www.elsevier.com/locate/meegid

Infection, Genetics and Evolution 8 (2008) 94–99

Short communication

Genetic diversity of the VP4 and VP7 genes affects the

genotyping of rotaviruses: Analysis of Paraguayan strains

Emilio E. Espınola a,1, Gabriel I. Parra a,b,1,*,Graciela Russomando a, Juan Arbiza b

a Departamento de Biologıa Molecular, Instituto de Investigaciones en Ciencias de la Salud,

Universidad Nacional de Asuncion, Asuncion, Paraguayb Seccion Virologıa, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay

Received 4 June 2007; received in revised form 21 August 2007; accepted 21 August 2007

Available online 25 August 2007

Abstract

The introduction of different multiplex RT-PCR strategies for the characterization of field rotavirus strains has led to improvements of

surveillance systems worldwide. Nevertheless, the failure or incorrect characterization of rotavirus strains by these PCR strategies, mainly due to

accumulation of point mutations in the VP4 and VP7 genes, has been reported. In this work, sequence analyses of the VP4 and VP7 genes from

Paraguayan G1P[8] and G4P[8] strains revealed that the high degree of similarity with the primers pNCDV and ET10 could lead to the incorrect

characterization of these strains as P[1] and G10 types. Moreover, the nucleotide diversity of the VP4 gene at the 1T-1 primer binding site could be

one, although not the only, reason of the failure of the P[8] typing. Therefore, the typing methods utilized by surveillance programs should be

constantly evaluated and sequencing of atypical strains should become a current practice in order to confirm their real nature.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Rotavirus; Genetic diversity; Genotyping

1. The study

Group A rotaviruses are the leading cause of severe

gastroenteritis in infants and young children worldwide,

causing more than 450,000 deaths per year (Parashar et al.,

2006). The rotavirus genome contains 11 segments of double-

stranded RNA (dsRNA), which are packaged within a triple-

layer capsid. The two outermost proteins, i.e. VP4 and VP7,

specify the P and G types, respectively. To date, 12 P and 11 G

types have been detected in humans; of them, G1–G4 are

considered the most prevalent types worldwide, generally

combined with P[8] (G1, G3, and G4) or P[4] (G2)

(Desselberger et al., 2001; Gentsch et al., 2005; Martella

et al., 2005, 2006b; Santos and Hoshino, 2005).

* Corresponding author. Present address: Department of Neurosciences/

NC30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500

Euclid Avenue, Cleveland, OH 44195, USA. Tel.: +1 216 444 6149;

fax: +1 216 444 7927.

E-mail address: gabriel_parra@hotmail.com (G.I. Parra).1 These authors contributed equally to this work.

1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2007.08.003

Since there is a limited availability of reagents for rotavirus

serotype characterization, the introduction of molecular

techniques for genotype characterization has improved the

surveillance systems (Fischer and Gentsch, 2004). Thus,

genotypes commonly found in animals have been detected

in humans worldwide and new data about rotavirus epidemiol-

ogy are available (Desselberger et al., 2001; Gentsch et al.,

2005; Martella et al., 2005, 2006b; Santos and Hoshino, 2005).

Among the molecular techniques available, four strategies of

multiplex reverse transcription (RT)-PCR for G typing and one

for P typing have been widely used for human strains

characterization (Das et al., 1994; Fischer and Gentsch,

2004; Gentsch et al., 1992; Gouvea et al., 1990; Iturriza-

Gomara et al., 2004; Taniguchi et al., 1992). However, failure or

incorrect characterization of field rotavirus strains by these RT-

PCR strategies and/or serotyping methods, mainly due to the

accumulation of point mutations in the VP4 and VP7 genes and

to the diversification on specific lineages, has been reported

(Adah et al., 1997; Arista et al., 2005; Banyai et al., 2004;

Cunliffe et al., 2001; Das et al., 2004; Gomara et al., 2001;

Iturriza-Gomara et al., 2000, 2004; Martella et al., 2006a, 2004;

E.E. Espınola et al. / Infection, Genetics and Evolution 8 (2008) 94–99 95

Page and Steele, 2004; Parra and Espinola, 2006; Phan et al.,

2007; Rahman et al., 2005; Santos et al., 2003).

