Molecular analysis of VP4, VP7, and NSP4 genes of P[6]G2 rotavirus genotype strains recovered from neonates admitted to hospital in Belém, Brazil

Journal of Medical Virology 78:281–289 (2006)

Molecular Analysis of VP4, VP7, and NSP4 Genes ofP[6]G2 Rotavirus Genotype Strains Recovered FromNeonates Admitted to Hospital in Belem, Brazil

Joana D’Arc P. Mascarenhas,1* Alexandre C. Linhares,1 Amanda Patrıcia G. Bayma,1

Jackson C. Lima,1 Maisa S. Sousa,1 Irene T. Araujo,2 Marcos B. Heinemann,2

Rosa Helena P. Gusmao,3 Yvone B. Gabbay,1 and Jose Paulo G. Leite2

1Virology Section, Instituto Evandro Chagas, Secretaria de Vigilancia em Saude, Ministerio da Saude,Ananindeua, Brazil2Department of Virology, Instituto Oswaldo Cruz, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil3University of Para State, Belem, Brazil

This investigation describes the molecular char-acterization of P[6]G2 rotavirus strains fromhospitalized neonates with community-acquireddiarrhea (CAD), nosocomial diarrhea (ND),and asymptomatic nosocomial infection (ANI) inBelem, Brazil. Twenty-six rotavirus strainswith P[6]G2 genotype were sequenced to genescoding for VP4, VP7, and NSP4 proteins. Phylo-genetic analysis of the VP4 gene, includingprototype strains RV3, ST3, M37, and U1205,showed that local P[6]G2 strains clustered form-ing a distinct lineage (bootstrap of 99%). BrazilianP[6]G2 strains had the highest homology (ran-ging from 96.0%–98.3%) with the Africanstrain GR1107, G4P[6]. Phylogenetic tree for VP7gene was constructed including old and newG2 African strains SA3958GR/97, SA356PT/96,SA514GR/87, SA4476PT/97, BF3676/99, GH1803/99, and representative strains of G1, G3, G4, G5,G8, and G9 genotypes. The Brazilian P[6]G2samples fell into a distinct group (bootstrap valueof 97%) and showed homology rates rangingfrom 92.1% to 93.5% with P[6]G2 Africanstrains BF3676/99, GH1803/99, and SA3958GR/97. Nucleotide sequence analysis of the NSP4gene, including human prototype strains S2,KUN, DS-1, RV5, RV3 and ST3, and animalprototype OSU, showed that all neonatal isolatesfell into genotypeA andclusteredwith a bootstrapvalue of 100%, with in-group similarities rangingfrom 99.3% to 100%. In this study no significantdifferences in nucleotide sequences of the VP4,VP7, and NSP4 genes could be observed whencomparing diarrheic (CAD and ND) and non-diarrheic (ANI) babies. Monitoring of rotavirusstrains in hospital environments is of particularimportance, since it is claimed currently that anefficacious rotavirus vaccine, when available forroutine use, will determine an impact on hospital-

acquired rotavirus disease. J. Med. Virol.78:281–289, 2006. � 2005 Wiley-Liss, Inc.

KEY WORDS: unusual P[6]G2 genotype; neo-natal rotavirus strains; VP4gene; VP7 gene; NSP4 gene

INTRODUCTION

Group A rotaviruses are the most common cause ofacute viral diarrhea in humans and animals throughoutthe world [Kapikian et al., 2001]. Rotaviruses are amember of the Reoviridae family. Complete virusparticles have a triple-layered protein capsid surround-ing a genome consisting of 11 segments of double-stranded RNA (dsRNA), which encode six structural(VP1-VP4,VP6, andVP7) and sixnon-structural (NSP1-NSP6) proteins [Kapikian et al., 2001]. Group A rota-viruses have been classified into three genogroups basedon hybridization with probes prepared from Wa, DS-1,and AU-1 rotavirus strains. Members of the samegenogroup share a high degree of genetic relatednessbut significantly less homology with members of othergenogroups [Nakagomi and Nakagomi, 1993].

Serotype designations are currently based on inde-pendent neutralization determinants on the outercapsid proteins VP7 (G serotype) and VP4 (P serotype)[Estes, 2001].On the basis of theneutralizing antibodies

Grant sponsor: COTEC/SECTAM/PA; Grant sponsor: IEC/SVS;Grant sponsor: FIOCRUZ; Grant sponsor: CNPq.

*Correspondence to: Joana D’Arc P. Mascarenhas, VirologySection, Instituto Evandro Chagas, Secretaria de Vigilancia emSaude, Rodovia BR 316–KM 07, S/N, Levilandia, 67.030-000,Ananindeua, Para, Brasil.E-mail: [email protected]

Accepted 10 October 2005

DOI 10.1002/jmv.20537

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2005 WILEY-LISS, INC.

and the oligonucleotide sequence, at least 15Gserotypesand 25 P genotypes, have been identified for group Arotaviruses [Estes, 2001; Kapikian et al., 2001; Rahmanet al., 2005].Studies conducted in hospital nurseries around the

world have suggested that a single rotavirus strain canpersist over long periods in neonatal wards [Rodgeret al., 1981; Perez-Schael et al., 1984; Steele et al., 1992;Pager et al., 2000]. Furthermore, the apparent stabilityof rotavirus neonatal strains seems to differ from that ofrotaviruses circulating in the community; the latterstrains usually showing a higher genomic variability[Rodger et al., 1981; Steele et al., 1992].Rotavirus strains infecting neonates were originally

associated with an avirulent P[6] genotype recoveredfrom asymptomatic subjects excreting rotavirus strainsbearing G1, G2, G3, and G4-type specificities [Hoshinoet al., 1985; Flores et al., 1986; Gorziglia et al., 1988].However, Steele et al. [1995], Pager et al. [2000], andCunliffe et al. [2002] have reported the detection of P[6]genotype associated with G4 and G8 rotavirus strainscausing symptomatic and asymptomatic infections inAfrican neonates.This same P[6] genotype was detected in children

suffering from acute diarrhea associated with serotypesG1,G2, G3,G8, andG9 all over Africa [Adah et al., 2001;Salu et al., 2003; Steele and Ivanoff, 2003; Page andSteele, 2004a,b].The non-structural protein NSP4, encoded by gene

