Molecular characterization of VP7 gene of human rotaviruses from Bangladesh

Molecular characterization of VP7 gene of human rotavirusesfrom Bangladesh

Kamruddin Ahmed • Selim Ahmed • Marcelo Takahiro Mitui •

Aminur Rahman • Luthful Kabir • Abdul Hannan •

Akira Nishizono • Osamu Nakagomi

Received: 30 August 2009 / Accepted: 20 February 2010 / Published online: 10 March 2010

� Springer Science+Business Media, LLC 2010

Abstract This study was carried out during July 2005–

June 2006, to characterize rotaviruses circulating in Ban-

gladeshi children less than 5 years attended a peri-urban

hospital. The proportion of rotavirus diarrhea was 39.5%.

Genotype G2 was dominant (45.5%) followed by G1

(24.8%), G12 (9.6%), G9 (8.5%), and G4 (2.1%). G2 were

mainly in combination with P[4], G1 and G9 with P[8], and

G12 with P[6]. Phylogenetically Bangladeshi G1, G2, and

G12 were closely related with the respective types from

India, whereas Bangladeshi G9s of lineage III were with

strains from Belgium and Australia. A G9 strain of lineage

IV was clustered with strains from Sri Lanka and Turkey.

Compared with prototype rotaviruses, Bangladeshi strains

showed several amino acid substitutions at the antigenic

sites of VP7. This study showed that the generation of

diverse strains continued as evidenced by long G2, short

G1 and G9 strains, and various combinations of G and P

types.

Keywords Rotavirus � Genotypes � Electropherotypes �Bangladesh

Introduction

Among the diverse pathogens causing diarrhea, rotavirus

has been firmly established as the most important cause of

severe diarrhea in children, accounting for an average of

39% of severe diarrheal cases [1]. Globally there are

approximately 454,000–705,000 deaths attributable to

rotavirus infection each year. Bangladesh is no exception

and is severely affected by this viral agent causing an

estimated 5,600–9,400 children death annually [2].

Currently two vaccines are available for rotavirus and

both of them showed significant effectiveness in reducing

severe rotavirus diarrhea in children [3, 4]. Keeping in

sight the global effort of introducing rotavirus vaccines in

developing countries, several studies in Bangladesh have

generated valuable information on rotavirus epidemiology

focused on particular localities or hospitals. Information

regarding rotavirus in other parts of the country remained

largely unknown. Since rotavirus vaccines become avail-

able therefore to monitor, the trend of antigenic variation of

rotaviruses across the country became necessary and

important. Thus, comprehensive data generated will have

paramount importance for the evaluation of viral ecology

before and after vaccine introduction.

Rotavirus belongs to the family Reoviridae, its genome

contains 11 segments of double-stranded linear RNA (dsRNA).

Rotavirus strains are classified into electropherotypes based

K. Ahmed (&)

Division of Infectious Diseases, Department of Social

and Environmental Medicine, Institute of Scientific Research,

Oita University, Yufu, Oita, Japan

e-mail: [email protected]

S. Ahmed � L. Kabir � A. Hannan

Department of Paediatrics, The Institute of Child and Mother

Health, Matuail, Dhaka, Bangladesh

M. T. Mitui � A. Nishizono

Department of Microbiology, School of Medicine, Oita

University, Yufu, Oita, Japan

A. Rahman

Centre for Injury Research and Prevention Bangladesh, Dhaka,

Bangladesh

O. Nakagomi

Division of Molecular Epidemiology, Department of Molecular

Microbiology and Immunology, Graduate School of Biomedical

Sciences, Global Center of Excellence, Nagasaki University,

Nagasaki, Japan

123

Virus Genes (2010) 40:347–356

DOI 10.1007/s11262-010-0463-x

on the differences in the relative migration rates of geno-

mic segments in polyacrylamide gel electrophoresis

(PAGE), as a result, opening more opportunities for

detection of strain diversification [5–7]. The most common

electropherotype patterns are designated ‘‘long’’ and

‘‘short’’ based on the fact that short electropherotype

strains have a remarkable reduction in the migration rate of

segment 11 due to the insertion of AT rich sequences in the

30 terminal non-coding region of segment 11 [8, 9]. Thus,

there is an inversion of the migration order of gene seg-

ments 10 and 11. Genotype G2 strains generally have short

RNA patterns, whereas genotype G1, G3, and G4 strains

almost always have long RNA patterns [10–12]. Most

strains of genotype G9 and G12 rotaviruses also have long

RNA patterns.