Recently, our group has published the molecular character-

ization and diversity of rotavirus strains that circulated in

Paraguay during 1998–2005. We found that (i) the predominant

G types (i.e. G1, G4 and G9) change from season to season, (ii)

the predominance of a specific G type is associated with the

emergence of atypical VP7 lineages, (iii) mixed infections are

detected only in years during which no strain predominated,

and (iv) there could be an across-border migration of rotavirus

strains between Paraguay and its neighboring countries (Parra

et al., 2005, in press; Stupka et al., 2007).

Since another survey of rotavirus strains circulating in

Paraguayan children in 1999 showed the presence of rotavirus

genotypes G10 and P[1] (Coluchi et al., 2002), and since,

during our survey, 16.9% (44/260) of the rotavirus-positive

samples were untypeable for P or G type or both, in this study

we included primers to detect the genotypes G10 and P[1] and

performed sequence analysis to assess the molecular char-

acterization of these untypeable strains.

Thirty-one out of the 44 rotavirus-positive faecal samples

untypeable for P or G type or both were available in sufficient

quantities to develop viral RNA extraction. Moreover, 39 faecal

samples fully characterized for the P and G type were used (11

G1P[8], 3 G2P[4], 7 G4P[8] and 18 G9P[8]). All the samples

were collected from Paraguayan children under 5 years old with

acute diarrhoea, from 1998 to 2005.

The viral RNA was extracted from 10% faecal suspensions

using TRIZOL1 (Invitrogen, Inc., Carlsbad, CA) or the silica

powder method (Boom et al., 1990). G and P types were

characterized by two rounds of PCR strategies. In the first

round, the gene 9 (VP7) was reverse-transcribed and amplified

using a pair of generic primers (Beg and End), corresponding to

highly conserved portions of the 50 and 30 ends of the gene

(Gouvea et al., 1990). The second round of PCR was performed

with a pool of internal primers (9T1-1, 9T1-2, 9T1-3P, 9T1-4,

FT5, 9T-9B, ET10 and 9Con1) (Das et al., 1994; Gouvea et al.,

1994a). A similar strategy was used to determine the P types,

using the primers Con2, Con3, 1T-1, 2T-1, 3T-1, 4T-1, 5T-1 and

pNCDV (Gentsch et al., 1992; Gouvea et al., 1994b). Therefore,

these pools of primers were designed to detect G1, G2, G3, G4,

G5, G9 or G10 and P[1], P[4], P[6], P[8], P[9] or P[11] strains.

PCR products were analysed by 2% agarose gel electrophoresis

and ethidium bromide staining. The molecular size of the

amplified products was confirmed by polyacrylamide gel

electrophoresis, which consisted of a stacking gel (4.5%) and a

running gel (7.5%) followed by silver staining. The DNA

sequencing was performed from the first round amplicons in an

automatic sequencer, as was described previously (Parra et al.,

2005). The nucleotide sequences obtained were analysed with

BioEdit and MEGA v3 (Hall, 1999; Kumar et al., 2004).

Twenty (64.5%) out of the 31 untypeable strains analysed

were successfully typed. The VP8* amplicons of 14 of them

were obtained with sufficient DNA concentration for nucleotide

(nt) sequencing and thus characterized as P[8] type. In addition,

two G1P[8], one G4P[8] and three G9P[8] strains were fully

characterized based on PCR-typing. It is noteworthy that one of

the two newly characterized G1P[8] strains, named Py99472,

presented an amplicon of the P[1] type.

The alignment of the VP4 gene from the Paraguayan strains

and the primer 1T-1, used for P[8] typing, showed from 3 to 6 nt

mismatches. Two G1 strains, collected in 1999, and two G9

strains – all of them with an untypeable P type – showed five

mismatches between nt 6 and 11 from the 30 end of the primer.

The three G4 strains with untypeable P type showed four or five

mismatches between nt 6 and 11 plus one at nt 1 (C:A) from the

30 end (Fig. 1c). Moreover, all the G1 strains collected during

2002–2003 showed a mismatch (G:T) at nt 4 from the 30 end of

the primer plus two to four mismatches between nt 6 and 13

(Fig. 1c).

In order to evaluate how many P[8] sequences available in

the GenBank database have these mismatches, and its

clustering within the four lineages previously described, we

collected 145 VP8* sequences from the GenBank database

Release 160.0 (alignments are available from the authors upon

request). The phylogenetic tree was constructed using neighbor

joining with Kimura 2-parameter as a model of nucleotide

substitution. The statistical significance of the tree was

performed by bootstrapping, using 1000 pseudo-replicates

data sets.

