VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR

VP7 gene polymorphism of serotype G9 rotavirus strains and itsimpact on G genotype determination by PCR�

Norma Santos a,*, Eduardo M. Volotao a, Caroline C. Soares a, Maria CarolinaM. Albuquerque a, Fabiano M. da Silva a, Vladimir Chizhikov b, Yasutaka Hoshino c

a Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS-Bl. I, Ilha do Fundao, Rio

de Janeiro, RJ 21.941-590, Brazilb Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, MD 20895, USA

c Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Received 24 January 2002; received in revised form 24 May 2002; accepted 24 May 2002

Abstract

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide.

Surveillance of rotavirus serotypes/genotypes (both VP7[G] and VP4[P]) is in progress globally in which polymerase chain reaction

(PCR) has been the assay of choice. We investigated polymorphism of the VP7 gene of serotype G9 rotavirus strains and its impact

on the determination of VP7 gene genotype by PCR assay. By VP7 gene sequence analysis, we and others have previously shown

that the G9 rotavirus strains belong to one of three VP7 gene lineages. By PCR assay using three different sets of commonly used

primers specific for G1-4, 8 and 9, 23 Brazilian G9 strains and 5 well-characterized prototype G9 strains which collectively

represented all three VP7 gene lineages were typed as: (i) G3; (ii) G4; (iii) G9; (iv) G3 and G9; or (v) G9 and G4 depending on a

primer pool employed. This phenomenon appeared to be due to: (i) a VP7 gene lineage-specific polymorphism, more specifically

mutation(s) in the primer binding region of the VP7 gene of G9 strain; and (ii) the magnitude of difference in nucleotide homology at

respective primer binding site between homotypic (G9) and heterotypic (G3 or G4) primers present in a primer pool employed.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Rotavirus; PCR G genotyping; VP7 gene polymorphism; Gastroenteritis

1. Introduction

It has been well established that virtually every child

becomes infected with a rotavirus at least once by 3

years of age (Bern and Glass, 1994; Kapikian et al.,

2001). The consequences of rotavirus infection, how-

ever, are markedly different depending on the economic

status of each country. In the U.S., for example,

rotavirus gastroenteritis is estimated to be responsible

for more than 500,000 physician visits, 50,000 hospita-

lizations, approximately 20 deaths and over $1 billion of

societal expenses each year (Parashar et al., 1998a,b;

Tucker et al., 1998). In developing countries, estimates

indicate that rotavirus diarrhea kill 1600�/2400 children/

day, or 600,000�/870,000 children/year (Glass et al.,

1999). Thus, the development and universal implemen-

tation of a safe and effective rotavirus vaccine is a global

public health goal (Bresee et al., 1999; Kapikian et al.,

2001).

Rotavirus is a member of the Reoviridae family which

includes seven groups (rotavirus A�/G). A complete

virus particle possesses 11 double-stranded (ds) RNA

segments surrounded by three concentric protein layers.

The outermost layer is composed of two proteins, VP7

(which defines G serotypes) and VP4 (which defines P

serotypes) that independently evoke neutralizing anti-

bodies. There are 14 G serotypes, however, types G1�/

G4 are the most common globally and, therefore, are

the targets for current vaccine development strategies

(Hoshino and Kapikian, 2000). More recently, various

epidemiologic studies have shown the emergence/re-

�PII of original article: S 0 1 6 8 - 1 7 0 2 ( 0 2 ) 0 0 1 0 6 - 5

* Corresponding author. Tel.: �/55-21-260-9311; fax: �/55-21-560-

8344.

E-mail address: [email protected] (N. Santos).

Virus Research 93 (2003) 127�/138

www.elsevier.com/locate/virusres

0168-1702/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1702(02)00318-0

emergence of rotavirus serotype G9 as an important

human pathogen worldwide and, it is likely to become

the fifth globally important strain (Urasawa et al., 1992;

Ramachandran et al., 1996, 1998; Cunliffe et al., 1999;Unicomb et al., 1999; Bon et al., 2000; Cubitt et al.,

2000; Griffin et al., 2000; Maneekarn and Ushijima,

2000; O’Halloran et al., 2000; Oka et al., 2000; Palombo

et al., 2000; Widdowson et al., 2000; Adah et al., 2001;

Araujo et al., 2001; Santos et al., 2001; Van Man et al.,

2001; Zhou et al., 2001; Bok et al., 2001). In addition,

sequence analysis of the VP7 gene of various G9

rotavirus isolates carried out by us and others haveshown the existence of at least three phylogenetic VP7

gene lineages (I�/III) (Ramachandran et al., 2000; Oka et

al., 2000; Bok et al., 2001; Santos et al., 2001; Zhou et

al., 2001).

