BRIEF REPORT

Molecular evolution of the VP7 gene of Japanese G2 rotavirusesbefore vaccine introduction

L. P. Do • T. Nakagomi • Y. H. Doan •

Y. Kitahori • O. Nakagomi

Received: 10 May 2013 / Accepted: 20 June 2013

� Springer-Verlag Wien 2013

Abstract Changes in the prevalence of G2 rotavirus after

vaccine introduction are an important issue. However, such

changes in a given country should be interpreted in the

global context over time. We determined 35 Japanese G2

sequences and compared them with 508 globally collected

G2 sequences. The D96N substitution, a substitution

known to be associated with an abrupt increase in G2

strains and antigenic changes, emerged in those strains that

formed a nascent lineage outside of the currently pre-

dominant lineage (sublineage IVa). Further studies are

warranted to monitor the potential of their global spread,

since they also appeared in Europe and Australia.

Rotavirus A is the most important etiological agent of severe

acute gastroenteritis in infants and young children world-

wide. Two outer capsid proteins, VP7 and VP4, are inde-

pendently involved in virus neutralization and define the G

type and the P type, respectively. More than three quarters of

human rotaviruses carry G1P[8], G2P[4], G3P[8], G4P[8]

and G9P[8], of which approximately 12 % are G2P[4] [2].

Currently, two globally licensed rotavirus vaccines are

available: a monovalent G1P[8] human rotavirus vaccine

(Rotarix, GlaxoSmithKline Biologicals, Rixensart, Bel-

gium) and a pentavalent bovine-human reassortant rotavirus

vaccine (RotaTeq, Merck and Co., Whitehouse Station, PA,

USA). A marked increase and the predominance of G2P[4]

rotavirus strains were observed concurrently with the intro-

duction of the universal mass vaccination program with the

monovalent vaccine in Brazil [6, 12, 14]. In Australia, where

two rotavirus vaccines were used in different regions, fol-

low-up studies after vaccination showed that in the states

using RotaTeq, G1P[8] was predominant, followed by

G2P[4], whereas in the states using Rotarix, G2P[4] was

predominant, followed by G1P[8] [9, 17]. On the other hand,

a similarly high prevalence of G2P[4] was observed in other

countries where no rotavirus immunization program was

implemented, such as Argentina and Paraguay, two neigh-

boring countries of Brazil [5, 10].

Thus, it is important to interpret the changes in the

prevalence of G2 strains after vaccine introduction in a

given country from the phylogenetic perspective over time.

In Japan, Rotarix was introduced into the private market as

the first rotavirus vaccine in November 2011, and RotaTeq

followed in July 2012. Because there were only 20 Japa-

nese G2 VP7 gene sequences available in the GenBank

database, this study was undertaken to determine the VP7

sequence of the G2 rotavirus strains collected in Japan over

the last 31 years (1980 to 2011) to understand how G2

rotavirus strains evolved over time under unvaccinated

conditions. In the phylogenetic analysis, we placed a spe-

cial emphasis on the amino acid substitution at position 96

because there was evidence that a D96N substitution in

VP7 was related to an increase or the re-emergence of G2

strains, as reported from Taiwan, Thailand, Bangladesh,

South Africa, the United Kingdom, and Nepal [1, 4, 7, 13,

19, 20]. Furthermore, Doan et al. [4] clarified that the

D96N substitution is the salient feature of virtually all G2

strains circulating in the world for the last decade.

