RESEARCH ARTICLE
Whole Genomic Analysis of Human G12P[6]
and G12P[8] Rotavirus Strains that Have
Emerged in Myanmar
Tomihiko Ide1, Satoshi Komoto1*, Kyoko Higo-Moriguchi1, KhaingWin Htun2, Yi Yi Myint3,
Theingi Win Myat4, Kyaw Zin Thant4, Hlaing Myat Thu4, Mo MoWin4, Htun Naing Oo5,
Than Htut6, MitsutakaWakuda1, Francis Ekow Dennis7,8, Kei Haga7, Yoshiki Fujii7,
Kazuhiko Katayama7, Shofiqur Rahman9, Sa Van Nguyen9, Kouji Umeda9, Keiji Oguma10,
Takao Tsuji11, Koki Taniguchi1
1 Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi,
Japan, 2 Nay Pyi Taw General Hospital (Central Myanmar), Nay Pyi Taw, Myanmar, 3 Department of
Medical Research (Upper Myanmar), Pyin Oo Lwin, Myanmar, 4 Department of Medical Research (Lower
Myanmar), Yangon, Myanmar, 5 Department of Traditional Medicine (Central Myanmar), Nay Pyi Taw,
Myanmar, 6 Ministry of Health (Central Myanmar), Nay Pyi Taw, Myanmar, 7 Department of Virology II,
National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan, 8 Department of Electron
Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health
Sciences, University of Ghana, Legon, Ghana, 9 Immunology Research Institute in Gifu, EW Nutrition Japan,
OPEN ACCESS Gifu, Japan, 10 Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry,
and Pharmaceutical Sciences, Okayama, Japan, 11 Department of Microbiology, Fujita Health University
Citation: Ide T, Komoto S, Higo-Moriguchi K, Htun School of Medicine, Toyoake, Aichi, Japan
KW, Myint YY, Myat TW, et al. (2015) Whole
Genomic Analysis of Human G12P[6] and G12P[8] * satoshik@fujita-hu.ac.jp
Rotavirus Strains that Have Emerged in Myanmar.
PLoS ONE 10(5): e0124965. doi:10.1371/journal.
pone.0124965
Abstract
Academic Editor: Karol Sestak, Tulane University,
UNITED STATES G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and
Received: January 20, 2015 young children worldwide. However, the whole genomes of only a few G12 strains have
Accepted: March 20, 2015 been fully sequenced and analyzed. In this study, we sequenced and characterized the
Published: May 4, 2015 complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/
Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/
Copyright: © 2015 Ide et al. This is an open access
article distributed under the terms of the Creative Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/
Commons Attribution License, which permits Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute
unrestricted use, distribution, and reproduction in any gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains
medium, provided the original author and source are were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1
credited.
for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for
Data Availability Statement: The nucleotide strain A23. Phylogenetic analysis showed that most genes of the six strains examined in
sequence data presented in this paper have been
deposited in the DDBJ and EMBL/GenBank data this study were genetically related to globally circulating human G1, G3, G9, and G12
libraries. The accession numbers for the nucleotide strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with
sequences of the VP1-4, VP6-7, and NSP1-5 genes the cognate genes of human-like bovine strains as well as human strains, suggesting the
of strains A14, A23, A25, P02, P39, and P43 are
occurrence of reassortment between human and bovine strains. Furthermore, strains A14,
LC019041-LC019051, LC019052-LC019062,
LC019063-LC019073, LC019074-LC019084, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene seg-
LC019085-LC019095, and LC019096-LC019106, ments, indicating derivation of the five strains from a common origin. On the other hand,
respectively. strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a
Funding: This study was supported in part by the distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our
MEXT-Supported Program for the Strategic Research
PLOSONE | DOI:10.1371/journal.pone.0124965 May 4, 2015 1 / 24
Whole Genomes of Human G12 Rotaviruses in Myanmar
Foundation at Private Universities, 2010-2014 (KT), knowledge, this is the first report on whole genome-based characterization of G12 strains
and Grants-in-Aid for Research on Emerging and Re- that have emerged in Myanmar. Our observations will provide important insights into the
emerging Infectious Diseases from the Ministry of
Health, Labor, and Welfare of Japan (KT). The evolutionary dynamics of spreading G12 rotaviruses in Asia.
