ORIGINAL ARTICLE

Sequence and phylogenetic analysis of the VP4 gene of humanrotaviruses isolated in Paraguay

E. E. Espınola Æ A. Amarilla ÆJ. Arbiza Æ G. I. Parra

Received: 9 December 2007 / Accepted: 18 March 2008 / Published online: 8 May 2008

� Springer-Verlag 2008

Abstract Nucleotide and amino acid analyzes of the VP4

gene of human rotaviruses isolated both in Paraguay and

worldwide were carried out in order to increase our

knowledge about the complex pattern of evolution of this

virus in nature. Paraguayan strains bearing the P[8] geno-

type were grouped in the lineages P[8]-1, P[8]-2, and P[8]-

3. Regardless of the year of detection, all of the G4 and G9

strains were related to lineage P[8]-3, whereas the G1

strains were related to the three lineages detected in Par-

aguay; this fact reinforces the notion of the existence of

constraints within specific populations of rotavirus strains

except for the G1 strains. In addition, we propose a phy-

logenetic classification for the P[4] strains in five different

lineages (i.e. P[4]-1 to P[4]-5). The findings presented in

this paper reinforce the importance of a continuous sur-

veillance of rotavirus strains in order to predict the possible

variants that will circulate in a country, and ultimately

improve current vaccination programs.

Introduction

Rotaviruses are considered as the leading cause of severe

gastroenteritis among children worldwide, with more than

600,000 annual deaths [32]. They belong to the family

Reoviridae, with a genome composed of 11 segments of

double-stranded RNA (dsRNA), and surrounded by a tri-

ple-layered protein capsid. The outermost layer is formed

by two proteins: the spike protein (VP4) and the coat

protein (VP7) [18]. Based on antigenic and genetic dif-

ferences of these proteins, rotaviruses can be classified into

P- and G-types, respectively. To date, 16 G- and more than

27 P-types have been detected [20, 35].

As with other genome-segmented-viruses, rotaviruses

can reassort their genes independently [23], leading to

different combinations of G- and P-types [18]. Despite this

intrinsic property, five combinations of genotypes (i.e.

G1P[8], G3P[8], G4P[8], G9P[8], and G2P[4]) comprise

more than 90% of the human cases detected worldwide

[36].

VP4 is a trimeric protein [15] involved in cell attach-

ment and membrane penetration. In order to achieve

efficient cell entry, the virion must be activated by trypsin.

Thus, the immature VP4 is cleaved by trypsin into an

N-terminal fragment, VP8*, and a C-terminal fragment,

The partial nucleotide sequences of the VP4 gene of human

rotaviruses obtained in this study were deposited in the GenBank,

EMBL, and DDBJ databases, under the accession numbers

EU045214–EU045254.

E. E. Espınola (&) � A. Amarilla � G. I. Parra

Departamento de Biologıa Molecular, Instituto de

Investigaciones en Ciencias de la Salud, Universidad Nacional

de Asuncion, Rıo de la Plata y Lagerenza,

Asuncion 2511, Paraguay

e-mail: emilioespinola@hotmail.com

J. Arbiza � G. I. Parra

Seccion Virologıa, Facultad de Ciencias,

Universidad de la Republica,

Montevideo, Uruguay

Present Address:

A. Amarilla

Centro de Pesquisa em Virologia, Faculdade de Medicina de

Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto,

SP 14049-900, Brazil

Present Address:

G. I. Parra

Department of Neurosciences, Lerner Research Institute,

The Cleveland Clinic Foundation, 9500 Euclid Avenue,

Cleveland, OH 44195, USA

123

Arch Virol (2008) 153:1067–1073

DOI 10.1007/s00705-008-0096-8

VP5*. The VP8* fragment is the viral hemagglutinin [19],

while the VP5* fragment is involved in the permeabiliza-

tion of membranes [16]. To date, the study of the VP8*

fragment of the P[8] genotype has revealed four distinct

phylogenetic lineages (i.e. P[8]-1, -2, -3, and -4) [5, 12,

26]. Nevertheless, few studies have been carried out

concerning the degree of genetic variation of the P[4]

genotype, and available phylogenetic studies are only

based on temporal distributions of strains for specific

countries [6, 27].

