rare RVAs and have never been reported previously in human in Japan. Furthermore, the complete genome sequence analyses of
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these two strains showed new genotype constellations. Molecular characterization demonstrated that the strains 12597 and 12638 detected in this study possessed the
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backbone of the G8P[14] and G3P[3] genotype constellation, respectively, with some gene reassortments. The NSP3 gene segment of the G8P[14] strain 12597 belongs to an
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uncommon T9 genotype, and it is the second human strain with the T9 genotype reported. Analyses of the nucleotide sequences of VP1 to VP7 and NSP4 of 12638 G3P[3] strain revealed that it was a reassortant strain between the Cat-97 genogroup and AU-1 genogroup, however, the other NSP1 to NSP3 gene segments were closely related to that of the bat RVA. Taken together, the phylogenetic analyses showed that genetic diversity of the strain 12638 could be resulted from interspecies transmission and multiple reassortment events.
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Acknowledgements This work was supported by the Japan Society for the Promotion of Science JSPS Kakenhi (16H05360), and the Public Foundation of the Vaccination Research Center
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(2015-34).
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Mijatovic-Rustempasic, S., Roy, S., Sturgeon, M., Rungsrisuriyachai, K., Reisdorf, E.,
AC C
Cortese, M.M., Bowen, M.D., 2015. Full-Genome Sequence of the First G8P[14] Rotavirus Strain Detected in the United States. Genome Announc 3, e00677-00615. Nakagomi, T., Nakagomi, O., 1989. RNA-RNA hybridazation identifies a human rotavirus that is genetically related to feline rotavirus. J. Clin. Microbiol. 63, 1431-1434. Papp, H., Mihalov-Kovacs, E., Doro, R., Marton, S., Farkas, S.L., Giammanco, G.M., De Grazia, S., Martella, V., Banyai, K., 2015. Full-genome sequencing of a Hungarian canine G3P[3] Rotavirus A strain reveals high genetic relatedness with a historic Italian human strain. Virus genes 50, 310-315.
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Rahman, M., Matthijnssens, J., Yang, X., Delbeke, T., Arijs, I., Taniguchi, K., Iturriza-Gomara, M., Iftekharuddin, N., Azim, T., Van Ranst, M., 2007. Evolutionary history and global spread of the emerging g12 human rotaviruses. J. Virol. 81, 2382-2390.
PT
RCWG. Rotavirus Classification Working Group: RCWG, List of accepted genotypes.
RI
https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg. (accessed 24 March 2018)
SC
Rojas, M.A., Goncalves, J.L.S., Dias, H.G., Manchego, A., Santos, N., 2017.
NU
Identification of two novel Rotavirus A genotypes, G35 and P[50], from Peruvian alpaca faeces. Infect.Genet. Evol. 55, 71-74.
MA
Ruuska, T., Vesikari, T., 1990. Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes. Scand. J. Infect. Dis. 22,
ED
259-267.
Sachsenroder, J., Braun, A., Machnowska, P., Ng, T.F., Deng, X., Guenther, S.,
EP T
Bernstein, S., Ulrich, R.G., Delwart, E., Johne, R., 2014. Metagenomic identification of novel enteric viruses in urban wild rats and genome
AC C
characterization of a group A rotavirus. J. Gen. Virol. 95, 2734-2747. Swiatek, D.L., Palombo, E.A., Lee, A., Coventry, M.J., Britz, M.L., Kirkwood, C.D., 2010. Characterisation of G8 human rotaviruses in Australian children with gastroenteritis. Virus Res. 148, 1-7. Thongprachum, A., Khamrin, P., Pham, N.T., Takanashi, S., Okitsu, S., Shimizu, H., Maneekarn, N., Hayakawa, S., Ushijima, H., 2017. Multiplex RT-PCR for rapid detection of viruses commonly causing diarrhea in pediatric patients. J. Med. Virol. 89, 818-824. Tsugawa, T., Hoshino, Y., 2008. Whole genome sequence and phylogenetic analyses
ACCEPTED MANUSCRIPT
reveal human rotavirus G3P[3] strains Ro1845 and HCR3A are examples of direct virion transmission of canine/feline rotaviruses to humans. Virology 380, 344-353. Tsugawa, T., Rainwater-Lovett, K., Tsutsumi, H., 2015. Human G3P[9] rotavirus strains possessing an identical genotype constellation to AU-1 isolated at high prevalence
PT
in Brazil, 1997-1999. J. Gen. Virol. 96, 590-600.