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Group A rotavirus (RVA), a member of the family Reoviridae, is the major cause of severe de-
hydrating diarrhea in the young of humans and many animal species worldwide. In humans,
RVA infections are associated with high morbidity and mortality, being responsible for an esti-
mated annual 453,000 deaths among children<5 years of age [1]. More than half of these
deaths were estimated to occur in developing countries in Asia and Africa [1, 2]. In Myanmar,
a high disease burden of RVA infection in children (<5 years old) was found in studies con-
ducted in Yangon, RVAs being identified in 53–57% of fecal samples from diarrheal disease
cases in the years 2002–2005 [3, 4].
The RVA virion is a triple-layered, non-enveloped icosahedron that encloses an 11-segment
genome of double-stranded (ds)RNA [5]. RVA has two outer capsid proteins, VP7 and VP4,
which are implicated independently in neutralization, and define the G and P genotypes, re-
spectively. To date, RVAs have been classified into at least 27 G and 37 P genotypes [6, 7].
Among them, 5 G (G1-4 and G9) and 3 P (P[4], P[6], and P[8]) genotypes are commonly asso-
ciated with human infections [8, 9]. Over the last decade, the global emergence of unusual G12
strains has been a matter of concern, and G12 seems to be the sixth major human G genotype
[9–11].
The first G12 strain, L26 (G12P[4]), was detected in children<2 years old with acute diar-
rhea in the Philippines in 1987 [12, 13]. More than 10 years later, G12 strains began to emerge
globally, predominantly in combination with either a P[6] or P[8] genotype and less commonly
with a P[4] or P[9] genotype [9–11, 14–17]. In Asia, G12 strains are being increasingly identi-
fied in diarrheic children in several countries; Bangladesh [10, 18–22], Bhutan [23], India [24–
33], Indonesia [34], Japan [16], Kazakhstan [35], Korea [36], Kyrgyzstan [35], Nepal [37–40],
Pakistan [41, 42], Sri Lanka [43], Thailand [14, 15, 44–46], and Vietnam [47], indicating the
continued spread of G12 strains in Asia. In 2011, we detected 29 G12 strains from diarrheic
children in Central Myanmar enrolling a total of 54 RVA-positive stool specimens by PCR-
based G and P genotyping (Ide et al., in preparation).
A whole genome-based genotyping system was recently proposed for RVAs based on as-
signment to all the 11 gene segments (i.e., G/P and non-G/P genes) [48]. In the new genotyp-
ing system, the acronym Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x is an integer,
defines the genotype of the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5
genes of a given RVA strain. The Wa-like strains are characterized by non-G/P genotypes
(I1-R1-C1-M1-A1-N1-T1-E1-H1), and tend to have G/P genotypes G1P[8], G3P[8], G4P[8],
and G9P[8] [49]. In contrast, the DS-1-like strains are characterized by non-G/P genotypes
(I2-R2-C2-M2-A2-N2-T2-E2-H2), and tend to have G/P genotype G2P[4]. The third minor
AU-1-like strains are characterized by non-G/P genotypes (I3-R3-C3-M3-A3-N3-T3-E3-
H3), and tend to have G/P genotype G3P[9]. Whole genome-based analysis is a reliable
method for obtaining conclusive data on the origin of an RVA strain, and for tracing its evo-
lutionary pattern [48, 50]. To date, the whole genome sequences of only a few G12 strains
have been fully sequenced and characterized, which provided evidence that the recently de-
tected Asian G12 strains with the Wa-like genotype backbone are distantly related to
PLOSONE | DOI:10.1371/journal.pone.0124965 May 4, 2015 2 / 24
Whole Genomes of Human G12 Rotaviruses in Myanmar
prototype Asian G12 strains L26 (G12P[4]) and T152 (G12P[9]) [14] having the DS-1-like
and AU-1-like genotype backbone, respectively [10]. However, as the overall genomic con-
stellation and the exact evolutionary pattern of Asian G12 strains remain to be elucidated,
whole genomic analysis of Myanmarese G12 strains might be useful for obtaining a more pre-
cise understanding of the evolutionary pattern of emerging G12 strains in Asia. In the present
study, we analyzed the whole genomes of six G12 strains that have emerged in Myanmar.