segment 10, is a glycoprotein anchoring in the mem-brane of the endoplasmic reticulum [Estes, 2001]. Thisprotein has been studied extensively due to its role inviral morphogenesis and its potential enterotoxigenicproperty [Tian et al., 1995; Ball et al., 1996; Estes, 2001].TheNSP4 gene has been classified into three groups (A,B, andC). GroupAhas been genetically classified into atleast five genotypes comprising the following strains: A(KUN), B (Wa), C (AU-1), D (EW), and E (avian-like)[Ciarlet et al., 2000; Ito et al., 2001; Mori et al., 2002].NSP4, particularly a 22-amino acid (aa) syntheticpeptide corresponding to residues 114–135, has beenshown to trigger a signal transduction pathway thatincreases the intracellular calcium levels inducingdiarrhea in young mice [Tian et al., 1995; Ball et al.,1996]. The ‘‘toxigenic’’ region seems to be extended fromaa 114 to 140, as reported with both virulent andattenuated OSU strains [Zhang et al., 1998].Studies conducted in Belem, Brazil reported the

first nosocomial transmission of P[6]G2 rotavirusstrains among symptomatic and asymptomatic neo-nates [Linhares et al., 2002]. In addition, P[6] genotypespecificitywas found in associationwithG1,G2,G4, andG5 genotypes among children with diarrhea with agesranging from 9 to 24 months who participated in a trialwith the Rhesus-human reassortant tetravalent rota-virus vaccine (RRV-TV) during 1990–1992 in Belem,Brazil [Mascarenhas et al., 2002].The aim of the present study was to determine

the genetic diversity among P[6]G2 rotavirus strainsrecovered fromBrazilian neonates with either asympto-

matic or symptomatic rotavirus infection, based onphylogenetic analysis of sequence of genes coding forVP4, VP7, and NSP4 proteins.

MATERIALS AND METHODS

Patients and Specimens

Surveillance for rotavirus infection was conductedfromMay 1996 toMay 1998 in neonatal care unit wardsat a public hospital in Belem, Brazil. A total of 614 stoolsamples were obtained from 437 premature/sick new-born babies (NB) with ages from 1 to 28 days whodeveloped community-acquired diarrhea (CAD), noso-comial diarrhea (ND), and asymptomatic nosocomialinfection (ANI). All samples were screened for thepresence ofGroupA rotavirus antigenusingmonoclonalantibodies by a commercially available enzyme-linkedimmnosorbent assay (ELISA) kit (Dakopatts, Denmark)according to the manufacturer’s instructions. Overall,51 (11.7%) hospitalized neonates were excreting rota-virus, 42 (82.3%) had ANI, 5 had ND, and 4 babies werefound to haveCAD, as reported byLinhares et al. [2002].Fifty (98%) and 1 (2%) rotavirus strains displayed shortand long electropherotypes, respectively. In this study26 rotavirus-positive samples having P[6]G2 specificitywere recovered from ANI (n¼18), ND (n¼5), and CAD(n¼3) patients, and were sequenced including thosedisplaying short genomic profile and being available insufficient amount.

RT-PCR Amplification

Rotavirus dsRNA was extracted from fecal suspen-sions using silica powder glass extraction as describedpreviously by Boom et al. [1990]. The purified viraldsRNA was denatured at 988C for 7 min and then usedas a template for the reverse transcription PCR (RT-PCR). The reverse transcription of dsRNA was carriedout with SuperScriptTM (Invitrogen, Carlsbad, CA ) andPCR amplification was performed with Taq DNA poly-merase recombinant (Invitrogen).The full-length738bpgene, encoding the NSP4 protein, was amplified usingprimers and PCR methods described by Cunliffe et al.[1997]. The Nested-PCR primers and conditions for theamplification of a 244 bp fragment for VP7 gene, and a267 bp fragment forVP4 genewere previously describedby Linhares et al. [2002]. All PCR products (NSP4, VP4,and VP7) were purified by using QIAquick1 PCR puri-fication kit (Qiagen, Hilden, Germany) or QIAquick1

Gel Extraction kit (Qiagen).

DNA Sequencing and Phylogenetic Analysis

The purified PCR products from asymptomatic andsymptomatic neonateswereautomatically sequencedbyusing the con3 forward and P6 reverse primers (VP4gene) described by Gentsch et al. [1992], 9con1 forward,andG2reverse primers (VP7gene) reported byDas et al.[1994], and jrg30 forward and jrg31 reverse primers forNSP4 gene [Cunliffe et al., 1997]. Sequencing was

J. Med. Virol. DOI 10.1002/jmv

282 Mascarenhas et al.

carried out by the dideoxynucleotide chain terminatormethod on a ABI Prism 3100 automatic sequencer(Applied Biosystems, Foster City, CA), using the ABIPrism Big Dye Terminator cycle sequencing ReadyReaction Kit (Applied Biosystems). The products werefurther purified by ethanol precipitation and resus-pended in formamide. The sequences obtained for theVP4, VP7, and NSP4 genes were aligned and editedusing a BioEdit Sequence Alignment Editor (version6.0). Phylogenetic analysis was performed usingMEGApackage (versions 2.0 and 3.0). Distances betweensequences were analyzed using the neighbor-joiningalgorithmbasedon theKimura twoparametersdistanceestimative method for nucleotide [Kimura, 1980]. Thesecondmethodwasmaximumparsimonywith heuristicor branch-and-bound search. Bootstrap resampling(over 1,000 replicates) was performed for neighbor-joining analysis of sequences obtained of genes VP4,VP7, and NSP4.

Prototype strains for VP4, VP7, and NSP4genes analysis were obtained from GenBank at theNational Center for Biotechnology Information,USA (www.ncbi.nlm.nih.gov), by conducting Blast andnucleotide search.

Nucleotide Sequence Accession Numbers

The nucleotide sequence data reported in thispaper were submitted to GenBank with accessionnumbers (VP4 gene) DQ070447–DQ070472; (VP7 gene)DQ070421–DQ070446; and (NSP4 gene) DQ070395–DQ070420.

RESULTS

Characterization of Rotavirus Strainsby PAGE

PAGE analysis of strains with P[6]G2 specificity fromneonates with CAD and ND has evidenced a strongstrain identity, since all rotavirus strains displayed thesame electropherotype (Fig. 1).