RI
Ward, M.L., Mijatovic-Rustempasic, S., Roy, S., Rungsrisuriyachai, K., Boom, J.A., Sahni, L.C., Baker, C.J., Rench, M.A., Wikswo, M.E., Payne, D.C., Parashar, U.D.,
SC
Bowen, M.D., 2016. Molecular characterization of the first G24P[14] rotavirus
AC C
EP T
ED
MA
NU
strain detected in humans. Infect. Genet. Evol. 43, 338-342.
ACCEPTED MANUSCRIPT
Table 1. Nucleotide sequence identities of the G8P[14] strain 12597 (A) and the G3P[3] strain 12638 (B) with the most closely related reference strains. (A) M ost Gene
closely
related
Genotype
% Host Identity
Tokushima9503
Cow
VP4
P[14]
Tottori-SG
Cow
VP6
I2
D110-15
Roe deer
R2
CK-20039
Human
96.1
97.3
Vaccine
MA
Rota Teq-WI-79-4 C2
O'Agent
Cow
96.0
VP3
M2
FRV537
Cat
97.0
NSP1
A3
PTRV
Rhesus
97.0
NSP2
N2
RRV
Simian
97.6
T9
2012741499
Human
97.5
E2
RF
Cow
98.1
88977
Dog
98.3
EP T
AC C
NSP4
ED
VP2
NSP3
(B)
96.8
Human
NU
VU12-13-176 VP1
98.8
RI
G8
SC
VP7
PT
strains
NSP5
H3
Gene
Genotype
M ost
closely
related
% Host
strains
Identity
VP7
G3
R2638
Human
97.7
VP4
P[3]
RV198-95
Dog
96.7
VP6
I3
PA260-97
Human
97.0
VP1
R3
1256A
Human
96.9
VP2
C3
T152
Human
96.1
1257A
Human
1701SR
Human
98.3
1259A
Human
PT
ACCEPTED MANUSCRIPT
M3
A9
LUS12-14
Bat
NSP2
N2
LUS12-14
Bat
NSP3
T3
LUS12-14
NSP4
E3
Bat
H6
M 2-102
ED EP T AC C
96.6
98.4
Human
MA
NSP5
98.5
Dog
NU
RV52-96 R1486
98.5
SC
NSP1
RI
VP3
Human
97.6
ACCEPTED MANUSCRIPT
Table 2. Complete genomic constellation of the G8P[14] strain 12597 and the reference strains VP
V
V
V
V
NS
NS
NS
NS
NS
4
P6
P1
P2
P3
P1
P2
P3
P4
P5
P
I
R
C
M
A
N
P[ G8
14/G8P[14]
14]
RVA/Human-wt/ITA/PR1300/20
P[1 G8
04/G8P[14]
I2 4] P[1
G8 4]
RVA/Human-wt/USA/20127414
P[1
EP T
4]
P[1
G8
/2015/G8P[14]
AC C
RVA/Sheep-tc/ESP/OVR762/200
2
R
C
A3
P[1
G8
2
2
2
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
2/G8P[14]
4]
2
2
2
RVA/Rhesus-tc/USA/PTRV/199
P[1
R
C
M
I2
0/G8P[1]
]
2
2
2
RVA/Human-tc/KEN/B12/1987/
P[1
R
C
M
2
2
2
R
C
M
2
2
2
G8 G8P[1]
I2 ]
RVA/Human-tc/ITA/PA169/1988
P[1 G6
/G6P[14]
I2 4]
genotypes
T9
E2
H3
A3
N2
T6
E2
H3
10
A3
N2
T6
E2
H3
10
A3
N2
T9
E2
H3
10
A3
N2
T6
E2
H3
10
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H3
9
A1
I2
G8
Shared H
M
I2 4]
N2
E
2
I2
ED
G24
RVA/Roe_deer-wt/SLO/D110-15
2
M
I2
09/G8P[14]
99/2012/G24P[14]
C
MA
RVA/Human-wt/ITA/PR1973/20
R I2
T
RI