Rotavirus is non-encapsulated triple-layered particle.

The middle layer is formed by inner capsid protein VP6,

which serves as group and subgroup-specific antigens. The

external capsid comprised of two proteins, VP4, a protease

sensitive hemagglutinin and VP7, a glycoprotein. Based on

the variability of VP7 and VP4 antigens, rotavirus is

classified into G and P types and expressed as combining

the two types. Currently there are 15 G and 27 P types [13,

14]. Theoretically many G/P combinations are possible

within the binary system utilized to classify genotypes;

however, G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8] are

the most commonly circulating types globally [15]. For the

past several years G12P[8] and G12P[6] have been

emerging in different countries [16]. Studies have identi-

fied regional variations in the genotype distribution of

rotavirus strains such as G5 in Brazil and G8 in Malawi

[17, 18]. Both VP7 and VP4 antigens of rotavirus elicit

neutralizing antibodies; although antibodies against both

are important however, the antibodies against VP7 play a

greater role than antibodies against VP4 for immunity after

natural rotavirus infections as well as after vaccination [19,

20]. Studies with monoclonal antibody neutralization-

resistant escape mutants have identified antigenic regions

A (aa 87–101), B (aa 143–152), C (aa 208–223), and F (aa

235–242) [21–23] on VP7 that are involved in neutraliza-

tion. As a result VP7 diversity has significant implications

on rotavirus vaccination. Therefore, in this study we

assessed the genetic variation of the VP7 of rotavirus

circulating among children at the peri-urban area of Dhaka.

Methods

Collection of stool specimens

During July 2005–June 2006, stool samples were collected

prospectively from children less than 5 years attended at

the affiliated hospital of the Institute of Child and Mother

Health (ICMH), Matuail, Dhaka, Bangladesh. A case of

diarrhea was defined as three looser than normal stool

during a 24-h period. The ethical review board of ICMH

approved the study. The verbal consent of child’s guardian

was obtained prior to sample collection.

Detection of rotavirus and determination

of electropherotypes

Rotavirus antigens were detected by enzyme immunoassay

(Rotaclone, Meridian Diagnostics, Inc., Cincinnati, OH,

USA), according to manufacturer’s instruction. The geno-

mic dsRNA was extracted from rotavirus-positive samples

and electropherotype was determined by running the

extracted dsRNA through a PAGE according to previously

published method [24, 25]. In brief, 5 ll of extracted

dsRNA mixed with 5 ll of loading buffer was loaded in

each lane of a 10% polyacrylamide gel and run for 16 h at

constant current of 8 mA. Numbering of electropherotypes

was arbitrarily assigned and based upon distinct changes in

the migration patterns within at least one of the four groups

of segments, i.e., segments 1 to 4, 5 and 6, 7 to 9, or

10 and 11.

Determination of types and nucleotide sequences

The G and P genotypes of rotavirus-positive specimens

were determined as follows [26–28]; the VP7 gene was

amplified with consensus primers Beg9 and End9. The G

type was identified with genotype-specific primers for G1,

G2, G3, and G4. The VP4 gene was amplified with con-

sensus primers con-2 and con-3, and the P type was iden-

tified with genotype specific primers for P[8], P[4], P[6],

and P[9] as described previously with a few modifications

as follows [28, 29]. For VP7 and VP4 gene amplification,

reverse transcriptase–polymerase chain reaction (RT–PCR)

was done with AccessQuick RT–PCR (Promega Corpora-

tion, Madison, WI, USA). For G and P-specific genotyping,

PCR Master Mix (Promega) was used. Samples which

could not be typed were subjected to nucleotide sequencing

[29]. Nucleotide sequencing of the VP7 gene was also done

on selected samples. The nucleotide sequence of the full-

length VP7 gene was determined by BigDye terminator

v3.1 cycle sequencing kit (Applied Biosystems, Foster

City, CA, USA) according to manufacturer’s instruction

and the product was subjected to an ABI Prism 3100

Genetic Analyzer (Applied Biosystems).

