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564
24
565
Figure Legends
566
Fig. 1. Phylogenetic tree analysis of VP7 genes for G12 RVA strains detected in five
567
samples collected in Surabaya, Indonesia. Black inverted triangles and Bullets: The RVA
568
strains sequenced in this study. The reference strains obtained from the GenBank database
569
are represented by the accession number, strain name, country, and year of isolation. Scale
570
bar: 0.050 substitutions per nucleotide.
571
572
Fig. 2. Phylogenetic tree analysis of VP4 genes detected in five samples collected in
573
Surabaya, Indonesia. Black inverted triangles and Bullets: The G12 and G1 RVA strains
574
sequenced in this study, respectively. The reference strains obtained from the GenBank
575
database are represented by the accession number, strain name, country, and year of isolation.
576
Scale bar: 0.020 substitutions per nucleotide.
577
578
Fig. 3. The VP7 genotyping of STM430.
579
To confirm the mixed infection for STM430 sample, the RNA was re-extracted from original
580
clinical sample. RVA genotyping using the semi-nested multiplex PCR were performed for
581
both 1st and 2nd extracted RNA of STM430 together with other G1 (STM387) and G12
582
(STM413) samples. The size of PCR products was determined by D1000 ScreenTape assay
583
on 4150 TapeStation (Agilent Technologies).
584
25
Table 1. RVA genotype constellations of five human G12 RVA study strains compared to the published human G12 RVA strains
G12 strain
Year
Genogroup
VP7
(Lineage)
VP4
VP6
VP1
VP2
VP3
NSP1
NSP2
NSP3
NSP4
NSP5
L26
1987
DS-1-like
G12 (I)
P[4]
I2
R2
C2
M1/M2
A2
N1
T2
E2
H1
T152
1998
AU-1-like
G12 (II)
P[9]
I3
R3
C3
M3
A12
N3
T3
E3
H6
RV161-00
2000–2002
DS-1-like
G12 (III)
P[6]
I2
R2
C2
M2
A2
N2
T2
E1
H2
RV176-00
2000–2002
DS-1-like
G12 (III)
P[6]
I2
R2
C2
M2
A2
N2
T2
E6
H2
N26-02
2000–2002
DS-1-like
G12 (III)
P[6]
I2
R2
C2
M2
A2
N1
T2
E6
H2
Dhaka25-02
2002–2005
Wa-like
G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
Dhaka12-03
2002–2005
Wa-like
G12 (III)
P[6]
I1
R1
C1
M1
A1
N1
T1
E1
H1
Matlab13-03
2002–2005
Wa-like
G12 (III)
P[6]
I1
R1
C1
M1
A1
N1
T2
E1
H1
SOEP379-18
2017–2018
Wa-like
G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
STM371-17
2017–2018
Wa-like
G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
STM413-17
2017–2018
Wa-like
G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
STM433-17
2017–2018
Wa-like
G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
STM430-17
2017–2018
Wa-like
G1/G12 (III)
P[8]
I1
R1
C1
M1
A1
N1
T1
E1
H1
Reference
Previous
study16
Present
study
The Wa-, DS-1-, and AU-1-like genogroups were assigned to human strains if at least seven gene segments belonged to the Wa-, DS-1, and AU-1-like
genotype, respectively. Colors were added to visualize patterns or gene constellations. Green: Wa-like, red: DS-1-like, and yellow: AU-like gene segments.
Gray: Subtype 4A, and blue: RVA gene from animals.
*Green is Wa-like strain. *Red is DS-1-like strain. *Yellow is AU-1-like strain. *Gray is an ST3-like strain. *Blue is a RVA gene from an animal.
Table 2. Nucleotide sequence identity between G12 RVA genes obtained from Illumina MiSeq sequencing and the closest strains isolated from
references based on NCBI homology BLAST
Gene
VP7
Accession
no.