RVA/Human-wt/JPN/12597/20
SC
G
NU
Strain
PT
VP7
1
ACCEPTED MANUSCRIPT
90/2009/G8P[14]
4]
RVA/Human-wt/HON/20118253
R
C
M
A1
2
2
2
3
R
C
M
I2
P[1 G10
I2
63/2011/G10P[14]
4]
2
2
2
RVA/Human-wt/USA/20128411
P[1
R
C
M
3
2
2
R
C
M
2
2
R
C
G8
I2 4] P[1
G15 13/G15P[14]
I2 4]
RVA/Human-wt/HUN/BP1062/2
P[1 I2
E2
H3
9
A3
N2
T6
E2
H3
9
A3
N2
T6
E2
H2
9
T6
E2
H2
9
N2
T6
E2
H3
9
N2
T6
E2
H3
8
N2
T6
E2
H3
8
N2
T6
H3
8
A3
M
A1
004/G8P[14]
4]
2
2
2
1
RVA/Antelope-wt/ZAF/RC-18/2
P[1
R
C
M
A1
G6 008/G6P[14]
I2
4]
RVA/Human-wt/HUN/BP1879/2
P[1
003/G6P[14]
4]
EP T
RVA/Guanaco-wt/ARG/Chubut/
P[1
G8
1999/G8P[14]
AC C
RVA/Vaccine/USA/RotaTeq-WI7
2
2
2
1
R
C
M
A1
2
2
2
1
R
C
M
I2
ED
G6
I2 4] P[8
G6
E1 A3
5
2
2
R
C
M
I2
2
A3
N2
T6
E2
H3
8
A3
N2
T6
E2
H3
8
N2
T6
E2
H3
8
N2
T6
E2
H3
8
9-4/1992/G6P1A[8]
]
2
2
2
RVA/Human-wt/AUS/CK20039/
P[8
R
C
M
2
2
2
R
C
M
A1
2
2
2
1
R
C
M
G1
2008/G1P[8]
I2 ]
RVA/Cow-tc/ZAF/'O'
P[1 G8
Agent/1965/G8P[1]
I2 ]
RVA/Cow-tc/FRA/RF/1982/G6P
P[1 G6
[1]
I2 ]
A3 2
2
N2
2
MA
G8
T6
SC
RVA/Cow-wt/JPN/Tottori-SG/20
NU
74/2012/G8P[14]
N2
PT
P[1 G8
RI
RVA/Human-wt/GTM /20097267
2
ACCEPTED MANUSCRIPT
24P[33]
3]
RVA/Cow-wt/JPN/AzuK-1/2006/
R
C
M
A1
2
2
2
3
R
C
M
A1
I2
P[2 G21
I2
G21P[29]
9]
2
2
2
RVA/Goat-wt/ARG/0040/2011/G
P[8
R
C
M
5
2
2
R
C
M
2
2
R
C
G8 8P[1]
I2 ]
RVA/Human-wt/USA/VU12-13-
G1,
P[8
RVA/Cat-tc/JPN/FRV537/2004/
] P[5 I2
M
]
2
2
2
RVA/Human-wt/JPN/To14-0/201
P[8
R
C
M
G8
I2
]
RVA/Guanaco-wt/ARG/Rio_Neg
P[1
ro/1998/G8P[1]
]
EP T
RVA/Human-tc/USA/DS-1/1976/
P[4
G2
G2P[4]
AC C
RVA/Simian-tc/USA/RRV/1975/
P[3
G3
]
RAV/Dog-wt/GER/88977/2013/
P[1 G8
G8P[1]
2
R
C
M
5
2
2
R
C
M
2
2
2
R
C
M
2
I2
P[14]
8
H3
7
N2
T6
N2
T6
E2
H3
7
N2
T6
E2
H3
7
N2
T2
E2
H2
7
N2
T6
H3
6
3
A1
E1
1
2
A2
N2
T2
E2
H2
6
A9
N2
T3
E3
H6
3
A3
N2
T6
E2
H3
-
-
-
-
-
-
-
-
-
-
3
-
-
R
C
M
2
2
2
-
-
-
I2 4]
RVA/Human-wt/AUS/WAG8.2/2
H3
2
P[1 G8
3
E2
C
]
RVA/Cow-tc/JPN/Sun9/2000/G8
P[1 G8
003/G8P[14]
2
I2
G3P[3]
T9
A1
A2
2
I2 ]
N2
2
I2
ED
G8
8
E1
A3
G6P[5]
4/G8P[8]
H3
1
MA
G6
E2
SC
G6*
NU
176/2013/G1G6P[8]
T9
3
A3
I2
N2
PT
P[3 G24
RI
RVA/Cow-tc/JPN/Dai-10/2008/G
4]
ACCEPTED MANUSCRIPT
RVA/Cow-tc/JPN/Tokushima950
P[1 G8
3/xxxx/G8P[11]
-
-
-
-
-
-
-
-
-
1]
Gray color indicates the strains that sharing the genotype with the strain 12597. -: Genotypes are not available in the GenBank database.