Moreover, in this study, deep sequencing with the next generation sequencing (NGS) Illu-
mina MiSeq platform was carried out to obtain the complete nucleotide sequences of the
whole genomes of these six G12 strains.
Materials and Methods
Ethics statement
The study was approved by the Ethical Committee on Medical Research Involving Human
Subjects of the Department of Medical Research (Central Myanmar). In this study, written in-
formed consent for the testing of stool samples for RVAs and characterization of identified
RVA strains was obtained from children’s parents/guardians.
Virus strains
The full-genomic sequences were determined for strains A14, A23, A25, P02, P39, and P43,
which were identified as the pathogens causing diarrhea in six stool specimens from children
with acute diarrhea during the RVA strain surveillance in the Pediatric Infectious Disease
Wards of the Defense Services Obstetrics, Gyneacology and Children’s Hospital, Central
Myanmar in 2011, which involved a total of 54 RVA-positive fecal samples (Ide et al., in prepa-
ration). Stool samples containing strains A14, A23, A25, P02, P39, and P43 were kept at −30°C
until use.
Virus isolation
Stool samples suspended in PBS containing 0.5 mMMgCl2 and 1 mM CaCl2 were inoculated
onto monkey kidney cell line MA104 for virus isolation [51], and the cultures were serially pas-
saged two more times in MA104 cells. The viral dsRNAs were extracted from the cell cultures
using a QIAamp Viral RNAMini Kit (Qiagen). The extracted dsRNAs were used for (i) poly-
acrylamide gel electrophoresis (PAGE) analysis, and (ii) whole genomic analysis. For PAGE
analysis, the dsRNAs were electrophoresed in a 10% polyacrylamide gel for 16 h at 20 mA at
room temperature, followed by silver staining [52] to determine the genomic dsRNA profiles.
For whole genomic analysis, viral dsRNAs were subjected to Illumina MiSeq sequencing as
described below.
cDNA library building and Illumina MiSeq sequencing
Preparation of a cDNA library and Illumina MiSeq sequencing were performed as reported
previously [49, 53]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was con-
structed for individual strains using an NEBNext Ultra RNA library Prep Kit for Illumina v1.2
(New England Biolabs) according to the manufacturer’s instructions. Library purification was
performed using Agencourt AMPure XP magnetic beads (Beckman Coulter). The quality of
the purified cDNA libraries was assessed on a MultiNAMCE-202 bioanalyzer (Shimadzu). Nu-
cleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq
Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was carried out using
CLC Genomics Workbench v7.0.3 (CLC Bio). Contigs were assembled from the obtained
PLOSONE | DOI:10.1371/journal.pone.0124965 May 4, 2015 3 / 24
Whole Genomes of Human G12 Rotaviruses in Myanmar
sequence reads by de novo assembly. Using the assembled contigs as query sequences, the Basic
Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to ob-
tain the full-length nucleotide sequence of each gene segment of the six strains. To fill in miss-
ing sequence gaps for the NSP2 gene of strain A23, and the NSP5 genes of strains A14 and
A25, specific primers were used to process viral dsRNAs of the three strains using Sanger se-
quencing [54]. The primer pairs used for cDNA amplification of the NSP2 and NSP5 genes are
(+) 5’-GGCTTTTAAAGCGTCTCAGTC-3’ and (-) 5’-GGTCACATAAGCGCTTTCTATTC
-3’, and (+) 5’-GGCTTTTAAAGCGCTACAGTG-3’ and (-) 5’-GGTCACAAAACGGGAG
TGGGG-3’, respectively [54]. The final full-length genomes for the six strains were assembled
using a combination of Illumina MiSeq and Sanger sequencing data. The nucleotide sequences
were translated into amino acid sequences using GENETYX v11 (GENETYX).
SNP detection
Single nucleotide polymorphisms (SNPs) were called using CLC Genomics Workbench v7.0.3
using all available sequencing data with at least 100 sequence reads covering each nucleotide
position. A variant frequency threshold of
1% per site was used and approximate variant
P-values were calculated.
Determination of RVA genotypes
The genotype of each of the 11 gene segments of the six strains was determined using the
RotaC v2.0 automated genotyping tool (http://rotac.regatools.be/) [55] according to the guide-
lines proposed by the Rotavirus Classification Working Group (RCWG).