Polymerase chain reaction (PCR) methodologies for

rotavirus genotyping were developed over a decade ago.

Because available VP7 gene sequences were rather

limited then, selection of type-specific primers for Ggenotyping was also limited. Moreover, since rotaviruses

were demonstrated to undergo constant genetic varia-

tion via sequential point mutations or ‘antigenic drift’,

genetic reassortment or ‘antigenic shift’, genomic re-

arrangement or intragenic recombination (Gouvea and

Brantly, 1995; Desselberger, 1996; Iturriza-Gomara et

al., 2001; Suzuki et al., 1998), the emergence of strains

that could not be easily identified by this method wasnot completely unexpected. Since neutralizing antibo-

dies and their type specificities appear to play an

important role in protection against rotavirus disease

and/or infection (Hoshino and Kapikian, 2000), epide-

miologic surveillance of rotavirus serotypes/genotypes is

underway throughout the world. However, a potential

impact of rotavirus genome polymorphism on G

genotype identification by PCR has just begun to befully explored (Nakagomi et al., 1991; Adah et al., 1997;

Iturriza-Gomara et al., 2000; Cunliffe et al., 2001a).

Here we describe: (i) a genomic variation of the VP7

gene of rotavirus serotype G9 strains isolated in Brazil

in comparison with that of G9 strains isolated in other

parts of the world which collectively represent all three

phylogenetic VP7 gene lineages; and (ii) the impact of

such variation on G genotype identification by PCR.

2. Materials and methods

2.1. Viruses

Twenty-three G9 strains were derived from children

under 5 years of age with acute diarrhea detected during

epidemiologic studies in Brazil from 1997 to 1999 aspreviously reported (Santos et al., 2001). The prototype

G9 strains WI61 (Clark et al., 1987), F45 (Akatani and

Ikegami, 1987), AU32 (Nakagomi et al., 1990), 116E

(Das et al., 1994) and US1205 (Ramachandran et al.,

1998) belonged to the rotavirus collection of the

Laboratory of Infectious Diseases, National Institutes

of Health, Bethesda, USA.

2.2. RNA extraction and RT-PCR

Rotavirus dsRNA was extracted from stool samples

or infected cell culture lysates by use of guanidine

isothiocyanate method (Santos and Gouvea, 1994) or

TRIzol method (Life Technologies Inc., Grand Island,

NY) and precipitated with ethanol. Reverse transcrip-tion (RT)-PCR for determination of rotavirus G types

was performed by using primers specific for genotypes

G1 to G6, G8 to G10 and G11 (Gouvea et al., 1990;

Taniguchi et al., 1992; Isegawa et al., 1993; Das et al.,

1994; Gouvea et al., 1994). Four different sets of G

typing primers were used (Fig. 1): (1) H1 pool which

contained the original set of primers described by

Gouvea et al. (1990), specific for G1 to G4, G8 andG9 genotypes; (2) H2 pool in which Gouvea’s primers

aET3, aDT4 and aAT8 (G3-, G4-, and G8-specific,

respectively) were replaced by S3, S4, and S8 primers

described by Taniguchi et al. (1992); (3) C pool reported

by Das et al. (1994), specific for G1 to G4 and G9

genotypes; and (4) A pool described by Gouvea et al.

(1994) which contained primers specific for G5, G6, G8,

and G11 genotypes in which the ET10 primer (G10-specific) was replaced by G10 primer described by

Isegawa et al. (1993). The dsRNA samples were

subjected to one cycle of reverse transcription (42 8C,

45 min) followed by 30 cycles of PCR. Each PCR cycle

for G or P genotyping contained steps of 1 min at

94 8C, 2 min at 50 8C and 3 min at 72 8C.

2.3. Sequence analysis

The VP7 gene of six Brazilian G9 strains (R44, R136,

R143, R146, R160 and R172) was sequenced and then

analyzed by using the DNASTAR program. The VP7 gene

sequence of selected G3, G4 and other G9 rotavirus

strains was obtained from GenBank.