Phylogenetic analysis

Multiple sequence alignment was done by ClustalW and

phylogenetic tree of the deduced amino acid sequences

of the VP7 gene was constructed using neighbor-joining

348 Virus Genes (2010) 40:347–356

123

method [30]. Bootstrap analysis of 1,000 replicates was

done to find the significance of branching.

Results

Prevalence and seasonality

Of 656 samples, 259 (39.5%) were rotavirus-positive.

Rotavirus mainly affected 3–23-month-old children and

peaked in 6-11-month-old. Rotavirus infection was pre-

valent throughout the year, however found higher in dry

than rainy months (Fig. 1). In percentage it remained

essentially similar, relatively less at the beginning of rainy

months.

Electropherotyping

In 111 (46.6%) of 238 samples, the segments of rotavirus

genome were visible. In 21 samples, the staining of seg-

ments was not clear enough to permit their assignment

to specific electropherotype. In 88 (37.0%) samples, 15

electropherotypes, E1–E15 were identified (Fig. 2).

Electropherotypes E1, E4–E6, E8, and E10–E14 were of

long pattern and E2, E3, E7, E9, and E15 were of short

pattern. The segments 7, 8, 9 of electropherotype E5, E8,

0

20

40

60

80

100

120

Jul05

Aug05

Sept0

5

Oct05

Nov05

Dec05

Jan0

6

Feb06

Mar

06

Apr06

May

06

Jun0

6

Months

Nu

mb

er/p

erce

nta

ge

of

sam

ple

s

Fig. 1 The monthly occurrence of rotavirus diarrhea among the

children in Bangladesh. The monthly occurrence is represented by

number (hatched bar) and the percentage (filled bar) of rotavirus

cases detected among the diarrheal cases (unfilled bar) of each month

Fig. 2 The electropherotypes of rotaviruses identified in Bangladesh.

A total of 34 samples belonged to electropherotypes E2, 7 to E1, 6 to

E3, E4 and E15, 5 to E5 and E8, 4 to E6, E13 and E14, 3 to E12, and 1

to E7, E9, E10 and E11. E1, E4–E6, E8 and E10–E14 were of long

electropherotype. Others were of short electropherotype. Electropher-

otype named E2 was dominant contained G2P[4] rotaviruses

Table 1 Numbers and percentages of rotaviruses with different G

and P genotypes combination detected in Bangladesh

Type Number Percentage of the total

G2P[8] 3 2.1

G2P[4] 57 39.3

G2P[6] 1 0.7

G2P[4]&P[6] 2 1.4

G2P[4]&P[8] 1 0.7

G2P[4]&P[6]&P[8] 1 0.7

G2P[nt] 1 0.7

SubtotalG2 66 45.5

G1P[8] 24 16.5

G1P[6] 2 1.4

G1P[4] 3 2.1

G1P[4]&P[8] 4 2.7

G1P[6]&P[8] 1 0.7

G1P[4]&P[6] 1 0.7

G1P[nt] 1 0.7

SubtotalG1 36 24.8

G12P[8] 2 1.4

G12P[6] 12 8.3

SubtotalG12 14 9.6

G9P[8] 8 5.5

G9P[4] 3 2.1

G9P[4]&P[8] 1 0.7

SubtotalG9 12 8.5

G4P[8] 1 0.7

G4P[4] 2 1.4

SubtotalG4 3 2.1

G1&G2P[6] 1 0.7

G1&G2P[nt] 1 0.7

SubtotalG1&G2 2 1.4

GntP[8] 4 2.7

GntP[6] 4 2.7

GntP[4] 2 1.4

GntP[4]&P[8] 1 0.7

GntP[nt] 1 0.7

SubtotalGnt 12 8.3

Total 145 100

Gnt and P[nt] indicate G non-typable and P non-typable, respectively

Virus Genes (2010) 40:347–356 349

123

E10, E12, and E13 were closer when compared with that of

electropherotypes E1, E6, and E10. Three bands of seg-

ments 7, 8, 9 of E4, E11, and E14 were clearly visible

compared with that of other electropherotypes of long

pattern. Compared with E1 in E6 and E10 there were dif-

ferences in the mobility of segment 4 and segments 5, 6,

Strain184(Bangladesh)

MMC88(Bangladesh)



NG4585/99(Nigeria)

MR4717/00(Mauritius)


SC-4(Kolkata India)

MMC6(Bangladesh)

IS2(India)

GH1803/99(Ghana)

TN1529/99(Tunisia)