Phylogenetic analyses
Sequence comparisons were carried out as described previously [54]. Briefly, multiple align-
ment of each viral gene was performed using CLUSTALW [56]. Phylogenetic trees were con-
structed using the maximum likelihood method and the Kimura 2-parameter substitution
model using MEGA6.06 [57]. The reliability of the branching order was estimated from 1000
bootstrap replicates [58]. The results of phylogenetic analyses were validated using several
other genetic distance models, such as the Jukes-Cantor, Tamura 3-parameter, Hasegawa-
Kishino-Yano, and Tamuta-Nei ones (data not shown).
Nucleotide sequence accession numbers
The nucleotide sequence data presented in this paper have been deposited in the DDBJ and
EMBL/GenBank data libraries. The accession numbers for the nucleotide sequences of the VP1-4,
VP6-7, and NSP1-5 genes of strains A14, A23, A25, P02, P39, and P43 are LC019041-LC019051,
LC019052-LC019062, LC019063-LC019073, LC019074-LC019084, LC019085-LC019095, and
LC019096-LC019106, respectively.
Results and Discussion
Isolation of strains A14, A23, A25, P02, P39, and P43 in cell culture
To obtain enough genomic material for whole genome sequencing of the emerging G12 strains
in Myanmar, we primarily attempted to isolate strains A14, A23, A25, P02, P39, and P43 using
the MA104 cell line; all six strains could be cell culture-adapted. Virion dsRNAs were extracted
and then analyzed by PAGE. Fig 1 shows the profiles of the viral dsRNAs from human strain
KU (G1P[8]), as a reference (lane 1), and strains A14 (lane 2), A23 (lane 3), A25 (lane 4), P02
(lane 5), P39 (lane 6), and P43 (lane 7) from the cell cultures. The individual dsRNA migration
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 1. Genomic dsRNA profiles of strains A14, A23, A25, P02, P39, and P43. Lane 1, dsRNAs of strain
KU (G1P[8]) extracted from a cell culture; lanes 2–7, dsRNAs of strains A14 (lane 2), A23 (lane 3), A25 (lane
4), P02 (lane 5), P39 (lane 6), and P43 (lane 7) extracted from cell cultures. The numbers on the left indicate
the order of the genomic dsRNA segments of strain KU.
doi:10.1371/journal.pone.0124965.g001
pattern in PAGE of cell culture-adapted strains A14, A23, A25, P02, P39, and P43 was identical
to that of the original strains A14, A23, A25, P02, P39, and P43 present in stool samples, re-
spectively (data not shown). They all showed a long electropherotype. Cell culture-adapted
strains A14, A23, A25, P02, P39, and P43 were named RVA/Human-tc/MMR/A14/2011/G12P
[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8],
RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and
RVA/Human-tc/MMR/P43/2011/G12P[8], respectively, according to the guidelines for the
uniformity of RVAs proposed by the RCWG. Of note was that strains A14, A25, P02, P39, and
P43 showed an almost identical electropherotype, suggesting a close genetic relatedness among
the five strains.
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Nucleotide sequencing and whole-genome-based genotyping of strains
A14, A23, A25, P02, P39, and P43
In order to gain an insight into the genetic variability among strains A14, A23, A25, P02, P39,
and P43, and the genetic relatedness with other RVA strains worldwide, the full-genome se-
quences of all the 11 segments of these six strains were determined using the NGS Illumina
MiSeq platform. The whole genomes of the six strains were amplified using a sequence-inde-
pendent primer set and then sequenced successfully. Illumina MiSeq sequencing yielded 2.0 x
106 reads (~143 bp average length), 1.9 x 106 reads (~144 bp average length), 1.7 x 106 reads
(~144 bp average length), 1.8 x 106 reads (~145 bp average length), 1.7 x 106 reads (~142 bp av-
erage length), and 1.7 x 106 reads (~145 bp average length) for strains A14, A23, A25, P02, P39,
and P43, respectively. Missing sequence information in the contigs of the NSP2 gene of strain
A23, and the NSP5 genes of strains A14 and A25 was filled in by Sanger sequencing of
RT-PCR products. This approach allowed determination of the complete or nearly complete
nucleotide sequences of all the 11 segments of the six strains, while deep sequencing revealed
the presence of multiple SNPs at<1.8% frequency in the six strains.
