lsevier.com/locate/yviro

Virology 346 (200

Identification of a novel VP4 genotype carried by a serotype

G5 porcine rotavirus strain

V. Martella a,*, M. Ciarlet b, K. Banyai c, E. Lorusso a, A. Cavalli a, M. Corrente a,

G. Elia a, S. Arista d, M. Camero a, C. Desario a, N. Decaro a, A. Lavazza e, C. Buonavoglia a

a Department of Animal Health and Well-being, University of Bari, Valenzano, Bari, Italyb Biologics–Clinical Research, Merck and Co., Inc., Blue Bell, PA 19422, USA

c Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pecs, Hungaryd Department of Hygiene and Microbiology, University of Palermo, Palermo, Italye Istituto Zooprofilattico Sperimentale di Lombardia/Emilia Romagna, Brescia, Italy

Received 26 May 2005; returned to author for revision 21 August 2005; accepted 2 November 2005

Available online 20 December 2005

Abstract

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy

was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-

length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine

strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage

fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain

MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15

shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and

NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6

genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be

considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A

rotaviruses.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Rotavirus; Diarrhea; Pigs; VP4; P genotype; Genetic diversity

Introduction

Group A rotaviruses are the main cause of acute dehydrating

diarrhea in children and are associated with 400,000–500,000

deaths annually, mainly in the developing countries (Parashar

et al., 2003). Group A rotaviruses are non-enveloped icosahe-

dral particles composed of 11 segments of double-stranded

(ds)RNA enclosed in a triple layered protein capsid (Ciarlet

and Estes, 2002). The inner capsid protein VP6 bears the

subgroup (SG) specificities that allow the classification of

0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2005.11.001

* Corresponding author. Dipartimento di Sanita e Benessere Animale, Facolta

di Medicina Veterinaria di Bari, S.p. per Casamassima km 3, 70010 Valenzano,

Bari, Italia. Fax: +39 080 4679843.

E-mail address: v.martella@veterinaria.uniba.it (V. Martella).

group A rotaviruses into SG I, SG II, both SG I and II, or into

neither SG based on reactivity with SG-specific monoclonal

antibodies (MAbs) (Ciarlet and Estes, 2002). The non-

structural glycoprotein NSP4 has been studied extensively

because of its multiple functions in rotavirus morphogenesis

and pathogenesis and its enterotoxic activity (Ciarlet and Estes,

2002). Sequence analyses of the NSP4 gene from human and

animal rotavirus strains have revealed the presence of five

distinct NSP4 genogroups, KUN-(A), Wa-(B), AU-1-(C), EW-

(D), and avian-like (E) (Ciarlet et al., 2000; Horie et al., 1997;

Ito et al., 2001; Mori et al., 2002).

The two outer capsid proteins, VP4 and VP7, independently

elicit neutralizing antibodies, induce protective immunity, and

are used to classify rotavirus strains into P (for protease-

sensitive) and G (for glycoprotein) serotypes (Ciarlet and Estes,

6) 301 – 311

www.e

V. Martella et al. / Virology 346 (2006) 301–311302

2002). Fifteen G genotypes (defined by sequence analysis and/

or nucleic acid hybridization data) have been identified and

shown to belong to distinct G serotypes (defined by serology

data). Usually, rotavirus strains within a G serotype share at

least 90–91% VP7 amino acid (aa) sequence identity (Green

et al., 1988). Out of 25 different P genotypes (designated in

brackets), only 14 P serotypes have been identified with

available antisera or anti-VP4 MAbs (Hoshino et al., 2002b;

Liprandi et al., 2003; Martella et al., 2003a; McNeal et al.,

2005; Rahman et al., 2005). Rotavirus strains sharing >89%

VP4 amino acid (aa) sequence identities are considered to

belong to the same P genotype, while those sharing VP4 aa

sequence identities of <89% belong to different genotypes

(Estes, 2001; Gorziglia et al., 1990). Moreover, the greatest aa

divergence in VP4 is seen between aa 71 and 204 of the VP4

trypsin cleavage product VP8*, which correlates with VP4

genotype specificity (Larralde and Gorziglia, 1992; Larralde et

al., 1991). The distribution of the various P genotypes across

the various animal species is summarized in Table 1.