A total of 35 rotavirus strains analyzed in this study

were isolated from archival stool specimens obtained from

L. P. Do � T. Nakagomi � Y. H. Doan � O. Nakagomi (&)

Department of Molecular Microbiology and Immunology,

Graduate School of Biomedical Sciences, Nagasaki University,

1-12-4 Sakamoto, Nagasaki 852-8523, Japan

e-mail: onakagom@nagasaki-u.ac.jp

Y. Kitahori

Nara Prefectural Institute for Hygiene and Environment,

Nara, Japan

123

Arch Virol

DOI 10.1007/s00705-013-1804-6

MMA08-4/Myanmar/2008 mani-268/India/2007

Matlab6/Bangladesh/2004 CU110-BK/Thailand/2008

05N011/Nepal/2005 16054 09MA/Brazil/2009 LB2764/USA/2005-06

GER31/Germany/2008 DC2020/Madagascar/2004

Seoul-397/South Korea/2009 B110056/Japan/2011

MR 4717/Mauritius/2000 XJ00-486/China/2000

Bangla66/Bangladesh/1993 NG5113/Negeria/1999

GH1803/Ghana/1999 4476PT/South Africa/1997

80123/Japan/2008 80120/Japan/2008

CH-130/China/2003 90096/Japan/2009

GER84/Germany/2008 26-Palermo/Italy/2003 07Y003/Japan/2007

80004/Japan/2008 Arg2395/Argentina/2007

Seoul-556/South Korea/2009 LB2772/USA/2005-06

14322 07MG/Brazil/2007 01P017/Japan/2001

01P007/Japan/2001 01P012/Japan/2001

01P129/Japan/2001 01P014/Japan/2001 01R137/Japan/2001

KO2/Japan/2001 J-4787/Japan/2001-2003

J-4708/Japan/2001-2003 J-4709/Japan/2001-2003

VN-11/Vietnam/2002 J-4765-Japan-2001-2003

J-4777/Japan/2001-2003 Py40699/Paraquay/1999

KY3103/Kenya/1999 01P008/Japan/2001 CMH277/Thailand/2001

TD64/Taiwan/1992 CIT-220RV/Ireland/2000

Mvd9707/Uruguay/1997 Bangla84/Bangladesh/1994

SL244/Sierra Leone/2005 356PT/South Africa/1996

87H038/Japan/1987 90Y289/Japan/1990

89A049/Japan/1989 90H377/Japan/1990

90H415/Japan/1990 88H449/Japan/1988 89H354/Japan/1989

92H085/Japan/1992 92H102/Japan/1992

JAPAN085/Japan/1993 CHIN8/China/1990

JAPAN076/Japan/1993 86Y1329/Japan/1986

86Y187/Japan/1986 Japan0022/Japan/1983

Japan38/Japan/1983 KY3303/Kenya/1999

PAK426/Pakistan/1993 TB-Chen/China/1996

S2/Japan/1981 TA81/Taiwan/1981

89Y1520/Japan/1989 90Y593/Japan/1990

TE29/Taiwan/1993 90H094/Japan/1990

90H115/Japan/1990 92H006/Japan/1992

87Y003/Japan/1987 88Y1112/Japan/1988

JAPAN137/Japan/1992 JAPAN58/Japan/1992

TWCH105/Thailand/1994 88F007/Japan/1988

85A21410/Japan/1985 86A005/Japan/1986

88Y259/Japan/1988 89H279/Japan/1988

87Y1438/Japan/1987 BE1248/Belgium/2009

GER177/Germany/2008 B110119/Japan/2011

B110005/Japan/2011 CK20047/Australia/2011

KUN/Japan/1980 TMCII/Japan/1980

83A001/Japan/1983 DS-1/USA/1976

TA3/Taiwan/1981 GR410/South Africa/1985

Hu5/Australia/1977 1831GR/South Africa/1993

92A/Australia/1992 Rj5619/Brazil/2002

H105-Palermo/Italy/1996 Nov04-H383/Russia/2004

90

99

99

94

99

85

88

92

91

88

8888

97

70

73

77

9890

84

9793

93

98

99

74

93

99

82

99

84

99

94

0.01

Lineage II

Lineage I

Lineage III

Group IV except IVa

Sub-lineage IVa

Lineage IVIVa-1

IVa-2

IVa-3

Fig. 1 A phylogenetic tree

based on the Japanese G2 VP7

nucleotide sequences

determined in this study

(indicated in boldface with

closed circular dots) along with

other Japanese G2 sequences (in

boldface) and representative

sequences obtained from the

GenBank database. The tree was

constructed using the neighbor-

joining method with bootstrap

probabilities after 1,000

replicate trials. The genetic

distance is indicated at the

bottom. Percent bootstrap

support is indicated by the value

at each node where the value is

70 % or larger. Five porcine G2

strains (accession numbers

DQ534015, AY766085,

JN974821, JX498969, and

JN974817) were used as the

outgroup, which are not shown

in the tree. Only the node where

the root is located is shown

L. P. Do et al.

123

children in Akita or Yamagata Prefecture between 1983

and 2007, and in Nara Prefecture between 2008 and 2011.

The VP7 nucleotide sequence for these 35 G2 strains were

determined essentially as described previously [4] except

that an additional primer pair G2VP7-14F (5’-AGA ATT

TCC GTC TGG CTA G-3’) and G2VP7-998R (5’-AAT

GAT CTT GAC CGT TTG GAC-3’) was used for cDNA

amplification.