Genotype
Samples
LC581270
LC581271
LC581272
LC581273
LC581274
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
LC581275
G1P[8]
STM430
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
LC721862
LC581261
LC581262
LC581263
LC721864
Closest strain of NCBI
homology
(% nt identity)
99.14%
99.24%
RVA/Human99.22% wt/USA/20137741
99.24%
66/2013/G12P[8]
99.24%
RVA/Human99.81%
wt/IDN/STM457/2
018/G1P[8]
99.22%
99.08%
RVA/Human99.35%
wt/BGD/Bang99.26%
065/2008/G9P[8]
99.26%
VP4
NSP1
Accession
no.
Genotype
Samples
LC721867
LC581221
LC581222
LC581223
LC581224
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
LC722500
G1P[8]
STM430
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
LC721868
LC581226
LC581227
LC581228
LC581229
Closest strain of NCBI
homology
(% nt identity)
99.45%
99.38%
Hungary human
99.52%
strain ERN5009
99.59%
isolated 2012
99.52%
RVA/Human99.60% wt/IDN/STM457/201
8/G1P[8]
98.80%
98.84%
RVA/Human98.88% wt/USA/2013774165
98.94%
/2013/G12P[8]
98.94%
NSP2
G1P[8]
STM430
100%
RVA/Humanwt/IDN/STM387/201
7/G1P8 and
RVA/Humanwt/IDN/STM453/201
7/G1P[8]
LC721869
LC581231
LC581232
LC581233
LC581234
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
98.93%
98.77%
99.28%
98.88%
98.71%
RVA/Humanwt/THA/DBM2018111/2018/G9P[8]
LC722502
G1P[8]
STM430
99.91%
RVA/Humanwt/IDN/STM457/201
7/G1P[8]
RVA/Humanwt/IDN/STM457/2
017/G3P[8]
LC722501
99.76%
99.76%
99.76%
99.76%
99.82%
RVA/human/SVK/
2451/I1
99.77%
MN304727.1
RVA/Humanwt/NGR/2017
G1P[8]
STM430
99.83%
LC721863
LC581266
LC581267
LC581268
LC581269
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
LC722499
G1P[8]
STM430
LC722498
VP6
Gene
NSP3
VP1
VP2
LC721864
LC581246
LC581247
LC581248
LC581249
STM430
SOEP379
STM371
STM413
STM433
99.25%
99.26%
99.33%
99.23%
99.26%
G12P[8]
LC722495
G1P[8]
STM430
99.88%
LC721865
LC581251
LC581252
LC581253
LC581254
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
99.10%
99.19%
99.44%
99.40%
99.33%
LC722496
G1P[8]
STM430
99.89%
STM430
(like G1)
99.81%
SOEP379
STM371
STM431
STM433
98.44%
98.33%
98.48%
98.48%
STM430
(like G12)
97.59%
LC721866
VP3
LC581256
LC581257
LC581258
LC581259
G12P[8]
LC722497
G1P[8]
LC721870
RVA/Humanwt/USA/VU12-1339/2013/G12P[8]
NSP4
RVA/Humanwt/IDN/STM453/2
018/G1P[8]
RVA/Humanwt/BGD/Bang015/2008/G12P[6]
RVA/Humanwt/IDN/STM453/2
018/G1P8
RVA/Humanwt/IDN/STM387/2
018/G1P[8]
RVA/Humanwt/USA/VU08-096/2008/G12P[8]
RVA/Humanwt/USA/VU08-096/2008/G12P[8]
STM430
(like G1)
99.86%
RVA/Humanwt/IDN/STM457/201
8/G1P[8]
SOEP379
STM371
STM413
STM433
99.46%
99.47%
99.31%
99.18%
RVA/Cowwt/UGA/BUW-14A035/2014/G12P[8]
LC581236
LC581237
LC581238
LC581239