We have recently reported the genotype diversity, and the

VP7 phylogenetic relationship of rotavirus strains detected

over 7 years in Paraguay [33, 34]. In the present report we

describe the VP8* genetic variation of the most common P

genotypes of human rotavirus strains circulating in Para-

guay, and compare them to those that present a worldwide

distribution. In addition, we propose a phylogenetic classi-

fication for the P[4] strains isolated worldwide.

Materials and methods

Samples

Four hundred and seventy-five fecal samples positive for

group-A rotaviruses were obtained from children under

5 years of age, and 155 from adults over 18 years old with

acute diarrhea, as described previously, from August 1998

to August 2000 [10] and from August 2002 to December

2005 [2]. The genomic dsRNA pattern was resolved by

polyacrylamide gel electrophoresis (PAGE), formed by a

stacking gel (4.5%) and a running gel (7.5%), followed by

silver staining.

G and P genotyping

Rotavirus dsRNA was extracted from 10% fecal suspen-

sion by using TRIzol� reagent (Invitrogen, Carlsbad, CA)

according to the manufacturer’s instructions. The geno-

typing was carried out using specific multiplex-PCRs for

the VP7 [13, 22] and VP4 genes [21].

RT-PCR and purification of amplicons

The VP8* region of the VP4 gene of the samples was

reverse-transcribed and amplified by polymerase chain

reaction (RT-PCR), using the consensus primers Con3–

Con2, as described previously [21]. The amplicons

obtained in sufficient concentration were purified from

agarose gels using QIAquick Gel Extraction Kit (Qiagen,

Valencia, CA), and directly sequenced (both strands) in an

ABI PRISM 37309l DNA analyzer (Applied Biosystems,

Foster City, CA).

Sequence and phylogenetic analysis

The raw chromatograms were displayed using Chromas

2.31 (Technelysium Pty Ltd., Helensville, Queensland,

Australia) for correction. Nucleotide sequences were

aligned with CLUSTAL W [38] and edited using the

BioEdit software v7.0.0 [24]. Prediction of protein sec-

ondary structure was done using the PSIPRED v2.5 server

[30], and the comparison was performed with the VP8*

x-ray structure of the P[8] Wa strain [9]. Phylogenetic

relationships between strains were reconstructed by the

neighbor-joining method with Kimura’s two-parameter

model as the model of nucleotide substitution and bootstrap

analysis of 1,000 replicates incorporated in the MEGA v3.1

analytical package [28]. For the sequence and phylogenetic

analysis of the P[8] and P[4] genotypes, the region from

amino acids 12 to 279 of the VP4 gene (nucleotide position

43 to 846) was included according to the reference strain

Wa (GenBank accession number L34161). The search for

other rotavirus strains circulating worldwide was carried

out using the BLAST algorithm [1] from the National

Center for Biotechnology Information (NCBI) and down-

loaded from the GenBank database, release 160.0.

Results

The study

To assess the evolutionary relationships between the P[8]

and P[4] genotypes of human rotaviruses isolated in Par-

aguay, the VP8* region of 41 strains was sequenced for

comparison analysis with worldwide strains deposited in

the GenBank database. Thirty-four of these strains belon-

ged to the P[8] genotype (with a long dsRNA migration

pattern, and G1, G4, or G9 specificities), and seven to the

P[4] genotype (with a short dsRNA migration pattern, and

G2 specificity). The samples were chosen based on the year

of detection as well as on the current genotype; thus, we

covered a great diversity of samples.

Sequence and phylogenetic analysis of P[8] strains

The phylogenetic analysis of the P[8] strains clustered the

Paraguayan strains in three distinct evolutionary lineages

(i.e., P[8]-1, P[8]-2, and P[8]-3), from a total of four dif-

ferent lineages described to date for this genotype (Fig. 1).

The main cluster was represented by the P[8]-3 lineage.

