Virus Research 137 (2008) 163–167

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Short communication

Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirusstrain detected in an infant with gastroenteritis in Italy

Maria Cristina Medici ∗, Laura Anna Abelli, Monica Martinelli, Giuseppe Dettori1, Carlo Chezzi1

Section of Microbiology, Department of Pathology and Laboratory Medicine, Section of Microbiology,University of Parma School of Medicine, viale Antonio Gramsci, 14, 43100 Parma, Italy

a r t i c l e i n f o

Article history:Received 23 April 2008Received in revised form 1 July 2008Accepted 3 July 2008Available online 15 August 2008

Keywords:Rotavirus G8Rotavirus P[14]Phylogenetic analysisHuman–animal reassortant

a b s t r a c t

In this study, the molecular characterization of a rare G8P[14] group A rotavirus (GARV) strain detectedin Northern Italy during the 2004–2005 epidemiological rotavirus season is described.

Two hundred and seventy three rotavirus-like particle positive stools out of 856 stools from children(31.9%) hospitalized with gastroenteritis were analyzed using polyacrilamide gel electrophoresis and 271GARVs were genotyped by VP7 and VP4 specific RT–PCRs. One strain (PR/1300/04) with a long electro-pherotype (e-type) displayed the G8 specificity and was VP4 un-typeable. The P and the subgroup (SG)specificities were determined by sequencing the VP4 and the VP6 gene, respectively. The PR/1300/04strain exhibited P[14] and SGI specificities. By sequence and phylogenetic analyses of the VP4, VP6 andVP7 amplicons, the PR/1300/04 VP4 and VP6 genes were demonstrated to be of human rotavirus origin,with the VP4 gene closely related to the human Italian PA169 strain (G6P[14]), while the VP7 gene wasof animal origin (bovine). These data suggest that the Italian PR/1300/04 strain could be a reassortant

between a PA169-like Italian strain with P[14] specificity, long e-type and SGI, and a G8 animal strain.The increasing number of reports of atypical GARVs in humans suggests that interspecies transmission of

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Group A rotaviruses (GARVs) are the most common viral agentsf acute gastroenteritis in humans and in a large variety of animalsorldwide (Estes, 2001; Gentsch et al., 2005; Santos and Hoshino,005).

The GARV genome consists of 11 segments of double strandedNA that migrate in polyacrilamide gel electrophoresis (PAGE)esulting in typical electropherotypes (e-types). Genotype isefined by the nucleic acid sequences of genes encoding the viralapsid proteins VP7 (G type) and VP4 (P type) (Estes, 2001; Kapikiant al., 2001). A serological classification system exists for the innerapsid protein VP6, based on the reactivity with two monoclonalntibodies (Greenberg et al., 1983), but by molecular analysis ofhe VP6 gene, two subgroup (SG) specificities have been identified.ccording to the classification system proposed by Matthijnssenst al. (Matthijnssens et al., 2008), 19 G, 27 P and 11 VP6 geno-

ypes (I-genotypes) in humans and animals are known, togetherith genotypes for all the other RV genes. Human GARVs usually

xhibit the G1, G3, G4, and G9 genotypes in association with the[8] genotype, SGII specificity and long e-type, while G2 GARVs

∗ Corresponding author. Tel.: +39 0521 988885; fax: +39 0521 993620.E-mail address: mariacristina.medici@unipr.it (M.C. Medici).

1 C. Chezzi and G. Dettori equally contributed to the co-ordination of the study..

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168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2008.07.005

GARV genetic evolution.© 2008 Elsevier B.V. All rights reserved.

re more often associated with the P[4] genotype, SGI specificitynd short e-type (Estes, 2001; Kapikian et al., 2001; Gentsch et al.,005; Santos and Hoshino, 2005). The G and P types are pecu-

iarly distributed across the various animal species (Santos andoshino, 2005), suggesting host species barriers, although a num-er of strains with unusual G and P types, regarded as animal-liketrains, have been sporadically identified in humans in differentart of the world (Gentsch et al., 2005; Santos and Hoshino, 2005)nd have acquired epidemiological relevance in some geographicalreas (Iturriza-Gómara et al., 2004).