2.4. Neutralization assay

Antigenic characterization of selected G9 isolates was

performed by a 60% plaque reduction neutralization

(PRN) assay using guinea pig hyperimmune antiserum

raised against each of G1 to G14 rotavirus prototype

strain as described previously (Wyatt et al., 1982).

2.5. Analysis of rotavirus dsRNA by polyacrylamide gel

electrophoresis

Rotavirus genomic dsRNA was extracted using

standard phenol�/chloroform methods and precipitated

Erratum128

with ethanol. Each RNA sample was analyzed on a

standard discontinuous 10% acrylamide slab gel with a

3.5% acrylamide stacking gel. After electrophoresis,dsRNAs were visualized by staining gel with silver

nitrate (Herring et al., 1982).

3. Results

3.1. G genotype variability displayed by G9 rotavirus

strains against three different primer sets

When analyzed by RT-PCR, each of 23 Brazilian G9

strains displayed different reactivity pattern against

primer pool H1, H2 or C and fell into one of three

distinct reactivity groups (Table 1). One group consist-

ing of 17 strains (represented by R44 strain) was typed

as G3 versus (vs) H1 pool, G4 vs H2 pool and G9 vs C

pool. Second group (strain R179) was typed as G3 vs H1

pool, G4 and 9 vs H2 pool and G9 vs C pool. Thirdgroup of five strains (represented by R143 strain) were

typed as G3 and 9 vs H1 pool, G9 and 4 vs H2 pool and

G9 vs C pool. None of the samples yielded positive

reactivity with A pool (data not shown) and only the C

pool provided a reliable detection of the G9 strains.

A panel of five prototype G9 rotavirus strains (the US

strains US1205 and WI61, the Japanese strains F45 and

AU32, and the Indian strain 116E) was also analyzed byRT-PCR against the same four sets of G typing primers

(Table 1). Strains WI61, F45 and AU32 were typed as

G9 vs H1, H2 or C pool. Strains US1205 and 116E

showed reactivity pattern similar to that of R143 strain

against three primer sets (H1, H2 and C). The A pool

did not produce any reactivity against any of the fiveprototype G9 strains (data not shown).

3.2. Reactivity of selected G9 rotavirus strains vs G3-,

G4- or G9-specific primer in H1 pool

Six Brazilian strains (R44, R136, R172, R143, R146

and R160) and one US strain (US1205), belonging to

the VP7 gene lineage III, had identical five nucleotide

mismatches (76.2% nucleotide [nt] homology) at theaET3 primer binding site (nt 689�/709) and all of them

were amplified (Table 2). Strain 116E belonging to

lineage II which had six nucleotide mismatches (71.4%

nucleotide homology) was amplified weakly with the

primer. The primer binding site of strain WI61, F45 or

AU32 belonging to lineage I had six identical nucleotide

mismatches (71.4% homology) different from those

found in lineage II virus and was not primed with theaET primer. All 11 G9 rotavirus strains analyzed had 9�/

12 nucleotide mismatches (36.8�/52.6% homology) at the

aDT4 primer binding site and none were amplified

(Table 2).

At the aFT9-specific binding site (nt 757�/776) strains

R143, R146, R160 and US1205, which presented three

or four mismatches (85.0 and 80.0% nucleotide homol-

ogy, respectively), were weakly amplified (Table 2). TheBrazilian strain R44, R136 or R172 belonging to lineage

III which presented four identical mismatches (80.0%

nucleotide homology) was not amplified by primer

Fig. 1. The position and directions of primers in H-1, H-2, C or A primer pool used in this study in relation to the schematic VP7-encoding gene.

Variable regions A�/F on the gene are denoted by black boxes. 1, Primers described by Gouvea et al. (1990); 2, Primers described by Gouvea et al.

(1990) and Taniguchi et al. (1992); 3, Primers described by Das et al. (1994); 4, Primers described by Gouvea et al. (1994) and Isegawa et al. (1993).

Erratum 129

aFT9. The Indian strain 116E belonging to lineage II

that had a set of four mismatches different from those

found in lineage III viruses was amplified. Each of three

prototype strains WI61, AU32, and F45 belonging to

lineage I showed perfect matches (100.0% nucleotide

homology) at the primer binding site.