AC C
EP T
ED
MA
NU
SC
RI
PT
*: The VU12-13-176 strain was reported to possess mixed genotype of VP
ACCEPTED MANUSCRIPT
Table 3. Complete genomic constellation of the G3P[3]strain 12638 and the reference strains VP
V
V
V
V
NSP
NS
NS
NS
NS
4
P6
P1
P2
P3
1
P2
P3
P4
P5
P
I
R
C
M
A
N
P[3 G3
/2014/G3P[3]
]
RVA/Dog-wt/HUN/135/2012
P[3 G3
/G3P[3]
RVA/Dog-tc/ITA/RV198-95/
P[3
ED
EP T
AC C
R
C
M
A9
3
3
C
M
3
3
3
R
C
M
3
2
3
R
C
M
3
2
3
R
C
M
I3
P[3
I3
]
RVA/Dog-tc/USA/CU-1/198
3
R
]
G3
996/G3P[3]
3
MA
]
RVA/Dog-tc/ITA/RV52-96/1
3
I3
7/1997/G3P[3]
G3
M
3
P[3 G3
1995/G3P[3]
C
I3 ]
RVA/Human-tc/ITA/PA260-9
R I3
P[3
G3
I3
2/G3P[3]
]
3
2
3
RVA/Dog-tc/AUS/K9/1981/
P[3
R
C
M
G3
I3
G3P[3]
]
3
2
3
RVA/Dog-tc/USA/A79-10/19
P[3
R
C
M
3
2
3
R
C
M
3
2
3
G3 79/G3P[3]
I3 ]
RVA/Cat-tc/AUS/Cat97/1984
P[3 G3
/G3P[3]
I3 ]
T
RI
RVA/Human-wt/JPN/12638
N2
SC
G
E
Shared H genotypes
T3
E3
H6
A15
N2
T3
E3
H6
10
A15
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
A9
N2
T3
E3
H6
10
NU
Strain
PT
VP7
ACCEPTED MANUSCRIPT
1985/G3P[3]
]
RVA/Human-tc/USA/HCR3
R
C
M
3
2
3
R
C
M
I3
P[3 G3
I3
A/1984/G3P[3]
]
3
2
3
RVA/Horse-wt/ARG/E3198/
P[3
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
G3
I3 ] P[3
G3 2/2014/G3P[3]
I3 ]
RVA/Bat-tc/CHN/M SLH14/2
P[3 G3
I8 3
RVA/Simian-tc/USA/RRV/19
P[3
R
P[3
94/G3P[3]
]
P[9
EP T
RVA/Human-tc/CHN/L621/2
G3
006/G3P[9]
AC C
P[9
G3
/2011/G3P[9]
H6
10
A9
N2
T3
E3
H6
10
A9
N3
E3
H6
10
T3
E3
H6
10
2
3
3
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
I3
T3
E3
H6
9
A9
N2
T3
E3
H6
9
A15
N3
T3
E3
H6
9
A3
N3
T3
E3
H6
8
A3
N3
T3
E3
H6
8
N3
T3
E3
H6
8(7)
A9
N2
T3
E2
H3
7
A3
N3
T3
E3
H3
7
3
3
R
C
M
A3,
3
3
3
A1*
R
C
M
2
2
3
R
C
M
P[9
2009/G3G4P[9]
G4*
]
I3
RVA/Bat-wt/ZM B/LUS12-14
P[3 G3
/2012/G3P[3]
I3 ]
RVA/Human-tc/JPN/AU-1/1
P[9 G3
I3 ]
3
3
N3
N3
3
G3,
3
T3
A9
]
RVA/Human-wt/PRY/1256A/
982/G3P[9]
M
I3
]
RVA/Human-wt/CHN/E2451
C
I3
ED
G3
3
I2 ]
RVA/Cat-tc/JPN/FRV348/19
3
MA
]
G3
E3
A9
012/G3P[3]
75/G3P[3]
T3
SC
RVA/Human-wt/CHN/M 2-10
N2
NU
2008/G3P[3]
A9
PT
P[3 G3
RI
RVA/Human-tc/ISR/Ro1845/
ACCEPTED MANUSCRIPT
998/G12P[9]
]
RVA/Human-wt/PRY/1259A/
R
C
M
3
3
3
R
C
M
I3
P[9 G12
I3
2009/G12P[9]
]
3
3
3
RVA/Human-wt/PRY/1701S
P[9
R
C
M
3
3
3
R
C
M
3
3
3
R
C
M
G1
I3 ] P[9
G1 2009/G1P[9]
I3 ]
RVA/Cat-tc/AUS/Cat2/1984/
P[9 G3
I3 3
RVA/Rhesus-tc/USA/TUCH/
P[2
R
P[9
5/G3P[9]
]
P[9
EP T
RVA/Human-wt/ITA/PAI58/1
G3
996/G3P[9]
AC C
P[9
G3
6/1996/G3P[9]
]
RVA/Human-wt/FRA/R1486/
P[3 G3
2007/G3P[3]
H6
7
A3
N3
T3
E3
H6
6
A3
N3
E3
H6
6
T3
E3
H6
6
3
3
3
R
C
M
2
2
2
R
C
M
2
2
2
R
C
M
I2
T3
N3
A3
N1
T6
E3
H3
5
A9
N1
T1
E1
H1
5
A3
N1
T3
E2
H3
2
A3
N2
T6
E2
H3
2
A3
N1
T6
E2
H3
2
2
2
2
I3
-
-
-
-
-
-
E3
-
I8
-
-
-
-
-
-
E3
-
-
-
-
-
-
-
-
-
-
]
RVA/Human-wt/THA/CM H2
P[3 G3
22/2001/G3P[3]
]
RVA/Human-wt/BRA/R2638
P[3 G3
/2011/G3P[3]
M
I2
]
RVA/Human-wt/ITA/PAH13
C
I2
ED
G3
3
I9 4]
RVA/Cat-wt/ITA/BA222/200
2
MA
]
G3
E3
A3
G3P[9]
2002/G3P[24]
T3
SC
RVA/Human-wt/PRY/1257A/
N3
NU
R/2009/G1P[9]
A12
PT
P[9 G12
RI