We compiled the G2 VP7 nucleotide sequences that

were determined in this study with those available in the

GenBank database by using the BLAST program (http://

blast.ncbi.nlm.nih.gov/), with the DS-1 VP7 sequence as a

query sequence. We retrieved more than 1000 sequences to

include all possible G2 VP7 sequences but used only

sequences that were at least 615 nucleotides in length,

spanning positions from 307 to 921 (corresponding to

amino acid residues 87 to 291). The sequences of reas-

sortant vaccine strains were also excluded. To distinguish

the sequences of G2 strains from those of other genotypes,

a preliminary phylogenetic tree was constructed using the

MEGA 5.0 software package [18]. Porcine G2 strains are

known to have the highest sequence similarities to human

G2 strains among all rotavirus A strains previously repor-

ted [8]. With the porcine G2 strains as an indicator as the

closest outgroup, we detected a large cluster formed by G2

strains from humans in the preliminary phylogenetic tree,

enabling the removal of strains outside of this large cluster

as non-G2 strains. Thus, we obtained 509 nucleotide

sequences of human G2 strains, of which 21 sequences

were from the strains detected in Japan. The AU605

sequence was excluded because the original stool specimen

(86A005) from which AU605 was isolated in cell culture

was used in this study to determine the VP7 sequence.

Thus, a total of 543 human G2 sequences, including 35

sequences determined in this study and 508 sequences

downloaded from the GenBank database were analyzed

together with 20 Japanese G2 sequences.

While the analysis was done by constructing a phylo-

genetic tree containing all 543 human G2 sequences

detected between 1980 and 2011, for the sake of simplicity,

an abridged tree is presented in Fig. 1. All 55 Japanese G2

sequences are included in this tree, but only selected ref-

erence sequences are shown to reflect the basic topology of

the original tree. The Japanese G2 sequences clustered into

three lineages, I, III and IV, but there was no sequence

belonging to lineage II (Fig. 1). Lineage I, to which the

prototype DS-1 strain belonged, contained only one Japa-

nese strain detected in 1983 (83A001) (Figs. 1, 2). The

nucleotide sequence identities between 83A001 and other

G2 VP7 sequences in this lineage including those from

Taiwanese, South African and Australian strains were

[97.9 %. With respect to the four amino acid positions in

the VP7 antigenic regions previously described as potential

epitopes for G2 strains [4], the 83A001 strain had an amino

acid constellation of A-D-N-N at positions 87, 96, 213 and

242, respectively, which was identical to that of the pro-

totype DS-1 strain (Fig. 3).

Lineage III contained only two Japanese strains detected

in a single year, 1980 (Fig. 2), and they were the only

strains in the world that belonged to lineage III (Fig. 1).

They had A87T and N242S substitutions from the amino

acid constellation of the lineage I strains; hence, an amino

acid constellation of T-D-N-S (Fig. 3). From the evolu-

tionary perspective, it was noted that G2 strains with lin-

eages I and III never appeared again since the early 1980s,

when they were detected for the first time in Japan (Fig. 2).

Lineage IV contained 52 Japanese strains. Within line-

age IV, however, there was sublineage IVa, with a bootstrap

value of 89 % (Fig. 1), which comprised 90 % of G2 strains

circulating after 2000 in Japan (19/21) (Fig. 2) as well as

[95 % of the global collection. The salient feature of the

strains that belong to this currently and globally predomi-

nant sublineage IVa is the presence of a D96N substitution

[4]. The remaining strains within lineage IV (33) clustered

into a group that was named ‘‘group IV except IVa’’