The 11 genes of strains A14, A23, A25, P02, P39, and P43 were assigned as G12-P[8]-
I1-R1-C1-M1-A1-N1-T1-E1-H1 (strains A14, A25, P02, P39, and P43), and G12-P[6]-
I1-R1-C1-M1-A1-N1-T1-E1-H1 (strain A23) (Fig 2). Strains A14, A23, A25, P02, P39, and P43
were confirmed to have the G12P[8], G12P[6], G12P[8], G12P[8], G12P[8], and G12P[8] geno-
types, respectively, as determined by PCR-based genotyping (Ide et al., in preparation). Compari-
son of the complete genotype constellations of strains A14, A23, A25, P02, P39, and P43 with
those of other G12 and non-G12 strains is shown in Fig 2. All the six Myanmarese G12 strains
exhibited typical Wa-like genotype constellations, which are commonly found in the G12 strains
recently detected worldwide (Rahman et al., 2007). Furthermore, as suggested by the genomic
dsRNA profiles observed on PAGE analysis (Fig 1), strains A14, A25, P02, P39, and P43 exhib-
ited extremely high nucleotide sequence identities (99.1–100%) to one another for all the 11 gene
segments. On the other hand, the nucleotide sequence similarities of the VP4 and other 10 gene
segments (VP7, VP6, VP1-3, and NSP1-5) of strain A23 to those of strains A14, A25, P02, P39,
and P43 were comparatively low (75.3 and 93.1–98.7%, respectively).
Phylogenetic analyses
We next constructed phylogenetic trees using the full-genome sequence for each of the 11 gene
segments because phylogenetic analysis of RVA nucleotide sequences provides direct evidence
of their relatedness to those of other strains, even within the same genotype [48].
The VP7 genes of strains A14, A25, P02, P39, and P43 exhibited the maximum nucleotide
sequence identities (99.7–99.8%) with that of Thai human strain CU331-NR (G12P[6]) [45]
(Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to form a
cluster with strain CU331-NR and several human G12 strains from Thailand in G12 lineage-3,
in which the majority of globally circulating G12 strains cluster [9, 10] (Fig 3). On the other
hand, the VP7 gene of strain A23 showed the highest nucleotide sequence similarity (99.1%)
with South African human strain MRC-DPRU4090 (G12P[6]) (Fig 2). Phylogenetically, strain
A23 was found to be closely related with strain MRC-DPRU4090 in a common branch with
German human strain GER172-08 (G12P[6]) [59] in G12 lineage-3 (Fig 3).
The VP4 genes of strains A14, A25, P02, P39, and P43 showed the highest nucleotide se-
quence identities (99.6%) with the cognate genes of Thai human strain CU769-KK (G1P[8])
[46], Russian human strain Nov09-B28 (G1P[8]), and/or Canadian human strain RT010-09
(G3P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 clustered near
these, and several human G1 and G3 strains from different countries of the world in P[8]
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Whole Genomes of Human G12 Rotaviruses in Myanmar
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 2. Genotype natures of the 11 gene segments of six Myanmarese G12 strains, A14, A23, A25, P02, P39, and P43, with those of selected human
and animal strains. Strains A14, A23, A25, P02, P39, and P43 are shown in red. Gray shading indicates the 10 gene segments (VP7, VP6, VP1-3, and
NSP1-5) with genotypes identical to those of strains A14, A23, A25, P02, P39, and P43. Green shading indicates the VP4 gene segments with a P[8]
genotype identical to those of strains A14, A25, P02, P39, and P43. Blue shading indicates the VP4 gene segments with a P[6] genotype identical to that of
strain A23. “−” indicates that no sequence data were available in the DDBJ and EMBL/GenBank data libraries. aThe gene segments that are most similar to
those of strain A14. bThe gene segments that are most similar to those of strain A23. cThe gene segments that are most similar to those of strain A25. dThe
gene segments that are most similar to those of strain P02. eThe gene segments that are most similar to those of strain P39. fThe gene segments that are
most similar to those of strain P43.
doi:10.1371/journal.pone.0124965.g002
lineage-3 (Fig 4). In contrast, the VP4 gene of strain A23 exhibited the maximum nucleotide se-
quence identity (99.2%) with South African human strain MRC-DPRU4090 (G12P[6]) (Fig 2).