The aim of the present study was to perform a molecular char-cterization and phylogenetic analysis of a rare G8P[14] GARVtrain detected in Parma, Northern Italy, during surveillance of the004–2005 epidemiological rotavirus (RV) season.

Two hundred and seventy three RV-like particle positive stoolsy electron microscopy out of 856 stools from children (range:

month–10 years and 4 months, median age: 1 year and 8onths) hospitalized with gastroenteritis at the University Hos-

ital of Parma, Italy (prevalence of infection: 31.9%), were analyzed

sing PAGE, as previously described (Medici et al., 2004).

To determine the G and P types of 271 GARV strains, the dsRNA,xtracted from 10% phosphate buffered saline stool suspensionsing the guanidine isothiocyanate/Glass Milk method (Gentscht al., 1992), was analyzed by RT–PCR and subsequent nested and

164 M.C. Medici et al. / Virus Research 137 (2008) 163–167

Table 1Percentage of nucleotide and amino acid identities of the group A rotavirus (GARV) strain PR/1300/04 VP4 gene with P[14] human and animal GARVs available in GenBank

Strain Origin Country GenBank accession no. %Identity

Nucleotide Amino acid

PA169 Human Italy L20874 96.0 99.1HAL1166 Human Finland L20875 88.3 97.9B4106 Human Belgium AY740738 81.6 93.6Hun5 Human Hungary AJ488139 95.9 98.9Mc35 Human Thailand D14032 85.6 94.8MG6 Human Australia U22012 89.4 97.8DG8 Human Australia AF034853 89.8 96.9EGY2295 Human Egypt AF104103 86.7 97.5EGY1850 Human Egypt AF104101 85.0 96.430/96 Lapine Italy DQ205224 81.7 93.0ALA Lapine United States U62149 81.5 93.0C-11 Lapine United States U62150 81.9 93.0R2 Lapine Japan U62151 81.0 92.0BAP2 Lapine United States U62152 84.3 93.3Cap455 Caprine South Africa AY128709 90.1 97.1S AK D

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un9 Bovine Japan8 Human Japan

he K8 strain with P[9] specificity was used as outgroup in the phylogenetic analys

emi-nested PCR typing strategies according to methods publishedlsewhere with minor modifications using different sets of G andtype-specific primers (Gentsch et al., 1992; Gouvea et al., 1990;as et al., 1994; Cunliffe et al., 1999; Iturriza-Gómara et al., 2000;artella et al., 2003, 2004).One strain with long e-type, named PR/1300/04, displayed the

8 genotype specificity and the VP4 gene was not typeable.

To confirm the G8 genotype and further investigate the P geno-

ype, the VP4 and VP7 first amplicons of the PR/1300/04 strain wereurified using the “Qiaquick Gel Extraction Kit” (QIAGEN, Italy),nd sequenced using the automated sequencer 3730 DNA Ana-yzer (Applied Biosystems, USA). The PR/1300/04 SG specificity was

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able 2ercentage of nucleotide and amino acid identities of the group A rotavirus (GARV) strain

train Origin Country

G8.01 Human AustraliaG8.01 Human Australia

I-885-06 Human SloveniaGY1850 Human EgyptGY2295 Human EgyptG8 Human AustraliaP409 Human IndiaR570 Human UKEH14262 Human UKP30 Human South AfricaAL1166 Human FinlandMG89 Human Niger9M Human IndonesiaRC88 Human Dem. Rep. CongoRC86 Human Dem. Rep. Congo-Agent Bovine South AfricaAG 87 Bovine JapanAG74 Bovine JapanAG75 Bovine Japan-8008 Bovine United Statesun9 Bovine Japanody Bovine United Statesiigata9801 Bovine Japanokushima9503 Bovine Japan78 Bovine UKRV 16 Bovine JapanRV RioNegro Lama guanicoe ArgentinaRV chubut Lama guanicoe ArgentinaCDV Bovine United States

he NCDV strain with G6 specificity was used as outgroup in the phylogenetic analysis. M

B158430 86.2 94.590260 78.1 86.3

or identities are marked in boldface while the minor ones are in italic.

etermined by RT–PCR and subsequent sequencing of a fragmentf the VP6 gene using the VP6F–VP6R primer pair (Iturriza-Gómarat al., 2002). Sequence and phylogenetic analyses were conductedsing MEGA version 2.1. The VP6, VP4 and VP7 nucleotide sequencesbtained in this study were deposited in GenBank under the acces-ion numbers EU835943, EU835944, and EU835945, respectively.