because of a low bootstrap value (26 % in the original

phylogenetic tree containing 543 G2 sequences). Strains

belonging to ‘‘group IV except IVa’’ had the substitutions

N213D and N242S from the amino acid constellation of the

lineage I strains; hence, an amino acid constellation of A-D-

D-S (Fig. 3). The strains carrying this constellation were

detected between 1981 and 1993 (Fig. 2). However, after an

interval of 18 years of non-detection, two strains (B110005

and B110119) belonging to this group emerged again in

2011, this time with an additional amino acid substitution,

D96N; hence, an amino acid constellation of A-N-D-S

(Figs. 2, 3). More importantly, however, these two strains

(B110005 and B110119) formed a nascent lineage with a

1980

1985

1990

1995

2000

2005

2010

2011

Lineage I

Lineage III

Group IV-except IVa

Sublineage IVa-1

Sublineage IVa-3

Fig. 2 The distribution of G2 strains in Japan by the year of detection

and the lineages to which they belonged. Each dot represents one

strain

Molecular evolution of Japanese G2 rotaviruses

123

high bootstrap probability of 99 % with three other G2

strains detected in Germany in 2008 (GER177-08), in

Belgium in 2009 (BE1248), and in Australia in 2011

(CK20047) (Fig. 1). The D96N substitution found in this

nascent lineage is the salient feature of sublineage IVa,

which has been shown to be strongly associated with an

abrupt increase and the re-emergence of G2 strains [4, 7, 8,

19, 20]. With respect to the nature of the amino acids, the

D96N substitution contains a radical change from a nega-

tively charged amino acid (aspartic acid) to an uncharged

amino acid (asparagine), thereby likely contributing to the

antigenic change [4]. Whereas the same D96N substitution

occurred in the VP7 of G3 strains, when there was an abrupt

increase (up to 95 %) during the 2003-2004 season in Japan

[15, 16], the G4 strains showed a conservative T96N

substitution unrelated to the increase. Thus, it remains to be

determined whether the D96N substitution would impart to

the strains belonging to the nascent lineage the potential to

spread globally in the near future.

The strains belonging to sublineage IVa were further

divided into sublineage IVa-1, with a bootstrap value of

94 %, and sublineage IVa-3, with a bootstrap value of

91 %. No Japanese G2 strain belonged to sublineage IVa-2

(Fig. 1). Sublineage IVa-1 strains appeared after 2000 and

were predominant until 2009 (Fig. 2). Their amino acid

constellation was T-N-D-S, which was completely differ-

ent from that of DS-1 (Fig. 3). One peculiar exception to

the amino acid constellation for sublineage IVa-1 strains

was a strain detected in Nara Prefecture in 2009, 90096,

which contained glycine at residue 96, a very unusual

A A C F A A C F A A C F(87) (96) (213) (242) (87) (96) (213) (242) (87) (96) (213) (242)

DS-1 USA 1976 A D N N DS-1 USA 1976 A D N N DS-1 USA 1976 A D N N

DS-1 USA 1976 A D N N S2 Japan 1981 A D D SHu5 Australia 1977 A D N N TA81 Taiwan 1981 T D D S TD64 Taiwan 1992 T N D STA3 Taiwan 1981 A D N N Japan022 Japan 1983 A D D S Bangla84 Bangladesh 1994 T N D S83A001 Japan 1983 A D N N Japan038 Japan 1983 A D D S 356PT South Africa 1996 T N D SGR410 South Africa 1985 A D N N 85A21410 Japan 1985 A D D S Mvd9707 Uruguay 1997 T N N S

86A005 Japan 1986 A D D S KY3103 Kenya 1999 T N D S86Y187 Japan 1986 A D D S Py40699 Paraquay 1999 T N D S

92A Australia 1992 A D S S 86Y1329 Japan 1986 A D D S CIT-220RV Ireland 2000 T N N S1831GR South Africa 1993 A D S S 87Y003 Japan 1987 A D D S KO2 Japan 2001 T N D SH105-Palermo Italy 1996 T D S S 87H038 Japan 1987 A D D S 01P007 Japan 2001 T N D SRj5619 Brazil 2002 T D N S 87Y1438 Japan 1987 A D D S 01P008 Japan 2001 T N D SNov04-H383 Russia 2004 T D S S 88F007 Japan 1988 A D D S 01P012 Japan 2001 T N D S

88Y259 Japan 1988 A D D S 01P014 Japan 2001 T N D S88H449 Japan 1988 A D D S 01P017 Japan 2001 T N D S

KUN Japan 1980 T D N S 88Y1112 Japan 1988 A D D S 01P129 Japan 2001 T N D STMCII Japan 1980 T D N S 89A049 Japan 1989 A D D S 01R137 Japan 2001 T N D S