3.3. Reactivity of selected G9 strains vs G3-, G4- or G9-

specific primer in H2 pool

With primer S3 (nt 481�/498), G3-specific, each of 11

G9 strains analyzed presented eight or nine mismatches

(55.6 and 50.0% nucleotide homology, respectively) and

none were amplified with this primer (Table 3). All seven

G9 strains belonging to lineage III which had identical

five nucleotide mismatches (75% homology) at the S4

primer binding site (nt 669�/688) were amplified (Table3). Although strain WI61, F45 or AU32 (lineage I) had

only four or five nucleotide mismatches (75�/80%

homology), none were amplified with the primer. Strain

116E (lineage II) which had four nucleotide mismatches

identical to those of F45 was weakly amplified.

Strains R44, R136 and R172 (lineage III) had four

identical nucleotide mismatches (80% homology) at the

aFT9 primer binding site and none were amplified.

Strains R143, R146, R160 and US1205 (lineage III) had

three nucleotide mismatches (85% homology) and wereamplified. Strains WI61, F45 and AU32 (lineage I)

which presented a 100% nucleotide homology at the

primer binding site were amplified, as expected. Strain

116E (lineage II) which had four nucleotide mismatches

(80% homology) different from those of lineage III

viruses and was still amplified.

3.4. Reactivity of selected G9 strains vs G3-, G4- or G9-

specific primer in C pool

All 11 G9 strains analyzed presented 7�/9 nucleotide

mismatches (50.0�/61.1% homology) with primer 9T-3P

(nt 484�/503) and none were amplified (Table 4). Strains

Table 1

Characteristics of selected Brazilian G9 and other G9 rotavirus strains as determined by various assays

Rotavirus strain PAGE pattern G genotype determined using primer pool G serotypea VP7 gene lineage

H1 H2 C

R44 Lb G3 G4 G9 G9 III

R113 L G3 G4 G9 NTd NT

R121 L G3 G4 G9 NT NT

R127 L G3 G4 G9 NT NT

R131 L G3 G4 G9 NT NT

R132 L G3 G4 G9 NT NT

R134 L G? G4 G9 NT NT

R135 L G3 G4 G9 NT NT

R136 L G3 G4 G9 NT III

R138 L G3 G4 G9 NT NT

R150 L G3 G4 G9 NT NT

R155 L G3 G4 G9 NT NT

R166 L G3 G4 G9 NT NT

R172 L G3 G4 G9 NT III

R178 Sc G3 G4 G9 NT NT

R185 L G3 G4 G9 NT NT

R191 L G3 G4 G9 NT NT

R179 L G3 G4/G9 G9 NT NT

R143 S G3/G9 G9/G4 G9 G9 III

R146 S G3/G9 G9/G4 G9 NT III

R160 L G3/G9 G9/G4 G9 NT III

R168 L G3/G9 G9/G4 G9 NT NT

R194 S G3/G9 G9/G4 G9 NT NT

US1205 S G3/G9 G9/G4 G9 G9 III

116E L G9/G3 G9/G4 G9 G9 II

WI61 L G9 G9 G9 G9 I

F45 L G9 G9 G9 G9 I

AU32 L G9 G9 G9 G9 I

a Determined by neutralization assay.b L, ‘long’ electropherotype.c S, ‘short’ electropherotype.d NT, not tested.

Erratum130

Table 2

VP7 gene primer binding site of selected G9 rotavirus strains: nucleotide homology (%) vs G3-, G4- or G9-specific primer in H1 pool