The nucleotide sequence identity between the Paraguayan

strains of this lineage was [97% (data not shown). The

Paraguayan strains typed as G1 (1998–1999), G4 (1998–

2000, 2002, 2005) and G9 (2000, 2002, 2004, 2005) were

grouped in this cluster. Only one Paraguayan strain was

1068 Arch Virol (2008) 153:1067–1073

123

identified as belonging to the P[8]-2 lineage. This strain,

which circulated in 1998, was typed as G1. Most Para-

guayan G1 strains were clustered in the P[8]-1 lineage. The

nucleotide sequence identity between the Paraguayan

strains of this lineage was [96% (data not shown). All of

the P[8]-1 Paraguayan strains circulated for a short period

of time, between 2002 and 2003. None of the Paraguayan

strains were grouped in lineage P[8]-4, which was com-

posed of the available G4 strains from Malawi [12]. Of

note are the P (isolated in USA in 1974) [39], and Ai-39

strains (isolated in Japan in 1983) [11], which were posi-

tioned in a separate branch between the cluster of lineages

P[8]-2/P[8]-3 and the cluster of lineage P[8]-4 in the

phylogenetic reconstruction (Fig. 1).

The alignment of the deduced amino acid sequences of

the VP8* Paraguayan strains with a set of strains circu-

lating worldwide showed that only the position 195 was

different between the four lineages. Lineage P[8]-1 pre-

sents an asparagine residue, lineage P[8]-2 an aspartic acid,

lineage P[8]-3 a glycine, and lineage P[8]-4 a serine

(Fig. 2).

Sequence and phylogenetic analysis of P[4] strains

The phylogenetic analysis of the P[4] strains isolated

worldwide revealed five lineages (i.e. P[4]-1 to P[4]-5;

Fig. 3). Lineage P[4]-1 grouped two G2 strains, i.e. DS-1

and RV-5, isolated in the late 1970s in the United States

and Australia, respectively. Lineage P[4]-2 grouped two

G8 strains isolated in Brazil (R291) and Malawi (MW333).

Lineage P[4]-3 corresponded to a G12 strain isolated in the

Philippines (L26). Lineage P[4]-4 grouped two Brazilian

G2 strains and two Italian G2 strains. And finally, lineage

P[4]-5 grouped strains isolated worldwide from the early

1990s until the present.

The lineages described above were supported by

nucleotide distances (Table 1). Thus, the mean nucleotide

distances within lineages ranged from 0.007 to 0.047, while

the mean nucleotide distances between the lineages ranged

from 0.051 to 0.077. Only one exception was found in the

range presented between lineages, i.e. 0.031, which was

present between strains from lineages P[4]-3 and P[4]-4.

Fig. 1 Neighbor-joining tree obtained from 84 nucleotide sequences

of the P[8] genotype isolated worldwide (including the 34 Paraguayan

strains reported in this study). Regions under comparison are the

VP8*, and the initial VP5* fragment. Bootstrap values above 65% are

shown at branch nodes. Paraguayan strains are indicated by a filledsquare. Each strain is denoted by an abbreviation of the country, G

genotype, and year of isolation (where available).The abbreviations

are as follow: Aus Australia, Bgd Bangladesh, Bel Belgium, BraBrazil, Chn China, Dnk Denmark, Ind India, Ita Italy, Jpn Japan, MwiMalawi, Py Paraguay, Sau Saudi Arabia, Svn Slovenia, Twn Taiwan,