G12P[8]
LC722503
G1P[8]
STM430
(like G12)
99.44%
LC722504
LC581241
LC581242
LC581243
LC581244
G12P[8]
STM430
SOEP379
STM371
STM413
STM433
99.39%
99.54%
99.52%
99.38%
99.54%
RVA/Humantc/USA/DC5685-40AG/1991/G1P[8]
LC721871
G1P[8]
STM430
100%
RVA/Humanwt/IDN/STM457/201
8/G1P[8]
RVA/Cowwt/UGA/BUW-14A035/2014/G12P8
NSP5
VP7
VP7_G12
100
VP7_G1
VP7_G3
0.050
100
RVA/Human/IDN/STM413/2017-2018/G12P[8][LC581273]
RVA/Human/IDN/STM371/2017-2018/G12P[8][LC581272]
83
RVA/Human//IDN/STM433/2017-2018/G12P[8][LC591274]
99
RVA/Human/IDN/STM430/2017-2018/G12P[8][LC581270]
RVA/Human/IDN/SOEP379/2017-2018/G12P[8][581271]
95
RVA/Human-wt/USA/2013774166/2013/G12P[8][KM116056]
RVA/Human-wt/USA/VU12-13-146/2013[KT919276]
95
RVA/Human-wt/USA/VU12-13-31/2013/G12P[8][KT919497]
76
96 RVA/Cow-wt/UGA/BUW-14-A035/2014/G12P[8][KX655533]
Human rotavirus A strain GER172-08 VP7[FJ747630]
RVA/Human-wt/BGD/SK327/2006/G12P[6][EU839944]
RVA/Human-wt/IND/ISO121/2005/G12P[x][EU016452]
75
RVA/Human-tc/MWI/MAL12/2007/G12P[6][JN591403]
RVA/Human-tc/MWI/MAL40/2007/G12P[6][AB938241]
RVA/Human-wt/SouthAFR/SA4727DGM04/G12P[6][EU284738]
RVA/Human-wt/IND/mani-74/2006/G12P[6][GQ229055]
RVA/Human-wt/BGD/Dhaka12/2003/G12P[6][DQ146665]
97
RVA/Human/BGD/Matlab13/2003/G12P[6][DQ146676]
RVA/Human-wt/SouthAFR/SA3332JHB04/G12P[6][EU284724]
RVA/Human-wt/UGA/MRC-DPRU3713/2010/G12P[6][KJ751872]
RVA/Human-wt/UGA/MUL-13-183/2013/G12P[6][KX632270]
77
RVA/Human-wt/ZMB/MRC-DPRU3491/2009/G12P[6][KF636151]
94
RVA/Human-wt/SRK/KUH407/2006/G12P[8][AB530274]
RVA/Human-wt/BR/RV98660/PA/2008/G12P[6][GU250829]
80 RVA/Human-wt/BGD/RV176/2000/G12P[6][DQ490556]
100 75 RVA/Human-wt/BGD/RV161/2000/G12P[6][DQ490550]
RVA/Human-wt/BGD/N26/2002/G12P[6][DQ145587]
RVA/Human-wt/BGD/Dhaka25/2002/G12P[8][DQ146654]
RVA/Human-wt/NPL/05NO65/2005/G12[AB275297]
RVA/Human-wt/SRK/05SLC033/2005/G12[AB306271]
RVA/Human-wt/THA/T152/1998/G12[AB071404]
91
78
RVA/Human-wt/JPN/K12/G12[AB186120]
RVA/Human-wt/ARG/Arg721-2A/2003/G12[EU496255]
RVA/Human-wt/JPN/CP1030/2003/G12[AB125853]
99
RVA/Human-wt/PRY/Py1135ASR07/2007/G12[FJ941102]
99 RVA/Human-wt/PRY/1135A/2007/G12P[9][KJ412846]
Human rotavirus A strain L26 neutralizing protein VP7[M58290]
100 RVA/Human-tc/PHL/L26/1987/G12P[4][EF672595]
RVA/Pig-wt/IND/RU172/2002/G12P[7][DQ204743]
RVA/Human/IDN/STM430/2017-2018/G1P[8][LC581275]
100
RVA/Human-wt/IDN/STM457/2018/G1P[8][LC434538]
RVA/Human-wt/IND/RV1326/2013/G1[KX638552]
RVA/Human-wt/IND/CMC/00033/2012/G1P[8][MN067085]
RVA/Human-tc/USA/Wa-20-AG/1974/G1P[8][KU861395]
RVA/Human-wt/AUS/D388/2013/G3P[8][KU059771]
Lineage III
Lineage II
Lineage I
Lineage IV
Fig. 1.