By sequence analysis, the PR/1300/04 VP4 gene was found to

ave the P[14] specificity and was closely related to the human6P[14] RV strains PA169 and Hun5, isolated in Italy and Hun-ary, respectively (Table 1). However, the VP7 gene displayedigh nucleotide and amino acid identities to bovine strains from

apan and South Africa, and Lama Guanicoe strains from Argentina

PR/1300/04 VP7 gene with G8 human and animal GARVs available in GenBank

GenBank accession no. %Identity

Nucleotide Amino acid

AF207060 88.6 98.8AF207061 88.6 98.1DQ995179 85.3 96.6AF104102 84.7 97.5AF104104 83.9 96.9AF034852 89.1 97.5AF141918 83.4 95.4AF143688 84.0 94.7AF143689 85.9 96.3AF143690 83.2 93.5L20882 87.5 98.8X98918 83.7 95.0Green et al. (1989) 88.3 98.1DQ005109 83.7 95.4DQ005120 83.8 95.4DQ838598 95.4 99.4AB077056 94.3 94.7AB077053 95.5 95.4AB077054 96.7 98.1U14998 94.6 96.3AB158431 95.5 97.8U14999 87 97.2AB044293 97.6 99.4AB044294 97.0 98.8L20883 88.4 98.1AB077058 97.9 98.1AF545859 97.5 98.8AF545860 97.4 97.5M12394 74.9 82.7

ajor identities are marked in boldface while the minor ones are in italic.

M.C. Medici et al. / Virus Research 137 (2008) 163–167 165

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ig. 1. Alignment of amino acid (aa) 243–369 sequence region of G8P[14] PR/1300/0he strain origin and the GenBank accession numbers are indicated. The aa residueor, porcine.

Table 2). By alignment of the deduced amino acid sequence withstablished SGI strains and with the SGII prototype strain Wa, allvailable in GenBank, the VP6 gene was found to contain residuesharacteristic for the GARV SGI specificity (Fig. 1). By sequence anal-sis, the PR/1300/04 VP6 gene displayed high nucleotide identitieso the human strain 1076 from Sweden and high amino acid identi-ies to both human (PA169 and 1076) and animal strains from cattle,orses and pigs (Table 3).

Phylogenetic analysis suggests a human origin for theR/1300/04 VP4 and VP6 genes and an animal origin for the VP7ene (Fig. 2): the VP4 and VP6 genes segregated in sublineagesopulated by human strains, resembling the results obtained bye Leener et al. (2004) for the VP4 gene of the PA169 strain and by

atthijnssens et al. (2008) for the VP6 gene of the 1076 strain, and

he VP7 gene segregated in the G8a VP7 genetic lineage, accordingo Fukai et al. (2004), which is populated only by animal strains.

Hence, the Italian PR/1300/04 strain was characterized as a long-type, G8P[14] genotype, and SGI specificity, a genetic constel-

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able 3ercentage of nucleotide and amino acid identities of the group A rotavirus (GARV) strain

train Origin Country

A169 Human ItalyCDV Bovine United States076 Human Swedena Human United States

S-1 Human United StatesU-1 Human JapanI-14 Equine United Statesottfried Porcine United StatesSU Porcine United StatesA11 Simian South AfricaH4 Equine JapanS5139 Human United StatesF Bovine FranceP140 Porcine India-1 Equine EnglandVR762 Ovine SpainO-13 Avian Japan210 Human AustraliaW Murine United Statesowden (group C) Porcine United States

he Cowden strain of group C was used as outgroup in the phylogenetic analysis. Major id

in and other established subgroup I strains and the subgroup II prototype strain Wa.ictive of subgroup specificity are marked. Abbreviations: Hu, human; Bov, bovine;

ation that is usually observed in animal-like viruses. Data fromiterature suggest that the presence of strains with the G8 and P[14]ntigens in humans might have been generated from exposure toovine and lapine RVs, respectively, and subsequent genetic reas-ortment with human strains (Gentsch et al., 2005). The G8 typean be found in ruminants at relatively high frequency (Pisanellit al., 2005). In humans, the G8 type was first identified in thearly 1970s in Indonesia, in association with P[10] genotype andn unusual “supershort” electrophoretic pattern (prototype strain9M) (Hasegawa et al., 1984). Since then, very few reports of suchG type have been published, except for some countries in Africahere it has been detected at an increasing frequency (Adah et