Epidemiological studies have demonstrated that five rotavi-

rus G serotypes (G1, G2, G3, G4, and G9) and two P serotypes

(P1A[8] and P1B[4]) are the most frequent VP7 and VP4 types

associated with human rotavirus (HRV) infection globally

(Hoshino et al., 2002a; Hoshino and Kapikian, 2000; Kapikian

et al., 2001; Santos and Hoshino, 2005). Unusual G and P types

may be identified in humans in different parts of the world

(Banyai et al., 2003; Cooney et al., 2001; Das et al., 1993;

Esona et al., 2004; Gerna et al., 1992; Laird et al., 2003; Okada

et al., 2000; Rahman et al., 2003; 2005) and reach an

epidemiological relevance in some geographical settings

(Cunliffe et al., 1999; Gouvea et al., 1994c; Iturriza Gomara

et al., 2004; Santos and Hoshino, 2005; Zhou et al., 2003). The

growing data on the onset of animal-like rotavirus strains in the

human population demonstrate the importance of direct

interspecies transmission of animal strains and genetic reassort-

ment (Nakagomi and Nakagomi, 2002; Palombo, 2002). Thus,

studying animal rotaviruses is paramount to acquire a more in-

depth understanding of rotavirus evolution and ecology, and

rotavirus surveillance programs have been established in

Table 1

Distribution of rotavirus P genotypes across the various animal species

Genotype 1 2 3 3 4 5 6 6 6 6 6 7 8 9 1

Serotype 6 5A 5A 5B 1B 7 2A 2B 2C 9 1A 3 4

Lineage I II III IV V

Species

Man o o . o . o o o . . .Pig o . . . . . oMonkey . .Cattle . o .Goat/Sheep . . oHorse

Dog .Cat . .Mouse

Rabbit

Birds

Full circle indicates that the P type is epidemiologically relevant for the species, o

sporadic description or low epidemiological relevance.

several countries. In Italy, a surveillance program has been

granted by the Ministry for Health that enlists Italian veterinary

and medical universities, hospitals, and institutions of the

Italian National Health System, to collect data on human and

animal rotavirus diversity, to prove the presence of common

and rare strains, and to monitor changes in strain distribution

patterns.

Among porcine rotaviruses, nine G types (G1, G2-like, G3–

6, and G9–11) have been described (Ciarlet et al., 1994, 1995,

1997; Ciarlet and Liprandi, 1994; Martella et al., 2001, 2005a;

Saif et al., 1994; Teodoroff et al., 2005). The most common

porcine rotavirus G serotypes are G3, G4, and G5, which are

usually associated with VP4 serotypes P9[7] or P2B[6]

(Liprandi et al., 1991; Saif et al., 1994). Additional P types

(P7[5], P1A[8], P[13], P12[19], P14[23]) have been described

in rotavirus strains isolated from or detected in porcine stool

samples (Burke et al., 1994; Huang et al., 1993; Liprandi et al.,

2003; Martella et al., 2001, 2005a; Santos et al., 1999;

Teodoroff et al., 2005). In addition, sequence analysis of

PoRVs detected in Brazil, Japan, and Italy has revealed the

existence of strains with genetically intermediate features

between porcine P[13] and lapine P[22] rotaviruses (Martella

et al., personal findings; Santos et al., unpublished data;

Teodoroff et al., 2005), while analysis of P[6] PoRVs identified

in Italy and Spain has revealed the existence of P[6] lineages

genetically related to either the major (I) or minor (V) human

P[6] lineages (Martella et al., in press). Altogether, analyses of

PoRV VP7 and VP4 proteins have revealed a complex genetic/

antigenic heterogeneity. In this report, we describe a novel VP4

genotype carried by a serotype G5 porcine rotavirus strain

identified in Italy.