Rotavirus strain Sequence (5?0/3?) % Nucleotide homology VP7 gene lineage

aET3/G3a C G T T T G A A G A A G T T G C A A C A G

R44 - A - - - - - - - - G - - G - - T - - - A 76.2 III

R136 - A - - - - - - - - G - - G - - T - - - A 76.2 III

R172 - A - - - - - - - - G - - G - - T - - - A 76.2 III

R143 - A - - - - - - - - G - - G - - T - - - A 76.2 III

R146 - A - - - - - - - - G - - G - - T - - - A 76.2 III

R160 - A - - - - - - - - G - - G - - T - - - A 76.2 III

US1205 - A - - - - - - - - G - - G - - T - - - A 76.2 III

WI61 - A - - - - - G - - - - - G - - C G - - A 71.4 I

F45 - A - - - - - G - - - - - G - - C G - - A 71.4 I

AU32 - A - - - - - G - - - - - G - - C G - - A 71.4 I

116E - A - - - - - - - - G - - A - - G - - G A 71.4 II

aDT4/G4a C G T T T C T G G T G A G G A G T T G

R44 T - A - - - A A C G T T A A - - C - A 36.8 III

R136 T - A - - - A A C G T T A - - - C - A 42.1 III

R172 T - A - - - A A C G T T A - - - C - A 42.1 III

R143 T - A - - - A A C G T T A - - - C - A 42.1 III

R146 T - A - - - A A C G T T A - - - C - A 42.1 III

R160 T - A - - - A A C G T T A - - - C - A 42.1 III

US1205 T - A - - - A A C G T T A - - - C - A 42.1 III

WI61 - - A - - - A A C A T T A - - - - - A 52.6 I

F45 - - A - - - A A C A T T A - - - - - A 52.6 I

AU32 - - A - - - A A C A T T A A - - - - A 52.6 I

116E T A A - - - A A C A T T A - - - - - A 42.1 II

aFT9/G9a C T A G A T G T A A C T A C A A C T A C

R44 - - T - - - - - G - - - - T - - A - - - 80.0 III

R136 - - T - - - - - G - - - - T - - A - - - 80.0 III

R172 - - T - - - - - G - - - - T - - A - - - 80.0 III

R143 - - T - - - - - G - - - - - - - A - - - 85.0 III

R146 - - T - - - - - G - - - - - - - A - - - 85.0 III

R160 - - T - - - - - - - - - - T - - A - - - 85.0 III

US1205 - - T - - - - - G - - - - T - - A - - - 80.0 III

WI61 - - - - - - - - - - - - - - - - - - - - 100.0 I

F45 - - - - - - - - - - - - - - - - - - - - 100.0 I

AU32 - - - - - - - - - - - - - - - - - - - - 100.0 I

116E - - T - - - - - - - - - - - C - A C - - 80.0 II

a Primer/G genotype specificity.

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Table 3

VP7 gene primer binding site of selected G9 rotavirus strains: nucleotide homology (%) vs G3-, G4- specific primer in H2 pool

Rotaviruss strain Sequence (5?0/3?) % Nucleotide homology VP7 gene lineage

S3/G3a G A C G C G A C G T T G C A A T T G

R44 - - T T - A - - - - - A A - G C - A 55.6 III

R136 - - T T - A - - - - - A G - G C - A 55.6 III

R172 - - T T - A - - - - - A G - G C - A 55.6 III

R143 - - T T - A - - - - - A G - G C - A 55.6 III

R146 - - T T - A - - - - - A G - G C - A 55.6 III

R160 - - T T - A - - - - - A G - G C - A 55.6 III

US1205 - - T T - A - - - - - A G - G C - A 55.6 III

WI61 - - T T - A - - A - - A G - G - - A 55.6 I

F45 - - T T - A - - A - - A G - G - - A 55.6 I

AU32 - - T T - A - - A - - A A - G - - A 55.6 I

116E A - T T - A - - A - - A G - G - - A 50.0 II

S4/G4a T C A A A C G A C A A A T A C A G C T A

R44 - A T T - - C - - - - - - - - - - - G - 75.0 III

R136 - A T T - - C - - - - - - - - - - - G - 75.0 III

R172 - A T T - - C - - - - - - - - - - - G - 75.0 III

R143 - A T T - - C - - - - - - - - - - - G - 75.0 III

R146 - A T T - - C - - - - - - - - - - - G - 75.0 III

R160 - A T T - - C - - - - - - - - - - - G - 75.0 III

US1205 - A T T - - C - - - - - - - - - - - G - 75.0 III

WI61 - A C - - - T - - - - - - - - - - - A - 80.0 I

F45 - A C - - - T - - - - - - - - G - - G - 75.0 I

AU32 - A C - - - T - - - - - - - - - - - A - 80.0 I

116E - A C - - - T - - - - - - - - G - - G - 75.0 II

aFT9/G9b

a Primer/G genotype specificity.b Results identical as described in Table 1.