253(India)

CMH277(Thailand)

TF85(Taiwan)

KO-2(Japan)

Sc27(India)

312(India)

CMH019/03(Thailand)

CMH041/03(Thailand)

CMH027/03(Thailand)

Mvd9716(Uruguay)

CHIN-1(China)

PAK426(Pakistan)

JAPAN0022(Japan)

TA20(Taiwan)

KY3303/99(Kenya)

TB-Chen(China)

KUN(Japan)

Lineage II

DS-1(USA)

Hu/5(Australia)

TA3(Taiwan)

TA6(Taiwan)

Lineage I

64SB/96(South Africa)

906SB/98(South Africa)

95A(Australia)

95B(Australia)

T79(China)

Lineage III

CMP034(Thailand)

34461-4(Spain)Lineage IV

KH210 (Human G5 as an outgroup)

56

99

94

91

85

79

83

66

62

52

65

57

62

89

90

64

84

84

99

97

0.05

Fig. 3 Phylogenetic tree

constructed with the deduced

amino acid sequences of the

VP7 gene of G2 strains. Human

rotavirus KH210 (G5) was used

as an outgroup. The numberadjacent to the node represents

the bootstrap value and values

lower than 50% have not been

indicated. Scale bar shows

genetic distance expressed as

amino acid substitutions per

site. The DNA Data Bank of

Japan/European Molecular

Biology Laboratory/GenBank

accessions nos. are AB534524

for Strain 184; AB534525 for

Strain174; AB534526 for

Strain325; AB534527 for

Strain258

350 Virus Genes (2010) 40:347–356

123

respectively. Segments 7, 8, 9 were closer in E12 when

compared with that of E5. Compared with E5 in E13

segments 7, 8, 9 were close and indistinguishable as three

segments. Between E5 and E8 there were differences in the

mobility of segments 2, 3, 4, 6, and 10. Compared with E4

there was differences in the mobility of segment 4 and

segments 7, 8, 9 in E11 and E14, respectively. Compared

with other electropherotypes of short pattern, three bands

of segments 7, 8, 9 were distinct in E7. In E2 and E15

segments 2, 3 were indistinguishable as two bands. There

were differences in the mobility of segments 7, 8, and 9 in

E2 and E15. Compared with E3 in E9 there was a variation

of the mobility of segments 7, 8, and 10. Two samples

showed more than 11 segments, indicating mixed infection

with different strains of rotavirus (data not shown in the

figure).

Genotypes distribution

Genotype G2 was dominant followed by G1, G12, G9, and

G4 (Table 1). Various combinations of G and P types were

detected. Among the successfully typed samples, genotype

G2P[4] was predominated followed by G1P[8]. Together

genotypes G2 and G1 were accounted for 70.3%. G12 and

G9 were almost in equal proportion circulating in this study

period. Type G3 was not found in this study. Non-typable

strains were 8.3%, which were mainly due to unable to

amplify the VP7 gene. A proportion of strains were in

combination with P[4], P[6], or P non-typable (P[nt]). Most

of the G12 strains were found in combination with P[6].

RT–PCR detected mixed G and P type in 2 and 12 samples,

respectively. Among them 2 samples were also detected as

mixed infection by PAGE, and the remaining 12 samples

were detected as mixed infection only by RT–PCR. These

12 samples accounted for 8.5% of the samples subjected to

RT–PCR. One G2P[4] and two G9P[4] strains had long

electropherotype. Short electropherotype of genotypes

G1P[8] and G9P[4] was found in one sample each.

Phylogenetic analyses

Phylogenetic tree showed that Bangladeshi G2 rotaviruses

of this and other studies belonged to lineage II and were

closely related to strains from India, Nigeria, and Mauritius

(Fig. 3). A 99–100% identity was found among Ban-

gladeshi strains detected in different areas. With other

strains of this lineage, Bangladeshi strains had 95–99%

amino acid identity, least identity (95–96%) with KUN,

and Chinese strain CHIN-1. The VP7 antigenic regions A,

B, C, and F of Bangladeshi G2 rotaviruses were analyzed.

In strains 258 and MMC6, substitutions (Asp ? Glu) and

(Asp ? Tyr) were found in residue 211 and 213 of anti-

genic region C. Compared with KUN, in Bangladeshi

strains amino acid substitutions (Fig. 4) were found in

antigenic regions A, 96 (Asp ? Asn); C, 211(Asp ? Glu),

213 (Asn ? Asp or Tyr), and F, 241 (Met ? Ile), 242

(Ser ? Asn).