RVA/Human-tc/THA/T152/1
]
Gray color indicates the strain that sharing the genotypes with the strain 12638.
ACCEPTED MANUSCRIPT
-: Genotypes are not available in the GenBank database.
AC C
EP T
ED
MA
NU
SC
RI
PT
*: The 1256A strain was reported to possess mixed genotypes of VP7 and NSP1.
ACCEPTED MANUSCRIPT
Figure legend. Fig 1. Phylogenetic trees of the viral structural protein VP7 and VP4 genes of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap
PT
values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of
RI
nucleotide substitutions per site. The strains 12597 and 12638 are indicated by the black dots. (A): VP7-G8 (841 nucleotides); (B): VP7-G3 (841 nucleotides); (C): VP4-P[14]
SC
(1975 nucleotides); 1D; VP4-P[3] (2290 nucleotides). Within 1B, arbitrary subclusters
NU
(b1 and B2) were indicated.
Fig 2. Phylogenetic trees of the vial structural protein VP 6, VP1, VP2, and VP3 genes
MA
of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap
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values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of nucleotide substitutions per site. (A): VP6 (1154 nucleotides); (B): VP1 (3208
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nucleotide); (C): VP2 (2631 nucleotides); (D): VP3 (2507 nucleotides). Fig.3. Phylogenetic trees of the non-structural protein NSP1, NSP2, NSP3, NSP4 and
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NSP5 genes of the rotavirus strains 12597 and 12638. A Kimura-2 parameter model was used for calculations of genetic distances using the maximum- likelihood method. Bootstrap values (1,000 replicates) greater 80% are shown. Scale bar indicates the number of nucleotide substitutions per site. The strains 12597 and 12638 are indicated by the black dots. (A): NSP1 (1477 nucleotides); (B): NSP2 (953 nucleotides); (C): NSP3 (942 nucleotides); (D): NSP4 (527 nucleotides); (E): NSP5 (599 nucleotides).
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Fig . 1 (C) VP4-P[14]
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Fig . 1 (D) VP4-P[3]
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(A) VP6
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(B) VP1
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(C) VP2
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(D) VP3
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(A) NSP1
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(B) NSP2
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(C) NSP3
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(D) NSP4
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(E) NSP5
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Highlights Detection of two rare rotavirus strains, G8P[14] and G3P[3], in Japanese children.
The G8P[14] strain 12597 possessed a rare T9 NSP3 genotype.
The G3P[3] strain 12638 was an intra-genotype reassortant strain.
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