Results and discussion

PoRV strain 134/04-15 was identified from a diarrheic piglet

of a farm in Reggio Emilia (Emilia Romagna), during a large

epidemiological study on the incidence of rotavirus infections

in several pig farms in Italy in 2003–2004. Initial screening by

PCR genotyping with multiple sets of G- and P-specific

0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

8 4 11 10 12 13 14

. . o .o . . . .

.. o o .. . o .

.

. .. .

.r that it has been described only in that species to date. Open circle indicates

Fig. 1. Phylogenetic tree of the VP6 (A), NSP4 (B) and VP7 (C), displaying the relationships between strain 134/04-15 and various rotavirus strains. The VP6 tree is

inferred on the nucleotide sequence, while the NSP4 and VP7 tree are inferred on the deduced amino acid sequence. The following abbreviations are used to identify

the species of origin of the strains: Si, simian; Eq, equine; Po, porcine; Bo, bovine; Hu, human; Ov, ovine; La, lapine; Fe, feline; Ca, canine; Mu, murine; Av, avian.

The strains labeled with an asterisk are from our laboratory collection.

V. Martella et al. / Virology 346 (2006) 301–311 303

Fig. 1 (continued ).

V. Martella et al. / Virology 346 (2006) 301–311304

Table 2

Percentage (%) amino acid (aa) sequence identity of the VP7 of the Italian

porcine strain 134/04-15 to selected rotavirus strains belonging to established G

typesa and to a selection of G5 rotavirusesa

Strain (origin) VP7 G type VP4 P type 134/04-15 aa%

KU (human) 1 1A[8] 79.31

S2 (human) 2 1B[4] 74.60

34461-4 (porcine) 2-like [23] 75.54

RRV (simian) 3 5B[3] 84.95

Y0 (human) 3 1A[8] 84.32

ST3 (human) 4 2A[6] 74.60

OSU (porcine) 5 9[7] 95.61

A46 (porcine) 5 [13] 95.29

JL94 5 [7] 95.90

H1 (equine) 5 9[7] 95.61

NCDV (bovine) 6 6[1] 80.87

Ch2 (avian) 7 [17] 58.30

B37 (human) 8 ? 78.99

116E (human) 9 8[11] 79.93

61A (bovine) 10 7[5] 79.62

YM (porcine) 11 9[7] 88.40

L26 (human) 12 1B[4] 79.62

L338 (equine) 13 [18] 76.80

CH3 (equine) 14 4[12] 77.74

Hg18 (bovine) 15 [21] 78.65

N.d.: not determined.a GenBank accession nos. of VP7 genes: KU (D16343); DS-1 (AB118023);

S2 (M11164); HN-126 (P11851); 7PE/89 (AY261339); BS3585/99

(AY261348); RRV (AF295303); Y0 (D86284); ST3 (P10501); OSU

(X04613); A46 (L35054); JL94 (AY538665); H1 (AF242393); NCDV

(M12394); Ch2 (X56784); B37 (J04334); 116E (L14072); 61A (X53403);

YM (M23194); L26 (M58290); L338 (D13549); CH3 (D25229); Hg18

(AF237666).

V. Martella et al. / Virology 346 (2006) 301–311 305

primers characterized the VP7 of strain 134/04-15 as G5

genotype, while the VP4 gene was untypeable. Despite

numerous efforts, PoRV strain 134/04-15 could not be adapted

to growth in MA104 cells.

The VP4, VP6, VP7, NSP4 genes of the PoRV strain 134/

04-15 were subjected to sequencing and molecular character-

ization to determine the molecular features of the non-

cultivable PoRV.

Comparative analysis of the deduced aa sequences of the

fragment of the VP6 (aa 241 to 367), known to correlate with

SG specificity (Iturriza Gomara et al., 2002), allowed

characterization of strain 134/04-15 as genogroup I, suggesting

that the PoRV strain 134/04-15 belongs to SGI (Fig. 1A). The

highest sequence identity (up to 99.0% nt and 100% aa) was

found to the VP6 of Italian porcine strains of our collection.