and Usa United States

c Py05AP85-G9-2005

Py05AP90-G9-2005

Py05AP97-G9-2005

BraRJ9473/04-G9-2004

Py04SR644-G9-2004

Py04SR525-G9-2004

Py04SR631-G9-2004

PY04SR521-G9-2004

BraRJ8207/04-G9-2004

Twn01TW1640-G9-2001

ItaBIA47-G1-2006

Twn01TW1291-G1-2001

Py00477-G9-2000

BelB4633-03-G12-2003

SauMD844-G12

Py00469-G9-2000

Py02SR47-G9-2002

Py02SR56-G9-2002

AusOBPerth-1-G9-2004

Svn761/06-G9-2006

Twn99TW1663-G1-1999

BraRJ7144/03-G9-2003

Jpn6226-2004/05

Py99365-G1-1999

Py99371-G1-1999

MwiOP351-G1-1998

DnkDK.V97-8405-G9-1997

Py02SR86-G4-2002

Py05AP99-G4-2005

HunBP641/01-G9-2001

Py00465-G9-2000

Py00466-G9-2000

BraRJ6906/03-G9-2003

Hun9-G9-1998

IndSc134-G4

BgdDhaka25-02-G12-2002

BraRJ5414/02-G9-2002

ItaCOS4-G9-2005

SvnMB7-G4-2006

Py9855-G1-1998

Py983-G4-1998

Py98102-G4-1998

Py99419-G1-1999

Py00464-G4-2000

Py99355-G4-1999

Py99449-G4-1999

P[8]-3

JpnKU-G1-1978

Bra1054-G5-1986

Py9856-G1-1998

JpnF45-G9-1986

BraH8-G5-1994

BraL5-G1-1996

BraIAL28-G5-1992

BraRJ25-G5-1996

BraRJ4883/01-G9-2001

BraRJ28-G5-1996

P[8]-2

JpnAi-39-G3-1983

UsaP-G3-1974

MwiOP530-G4-1999

MwiOP354-G4-1998

MwiMW670-G4-1999

P[8]-4

JpnITO-G3-1981

ItaVA70-G4-1975

UsaWa-G1-1974

JpnHochi-G4-1980

JpnOdelia-G4-1984

Py03SR124-G1-2003

HunBP1829/01-G9-2001

DnkDK.V00-2138-G9-2000

BraL8-G1-1996

ItaGHE44-G1-2006

Svn201/06-G1-2006

Py03SR234-G1-2003

Py03SR316-G1-2003

Py03IPN130-G1-2003

Jpn6690-2004/05

Py03SR170-G1-2003

Twn02TW498-G9-2002

Py02SR43-G1-2002

Py03SR139-G1-2003

Py02SR53-G1-2002

Py03SR305-G1-2003

HunBP785/00-G9-2000

P[8]-1

Gottfried-Porcine-G4P6

68

99

8267

83

92

85

82

96

80

74

84

77

69

93

80

70

67

97

75

72

65

0.05

Arch Virol (2008) 153:1067–1073 1069

123

Despite this exception, lineage P[4]-3 was maintained as an

independent lineage based on the phylogenetic analyzes

carried out using the neighbor-joining and parsimony

algorithms (data not shown). It is worth mentioning that

none of the amino acids were consistently recognized as

conserved within the lineages described for genotype P[4].

The seven P[4] Paraguayan strains sequenced clustered

within lineage P[4]-5 (Fig. 3). Nevertheless, they were

separated into two groups, i.e. (1) those strains isolated

from children (years 1999 and 2005), which were clustered

with strains from Italy [6], Japan [25], Taiwan [29], and

Vietnam; and (2) the ones isolated from adults in 2004 and

2005, which were clustered with strains from India.

Discussion

The study of the diversity of the strains circulating in a given

area and the analysis of the forces that drive the evolution of

rotaviruses (such as point mutations, rearrangements, reas-

sortments, recombinations, and interspecies transmission)

are gaining importance in the design and implementation of

rotaviral vaccines [18], mainly because these mechanisms

could produce natural vaccine-escape mutants [37].

In agreement with these mechanisms of rotavirus evo-

lution, different studies have reported the appearance of

different lineages within the P[8] genotype throughout the

world, mainly by the positive accumulation of point

mutations in the hypervariable region of the VP4 gene (i.e.

VP8*) [5]. Notably, these lineages are maintained over

time and throughout different geographical settings.

In this study, lineage P[8]-3 grouped most strains

circulating worldwide (Fig. 1). Thus, numerous P[8]

nucleotide sequences reported to date (including strains

reported in this study) are composed of strains of this

lineage [3, 4, 7]. This fact indicates that this lineage is

gaining importance as an emerging pathogen. Regardless

of the year of detection, all of the G4 and G9 Paraguayan

strains were related to lineage P[8]-3. This association has

been reported previously for both G4 and G9 strains [5, 8,

27]. Moreover, it is interesting to note that, except for

possible reassortant strains, all of the G9 strains circulating

worldwide were clustered in this lineage. This fact rein-

forces the existence of constraints within a population of

rotavirus strains bearing the combination of G9 and P[8]

specificities [8, 27].

The Paraguayan G1 strains from lineage P[8]-1 circu-

lated between 2002 and 2003, while the G1 strains from

lineage P[8]-3 circulated between 1998 and 1999 (Fig. 1).