89H279 Japan 1989 A D D S CMH277 Thailand 2001 T N D S89H354 Japan 1989 A D D S J-4708 Japan 2001-2003 T N D S89Y1520 Japan 1989 A D D S J-4709 Japan 2001-2003 T N D S90Y289 Japan 1990 A D D S J-4765 Japan 2001-2003 T N D S90H377 Japan 1990 A D D S J-4777 Japan 2001-2003 T N D S90H415 Japan 1990 A D D S J-4787 Japan 2001-2003 T N D SCHIN8 China 1990 A D D S VN-11 Vietnam 2002 T N D S90H094 Japan 1990 A D D S CH-130 China 2003 T N D S90H115 Japan 1990 A D D S 26-Palermo Italy 2003 T N D S90Y593 Japan 1990 A D D S SL244 Sierra Leone 2005 T N D S92H006 Japan 1992 A D D S LB2772 USA 2005-2006 T N D S92H085 Japan 1992 A D D S Arg2395 Argentina 2007 T N D S92H102 Japan 1992 A D D S 14322/07MG Brazil 2007 T N D SJAPAN58 Japan 1992 A D D S 07Y003 Japan 2007 T N D SJAPAN076 Japan 1993 A D D S 80004 Japan 2008 T N D SJAPAN085 Japan 1993 A D D S 80120 Japan 2008 T N D SJAPAN137 Japan 1992 A D D S 80123 Japan 2008 T N D SPAK426 Pakistan 1993 A D D S GER84 Germany 2008 T N D STE29 Taiwan 1993 A D D S Seoul-556 South Korea 2009 T N D STWCH105 Thailand 1994 A D D S 90096 Japan 2009 T G D STB-Chen China 1996 A D D SKY3303 Kenya 1999 A D D S

GH1803 Ghana 1999 T N D S4476PT South Africa 1997 T N D S

GER177 Germany 2008 A N D SBE1248 Belgium 2009 A N D SCK20047 Australia 2011 A N D S Bangla66 Bangladesh 1993 T N D NB110005 Japan 2011 A N D S NG5113 Negeria 1999 T N D NB110119 Japan 2011 A N D S MR 4717 Mauritius 2000 T N D N

XJ00-486 China 2000 T N D NDC2020 Madagascar 2004 T N D NMatlab6 Bangladesh 2004 T N D N05N011 Nepal 2005 T N D NLB2764 USA 2005-2006 T N D Nmani-268 India 2007 T N D NGER31 Germany 2008 T N D NMMA08-4 Myanmar 2008 T N D NCU110-BK Thailand 2008 T N D NSeoul-397 South Korea 2009 T N D N16054 09MA Brazil 2009 T N D NB110056 Japan 2011 T N D N

Sublineage IVa-1

Antigenic region(aa position)

Nascent lineage

Group IV except IVaLineage I

Sublineage IVa-2

Sublineage IVa-3

Antigenic region(aa position)

Antigenic region(aa position)

Lineage II

Lineage III

Sub-lineage IVa

Fig. 3 The amino acid constellation at residues 87, 96, 213 and 242 in the VP7 antigenic regions of various G2 strains detected in Japan between

1980 and 2011 as well as representative G2 strains used in Fig. 1 in relation to the lineages of the G2 VP7 gene

L. P. Do et al.

123

amino acid for a G2 strain at this position. To the best of

our knowledge, this was the only G2 strain that had glycine

at position 96 in the G2 VP7. However, glycine at position

96 is highly conserved in G1 strains except for only two

strains detected in Argentina [3].

Only one strain, B110056, detected for the first time in

Japan in 2011 belonged to sublineage IVa-3 (Figs. 1, 2).

However, this sublineage was shown to be a contemporary,

globally dominant sublineage. In Brazil, there was an

apparent shift of sublineages from IVa-1 to IVa-3 after the

introduction of a rotavirus vaccine [11]. Thus, it is specu-

lated that a shift from sublineage IVa-1 to IVa-3 will be

facilitated by the widespread use of rotavirus vaccines in

Japan.

In conclusion, this study provided the knowledge base

on the molecular evolution of Japanese G2 rotavirus strains

over a 31-year-period under unvaccinated conditions. The

results demonstrated the emergence of a nascent lineage

that had the D96N substitution outside of the currently and

globally predominant lineage. Further studies are therefore

warranted to monitor the potential of the global spread of

the emerging strains that have acquired the D96N substi-

tution, since they also appeared in Europe and Australia.

Acknowledgments We acknowledge Dr, Katsushima for providing

some of the stool specimens containing G2 rotaviruses that were used

in this study.

This study was supported in part by the Global Center of Excel-

lence Program on Integrated Global Control Strategy for Tropical and

Emerging Infectious Diseases from the Ministry of Education, Cul-

ture, Sports, Science and Technology, Japan. This study was also

supported in part by a grant-in-aid for scientific research from the

Ministry of Health, Labor, and Welfare, Japan.

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Molecular evolution of Japanese G2 rotaviruses

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