Seventy-six (52.4%) strains showed nt sequences associated

with genotyping failure; of them, 71 (49%) grouped within the

lineage III and 5 (3.4%) within the lineage I (Fig. 2). It is worth

of mention that the primer 1T1-1 was originally designed using

the KU strain that grouped within the lineage II (indicated by a

filled diamond, Fig. 2), and none nt sequence associated with

genotyping failure clustered within the lineage II (Fig. 2).

Therefore, these data show that the genotyping failure of P[8]

strains are associated with accumulation of point mutations and

diversification on specific lineages.

Of note is that these point mutations at the 1T-1 primer

binding site have been reported previously, as a possible cause

of the P typing failure in most of the tested samples (Cunliffe

et al., 2001; Iturriza-Gomara et al., 2000; Phan et al., 2007).

Since the same mutations were found in 20 Paraguayan strains

from which the P type was successfully characterized with the

primer 1T-1 (Fig. 1c), these nt mismatches do not appear to be

the only cause of the failure. Of note is that during a

surveillance carried out in Bangladesh in 2002, 75% of the G1

strains were unable to be typing using the Das’s RT-PCR

strategy, however, either the typed and the untyped G1 strains

showed the same nt sequence in the VP7 primer binding site

(Parra and Espinola, 2006). Therefore, the first round amplicon

concentration, the annealing temperatures used, and the

number, position and nature of the nt mismatches could be

other factors to be taken into account to explain these

observations.

Surprisingly, in addition to the strain Py99472, 11 fully

characterized strains – a few of them initially used as control of

the PCR reactions – showed amplicons of the genotypes G10

(4 strains) and P[1] (7 strains) (Fig. 1). Of note is that one of the

four G4P[8] strains that showed a G10 type amplicon did not

present sufficient DNA concentration for nt sequencing. There-

fore, in order to analyse the influence of nucleotide diversity in

Fig. 1. Alignment of the VP4 and VP7 genes from rotavirus Paraguayan strains at the ET10 (a), pNCDV (b), and 1T1-1 (c) primer binding sites. The DNA direction

and the sense of each primer are indicated. For each strain, the year of isolation is indicated by the first two numbers of its name (for instance, strains Py99 were

isolated in Paraguay in 1999). The results of the genotyping with each primer are shown on the right-hand side: +, positive;�, negative; +/�, weak amplicon; NT, not

tested.

E.E. Espınola et al. / Infection, Genetics and Evolution 8 (2008) 94–9996

the incorrect characterization of these strains, partial sequences

of the VP7 and VP4 genes from the Paraguayan strains were

compared against the primers ET10 and pNCDV, respectively

(Fig. 1a and b). It is worth of mention that none of the G2P[4]

strains was incorrectly characterized by the ET10 and pNCDV

primers.

The VP7 gene of the G1 and G9 Paraguayan strains showed

10–11 nt mismatches, but only 7 nt mismatches, most of them at

the 50 end of the primer were found in the G4 strains. Thus, the

30 end of the primer binding site from the G4 strains showed a

high degree of similarity (78%; 7/9) with the ET10-primer

(Fig. 1a). Even though it has been shown that a perfect match of

the last 3 nt at the 30 end in primers of 17–20 nt length is

necessary to achieve a successful annealing and amplification

(Sommer and Tautz, 1989), Kwok et al. (1990) showed that the

T:C mismatch does not have a significant effect on the

Fig. 2. Phylogenetic tree showing the four lineages described in genotype P[8]

of rotavirus strains. The symbols showing the nt sequences associated with the

genotyping failure are indicated on the right-hand side of Fig. 1c. The strain KU,

used for the P[8] primer design, is indicated by a filled diamond. The lineages

are represented in the tree as follows: lineage I (orange), lineage II (green),

lineage III (blue) and lineage IV (red).

E.E. Espınola et al. / Infection, Genetics and Evolution 8 (2008) 94–99 97

amplification of the target sequence (Fig. 1a). Therefore, this

could be one of the reasons why the ET10-primer annealed to

G4 strains, resulting in the amplification of the DNA fragments

corresponding to the size of G10 amplicons in four out of the

seven tested samples (Fig. 1a). Recently, Iturriza-Gomara et al.