Amplification of the VP4, VP7, andNSP4 Genes

Twenty six amplicons were obtained based on theamplification of the NSP4 protein [Cunliffe et al., 1997]and VP7 and VP4 genes, as described previously byLinhares et al. [2002].

Sequence Analysis of VP4, VP7, andNSP4 Genes

Twenty six rotavirus strains with P[6]G2 genotype-specificity were sequenced and analyzed to assessgenetic diversity among neonates with CAD (3 strains),ND (5 strains), and ANI (18 strains). Table I showsresults concerning the 26 P[6]G2 rotavirus strains,when comparing the nucleotide homologies of the threegroups studied (ANI, CAD, and ND), according to ageand gender of neonates. The homologies ranged from

99.9% to 100% (VP4 gene), 99.2%–99.4% (VP7 gene),and 99.8%–99.9% (NSP4 gene).

Phylogenetic analysis showed that the local P[6]strains clustered forming a distinct lineage (bootstrapof 99%), including prototype strains RV3, ST3,M37, andU1205 (Fig. 2a). When only local strains were analyzed,they clustered into a lineage with a 92% bootstrap,which has been classified as lineage 2A by Banyai et al.[2004]. Collectively, the Brazilian P[6]G2 strainsdemonstrated the highest homology (ranging from96.0%–98.3%) with the African strain GR1107, typedas P[6]G4. The porcine strain Gottfried (lineage 2B),the human strains BP1338 (lineage IV) and BP1227(lineage V), and the outgroup strain AU-1 (lineage P3)were included in this analysis showing nucleotidedivergence rates of 22.6%, 14.5%, 17.4%, and 53.2%,respectively. The divergence observed between Brazi-lian strains (lineage 2A) and GR1107, included in thisstudy, was 3.4%. Compared to Brazilian P[6] fromneonates, the samples BP1227 and Gottfried, lineagesV and 2B, respectively, diverged 18.2%.

Phylogenetic tree for VP7 gene (Fig. 2b) was con-structed by analyzing 26 G2 strains from Belemneonates, six G2 African strains described by Page andSteele [2004b], the prototype G2 strain, DS-1, andrepresentative strains of G1, G3, G4, G5, G8, and G9genotypes. The Brazilian P[6]G2 samples fell into adistinct group (bootstrap value of 97%). The P[6]G2African strainsBF3676/99,GH1803/99, andSA3958GR/97 showed homology rates ranging from 92.1% to 93.5%,when compared to the P[6]G2 strains from Belem,Brazil. The divergences between P[6]G2 from Belemand P[6]G2 and P[4]G2African strainswere of 6.3% and8.3%, respectively.

Comparison of nucleotide sequences of the NSP4genes from 26 P[6]G2 neonatal rotavirus strainswere made including human prototype strains S2,KUN, DS-1, RV5, RV3, and ST3, and animal prototypeOSU (Fig. 2c). All rotavirus neonatal samplesfrom Belem clustered with bootstrap value of 100%,showing in-group similarities ranging from 99.3% to100%. These isolates fell into a major group (bootstrapof 100%) designated genotype A by Lee et al. [2000],which contains human prototype strains S2, KUN, DS-1, and RV5. The diversities between genotypes A and B,B and C, and A and C were of 21.0%, 22.4%, and 25.1%,respectively. The mean genetic diversity in genotypes Aand B were of 1.7% and 5.8%, respectively. Theprototype AU-1 represents the NSP4 genotype C andwas included in this tree as an out group.

DISCUSSION

Several reports have documented the associationof neonatal P[6] genotype with different G serotypessuch as G8, G9, G4, and G1 in Guinea-Bissau, dur-ing 1996 and 1997 [Fischer et al., 2000], in Malawi,during 1997 and 1998 [Cunliffe et al., 1999], in Africa,during 1996 and 1999 [Pager et al., 2000; Steele andIvanoff, 2003], and in the United States, during 1996 to


Analysis of VP4, VP7, and NSP4 Genes from Neonatal Rotavirus Strains 283

1997 [Ramachandran et al., 1998]. In Brazil, P[6]rotavirus strains bearing G1, G2, G3, or G4 type-specificities have been reported to be a cause of acutegastroenteritis among infants and young children inSaoPaulo, Rio de Janeiro, and Belem [Timenetsky et al.,

1994; Leite et al., 1996; Mascarenhas et al., 2002]. InNigeria, the G2 serotype with P[6] genotype specificitywas isolated in a pediatric outpatient unit from childrenwith diarrhea under 7 years of age between November1999 and April 2000 [Adah et al., 2001]. Another studyconducted in Nigeria, among children less than 5 yearsof age with acute diarrhea, between January andDecember 2001, has also shown rotaviruses with thesame unusual P[6]G2 genotype [Salu et al., 2003].During 1997, P[6]G2 strains circulated among childrenwith diarrhea in Ga-Rankuwa, South Africa [Page andSteele, 2004a].

The Brazilian P[6]G2 strains are similar to thosereported by Adah et al. [2001], Salu et al. [2003],and Page and Steele, [2004a], who conducted studiesinvolving children with acute diarrhea in Nigeria,Ghana, Burkino Faso, and South Africa, respectively.In these studies, P[6]G2 strains typically sharedsubgroup I, VP7 serotype G2, and short RNA electro-pherotype.


TABLE I. Comparison Between Homologies of VP4, VP7, andNSP4 Genes from the Diarrheic (CAD and ND) and Non-Diarrheic (ANI) Neonates, According to Age and Gender in

Belem, Brazil

Genes CAD�ANI (%) CAD�ND (%) ND�ANI (%)

VP7 99.2 99.3 99.4VP4 99.9 100.0 99.9NSP4 99.9 99.8 99.9

Age and gender range in days.CAD, 16–28 days (75% female and 25% male); ANI, 8–22 days (61%female and 39%male); ND, 12–28 days (20% female and 80%male);þ,mean percentage homology was calculated by MEGA package(version 3.0).

Fig. 1. Polyacrylamide gel electrophoresis of rotavirus strains from diarrheic neonates (CADandND) inBelem,Brazil.Lane 1display SA11; lane 2 longRNApattern (control); lane 3 shortRNApattern (control);lanes 4 to 7, neonatal rotavirus strains.