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Table 4

VP7 gene primer binding site of selected G9 rotavirus strains: nucleotide homology (%) vs G3-, G4- or G9-specific primer in C pool

Rotavirus strain Sequence (5?0/3?) % Nucleotide homology VP7 gene lineage

9T-3P/G3a G C T A C A C T G C A A C T G G A C

R44 T - A - - G T - A A - G - - A - - T 50.0 III

R136 T - A - - G T - A G - G - - A - - T 50.0 III

R172 T - A - - G T - A G - G - - A - - T 50.0 III

R143 T - A - - G T - A G - G - - A - - T 50.0 III

R146 T - A - - G T - A G - G - - A - - T 50.0 III

R160 T - A - - G T - A G - G - - A - - T 50.0 III

US1205 T - A - - G T - A G - G - - A - - T 50.0 III

WI61 T - A - - - T - A G - G T - A - - - 55.5 I

F45 T - A - - - T - A G - G T - A - - - 55.5 I

AU32 T - A - - - T - A A - G T - A - - - 55.5 I

116E T - A - - - T - A - - - T - A - - T 61.1 II

9T-4/G4a A G A A T T T T C C A T C G A C C C

R44 T T C - - - C - - A - - T - - T - - 61.1 III

R136 T T C - - - C - - A - - T - - T - - 61.1 III

R172 T T C - - - C - - A - - T - - T - - 61.1 III

R143 T T C - - - C - - A - - T - - T - - 61.1 III

R146 T T C - - - C - - A - - T - - T - - 61.1 III

R160 T T C - - - C - - A - - T - - T - - 61.1 III

US1205 T T C - - - C - - A - - T - - T - - 61.1 III

WI61 T T C - - - - - - T - - T - - T - - 66.7 I

F45 T T C - - - - - - T - - T - - T - - 66.7 I

AU32 T T C - - - - - - T - - T - - T - - 66.7 I

116E T T C - - - - - - A - - - - - T - - 72.2 II

9T-9B/G9a G T G C A A T G G A C T T T A T A

R44 - - - - G - - - - - - - - - - - - 94.1 III

R136 - - - - G - - - - - - - - - - - - 94.1 III

R172 - - - - G - - - - - - - - - - - - 94.1 III

R143 - - - - G - - - - - - - - - - - - 94.1 III

R146 - - - - G - - - - - - - - - - - - 94.1 III

R160 - - - - G - - - - - - - - - - - - 94.1 III

US1205 - - - - G - - - - - - - - - - - - 94.1 III

WI61 - - - - - - - - - - - - - - - - - 100.0 I

F45 - - - - - - - - - - - - - - - - - 100.0 I

AU32 - - - - - - - - - - - - - - - - - 100.0 I

116E - - - - - - - - - - - - - - - - - 100.0 II

a Primer/G genotype specificity.

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3

belonging to lineage I or III presented six or seven

nucleotide mismatches (61.1 or 66.7% homology) at the

primer 9T-4 binding site (nt 423�/440) and none were

amplified. Although strain 116E (lineage II) presentedonly five nucleotide mismatches (72.2% homology), it

was not amplified with the primer.

Strains belonging to lineage III which presented one

identical nucleotide mismatch (94.1% homology) at the

9T-9B primer binding site (nt 131�/147) were amplified.

No mismatches were observed at the primer binding site

of strains belonging to lineage I or II.

3.5. Phylogenetic analysis of VP7 gene of Brazilian and

other G9 rotavirus strains

Phylogenetic comparison of the VP7 encoding gene of

selected strains belonging to G3, G4 or G9 showed that

the 12 G9 strains that were analyzed in this study

clustered together in G9 genotype which was distinct

from G3 or G4 genotype phylogenetically (Fig. 2), even

though such G9 strains showed a high degree of cross-

reactivity with G3- or G4-specific primer in PCR

amplification (Tables 2 and 3).The VP7 gene sequence analysis of the Brazilian as

well as other G9 strains from a global collection showed

the existence of at least three VP7 gene lineages among

them (Fig. 3): lineage I represented by strains WI61,

F45, and AU32; lineage II formed by the Indian strain

116E; and lineage III consisted of strains from Brazil,

U.S., India, Malawi, Bangladesh, Nigeria, UK, China

and Japan.

3.6. Characterization by neutralization of selected

Brazilian G9 rotavirus strains

Brazilian G9 strains R44 and R143 were cell culture-

adapted and characterized by PRN assay. Each rota-

virus strain was tested against guinea pig hyperimmune

antiserum raised to each of G1 to G14 rotavirus strains.

Both strains R44 and R143 were neutralized strongly

only by hyperimmune antiserum to WI61 or F45

prototype G9 strain.