An interesting finding related to the Paraguayan G1 strains

from this lineage was that most of them failed to be P[8]-

typed by using the primer 1T-1 [17]. It is worth mentioning

that the G1P[8]-1 strains predominated during 2003, and in

fact, represented a breakpoint in the continuous circulation

UsaWa-G1-1974 TPRATTDSSSTANLNNISITIHSEFYIIPRSItaVA70-G4-1975 ...............................JpnHochi-G4-1980 ...............................JpnOdelia-G4-1984 ...............................Py03SR124-G1-2003 .........N.T...................Py03SR234-G1-2003 ...............................Py03SR316-G1-2003 ...............................Py03IPN130-G1-2003 ...............................Py03SR170-G1-2003 ............................... JpnKU-G1-1978 .........N.....D...I...........Py9856-G1-1998 .........N.....D...V...........BraRJ4883/01-G9-2001 .........N.T...D...I........... Py983-G4-1998 .........N....DG...............Py9855-G1-1998 .........N....DG...............Py99355-G4-1999 .........N....DG...............Py99365-G1-1999 .........N.....G...............Py00464-G4-2000 .........N....DG...............Py00465-G9-2000 .........N.....G...............Py02SR47-G9-2002 .........N.....G...............Py02SR56-G9-2002 .........N.....G...............PY04SR521-G9-2004 .........N.....G...............Py04SR525-G9-2004 .........N.....G...............Py05AP85-G9-2005 .........N.....G...............Py05AP90-G9-2005 .........N.....G............... MwiOP530-G4-1999 ..G.V..G.N.SD.TS...I...........MwiOP354-G4-1998 ..G.V..G.N.SD.TS...I...........MwiMW670-G4-1999 ..G.V..G.N.SD.TS...I...........

P[8]-1

P[8]-2

P[8]-3

P[8]-4

180 190 200 210 |....|....|....|....|....|....|

Fig. 2 Alignment of deduced

amino acid sequences of

selected P[8] strains

corresponding to the four

described lineages. Secondary

structures corresponding to the

b-strands are indicated by

arrows, and coils are indicated

by lines, as previously described

[9]. Position 195 is denoted by a

gray column. Amino acids are

denoted by a one-letter

abbreviation, where asparagine

is N, aspartic acid is D, glycine

is G, and serine is S. Dotssymbolize the identity of amino

acids with the reference strain

Wa

1070 Arch Virol (2008) 153:1067–1073

123

Twn00TW1959-G2-2000

Twn02TW569-G9-2002

Twn97TW967-G2-1997

VnmVN594-2003

VnmVN322-2003

Twn98TW762-G2-1998

JpnKO-2-G2-2000

Py05SR1297-G2-2005

Py05SR1124-G2-2005

Py05SR1134-G2-2005

Py99406-G2-1999

Ita3-G2-2004

Ita1-G2-2003

Py05AP98-G2-2005

ThaCU127P4

ThaCU81P4

IndSC185

IndNR1

BgdDhaka4-03-G2-2003

IndIS-2-G2

IndRMC61

VnmVN271-2003

VnmVN580-2003

ThaCU100P4

Ind107E1B

IndRMC/G66

Py04ASR42-G2-2004

Py05ASR60-G2-2005

ChnCHW17-G3-1992

ItaH41-G2-1993

ChnTB-Chen-G2

P[4]-5

UsaDS-1-G2-1976

AusRV-5-G2P[4]-1

BraHFF10

BraRJ5619/02-G2-2002

BraRJ5323/02-G2-2002

ItaI200-G2-1997

ItaH93-G2-1996

PhlL26-G12-1987/88 P[4]-3

BraR291-G8

MwiMW333-G8-1997/98P[4]-2

Gottfried-Porcine-G4P6

98

73

86

76

93

100

77

100

85

93

90

85

88

88

88

0.05

P[4]-4

Fig. 3 Neighbor-joining tree

obtained from forty-two

nucleotide sequences of the P[4]

genotype isolated worldwide

(including the seven Paraguayan

strains reported in this study).