Molecular studies have identified three lineages ofgenotype P[6] VP4 gene, representing serologicallydistinct subtypes designated P2A[6] (M37-like) andP2C[6] (AU19-like), for the human strains and P2B[6](Gottfried-like) for the porcine strain [Gunasena et al.,

1993; Nakagomi et al., 1999]. Banyai et al., [2004] havesuggested that the variability within P[6} speci-ficity is larger than previously appreciated and haveestablished two new phylogenetic lineages designatedIV (BP1198-like) and V (BP720-like), distinguishable


NB-171 ND (03/1997)*NB-332 ANI (11/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-301 ANI (10/1997)

NB-211 ANI (05/1997)

NB-148 ND (02/1997)

NB-189 ANI (03/1997)

NB-157 ND (02/1997)

NB-175 ANI (03/1997)

NB-140 CAD (01/1997)

NB-209 ANI (04/1997)

NB-142 CAD (02/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-166 ND (02/1997)

NB-275 ANI (08/1997)

NB-156 ND (02/1997)

NB-162 CAD (02/1997)

NB-174 ANI (03/1997)

NB-207 ANI (04/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

GR1107/Hu/P[6]G4

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

M37/Hu/P[6]G1

US1205/Hu/P[6]G9

BP1338/Hu/P[6]G4

BP1227/Hu/P[6]G4

Gottfried/Po/P[6]G4

L26/Hu/P[4]

DS-1/Hu/P[4]G2

BP1744-01/Hu/P[8]G9

Kagawa90-554/Hu/P[8]G4

1054/Hu/P[8]G5

K8/Hu/P[9]

AU1/Hu/P[9]G3100

98

96

80

98

99

79

91

99

92

0.02

Lineage 2A

Lineage 2B

Lineage P3

Lineage IV

Lineage V

NB- D 1997)

NB-332 ANI (11/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-301 ANI (10/1997)

NB-211 ANI (05/1997)

NB-148 ND (02/1997)

NB-189 ANI (03/1997)

NB-157 ND (02/1997)

NB-175 ANI (03/1997)

NB-140 CAD (01/1997)

NB-209 ANI (04/1997)

NB-142 CAD (02/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-166 ND (02/1997)

NB-275 ANI (08/1997)

NB-156 ND (02/1997)

NB-162 CAD (02/1997)

NB-174 ANI (03/1997)

NB-207 ANI (04/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

GR1107/Hu/P[6]G4

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

M37/Hu/P[6]G1

US1205/Hu/P[6]G9

BP1338/Hu/P[6]G4

BP1227/Hu/P[6]G4

Gottfried/Po/P[6]G4

L26/Hu/P[4]

DS-1/Hu/P[4]G2

BP1744-01/Hu/P[8]G9


1054/Hu/P[8]G5

K8/Hu/P[9]

AU1/Hu/P[9]G3

- 1 97)

NB-332 ANI (11/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-301 ANI (10/1997)

NB-211 ANI (05/1997)

NB-148 ND (02/1997)

NB-189 ANI (03/1997)

NB-157 ND (02/1997)

NB-175 ANI (03/1997)

NB-140 CAD (01/1997)

NB-209 ANI (04/1997)

NB-142 CAD (02/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-166 ND (02/1997)

NB-275 ANI (08/1997)

NB-156 ND (02/1997)

NB-162 CAD (02/1997)

NB-174 ANI (03/1997)

NB-207 ANI (04/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

GR1107/Hu/P[6]G4

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

M37/Hu/P[6]G1

US1205/Hu/P[6]G9

BP1338/Hu/P[6]G4

BP1227/Hu/P[6]G4

Gottfried/Po/P[6]G4

L26/Hu/P[4]

DS-1/Hu/P[4]G2

BP1744-01/Hu/P[8]G9


1054/Hu/P[8]G5

K8/Hu/P[9]

AU1/Hu/P[9]G3

- 1 97)

NB-332 ANI (11/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-301 ANI (10/1997)

NB-211 ANI (05/1997)

NB-148 ND (02/1997)

NB-189 ANI (03/1997)

NB-157 ND (02/1997)

NB-175 ANI (03/1997)

NB-140 CAD (01/1997)

NB-209 ANI (04/1997)

NB-142 CAD (02/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-166 ND (02/1997)

NB-275 ANI (08/1997)

NB-156 ND (02/1997)

NB-162 CAD (02/1997)

NB-174 ANI (03/1997)

NB-207 ANI (04/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

GR1107/Hu/P[6]G4

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

M37/Hu/P[6]G1

US1205/Hu/P[6]G9

BP1338/Hu/P[6]G4

BP1227/Hu/P[6]G4

Gottfried/Po/P[6]G4

L26/Hu/P[4]

DS-1/Hu/P[4]G2

BP1744-01/Hu/P[8]G9


1054/Hu/P[8]G5

K8/Hu/P[9]

AU1/Hu/P[9]G3

Lineage 2A

Lineage 2B

Lineage P3

Lineage IV

Lineage V

a

Fig. 2. Phylogenetic analysis based on the VP4, VP7, and NSP4nucleotide sequences of neonate rotavirus strains, human, and animalprototypes. Phylogenetic trees (a) VP4, (b) VP7, and (c) NSP4 wereconstructed based on the neighbor joining method within MEGApackage.Bootstrapvalues greater than75%are indicatedat thebranch

nodes and scale bar is proportional to the genetic distance. Outgroupsequences AU-1 and WI61 were included to better understand thephylogenetic relationships among strains. ANI, asymptomatic nosoco-mial infection; ND, nosocomial diarrhea; CAD, community-acquireddiarrhea; *, month/year of collection.


from M37-like, AU19-like, andGottfried-like strains. Inthis study, all strains which formed a distinct lineageinto a major group clustered with lineages M37-like, asstatistically supported by bootstrap of 99%.