The highest nt identity to sequences published in the databases

was found to the PoRVs CN86 (90.7%) and YM (89.6%),

while the highest aa identity (99.1%) was found to the SGI

PoRV strains A253, YM, CRW8, and OSU. The residues 305

and 315 were Ala and Ile, respectively, a pattern that is

consistent with SGI specificity (data not shown). Phylogenetic

analysis clustered strain 34461-4 in a defined clade of PoRV

strains, within a group of porcine and human strains with SGI

specificity (Fig. 1A), with exception of the porcine strain A411,

that is SGII and that has Asn-305 instead of Ala.

The full-length NSP4 protein was compared with represen-

tative strains of NSP4 genogroups A, B, C, D, and E (Fig. 1B).

In this comparison, the strain 134/04-15 showed the highest aa

identity (95.4% aa) to PoRV strain A34 belonging to the NSP4

B (Wa-like) genotype.

The completely deduced amino acid sequence of the VP7

gene from the PoRV strain 134/04-15 was determined and

compared to those of reference rotavirus strains belonging to

all known G serotypes (Table 2 and Fig. 1C). The VP7 aa

sequence of strain 134/04-15 was 91.9 to 95.9% identical to

those of rotavirus strains exhibiting G5 serotype specificity,

and the highest aa identity was found to the G5 PoRV JL94,

isolated in China (Shi et al., unpublished). The VP7 protein of

strain 134-04/15 had a potential N-linked glycosylation site

located at aa 69 (Asn), which tends to be conserved among

most rotavirus strains (Ciarlet et al., 1995, 1997). The VP7

antigenic regions, A (aa 87–101), B (aa 143–152), C (aa 208–

223), and F (aa 235–242) (Ciarlet et al., 1997; Dyall-Smith et

al., 1986; Nishikawa et al., 1989) of the PoRV strain 134/04-15

also support its classification as serotype G5, even if the

changes 96-ThrYAla, 146-GlyYGlu, and 241-SerYAsn were

observed within regions A, B, and F, respectively, with respect

to strain OSU (data not shown).

Initial attempts to determine the P genotype of the PoRV

strain 134/04-15 by RT-PCR genotyping were unsuccessful. The

full-length VP4 gene of strain 134/04-15 was determined with

newly designed 5V and 3V end primers, which have been

developed on the basis of full-length VP4 sequences of strains

with different genotype specificities. The VP4 genome segment

of PoRV strain 134/04-15 was 2362 nucleotides in length, and

the deduced aa sequence was 776 aa long. The prolines at

residues 68, 71, 225, 226, 334, 390, 395, 435 451, 455, 475, 524

669, 716, 749, and 761, and the cysteines at positions 216, 318,

380, and 774, which are highly conserved among other P

genotypes, were maintained in the VP4 of strain 134/04-15 (Fig.

2). The potential trypsin cleavage sites, Arg-231 and Arg-241,

were conserved, while Arg-247 was replaced by Lys. Arg-247 is

required to enhance infectivity in vitro of strain SA114S (Arias

et al., 1996) and to induce syncytia in cholesterol-supplemented

cells by virus-like particles bearing the VP4 of strain RRV

(Gilbert and Greenberg, 1998), but it is not conserved in all the P

genotypes. The additional cleavage sites identified in the VP5*

Lys-259, Arg-583, and, putatively, Arg-467 (Crawford et al.,

2001), were also conserved in strain 134/04-15.

The VP8* subunit, which correlates with VP4 genotype

specificity (Larralde and Gorziglia, 1992; Larralde et al., 1991),

was compared with those of rotavirus strains representative of

all 25 P genotypes (Table 3). The aa sequence of the VP8* of

strain 134/04-15 shared low aa identity, ranging from 37.52%

(bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-

13, P[13]), with the homologous sequences of representative

strains of the remaining 25 P genotypes. The aa sequence of the

full-length VP4 protein showed aa identity values ranging from

59.7% (bovine strain KK3, P8[11]) to 86.05% (porcine strain

A46, P[13]). Since it has been established that rotavirus strains

that exhibit a VP4 aa identity of approximately >89% belong to

the same P genotype (Gorziglia et al., 1990), our results

indicate that the Italian PoRV strain 134/04-15 represents a

novel P genotype.