Phylogenetically, strain A23 was found to be closely related with strain MRC-DPRU4090 in a
Fig 3. Phylogenetic tree constructed from the nucleotide sequences of the VP7 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. In the tree, the positions of strains A14, A23, A25, P02, P39, and P43 are shown in red. Bootstrap values of <75% are not
shown. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g003
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 4. Phylogenetic tree constructed from the nucleotide sequences of the VP4 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g004
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Whole Genomes of Human G12 Rotaviruses in Myanmar
common branch with several human G12 strains from different parts of the world in P[6] line-
age-2 (Fig 4).
The VP6 genes of strains A14, A25, P02, P39, and P43 exhibited the highest nucleotide se-
quence identities (99.3–99.6%) with the VP6 gene of Australian human strain CK00089 (G1P
[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to be
closely related with strain CK00089 near several human G12 strains (Fig 5). On the other hand,
the VP6 gene of strain A23 showed the maximum nucleotide similarity (99.6%) with South Af-
rican human strain MRC-DPRU4090 (G12P[6]) (Fig 2). Phylogenetically, strain A23 was
found to be clustered with strain MRC-DPRU4090 and several human G1 strains (Fig 5).
The VP1 genes of strains A14, A25, P02, P39, and P43 showed the maximum nucleotide se-
quence identities (99.6–99.7%) with the cognate gene of Australian human strain CK00089
(G1P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to
be clustered near strain CK00089, and several human G1, G11, and G12 strains (Fig 6). On the
other hand, the VP1 gene of strain A23 exhibited the highest nucleotide sequence similarity
(99.1%) with South African human strains MRC-DPRU2130-05 (G12P[6]) and
MRC-DPRU840 (G1P[8]) (Fig 2). Phylogenetically, strain A23 clustered near these, and several
human G1 and G12 strains (Fig 6).
The VP2 genes of strains A14, A25, P02, P39, and P43 showed the highest nucleotide se-
quence similarities (99.6–99.7%) with the VP2 genes of Thai human strains CU331-NR (G12P
[6]) and/or CU460-KK (G12P[8]) [45] (Fig 2). On phylogenetic analysis, strains A14, A25, P02,
P39, and P43 were found to be closely related with these strains in a common branch with South
African human strain MRC-DPRU2132 (G1P[8]) (Fig 7). In contrast, the VP2 gene of strain
A23 exhibited the maximum nucleotide sequence identity (99.4%) with South African human
strain MRC-DPRU4090 (G12P[6]) (Fig 2). On phylogenetic analysis, strain A23 was found to be
closely related with strain MRC-DPRU4090 in a common branch with German human strain
GER172-08 (G12P[6]) and Bangladeshi human strain MMC71 (G1P[8]) [60] (Fig 7).
The VP3 genes of strains A14, A25, P02, P39, and P43 showed the maximum nucleotide se-
quence identities (99.7–99.8%) with those of Thai human strains CU331-NR (G12P[6]) and/or
CU460-KK (G12P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43
were found to be clustered with these strains, and several human G1 and G12 strains (Fig 8).
On the other hand, the VP3 gene of strain A23 exhibited the highest nucleotide sequence simi-
larity (99.5%) with Canadian human strain RT103-07 (G1P[8]) (Fig 2). Phylogenetically, strain
A23 was found to be closely related with South African human strain MRC-DPRU4090 (G12P
[6]) in a common branch with strain RT103-07 and several human G12 strains from Africa
(Fig 8).
The NSP1 genes of strains A14, A25, P02, P39, and P43 exhibited the highest nucleotide se-
quence identities (99.5–99.6%) with the cognate gene of Australian human strain CK00089
(G1P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to
be closely related with strain CK00089 in a common branch with Thai human strains
CU331-NR (G12P[6]) and CU460-KK (G12P[8]) (Fig 9). On the other hand, the NSP1 gene of
strain A23 showed the maximum nucleotide sequence similarity (99.0%) with Argentinian
human strains (Arg6627 (G12P[8]) and Arg7500 (G12P[8]) [61]), Paraguayan human strains
(1670SR (G1P[8]), 1679SR (G1P[8]), and 1692SR (G1P[8])), and South African human strain
MRC-DPRU799 (G1P[8]) (Fig 2). Phylogenetically, strain A23 was found to be clustered near
these strains, and several human G1 and G12 strains (Fig 9).