Phylogeneticanalysisof theVP7geneofG2strains fromBelem revealed a separated branch clustered togetherwith ‘‘new’’ lineages IIb, IIc, and IId described byPageandSteele [2004b].Thestrains from ‘‘old’’ lineageI, included in


NB-189 ANI (03/1997)*NB-211 ANI (05/1997)

NB-242 ANI (07/1997)

NB-175 ANI (03/1997)

NB-209 ANI (04/1997)

NB-157 ND (02/1997)

NB-174 ANI (03/1997)

NB-301 ANI (10/1997)

NB-207 ANI (04/1997)

NB-275 ANI (08/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-198 ANI (04/1997)

NB-166 ND (02/1997)

NB-162 CAD (02/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-208 ANI (04/1997)

NB-171 ND (03/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

NB-140 CAD (01/1997)

NB-148 ND (02/1997)

NB-332 ANI (11/1997)

NB-156 ND (02/1997)

NB-142 CAD (01/1997)

SA3958GR-97/Hu/P[6]G2

SA356PT-96/Hu/P[4]P[6]G2

SA4476PT-97/Hu/P[4]P[6]G2

BF3676-99/Hu/P[6]G2

GH1803-99/Hu/P[6]G2

SA514GR-87/Hu/P[4]G2

DS-1/Hu/P[4]G2

WI-61/Hu/G9

97CM113/Hu/G9

R160/Hu/G9

5001DB-97/Hu/P[6]G9

H8/Hu/G5

R291/Hu/G8

MaCH09004/Hu/G3

T108/Hu/G3

GR846-86/Hu/P[6]G4

Arg-928/Hu/P[8]G4

TE56/Hu/P[6]G1

B74-02/Hu/P[8]G1

T73/Hu/G1

100

100

100

7699

81

89

99

98

96

97

100

97

0.02

Lineage IIb

Lineage IIc

Lineage IId

Lineage I

NB-189 ANI (03/1997)NB-211 ANI (05/1997)

NB-242 ANI (07/1997)

NB-175 ANI (03/1997)

NB-209 ANI (04/1997)

NB-157 ND (02/1997)

NB-174 ANI (03/1997)

NB-301 ANI (10/1997)

NB-207 ANI (04/1997)

NB-275 ANI (08/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-198 ANI (04/1997)

NB-166 ND (02/1997)

NB-162 CAD (02/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-208 ANI (04/1997)

NB-171 ND (03/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

NB-140 CAD (01/1997)

NB-148 ND (02/1997)

NB-332 ANI (11/1997)

NB-156 ND (02/1997)

NB-142 CAD (01/1997)

SA3958GR-97/Hu/P[6]G2

SA356PT-96/Hu/P[4]P[6]G2

SA4476PT-97/Hu/P[4]P[6]G2

BF3676-99/Hu/P[6]G2

GH1803-99/Hu/P[6]G2


DS-1/Hu/P[4]G2

WI-61/Hu/G9

97CM113/Hu/G9

R160/Hu/G9

5001DB-97/Hu/P[6]G9

H8/Hu/G5

R291/Hu/G8

MaCH09004/Hu/G3

T108/Hu/G3

GR846-86/Hu/P[6]G4

Arg-928/Hu/P[8]G4

TE56/Hu/P[6]G1

B74-02/Hu/P[8]G1

T73/Hu/G1

0.02

NB-189 ANI (03/1997)NB-211 ANI (05/1997)

NB-242 ANI (07/1997)

NB-175 ANI (03/1997)

NB-209 ANI (04/1997)

NB-157 ND (02/1997)

NB-174 ANI (03/1997)

NB-301 ANI (10/1997)

NB-207 ANI (04/1997)

NB-275 ANI (08/1997)

NB-308 ANI (10/1997)

NB-287 ANI (09/1997)

NB-198 ANI (04/1997)

NB-166 ND (02/1997)

NB-162 CAD (02/1997)

NB-334 ANI (11/1997)

NB-321 ANI (10/1997)

NB-208 ANI (04/1997)

NB-171 ND (03/1997)

NB-220 ANI (05/1997)

NB-233 ANI (06/1997)

NB-140 CAD (01/1997)

NB-148 ND (02/1997)

NB-332 ANI (11/1997)

NB-156 ND (02/1997)

NB-142 CAD (01/1997)

SA3958GR-97/Hu/P[6]G2

SA356PT-96/Hu/P[4]P[6]G2

SA4476PT-97/Hu/P[4]P[6]G2

BF3676-99/Hu/P[6]G2

GH1803-99/Hu/P[6]G2


DS-1/Hu/P[4]G2

WI-61/Hu/G9

97CM113/Hu/G9

R160/Hu/G9

5001DB-97/Hu/P[6]G9

H8/Hu/G5

R291/Hu/G8

MaCH09004/Hu/G3

T108/Hu/G3

GR846-86/Hu/P[6]G4

Arg-928/Hu/P[8]G4

TE56/Hu/P[6]G1

B74-02/Hu/P[8]G1

T73/Hu/G1

0.02

Lineage IIb

Lineage IIc

Lineage IId

Lineage I

b

Fig. 2. (Continued)


this analysis, were more divergent as compared toneonatal strains (Fig. 2b). The P[6]G2 strains fromBelemclustered with the Africa P[6]G2 strains included in thisanalysis, as supported by a bootstrap of 100%.

The human rotaviruses NSP4 genotype A has beenassociated usually with SGI specificity, whereas NSP4genotype B with SGII specificity [Cunliffe et al., 1997;Kirkwood and Palombo, 1997]. Lee et al. [2000] have


NB-157 ND (02/1997)*NB-166 ND (02/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-211 ANI (05/1997)

NB-140 CAD (01/1997)

NB-220 ANI (05/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-332 ANI (11/1997)

NB-174 ANI (03/1997)

NB-142 CAD (01/1997)

NB-175 ANI (03/1997)

NB-171 ND (03/1997)

NB-301 ANI (10/1997)

NB-207 ANI (04/1997)

NB-275 ANI (08/1997)

NB-189 ANI (03/1997)

NB-156 ND (02/1997)

NB-209 ANI (04/1997)

NB-233 ANI (06/1997)

NB-148 ND (02/1997)

NB-321 ANI (10/1997)

NB-308 ANI (10/1997)

NB-162 CAD (02/1997)

NB-334 ANI (11/1997)