Phylogenetic tree (Fig. 5) showed that G1 rotaviruses of

this study belonged to lineage Ic and had 97–100% amino

acid identity with strains of this lineage. The strains were

closely related with Indian, Thai, and old (Dhaka8-02) and

new Bangladeshi strains from different areas. Lowest

identity was observed with representative strain Wa

(93–94%). Among Bangladeshi strains, a 99–100% identity

90 100 210 220 240 | | | | |Strain174 TEAKNEISDNEWENT KTTDVDTFEIVASSEK HKINISIN MMC88 ............... ................ ........ Strain325 ............... ................ ........ Strain184 ............... ................ ........ Strain258 ............... ...E............ ........ MMC6 ............... .....Y.......... ........ KUN .........D..... .....N.......... ......MS

90 100 150 210 220 | | | | |Strain408 TEASTQISDGEWKDS YDQNFELDM QTTNVDSFETVAENEK MMC56 ............... ....L.... ................ Dhaka8-02 ............... ....L.... ................ DH402 ............... ....L.... ................ Strain334 ............... ....L.... ................ Strain151 ............... ....L.... ................ Strain129 ............... ....L.... ................ Ban-48 ..........D.... ....L.... ...........T.... Wa .......N..D.... ...SL.... .........MI.....

Fig. 4 The upper panel shows

amino acid substitutions in the

antigenic regions A (aa

87–101), C (aa 208–223), and F

(aa 235–242) of the VP7 of

Bangladeshi G2 strains

compared with that of KUN,

whereas the lower panel shows

the amino acid substitutions in

the antigenic regions A, B (aa

143–152), and C of the VP7 of

Bangladeshi G1 strains

compared with that of Wa

Virus Genes (2010) 40:347–356 351

123

was found. Compared with other Bangladeshi strains, in

strain 408, one substitution (Leu ? Phe) was found at

residue 148 of antigenic region B. Compared with Wa, in

Bangladeshi strains substitutions (Fig. 4) were found at

residue 94 (Asn ? Ser) and 97 (Asp ? Glu) of antigenic

region A, at 147 (Ser ? Asn) of B, at 217 (Met ? Thr),

and 218 (Ile ? Val) of C.

Phylogenetic tree (Fig. 6) showed that Bangladeshi G12

strains belonged to lineage III along with strains from

India, Nepal, Slovenia, and the United States. All strains

had 99–100% identity among themselves except Slovenian

strains, which had 96–98% identity with other strains.

Compared with other strains, Dhaka 12-03, a strain of

2003, had one substitution (Thr ? Asn) at residue 91 of

antigenic region A.

Phylogenetic tree of G9 rotaviruses (Fig. 7) showed that

Bangladeshi strains belonged to lineage III and clustered

with strains circulating in Bangladesh, Belgium, and Aus-

tralia. The strains had a 99–100% identity among them-

selves. Compared with other strains, one substitution

(Ser ? Asn) was found at residue 221 of antigenic region

C of strain 285 and Dhaka 19-04. One strain of this study

belonged to the newly identified lineage IV [31], which

contained strains from Sri Lanka and Turkey. Other than

Dhaka8-02(Bangladesh)



MMC56(Bangladesh)



BD-23(Bangladesh)

BD-723(Bangladesh)

ISO-4(India)

DH402(Bangladesh)

43vp7n(Thailand)

Ban-48(Bangladesh)

VN-368(Vietnam)

Mvd9816(Uruguay)

97SZ29(China)

Lineage Ic

PA17c/86(Italy)

PA12.90(Italy)Lineage Ia

Lineage Ib PA19/01(Italy)

Wa(USA)

K8(Japan)Lineage III

80(Japan)

DC03(Taiwan)

G192B(Australia)

Lineage II

K54(Korea)

421(Japan)Lineage IV

PA10/90(Italy)

PA5/90(Italy)Lineage V

Lineage VI AU19(Japan)

T449(Bovine)

C60(Porcine)

C95(Porcine)

Lineage VII


99

97

95

88

84

75

67

60

58

56

51

52

85

63

0.05




VP7 gene of G1 strains.