The NSP2 genes of strains A14, A25, P02, P39, and P43 exhibited the maximum nucleotide
sequence identities (99.6–99.8%) with those of Thai human strains CU331-NR (G12P[6]) and
CU460-KK (G12P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43
were found to be closely related with these strains (Fig 10). On the other hand, the NSP2 gene
PLOSONE | DOI:10.1371/journal.pone.0124965 May 4, 2015 10 / 24
Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 5. Phylogenetic tree constructed from the nucleotide sequences of the VP6 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.02 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g005
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 6. Phylogenetic tree constructed from the nucleotide sequences of the VP1 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g006
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 7. Phylogenetic tree constructed from the nucleotide sequences of the VP2 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g007
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 8. Phylogenetic tree constructed from the nucleotide sequences of the VP3 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g008
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 9. Phylogenetic tree constructed from the nucleotide sequences of the NSP1 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g009
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 10. Phylogenetic tree constructed from the nucleotide sequences of the NSP2 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.02 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g010
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Whole Genomes of Human G12 Rotaviruses in Myanmar
of strain A23 showed the highest nucleotide sequence identity (99.8%) with Ugandan human
strain MRC-DPRU4616 (G12P[6]), and comparable identity (99.7%) with South African
human strain MRC-DPRU4090 (G12P[6]) (Fig 2). Phylogenetically, strain A23 was found to
be closely related with these strains in a common branch with German human strain GER172-
08 (G12P[6]) and Russian human strain Nov10-N921 (G9P[6]) [62] (Fig 10).
The NSP3 genes of strains A14, A25, P02, P39, and P43 showed the highest nucleotide se-
quence identities (99.3–99.5%) with the NSP3 gene of Thai human strain CU331-NR (G12P
[6]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to be
closely related with strain CU331-NR in a common branch with several human G1, G3, G9,
and G12 strains (Fig 11). In contrast, the NSP3 gene of strain A23 exhibited the maximum nu-
cleotide sequence identity (99.2%) with German human strain GER172-08 (G12P[6]), and
comparable identity (99.1%) with South African human strains MRC-DPRU4090 (G12P[6]),
MRC-DPRU799 (G1P[8]), and MRC-DPRU840 (G1P[8]) (Fig 2). On phylogenetic analysis,
strain A23 was found to be closely related with strain MRC-DPRU4090 in a common branch
with these, and several human G1 and G12 strains (Fig 11).
The NSP4 genes of strains A14, A25, P02, P39, and P43 showed the maximum nucleotide
sequence similarities (99.4–99.6%) with the cognate genes of Thai human strain CU769-KK
(G1P[8]), Chinese human strain LL11131077 (G9P[8]), and/or Russian human strain
Nov09-B28 (G1P[8]) (Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43
were found to be clustered near these, and several human G1, G3, G9, and G12 strains (Fig 12).
In contrast, the NSP4 gene of strain A23 exhibited the highest nucleotide sequence identity
(99.5%) with Indian human-like bovine strain B85-MP (G3P[3]), South African human strain
MRC-DPRU799 (G1P[8]), and American human strain USA2007719739 (G1P[8]) (Fig 2).
Phylogenetically, A23 was found to be clustered with these, and several human-like bovine and
human strains within the human-like E1 subcluster (Fig 12).
The NSP5 genes of strains A14, A25, P02, P39, and P43 exhibited the maximum nucleotide
sequence identities (99.6–99.8%) with that of Australian human strain CK00089 (G1P[8])
(Fig 2). On phylogenetic analysis, strains A14, A25, P02, P39, and P43 were found to be clus-
tered with strain CK00089, and several human G1 and G12 strains (Fig 13). On the other hand,
the NSP5 gene of strain A23 showed the maximum nucleotide sequence similarity (99.6%)
with South African human strains MRC-DPRU4090 (G12P[6]) and MRC-DPRU1191 (G12P
[8]) (Fig 2). On phylogenetic analysis, strain A23 was found to be clustered near these, and sev-
eral human G9 and G12 strains (Fig 13).