3.7. Characterization by PAGE of Brazilian and other

G9 rotavirus strains

Four of 23 Brazilian G9 rotavirus strains exhibited so-called ‘short’ (S) pattern and the remaining 19 strains

had a ‘long’ (L) pattern when analyzed by polyacryla-

mide gel electrophoresis (PAGE) (Table 1). These four

strains with S pattern may be naturally-occurring

reassortants. Four prototype G9 strains (WI61, F45,

AU32 and 116E) displayed in PAGE gel an L pattern

whereas strain US1205 exhibited a S pattern as de-

scribed previously.

4. Discussion

Sequence analysis of selected rotavirus genes has

provided information leading to substantial advances

in our understanding of: (i) the phylogenetic diversity of

this virus; and (ii) genetic and antigenic diversity of

outer capsid protective antigens VP7 and VP4, the

targets of current vaccine development strategies. Suchstudies have also provided new insight into genetic and

antigenic intratypic VP7 variation within a given human

rotavirus G serotype including G1 (Xin et al., 1993; Jin

et al., 1996; Maunula and von Bonsdorff, 1998; Diwa-

karla and Palombo, 1999), G2 (Wen et al., 1995;

Coulson et al., 1996; Zao et al., 1999), G3 (Wen et al.,

1997), G4 (Coulson et al., 1996; Palombo et al., 1997),

and G9 (Coulson et al., 1999; Ramachandran et al.,2000). Furthermore, for rotavirus serotypes G1, G2, G4

and G8, such VP7 gene variations were shown to be

associated with a failure in correct G type identification

by PCR (Nakagomi et al., 1991; Adah et al., 1997;

Cunliffe et al., 2001a,b). Genetic variation has also been

described for the most common human rotavirus VP4

serotype P1A[8], leading to a failure to P genotype such

Fig. 2. Phylogenetic tree of VP7 gene nucleotide sequences of rotavirus serotypes G3, G4 and G9 strains. Branch length is proportional to the

percentage of nucleotide substitution indicated in the scale.

Erratum134

isolates (Gouvea et al., 1999; Iturriza-Gomara et al.,

2000; Cunliffe et al., 2001a,b). Here we showed that the

Brazilian G9 strains as well as other well-characterized

G9 strains could mistakenly be genotyped as G3, G4 or

dual infection of G3/G9 or G9/G4 depending on the

primer pool employed in PCR. This phenomenon had

previously been observed in our laboratory: selected

Brazilian rotavirus strains previously typed as G10genotype by using just one set of primers (Santos et

al., 1998) turned out to belong to G3 genotype when a

different set of primers was employed. The G3 pheno-

type of such viruses was also confirmed by neutraliza-

tion assay.

Existence of at least three distinct phylogenetic VP7

gene lineages among G9 strains reported previously by

us (Santos et al., 2001) and others (Bok et al., 2001; Oka

et al., 2000; Ramachandran et al., 2000; Zhou et al.,2001) was confirmed and extended in this study (Fig. 3).

Sequence analysis of the VP7 gene of selected G9 strains

showed that genomic sequence variation, more specifi-

cally sequence mutation(s) in the primer binding site of

the gene, appeared to account for the observed G

genotype variability determined by PCR. Furthermore,

such sequence mutations(s) at the primer binding site

was shown to be VP7 gene lineage-specific. Thus, this

study showed that: (i) G9 strains in lineage I was typedcorrectly as G9 with any of the three primer pools (H1,

H2 and C) employed; (ii) lineage II G9 strain was typed

as G9/G3 vs H1 pool, G9/G4 vs H2 pool and G9 vs C

pool; and (iii) G9 strains belonging to lineage III were

typed as G3 or G3/G9 vs H1 pool, G4 or G9/G4 vs H2

pool and G9 vs C pool (Table 1). Although G9 strains

belonging to lineage II have been detected only in parts

of India, G9 strains in lineage III have been detected

throughout the world (Fig. 3). Therefore, caution must

be exercised when primers aET3 and/or S4 are used in

combination with primer aFT9 to G genotype rotavirus

field isolates. Since this G9 primer was designed fromthe sequence of only a few strains available at the time

and the G9 viruses have evolved throughout the years as

observed on Tables 2 and 4, some strains could be

mistakenly typed with this primer.