S2/Hu/P[4]G2

KUN/Hu/P[4]G2

DS-1/Hu/P[4]G2

RV5/Hu/P[4]G2

TA3/Hu/P[4]G2

Genotype A

RMC321/Hu/P[19]G9

OSU/Po/P[7]G5

TA371/Hu/P[8]G3

TA373/Hu/P[8]G3

CH631/Hu/P[8]G1

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

CH61/Hu/P[6]G1

CH2/Hu/P[6]G1

TE56/Hu/P[6]G1

TA79/Hu/P[8]G4

TA77/Hu/P[8]G4

TA74/Hu/P[8]G4

Genotype B

Genotype CAU-1/Hu/P[9]G3

100

89

7995

84

99

79

100

98

9999

100

94

100

0.02

NB-157 ND (02/1997)NB-166 ND (02/1997)

NB-242 ANI (07/1997)

NB-198 ANI (04/1997)

NB-211 ANI (05/1997)

NB-140 CAD (01/1997)

NB-220 ANI (05/1997)

NB-287 ANI (09/1997)

NB-208 ANI (04/1997)

NB-332 ANI (11/1997)

NB-174 ANI (03/1997)

NB-142 CAD (01/1997)

NB-175 ANI (03/1997)

NB-171 ND (03/1997)

NB-301 ANI (10/1997)

NB-207 ANI (04/1997)

NB-275 ANI (08/1997)

NB-189 ANI (03/1997)

NB-156 ND (02/1997)

NB-209 ANI (04/1997)

NB-233 ANI (06/1997)

NB-148 ND (02/1997)

NB-321 ANI (10/1997)

NB-308 ANI (10/1997)

NB-162 CAD (02/1997)

NB-334 ANI (11/1997)

S2/Hu/P[4]G2

KUN/Hu/P[4]G2

DS-1/Hu/P[4]G2

RV5/Hu/P[4]G2

TA3/Hu/P[4]G2

Genotype A

RMC321/Hu/P[19]G9

OSU/Po/P[7]G5

TA371/Hu/P[8]G3

TA373/Hu/P[8]G3

CH631/Hu/P[8]G1

RV3/Hu/P[6]G3

ST3/Hu/P[6]G4

CH61/Hu/P[6]G1

CH2/Hu/P[6]G1

TE56/Hu/P[6]G1

TA79/Hu/P[8]G4

TA77/Hu/P[8]G4

TA74/Hu/P[8]G4

Genotype B

Genotype CAU-1/Hu/P[9]G3

0.02

c

Fig. 2. (Continued)


described G1P[6] rotavirus strains recovered fromneonates that clustered into genotypeB.NSP4 genotypeAwas largelypredominant amongneonates innurseriesin Belem, Brazil, being associated with P[6]G2 specifi-city. The fact that all local strains had a 100%homology,regardless of being recovered from CAD, ND, or ANIgroup suggests that NSP4 gene may not be the soledeterminant for rotavirus virulence.Several studies have been conducted worldwide

with asymptomatic and symptomatic rotavirus infec-tions in neonatal wards, and the relationship betweenneonates and attenuated rotavirus strains are high-lighted by these studies [Rodger et al., 1981; Perez-Schael et al., 1984; Steele et al., 1992; Steele and Sears,1996]. In the local study, when comparing strains fromneonates who developed ANI, CAD, and ND, higherhomologies can be seen between VP4, VP7, and NSP4genes.The rotavirus strains infecting hospitalized neonates

are in general different from community strains infect-ing children at home [Steele et al., 1995; Kilgore et al.,1996]. Unlike this assessment, Linhares et al. [2002]have suggested that P[6]G2 neonatal strains may havebeen introduced into the hospital’s NCU from theexternal community, as suggested by the identical shortelectrophoretic profiles yielded from PAGE. This wasconfirmed in this study since two cases of P[6]G2rotavirus-related CAD (NB-140 and NB-142) wereadmitted to the ward around 01/1997, and the first ND(NB-148) case occurred on 02/1997. Strains from threecases were identical in their nucleotide and amino acidsequences (data, not shown).It has been suggested that neonatal host factors

may account for the adaptation of a strain to theintestine, leading it to become less virulent [Hoshinoet al., 1985]. Furthermore, rotavirus antibodies ofmaternal origin were claimed to have a variable effectin protecting neonates against different strains ofrotavirus [Snodgrass et al., 1977; Snodgrass andWells, 1978; Sheridan et al., 1984; Offit and Clark,1985]. This seems evident, as reported by Linhares et al.[2002], since symptomatic and asymptomatic P[6]G2infection were noted among breast-fed neonates.Five of eight neonates who developed CAD or NDreceived maternal milk only, of whom three presentedwith mild/moderate symptoms and two with severesymptoms. It is noticeable that among the threeneonates who received breastmilk plus formula, onedevelopedmild/moderate illness and the remainder hadsevere symptoms.Overall, no significant differences in nucleotide

sequences of the VP4, VP7, and NSP4 genes could beobserved when comparing diarrheic (CAD and ND) andnon-diarrheic (ANI) babies.Monitoring of rotavirus strains in hospital environ-

ments is of particular importance, since it is currentlyclaimed that an efficacious rotavirus vaccine, whenavailable for large-scale use,will determine a significantimpact in hospital-acquired rotavirus disease [Fischeret al., 2004].

Accession Numbers

The VP4 gene sequences used were GR1107(AF161830), RV3 (U16299), ST3 (L33895), M37(L20887), US1205 (AF079356), BP1338 (AJ621507),BP1227 (AJ621505), Gottfried (M33516), L26(M58292), DS-1 (AJ540227), BP1744/01 (AJ605318),Kagawa90–544 (AB039939), 1054 (U41004), K8(D90260), and AU1 (D10970). The VP7 gene sequencesused were SA3958 (AY261344), SA356 (AY261342),SA4476 (AY261343), BF3676 (AY261355), GH1803(AY261353), SA514 (AY261338), DS-1 (AB118023),WI61 (AB180969), 97CM113 (AY866505), R160(AF274971), 5001DB/97 (AF529864), H8 (U4105),R291 (AY855064), MaCH09004 (AY900173), GR846(AF161822), Arg928 (AF373918), TE56 (AF183856),B74-02 (AY635048), and T73 (AF450291). The NSP4gene sequences used were S2 (U59104), KUN (D88829),DS-1 (AF174305), RV5 (U59103), TA3 (AF174298),RMC321 (AF541921), OSU (D88831), TA371(AF173208), TA373 (AF173210), CH631 (AF173201),RV3 (U42628), ST3 (U59110), CH61 (AF173185), CH2(AF173179), TE56 (AF173200), TA79 (AF173215), TA77(AF173214), T74 (AF173213), and AU-1 (D89873).