VP4
72
89
0.020
RVA/Human//IDN/STM433/2017-2018/G12P[8][LC721864]
RVA/Human/IDN/STM430/2017-2018/G12P[8][LC721862]
RVA/Human/IDN/STM413/2017-2018/G12P[8][LC581263]
98
RVA/Human/IDN/SOEP379/2017-2018/G12P[8][LC581261]
88
RVA/Human/IDN/STM371/2017-2018/G12P[8][LC581262]
95 RVA/Human-wt/BGD/Bang-065/2008/G9P[8][KP882000]
93 RVA/Human-wt/CAN/RT060-09/2009/G9P[8][JQ069696]
RVA/Human-wt/BGD/DH396/2004/G9P[8][EU839959]
74
RVA/Human-wt/USA/CNMC15/2011/G12P[8][KT920863]
RVA/Human-wt/BGD/Dhaka25/2002/G12P[8][DQ146652]
RVA/Human-wt/JPN/UR14-24/2014/G1P[8][LC105501]
RVA/Human-wt/USA/3000390639/2015/G3P[8][MF997038]
90
97 RVA/Human-wt/JPN/S140023/2014/G3P[8][AB930192]
RVA/Human-wt/Vanderbilt/USA/VU05-06-74/2005/G12P[8][JF790309]
88
RVA/Human-wt/Vanderbilt/USA/VU05-06-72/2005/G12P[8][JF790298]
RVA/Human-wt/IDN/STM457/2018/G3P[8][LC430884]
98
RVA/Human/IDN/STM430/2017-2018/G1P[8][LC722498]
RVA/Human-wt/IDN/STM453/2018/G3P[8][LC430883]
97 RVA/Human-wt/JPN/Tokyo17-21/2017/G3P[8](Wa)[LC477393]
RVA/Human-wt/IND/CMC/00048/2013/G9P[8][MN066757]
RVA/Human-wt/DOM/3000503708/2015/G1P[8][MG652340]
RVAHuman-wt/USA/VU12-13-30/2013/G2P[4][MF168166]
RVA/Human-tc/USA/Wa/1974/G1P[8][JX406750]
99 RVA/Human-wt/JPN/MU14-16/2014/G2P[4][LC105164]
95
RVA/Human-wt/USA/VU12-13-30/2013/G2P[4][MF168166]
RVA/Human-wt/BEL/BE35/2007/G2P[4][KR705281]
98
RVA/Human-wt/PAK/3085/2010/G2P[4][KY497532]
RVA/Human-wt/JPN/K-3-16/2016/G2P[4][LC228351]
97
Human rotavirus A strain L26 outer capsid protein VP4[EF672591]
RVA/Human-tc/USA/DS-1/1976/G2P[4][HQ650119]
RVA/Human-wt/BGD/RV161/2000/G12P[6][DQ490548]
RVA/Human-wt/BGD/RV176/2000/G12P[6][DQ490554]
81 RVA/Human-wt/BGD/MMC29/2005/G12P[6][EU839947]
RVA/Human/BGD/Matlab13/2003/G12P[6][DQ146674]
RVA/Human-wt/BGD/Dhaka12/2003/G12P[6][DQ146663]
96
RVA/Human-wt/BGD/N26/2002/G12P[6][DQ146685]
RVA/Human-wt/SouthKOR/CAU/214/G12[EF059921]
99
RVA/Human-wt/UGA/MUL-12-117/2012/G3P[6][KX655476]
96 RVA/Human-wt/THA/T152/1998/G12[AB077766]
RVA/Human-wt/BRA/PE15776/2008/G12P[9][KF907295]
100
RVA/Human/AU-1/1994/VP4[D10970]
P[8] [Wa-like]
P[4] [DS-1-like]
P[6] [ST3-like]
P[9] [AU-1-like]
Fig.2.
D1000
Ladder
Positive control
G1
G12
STM
430
(1st)
STM
430
(2nd)
STM
387
(G1)
STM
413
(G12)
Neg
control
636 bp (G1)
264 bp (G12)
Fig. 3.
...
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