Bangladeshi rotaviruses strain

129, 151, 408, and 334 are from

this study. Human rotavirus

KH210 (G5) was used as an

outgroup. The number adjacent

to the node represents the

bootstrap value and values








accessions nos are AB534520

for Strain151, AB534521 for

Strain408, AB534522 for Strain

334, AB534523 for Strain129

352 Virus Genes (2010) 40:347–356

123

one substitution (Ala ? Val) at residue 156 of GUH13, no

amino acid substitution was found among them. When

compared with strains of lineage III, strains of lineage IV

had one substitution (Ser ? Asn) at residue 242 of anti-

genic region F. However, several substitutions were found

at residues 8 (Thr ? Ala), 40 (Phe ? Leu), 44 (Ala ?Val), 68 (Val ? Ala), and 287 (Val ? Ile) of strains of

lineage IV when compared with strains of lineage III.

Bangladeshi G9 rotaviruses of this study had 97–99%

identity among them. Compared with other strains, in strain

285 one substitution (Ser ? Asn) was found in residue 221

of antigenic region C.

Discussion

In this study, it was possible to reveal a comprehensive

picture of the genetic diversity of rotaviruses among Ban-

gladeshi children in a hospital of a peri-urban area. In this

study, only two samples of mixed infection were detected

by PAGE, which was significantly lower than found in a

previous study where 12.1% samples were of mixed

infection [32]. That is also higher than the percentage of

mixed infection detected in this study by RT–PCR.

RT–PCR is able to detect trivial amount of nucleic acid

which remain undetected by PAGE. This may indicate that

mixed infection with heavy viral load has decreased sig-

nificantly in Bangladesh. However, relatively low number

of mixed infection did not perturb the generation of vari-

ants of rotaviruses such as long G2 and short G1 and G9 or

combination of various G and P types. In our study the

prevalence of rotavirus was relatively higher when com-

pared with studies done in similar period [33–35] in other

areas of the country. This difference may be due to dif-

ferent site of study where population might have different

prevalence of rotavirus infection. However, we cannot rule

out the effect of different detection methods used in these

studies. However, all studies found G2 as dominating type

of rotavirus and G1, G3, G4, G9, and G12 circulating in

different proportion.

G2 rotavirus was the dominant type in Bangladesh

during the 1987–1989 season [36]. During this study period

this genotype crossed the peak (37%) of 1989 [37]. Fur-

thermore we identified G2P[8] and G2P[6] in Bangladesh

which have also been reported from Brazil [38, 39]. G2

rotaviruses have been reemerging as dominant type in

Paraguay, Thailand, and Nepal [40–42]. Phylogenetic

analysis revealed that Bangladeshi G2 strains belonged to

lineage II and in close association with strains from India,

Mauritius, and Nigeria.

ISO29(India)



05K021(Nepal)





MMC206(Bangladesh)


MMC29(Bangladesh)

ISO-01(India)

36B2(India)

Se585(USA)

14B2(India)


03N250(Nepal)

Lineage III

K12(Japan)

T152(Thailand)

Arg720(Argentina)

Lineage II

Lineage I L26(Philippines)

KH210(G5 out group)

88

66

99

66

59

51

93

0.02






203, 358, 409, 179, 170, and

180 are from this study. Human

rotavirus KH210 (G5) was used

as an outgroup. The numberadjacent to the node represents

the bootstrap value and values








accessions nos. are AB534531

for Strain348, AB534532 for

Strain203, AB534533 for




Strain179

Virus Genes (2010) 40:347–356 353

123

Type G1 was dominant in Bangladesh in 1988 and 2004

[43, 44]. We detected only G1 rotaviruses of lineage Ic,

which were possibly circulating in Bangladesh for several

years as the current and old strains clustered together in the

same Ic lineage [45]. The lack of heterogeneity in Ban-

gladeshi G1 may indicate why this type is gradually

decreasing. Children may have immunity against group Ic

rotavirus due to previous infection with same sublineage

therefore protecting from subsequent infection.

In Nepal and India 23 and 17% of the strains isolated

from children with rotavirus belong to genotype G12 [42,

46]. G12 never attained to that level in Bangladesh after its

first detection in 2000 [33]. However, unlike emerging

Slovenian G12 rotaviruses which are in combination with

P[8] [47] the G12 rotaviruses causing infection in the

subcontinent are in combination with P[6] [33, 42, 46, 48].