Fig. 2. Comparison of the deduced amino acid sequence of the VP4 spike protein of the Italian porcine rotavirus strain 134/04-15 with a selection of rotavirus strains representative of various P genotypes. The highly

conserved cysteine (.) and prolines (4) are indicated. Also, the highly conserved trypsin cleavage sites (˝) and the additional trypsin-susceptible sites (g) (Crawford et al., 2001) are shown. The number of amino

acids is based strain NCDV. For optimal alignment, gaps were introduced in the sequences. The GenBank Accession No. of the VP4 sequences are listed in Table 2. The following abbreviations are used to identify the

species of origin of the strains: Si, simian; Eq, equine; Po, porcine; Bo, bovine; Hu, human; Ov, ovine.

V.Martella

etal./Viro

logy346(2006)301–311

306

Table 3

Percentage (%) amino acid (aa) sequence identity of the VP8* trypsin-cleavage

product (aa 1 to 247) and of the full-length VP4 of the Italian porcine strain

134/04-15 to selected rotavirus strains belonging to established P genotypesa

Strain (origin) P genotype P serotype 134/04-15

VP8* VP4

NCDV (bovine) 1 6 69.60 80.24

SA11 (simian) 2 5B 68.76 80.76

RRV (simian) 3 5B 70.08 81.28

K9 (canine) 3 5A 67.88 80.76

GRV 3 n.d. 73.60 83.33

DS1 4 59.08 72.05

UK (bovine) 5 7 60.84 73.35

Gottfried (porcine) 6 2B 56.44 72.31

M37 (human) 6 2A 55.12 72.05

OSU (porcine) 7 9 72.72 81.41

KU (human) 8 1A 57.32 70.75

K8 (human) 9 3 55.56 68.77

69M (human) 10 4 67.00 81.50

KK3 (bovine) 11 8 38.84 59.70

F123 (equine) 12 4 67.00 78.94

MDR-13 (porcine) 13 n.d. 73.60 85.96

A46 (porcine) 13 n.d. 70.06 86.05

Mc35 (human) 14 11 58.64 70.10

Lp14 (ovine) 15 n.d. 67.88 79.98

EDIM (murine) 16 10 62.16 76.60

993/83 (bovine) 17 n.d. 37.52 61.52

L338 (equine) 18 n.d. 69.64 77.51

Mc345 (human) 19 12 57.32 73.35

EHP (murine) 20 13 67.00 78.03

Hg18 (bovine) 21 n.d. 63.92 77.38

160/01 (lapine) 22 n.d. 72.72 –

A34 (porcine) 23 14 68.76 –

TUCH (simian) 24 n.d. 69.20 81.28

Dhaka6 (human) 25 n.d. 65.82 –

N.d.: not determined.a GenBank accession nos. of VP4 genes: NCDV (AB119636); SA11

(M23188); RRV (M18736); K9 (D14725); GRV (AB055967); DS1

(P11196); UK (M22306); M37 (L20887); Gottfried (M33516); OSU

(X13190); KU (M21014); K8 (D90260); 69M (M60600); KK3 (D13393);

F123 (D16342); MDR-13 (L07886); A46 (AY05027); MC35 (D14032); LP14

(L11599); EDIM (AF039219); 993/83 (D16352); L338 (D13399); Mc345

(D38054); EHP (U08424); Hg18 (AF237665); 160/01 (AF526376); A34

(AY174094); TUCH (AY596189); Dhaka6 (AY773004).

V. Martella et al. / Virology 346 (2006) 301–311 307

Parsimony phylogenetic analysis of the deduced aa

sequences of the VP8* (Fig. 3) of PoRV strain 134/04-15

provided important clues to understand its genetic relatedness

to previously established rotavirus P genotypes. In the

phylogenetic tree, the VP8* of the strain 134/04-15 showed a

common evolutionary origin with porcine P[13] and lapine

P[22] rotavirus strains. A similar topology was obtained using

distance (neighbor joining) methods (data not shown). There-

fore, the Italian PoRV strain 134/04-15 qualifies as a new P

genotype, tentatively called P[26].