ACKNOWLEDGMENTS

This studywas supported in part by a grant fromParaState Secretary of Science and Technology (COTEC/SECTAM/PA). Amanda Patrıcia Gaudio Bayma andJackson Cordeiro Lima received a grant fellowship fromtheNationalCouncil for theDevelopment of Science andTechnology, Brasılia, Federal District, Brazil (CNPq).We gratefully acknowledge the valuable technicalsupport provided by Dr. Olinda Macedo, Dr. Darleisede Souza Oliveira, Dr. Luana Soares, and Euzeni Mariade Fatima Menezes.

REFERENCES

Adah MI, Wade A, Taniguchi K. 2001. Molecular epidemiology ofrotavirus in Nigeria: Detection of unusual strains with G2P[6] andG8P[1] specificities. J Clin Microbiol 39:3969–3975.

Ball JM, Tian P, Zeng CQ-Y, Morris AP, Estes MK. 1996. Age-dependent diarrhoea induced by a rotaviral nonstructural glyco-protein. Science 272:101–104.

Banyai K,Martella V, JakabF,MeleghBM, SzucsG. 2004. Sequencingandphylogenetic analysis of humangenotypeP[6] rotavirus strainsdetected in Hungary provides evidence for genetic heterogeneitywithin the P[6] VP4 gene. J Clin Microbiol 42:4338–4343.

Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van DillenPME, Van Der Noordaa J. 1990. Rapid and simple method forpurifications of nucleic acids. J Clin Microbiol 28:495–503.

Ciarlet M, Liprandi F, Conner ME, Estes MK. 2000. Species specificityand interspeccies relatedness of NSP4 genetic groups by compara-tive NSP4 analysis of animal rotaviruses. Arch Virol 145:371–383.

Cunliffe NA, Gondwe JS, Broadhead RL, Molyneux ME, Woods PA,Bresee JS, Glass RI, Gentsch JR, Hart CA. 1999. Rotavirus G and Ptypes in children with acute diarrhoea in Blantyre, Malawi, from1997 to 1998. J Med Virol 57:308–312.

Cunliffe NA, Rogerson S, Dove W, Thindwa BDM, Greensill J,Kirkwood CD, Broadhead RL, Hart CA. 2002. Detection andcharacterization of rotaviruses in hospitalized neonates inBlantyre, Malawi. J Clin Microbiol 40:1534–1537.

Cunliffe NA, Woods PA, Leite JPG, Das BK, Ramachandran M, BhanMK, Hart CA, Glass RI, Gentsch JR. 1997. Sequence analysis of



NSP4 gene of human rotavirus allows classification into two maingenetic groups. J Med Virol 53:41–50.

Das BK, Gentsch JR, Cicirello HG, Woods PA, Ramachandran M,Gupta A, Kumar R, Bhan MK, Glass RI. 1994. Characterization ofrotavirus strains from newborns in New Delhi, India. J ClinMicrobiol 32:1820–1822.

Estes MK. 2001. Rotaviruses and their replication. In: Knipe DM,HowleyPM,GriffinDE,LambRA,MartinMA,RoizmanB,StraisE,editors. Fields virology, 4th edition. Philadelphia, Pa: LippincottWilliams and Wilkins, pp 1747–1785.

Fischer TK, Bresee JS, Glass RI. 2004. Rotavirus vaccines and theprevention of hospital-acquired diarrhea in children. Vaccine6:S49–S54.

Fischer TK, Steinsland H, Molbak K, Ca R, Gentsch JR, Valentiner-Branth P, Aaby P, Sommerfelt H. 2000. Genotype profiles ofrotavirus strains from children in a suburban community inGuinea-Bissau, Western Africa. J Clin Microbiol 38:264–267.

Flores J, Midthun K, Hoshino Y, Green K, Gorziglia M, Kapikian AZ,Chanock RM. 1986. Conservation of the fourth gene amongrotaviruses recovered from asymptomatic newborn infants and itspossible role in attenuation. J Virol 60:972–979.

Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, DasBK,BhanMK.1992. Identification of groupArotavirus gene4 typesby polymerase chain reaction. J Clin Microbiol 30:1365–1373.

Gorziglia M, Green K, Nishikawa K, Taniguchi K, Jones R, KapikianAZ, Chanock RM. 1988. Sequence of the fourth gene of humanrotaviruses recovered from asymptomatic or symptomatic infec-tions. J Virol 62:2978–2984.

Gunasena S, Nakagomi O, Isegawa Y, Kaga E, Nakagomi T, Steele D,Flores J, Ueda S. 1993. Relative frequency of VP4 gene allelesamong human rotaviruses recovered over a 10-year period (1982–1991) from Japanese children with diarrhoea. J Clin Microbiol 31:2195–2197.

Hoshino Y, Wyatt RG, Flores J, Midthun K, Kapikian AZ. 1985.Serotypic characterization of rotaviruses from asymptomatichuman neonatal infections. J Clin Microbiol 21:425–430.

ItoH,SugiyamaM,MasubuchiY,MoriY,MinamotoN. 2001.Completenucleotide sequence of a group A avian rotavırus genome and acomparison with counterparts of mammalian rotaviruses. VirusRes 75:123–138.

Kapikian AZ, Hoshino Y, Chanock RM. 2001. Rotaviruses. In: KnipeDM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B,Strais E, editors. Fields virology, 4th edition. Philadelphia, Pa:Lippincott Williams and Wilkins, pp 1787–1833.

Kilgore PE, Unicomb LE, Gentsch JR, Albert MJ, McElroy CA, GlassRI. 1996. Neonatal rotavirus infection in Bangladesh: Straincharacterization and risk factors for nosocomial infection. PediatrInfect Dis J 15:672–677.

Kimura M. 1980. A simple method for estimating evolutionary rate ofbase substitution through comparative studies of nucleotidesequences. J Mol Evol 16:111–120.

Kirkwood CD, Palombo EA. 1997. Genetic characterization of therotavirus nonstructural protein, NSP4. Virology 236:258–265.