Among non-emerging G12 strains, Se585 had a P[6]

specificity [49] and in the phylogenetic tree clustered with

emerging G12 strains from India and Bangladesh.

G9 rotaviruses are being prevalent in Bangladesh after

its first detection in 1995 [50]. Bangladeshi G9 rotaviruses

belonged to lineage III which continues to expand in

phylogenetic diversity with strains from other parts of the

world. Only one Bangladeshi G9P[4] of short electro-

pherotype belonged to the recently identified new lineage

IV [31]. Sri Lankan [16] and Turkish strains of lineage IV

were of G9P[8] of long and short electropherotypes,

respectively. It is not known whether infection by strain of

lineage IV is protected by the antibodies generated from

pervious infection by strain from other lineages. This may

have implication on the spread of this strain among the

children and future G9 vaccine. G9 rotaviruses identified

from different countries exhibited a great variety of gen-

ome constellations, formed predominantly by reassortment

B485-03(Belgium1999-2003)

Dhaka37-03(Bangaldesh2003)

Strain263(Bangladesh2006)

DH375(Bangladesh2004)

AS151572(Australia2003)

Dhaka21-01(Bangladesh2001)

MMC24(Bangladesh2005)


Dhaka19-04(Bangladesh 2004)

AT649 (USA 2000)

AHP32(Turkey 2005)

R143 (Brazil 1999)

GH3550 (Ghana 1998-2000)

B4430 (Belgium 2003)

OM526 (USA 1998)

AP13 (India 1993)

R4 (Sweden 2001-2002)

US1205(USA 1996-1997)

95H115(Japan 1995)

Lineage III

05SLC051 (Sri Lanka 2005)



GUH13 (Turkey 2005)


Lineage IV

Lineage II 116E (India 1985)

WI61 (USA 1983)

AU32 (Japan 1985)Lineage I


99

95

57

92

65

64

67

71

64

55

65

0.02






285, 263, and 305 are from this

study. Human rotavirus KH210

(G5) was used as an outgroup.

The number adjacent to the

node represents the bootstrap

value and values lower than

50% have not been indicated.

Scale bar shows genetic

distance expressed as amino

acid substitutions per site. The

DNA Data Bank of Japan/

European Molecular Biology

Laboratory/GenBank accessions

nos. are AB534528 for



Strain305

354 Virus Genes (2010) 40:347–356

123

of the VP7 and VP4 genes into both long and short

electropherotypes [51]. G9P[6] long and short patterns and

G9P[8] long pattern have been reported from Bangladesh

[50]. As far as we are concerned, this is the first report of

G9P[4] with long and short electropherotypes from Ban-

gladesh. G9P[4] of long and short electropherotypes have

only been reported from Brazil and Thailand, respectively

[39, 52].

The antibody against P[8] component induced by rota-

virus vaccination is suggested to act against infection with

other G types. Currently available rotavirus vaccines do not

contain non-P[8] antigen prevalent in humans; however, as

reveled in this study 68.4% of the circulating rotavirus

strains were non-P[8]. Non-P[8] is not a common finding in

developing countries only, P[6] and P[9] have been

emerging in Ireland [53]. P[9] has been detected in com-

bination with a variety of G types and its detection recently

in Ireland is suggest to be a trend worldwide [53]. Type

P[9] has not yet been detected in Bangladesh. It might be of

significance to find out the consequence of current vaccines

on P-type selection.

Rotavirus vaccine has been approved in several coun-

tries and will be approved in many others. Before the

widespread introduction of this vaccine in a country like

Bangladesh, careful characterization of the circulating

strains is necessary. Compared with the standard rotavirus

strains a number of amino acid substitutions have been

found in the antigenic sites of the VP7 of Bangladeshi

strains. There is a possibility that variation in the antigenic

sites of the VP7 may help these strains to escape immunity

conferred by vaccine-induced antibodies. Although the

parameters of protection against rotavirus disease have not

been firmly established, it appears that type specificity of

anti-rotavirus antibodies plays an important role in pro-

tection against rotavirus disease [54]. Unless the currently

available rotavirus vaccines induce an adequate heterotypic

response, significant protection may not be achieved in

these populations.

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Documents

Molecular characterization of VP7 gene of human rotaviruses from Bangladesh