(2004) designed a new G10-primer to overcome the cross-

reaction with the genotype G3. Since this new primer presents

81% (17/21) of similarity with G4 strains (data not shown), it

would be necessary to test it in other geographical locations

with high prevalence of G4 strains.

The alignment between the primer pNCDV and the VP4

gene from the Paraguayan strains are depicted in Fig. 1b. The

G9, G4 and G1 strains showed between 11 and 13 nt, 12 nt, and

between 10 and 13 nt mismatches, respectively, when

compared with the pNCDV-primer. It is interesting to note

that all the strains shared the same nt mismatches at the 50 end of

the primer and the diversity was found at the last 10 nt of the 30

end of the primer (Fig. 1b). Thus, in that region, the G9 strains

showed between 3 and 5 nt mismatches, while all the G4 strains

and 5 out of the 13 G1 strains sequenced showed between 4 and

5 nt mismatches (Fig. 1b). Finally, nine of the G1 strains

showed only two or three mismatches in the last 10 nt region of

the 30 end of the primer. Therefore, since it has been shown that

in primers >20 nt length is needed only 2 nt matches at the 30

end to reach a successful amplification (Sommer and Tautz,

1989), and that the T:T mismatch does not affect the efficiency

of the amplification (Fig. 1b) (Kwok et al., 1990), these results

suggest that the high degree of similarity of the primer pNCDV

and the VP4 gene from six G1P[8] strains could explain the

cross-reaction with the P[1]-primer. It is noteworthy that a G:A

mismatch at nt 4 from the 30 end of the primer was found in all

the Paraguayan strains and that this mismatch has been reported

by Kwok et al. (1990) as a mismatch that reduces 100-fold the

efficiency of the amplification (Fig. 1b). Therefore, this could

be the cause that weak P[1] type amplicons were detected in

few samples. Of note is that 6 out of the 145 VP8* sequences

retrieved from the GenBank, showed the same nt sequence in

the P[1]-primer binding region presented by the G1P[8]

Paraguayan strains incorrect characterized as P[1]; all of them

clustered with a high bootstrap value (83%) (data not shown).

Moreover, these strains were isolated between 2000 and 2006,

reinforcing the notion that accumulation of point mutations

over the time could lead to incorrect characterizations of

rotavirus strains.

During a survey carried out in Paraguay in 1999, Coluchi

et al. (2002) detected the presence of the animal rotavirus

genotypes G10 and P[1], mainly associated to mixed infections

with G1P[8] and G4P[8] strains. Since in the present study we

only detected G1P[8] strains incorrectly associated with P[1]

type, reassortments of the VP4 or VP7 genes could explain the

presence of the G and P type combinations found by Coluchi

et al. (2002).

Since only 20 (64.5%) out of the 31 rotavirus-positive

samples untypeable for G or P type or both were successfully

characterized, the failure of the characterization could be due to

several reasons: (i) the fact that samples were stored at �20 8Cand submitted to freeze/thaw cycles several times, therefore

virus/RNA deterioration can be expected, (ii) low amount of

virus in the sample, (iii) the circulation of rotavirus strains with

genotypes not included in the pool of primers, or (iv) the

accumulation of point mutations in the binding sites of the

primers used for the reverse transcription.

Even though four genotypes (G1P[8], G2P[4], G3P[8] and

G4P[8]) appear to be the most prevalent in humans globally,

animal rotavirus strains, such as G5, G8, and G10, have been

shown to be epidemiologically important in many developing

countries (Araujo et al., 2002; Banerjee et al., 2006; Santos and

Hoshino, 2005; Santos et al., 1998). It is worth mentioning that

the increased prevalence of the genotype G10 in a community-

based study, carried out in India during 2002–2003, suggest it as

a potential emergent strain (Banerjee et al., 2006). Therefore,

since the efficacy of the available vaccines seems to be linked to

the rotavirus strains circulating in a given population, it is

important to collect well-documented data (i.e. correct

characterization of rotavirus field strains) during pre- and

post-vaccination programmes. Consequently, the typing meth-

ods utilized by surveillance programmes should be constantly

E.E. Espınola et al. / Infection, Genetics and Evolution 8 (2008) 94–9998

evaluated, and the sequencing of a percentage of the typed

strains should become a current practice in order to confirm

their real nature.

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