It has been shown that primers with a length between

17 and 20 nt long need at least three homologous

nucleotides at their 3? end for successful priming

(Sommer and Tautz, 1989). Although the VP7 gene of

Brazilian G9 rotavirus strain R44, R136, R143, R146,R160 or R172 presented an 80�/85% homology at the

primer binding site including three homologous nucleo-

tides at the 3? end with the G9-specific primer aFT9 (20

nt long) in primer pool H1 or H2, it was not amplified

correctly as G9. The priming process with the aFT9

appeared to get impaired in the presence: (i) G3-specific

aET3 primer in H1 pool (which presented 76.2%

homology at the primer binding site); or (ii) G4-specific

S4 primer in H2 pool (which presented 75% homologyat the primer binding site). Consequently, the VP7 gene

of each of the Brazilian G9 strains was amplified by

both the G9-specific primer and the G3- or G4-specific

primer (Tables 2 and 3). Perhaps, when using in PCR

Fig. 3. Phylogenetic analysis of the VP7 gene nucleotide sequence of rotavirus G9 strains from a global collection. The country of origin and the year

of strain isolation are indicated. Three clusters of G9 strains representing VP7 gene lineages I, II and III are evident.

Erratum 135

amplification a pool of two or more primers which have

a close nucleotide homology at each respective primer

binding site, the priming process may favor an annealing

of one primer over others regardless of the number and

position of nucleotide mismatches at the primer binding

site. Further studies need to be explored.

Of interest was the behavior in PCR of three Brazilian

strains (R143, R146 and R160) against G9-specific aFT9

primer (which showed an 85% homology at primer

binding site) present in H1 or in H2 pool (Tables 2 and

3). Data obtained in this study appear to suggest that at

an annealing temperature of 50 8C in PCR, if the VP7

gene nucleotide homology difference between homoty-

pic and heterotypic primers at each respective primer

binding site in a pooled primer set is: (i) higher than

20%, only the homotypic priming may take place; (ii)

lower than 5%, only the heterotypic priming may occur;

and (iii) between 5 and 10%, both homotypic and

heterotypic priming may be seen. More detailed studies

need to be performed.

Epidemiologic surveillance of rotavirus serotypes/

genotypes (both VP7[G] and VP4[P]) is underway

worldwide, because neutralizing antibodies appear to

play an important protective role against rotavirus

disease in a type-specific manner (Hoshino and Kapi-

kian, 2000). The advent of PCR-typing methodology

has greatly enhanced an efficiency of such epidemiologic

surveillance of rotavirus genotypes. Nonetheless, the

results described here reinforce the need for improve-

ment on such methodology. For example, the use of

more than one set of type-specific primer pools may be

necessary for an accurate and efficient genotype deter-

mination of rotavirus field isolates. In addition, since an

‘antigenic drift’ of the rotavirus VP7 gene has been

shown to occur as reported by us (this study) and others

(Palombo et al., 1997), continuous efforts need to be

made to design a type-specific primer which recognizes

as many rotavirus isolates as possible within a given G

genotype. Furthermore, since errors in PCR genotyping

due to cross-priming of multiplexed primers could

occasionally occur, selective confirmation of PCR

results by other assays will be needed including sequen-

cing, probe hybridization or serotyping with type-

specific antibodies.

The primer aFT9 which was designed from the VP7

gene sequence of lineage I G9 strains WI61, AU32 and

F45 isolated in the 1980s was shown to fail to prime

correctly more recent G9 isolates belonging to lineage II

or III. Whereas the primer 9T-9B designed from the VP7

gene sequence of more recent 116E strain (lineage II)

genotyped correctly all the G9 isolates analyzed. If in

the present study only H1 or H2 primer pool had been

used, Brazilian and Indian G9 strains would have been

typed wrongly. Actually, it is possible that the real

frequency of rotavirus G9 genotype in Brazil, and other

countries as well, could be underestimated due to the

phenomenon that we described in this study.

5. Nucleotide sequence accession numbers

The sequences were deposited in the GenBank under

the accession numbers AF438227, AF438228,

AF274969, AF274970 and AF274971. Nucleotide se-

quences of the VP7 gene from strains R172 and R136

are identical.

Acknowledgements

We thank Ronald Jones, Jerri Ross, and Mariam

Wagner for their expert technical assistance; and Albert

Kapikian for encouragement throughout the study. This

work was partially supported by CNPq, FINEP,

FAPERJ, FUJB, and CAPES Brazil, and TWAS, Italy.

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