Regions under comparison are

the VP8*, and the initial VP5*

fragment. Bootstrap values

above 65% are shown at branch

nodes. Paraguayan strains

isolated from children are

indicated by a filled square, and

those isolated from adults by a

filled circle. Each strain is

denoted by an abbreviation of

the country, G genotype, and

year of isolation (where

available).The abbreviations are

as follows: Aus Australia, BgdBangladesh, Bra Brazil, ChnChina, Ind India, Ita Italy, JpnJapan, Mwi Malawi, PhlPhilippines, Py Paraguay, TwnTaiwan, Tha Thailand, UsaUnited States, and Vnm Vietnam

Arch Virol (2008) 153:1067–1073 1071

123

of Paraguayan P[8]-3 strains from 1998 to 2005 (i.e. either

G1, G4 or G9 strains). Furthermore, it is interesting that the

VP7 of the G1P[8]-1 strains belongs to an atypical sub-

lineage, with amino acid substitutions also presented in G1

animal strains [34]. Therefore, taken together, these data

could suggest that a new strain (i.e. G1P[8]-1) of rotavirus

was introduced in the country and exposed to an immu-

nologically naive population.

An important aspect related to the P[8] genotype is the

possibility of co-circulation of different lineages at the

same time. This study shows the co-circulation of two

lineages (P[8]-2 and P[8]-3 in 1998) (Fig. 1), similar to that

observed in other countries such as Brazil in 2001 [4], or

Italy, with co-circulation of P[8]-1 and P[8]-3 [3, 7]. Co-

circulation of three lineages, involving P[8]-1, P[8]-2, and

P[8]-3 [26, 27] or P[8]-2, P[8]-3, and P[8]-4 [12] has also

been reported. So far, there are no reports showing co-

circulation of the four lineages or co-circulation for more

than three years, which suggests that the appearance and

disappearance of different lineages of P[8] strains is a

mechanism that could be used by rotaviruses to escape

from the herd immunity acquired by the populations due to

previous natural infections. It is worth mentioning that the

years when G4P[8]-3 strains predominated in Paraguay

(1998, 1999, 2002) were different from the ones when

G9P[8]-3 strains predominated (2000, 2004, 2005) [34],

reinforcing the notion of fluctuation of rotavirus strains in

order to infect naive populations.

In accordance with the accumulation of point mutations,

this study shows that amino acid position 195 is the only

site that changes systematically among the four described

lineages of the P[8] genotype. In fact, this position forms

part of a coil secondary structure, situated between the

b-strands of the VP8* core (formed by a b-sandwich fold)

(Fig. 2) [14, 31]. The variability observed in the coil sec-

ondary structure could be explained by the presence of low

structural constraints that allow the free interchange of

amino acids, contrary to the situation in a-helices and

b-strands.

Regarding the P[4] genotype, the study of the evolution

of this genotype could be affected by the limited number of

available sequences in public databases. Nevertheless,

according to the present data, five genetic lineages were

identified for this genotype (Fig. 3), which was supported

by nucleotide distance within lineages (Table 1). Three out

of the five lineages grouped G2 strains isolated in different

geographical regions, while the remaining two lineages

were associated with strains bearing different G genotypes,

i.e. lineage P[4]-2 grouped G8 strains and lineage P[4]-3

grouped a G12 strain, thus suggesting an independent

evolution of the P[4] strains.

Even though all of the Paraguayan strains clustered in

lineage P[4]-5, it is worth pointing out that the P[4] strains

isolated from children clustered separately from the P[4]

strains isolated from adults (Fig. 3). Due to the low number

of strains analyzed here, further studies should be under-

taken in order to find out whether differences in the VP4

protein are needed to infect different populations or age

groups.

The dispersion of rotavirus strains into different evolu-

tionary lineages is a fact that should not be underestimated.

As a result, a continuous surveillance of rotavirus strains is

essential in order to predict the possible variants that will

circulate in a country, and ultimately to improve current

vaccination programs.

Acknowledgments We wish to thank Dr. Graciela Russomando for

her constant support during the study, and Marıa E. Galeano, for

technical assistance.

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Table 1 P-distances between (lower-left) and within (bold) the pro-

posed lineages of the P[4] genotype

P[4]-1 P[4]-2 P[4]-3 P[4]-4 P[4]-5

P[4]-1 0.047

P[4]-2 0.077 0.019

P[4]-3 0.058 0.053 n/ca

P[4]-4 0.052 0.055 0.031 0.007

P[4]-5 0.07 0.074 0.051 0.053 0.026

a Not computable

1072 Arch Virol (2008) 153:1067–1073

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