In the present study, we analyzed the whole genomes of six Asian G12 strains that have
emerged in Myanmar (strains A14, A23, A25, P02, P39, and P43) identified in six stool speci-
mens from children with gastroenteritis. All six Myanmarese G12 strains were found to have a
Wa-like backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strains A14, A25, P02, P39,
and P43) and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strain A23). Whole genomic analy-
ses of currently circulating G12 strains from different parts of the world have revealed the pre-
dominant possession of a Wa-like genetic backbone by them [10]. Because a Wa-like genotype
backbone is believed to be one of the stable genetic constellations of human RVAs, the Wa-like
backbone might have conferred an evolutionary advantage on G12 strains, making it possible
for them to spread across the globe in an incredibly short time period [48, 50, 63]. Phylogenetic
analysis revealed that in most genes the six strains examined in this study were genetically re-
lated to globally circulating human G1, G3, G9, and G12 strains. Furthermore, strains A14,
A25, P02, P39, and P43 were very closely related to one another as to all the 11 genes, indicat-
ing the derivation of the five strains from a common origin. On the other hand, strain A23 con-
sistently formed distinct clusters for all the 11 gene segments, indicating its distinct origin from
that of strains A14, A25, P02, P39, and P43. While eight of the 11 gene segments (VP7, VP4,
PLOSONE | DOI:10.1371/journal.pone.0124965 May 4, 2015 17 / 24
Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 11. Phylogenetic tree constructed from the nucleotide sequences of the NSP3 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.05 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g011
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 12. Phylogenetic tree constructed from the nucleotide sequences of the NSP4 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.02 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g012
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Whole Genomes of Human G12 Rotaviruses in Myanmar
Fig 13. Phylogenetic tree constructed from the nucleotide sequences of the NSP5 genes of strains A14, A23, A25, P02, P39, and P43, and
representative RVA strains. See legend of Fig 3. Scale bars, 0.02 substitutions per nucleotide.
doi:10.1371/journal.pone.0124965.g013
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Whole Genomes of Human G12 Rotaviruses in Myanmar
VP2-3, and NSP1-4) of strains A14, A25, P02, P39, and P43 were very closely related to those
of Asian strains, all the 11 gene segments of strain A23 were very closely associated with Afri-
can strains. Thus, the G12 strains that have emerged in Myanmar appeared to have originated
from not a single, but at least two distinct ancestral G12 strains.
Notably, the NSP4 gene of strain A23 having E1 genotype showed the closest relationship
with the NSP4 genes of human-like bovine strains from India, as well as human strains
(Fig 12). Genotype E1 has been recently detected in bovines in India and Korea, in addition to
more common bovine genotype E2 [64, 65]. However, the exact origin of the NSP4 gene of
strain A23 could not be ascertained due to a lack of a sufficient number of representative
strains as references. In any case, this observation suggests the occurrence of reassortment be-
tween human and bovine strains. To our knowledge, this is the first description on full ge-
nome-based characterization of the G12 strains that have emerged in Myanmar.
The increasing detection of G12 strains in Asia implies the continued spread of G12 strains
in Asia. Because it has not been examined as to whether or not the two available RVA vaccines
(Rotarix (GlaxoSmithKline) and RotaTeq (Merck)) are effective for prevention against unusual
strains, such as G12P[6] strains that share no G/P genotype specificities with strains included
in these vaccines, continuing RVA surveillance of the G12 genotype may be required. Simulta-
neous monitoring of RVA strains in humans and animals is also essential for a better under-
standing of RVA ecology. Furthermore, whole genome-based analyses are essential to
understand the evolutionary dynamics of G12 strains spreading in Asia.
Acknowledgments
We wish to thank Mayuko Tomita for her technical assistance.
Author Contributions
Conceived and designed the experiments: TI SK KT. Performed the experiments: TI SK KHM
MWKT. Analyzed the data: TI SK KT. Contributed reagents/materials/analysis tools: KWH
YYM TWMKZT HMTMMWHNO TH FED KH YF KK SR SVN KU KO TT. Wrote the
paper: TI SK KT.
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