Lee C-N,Wang Y-L, Kao C-L, Zao C-L, Lee C-Y, Chen H-N. 2000.Nsp4gene analysis of rotaviruses recovered from infected children withand without diarrhoea. J Clin Microbiol 38:4471–4477.

Leite JPG,AlfieriAA,WoodsPA,GlassRI,GentschJR. 1996.RotavirusG and P types circulating in Brazil: Characterization by RT-PCR,probe hybridization, and sequence analysis. Arch Virol 141:2365–2374.

Linhares AC, Mascarenhas JDP, Gusmao RHP, Gabbay YB, FialhoAM, Leite JPG. 2002. Neonatal rotavirus infection in Belem,NorthernBrazil:Nosocomial transmission of aP[6]G2 strain. JMedVirol 67:418–426.

Mascarenhas JDP, Linhares AC, Gabbay YB, Leite JPG. 2002.Detection and characterization of rotavirus G and P types fromchildren participating in a rotavirus vaccine trial in Belem, Brazil.Mem Inst Oswaldo Cruz Rio de Janeiro 97:113–117.

Mori Y, BorganMA, ItoN, SugiyamaM,MinamotoN. 2002.Diarrhoea-inducing activity of avian rotavirus NSP4 glycoproteins, whichdiffer greatly from mammalian rotavirus NSP4 glycoproteins in

deduced amino acid sequence, in suckling mice. J Virol 76:5829–5834.

Nakagomi O, Nakagomi T. 1993. Interspecies transmission of rota-viruses genogroup. Microbiol Immunol 37:337–348.

NakagomiT,HorieY,KoshimuraY,GreenbergHB,NakagomiO.1999.Isolation of a human rotavirus strain with a super-short RNApattern and a new P2 subtype analysis. J Clin Microbiol 37:1213–1216.

Offit PA, Clark HF. 1985. Maternal antibody-mediated protectionagainst gastroenteritis due to rotavirus in newborn mice isdependent on both serotype and titer of antibody. J Infect Dis 152:1152–1158.

Page NA, Steele AD. 2004a. Antigenic and genetic characterization ofserotype G2 human rotavirus strains from South Africa from 1984to 1988. J Med Virol 72:320–327.

Page NA, Steele AD. 2004b. Antigenic and genetic characterization ofserotype G2 human rotavirus strains from the African Continent. JClin Microbiol 42:595–600.

Pager CT, Alexander JJ, Steele AD. 2000. South African G4P[6]asymptomatic and symptomatic neonatal rotavirus strains differ intheir NSP4, VP8*, and VP7 genes. J Med Virol 62:208–216.

Perez-Schael I, DaoudG,White L, UrbinaG, DaoudN, PerezM, FloresJ. 1984. Rotavirus shedding by newborn children. J Med Virol14:127–136.

Rahman M, Mattthijjnssens J, Nahar S, Podder G, Sack DA, Azim T,Van Ranst M. 2005. Characterization of a novel P[25]G11 humanGroup A rotavirus. J Clin Microbiol 43:3208–3212.

RamachandranM,GentschJR,ParasharUD, JinS,WoodsPA,HolmesJL, Kirkwood CD, Bishop RF, Greenberg HB, Urasawa S, Gerna G,Coulson BS, Tanaguchi K, Bresee JS,Glass RI. 1998. Detection andcharacterization of novel rotavirus strains in the United States. JClin Microbiol 36:3223–3229.

Rodger SM, Bishop RF, Birch C, McLean B, Holmes IH. 1981.Molecular epidemiology of human rotavirus in Melbourne, Aus-tralia from1973 to 1979 asdeterminedby electrophoresis of genomeribonucleic acid. J Clin Microbiol 19:272–278.

Salu OB, Audu R, Geyer A, Steele AD, Oyefolu AOB. 2003. Molecularepidemiology of rotaviruses inNigeria:Detection of unusual strainsG2P[6] and G8P[1] specificities. J Clin Microbiol 41:913–914.

Sheridan JF, Smith CC, Manak MM, Aurelian L. 1984. Prevention ofrotavirus-induced diarrhea in neonatal mice born to dams immu-nizedwith empty capsids of simian rotavirus SA-11. J Infect Dis 49:434–438.

Snodgrass DR, Madeley CR, Wells PW, Angus KW. 1977. Humanrotavirus in lambs: Infection and passive protection. Infect Immun16:268–270.

Snodgrass DR, Wells PW. 1978. Passive immunity in rotaviralinfections. J Am Vet Med Assoc 173:565–568.

Steele AD, Ivanoff B. 2003. Rotavirus strains circulating in Africaduring 1996-1999: Emergence of G9 strains and P[6] strains.Vaccine 17:361–367.

Steele AD, Sears JF. 1996. Characterisation of rotaviruses recoveredfrom neonates with symptomatic infection. S Afr Med J 86:1546–1549.

Steele AD, van Niekerk MC, Geyer A, Bos P, Alexander JJ. 1992.Further characterisation of human rotaviruses isolated fromasymptomatically infected neonates in South Africa. J Med Virol38:22–26.

Steele AD, van Niekerk MC, Mphalele MJ. 1995. Geographic distribu-tion of human rotavirus VP4 genotypes and VP7 serotypes in fiveSouth African regions. J Clin Microbiol 33:1516–1519.

Tian P, Estes MK, Hu Y, Ball JM, Zeng CQ-Y, Schilling WP. 1995. Therotavirusnonstructural glycoproteinNSP4mobilizesCa2þ from theendoplasmic reticulum. J Virol 69:5763–5772.

Timenetsky MCST, Santos N, Gouvea V. 1994. Survey of rotavirus Gand P types associated with human gastroenteritis in Sao Paulo,Brazil, from 1986 to 1992. J Clin Microbiol 32:2622–2624.

Zhang MC, Zeng QY, Dong Y, Ball JM, Saif LJ, Morris AP, Estes MK.1998. Mutations in rotavirus nonstructural glycoprotein NSP4 areassociated with altered virus virulence. J Virol 72:3666–3672.



Documents

Molecular analysis of VP4, VP7, and NSP4 genes of P[6]G2 rotavirus genotype strains recovered from neonates admitted to hospital in Belém, Brazil