l., 2001; Fischer et al., 2003). Interestingly, the majority of these

8 human strains present a great diversity of P type combinations

Santos and Hoshino, 2005). P[14] strains have been described inabbits, goats, Lama Guanicoe, and cattle and humans. The firstuman P[14] strain was reported in Italy with the G6 specificity,

ong e-type and SGI specificity (PA169 strain) (Gerna et al., 1992).

PR/1300/04 VP6 gene with other human and animal GARVs available in GenBank

GenBank accession no. %Identity

Nucleotide Amino acid

EF554130 87.6 99.2M12394 87.9 99.2D00325 94.7 98.4K02086 72.8 91.9DQ870507 85.4 97.6DQ490538 76.2 94.4D00323 75.4 91.9D00326 75.9 92.7AF317123 73.5 91.9M27824 82.0 96.0D82975 88.3 99.2EF426130 88.2 98.4K02254 87.6 98.4DQ003295 87.2 99.2AF242394 73.5 91.1EF554152 88.2 97.6D16329 61.6 75.0U36240 75.5 92.7U36474 73.7 95.2M94157 25.2 24.4

entities are marked in boldface while minor ones are in italic.

166 M.C. Medici et al. / Virus Research 137 (2008) 163–167

Fig. 2. Neighbor-joining phylogenetic trees based on partial nucleotide (nt) sequences of VP4 (nt 64–1050) (A), VP7 (nt 40–989) (B), and VP6 (nt 753–1123) (C) genes ofPR/1300/04 rotavirus strain and others established P[14] (A), G8 (B) and subgroup I and II (C) rotavirus strains. The numbers adjacent the nodes represent the percentage ofbootstrap support (of 1000 replicates). Bootstrap values lower than 50% are not shown. K8 strain with P[9] specificity, NCDV strain with G6 specificity and Cowden strain ofgroup C were used as outgroups in the VP4, VP7 and VP6 phylogenetic analyses, respectively. The VP7 lineages are according to Fukai et al. (2004). Abbreviations: Hu, human;Cap, caprine; Lap, lapine; Bov, bovine; Eq, equine; Ov, ovine; Por, porcine; Sim, simian; Mur, murine; Avi, avian.

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ubsequently, few observations of P[14] GARVs were reported inssociation with various G types (De Leener et al., 2004).

The data obtained in this report seem to suggest that the ItalianR/1300/04 strain could be the result of a reassortment betweenPA169-like strain with the P[14] specificity, long e-type and SGI,

lready circulating in Italy, and a G8 animal strain by the introduc-ion of a GARV VP7 gene from an unidentified animal source, mostikely bovine.

Interspecies transmission can lead to genetic reassortmentetween animal and human RV strains during co-infection. Thisnique mechanism can lead to progeny viruses with novel ortypical phenotypes. The increasing number of reports of atypi-al RV strains in humans suggests that interspecies transmissionf gene segments greatly contributes to the RV genetic evolution.lthough epidemiological investigations have demonstrated exam-les of intergenogroup reassortants (Ward et al., 1990; Nakagomit al., 1992; Medici et al., 2007), and a high rate of exposuref humans to animal RV strains is expected to exist in someeographical areas (Matthijnssens et al., 2006), there is a little evi-ence of interspecies transmission due to RV completely belongingo another species, resulting in clinical apparent infections. Pre-umably, when RVs cross the host species barrier, they are notaturally able to efficiently infect or spread in a new host, buthey tend to reassort with another RV inherent in that host species.hus, the resultant reassortant strains may have more chances tofficiently infect and spread among the population of the newost.

The molecular characterization of rare/new GARV strains inumans is determinant to provide a more in-depth understand-

ng of the natural mechanisms that drive GARV evolution and hostange restriction.

cknowledgements

This study was supported by grants from the research projectFondi di Ateneo per l’anno 2007 - Epidemiologia molecolare etudio dei meccanismi evolutivi di norovirus e rotavirus umani”.

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