Based on the VP8* nt alignment of rotaviruses representa-

tive of the various P genotypes, a primer specific (MV26) for

strain 134/04-15 was designed (Table 4) and used to screen

rotavirus-positive samples that were untypeable by both PCR-

genotyping and sequence analysis. Only one sample out of 35

untypeable samples was recognized by primer MV26, indicat-

ing that the new P genotype is rare, at least in the Italian

porcine herds.

In recent years, epidemiologic surveillance to monitor the

appearance of novel rotavirus antigenic types has intensified

throughout the world. Together with data presented in this

study, at least 26 rotavirus P genotypes and several novel

lineages within the epidemiologically relevant human VP4

genotypes (e.g., P[6] and P[8]) have been recognized, some of

which presumably account for novel antigenic P types or

subtypes (Arista et al., 2005; Banyai et al., 2004; Lee et al.,

2003; Liprandi et al., 2003; McNeal et al., 2005; Martella et al.,

2003a, 2003b; Okada and Matsumoto, 2002; Martella et al., in

press, 2005b; Rahman et al., 2005; Rao et al., 2000). This

magnitude of antigenic diversity is usually seen in those

viruses, which are under strong B cell/antibody mediated

control in the host (Bachmann and Zinkernagel, 1996), a

recognition that has implications for vaccine design and use.

Thus, rotavirus antigens that induce neutralizing antibodies

have played a central role in research and development of a

rotavirus vaccine, and a number of polyvalent vaccines,

bearing the most common VP7 serotype specificities, have

been constructed and evaluated (Santos and Hoshino, 2005).

To further broaden the spectrum of protection, the major human

VP4 specificity, P1A[8], has been introduced in a next-

generation (bovine WC3-based) rotavirus vaccine, now in

large phase III clinical trials (Desselberger, 2005) and waiting

for licensure in several countries. As there is still debate on the

possible implications of the observed genetic/antigenic diver-

sification of group A rotaviruses on the overall effectiveness of

current vaccination strategies, continued surveillance following

the introduction of rotavirus vaccines in national vaccination

programs would provide a chance to clarify this issue.

The present work describes a novel VP4 type, tentatively

called P[26], carried by an Italian PoRV strain. Further

investigations are warranted to determine if this new P type

is of epidemiological importance in pigs or if it can be detected

in other species, including humans. It would be helpful to

elucidate if the novel P type described herein represents a

newly emerged VP4 type, which could have evolved recently

by successive accumulation of mutations on VP4 due to

constant immunologic pressure from a relatively close P

genotype (i.e., P[13]) or whether it represents a P genotype

that had remained undetected until now. Regardless, the

detection of the new rotavirus VP4 type, exhibited by the

PoRV strain 134/04-15, extends the knowledge on the genetic/

antigenic diversity of group A rotaviruses. A better under-

standing of rotavirus epidemiology will contribute to the

optimization of current vaccines and prevention programs of

rotavirus diarrhea in humans and animals and will help in

understanding the global ecology of rotaviruses.

Materials and methods

Origin of the virus and virologic diagnostic tests

PoRV strain 134/04-15 was identified during a large

epidemiological survey in Italy. A total of 751 fecal samples

were collected from nursing and weanling pigs involved in

outbreaks of diarrhea at 74 farms in five regions (Lombardia,

Fig. 3. Phylogenetic tree of the VP8* of group A rotavirus displaying the relationships between porcine strain 134/04-15 and strains representative of the 25

genotypes recognized to date. The tree is drawn to scale, and it is inferred on the amino acid sequence. The GenBank Accession No. of the VP4 sequences are listed

in Table 2. The following abbreviations are used to identify the species of origin of the strains: Si, simian; Eq, equine; Po, porcine; Ca, canine; Bo, bovine; Mu,

murine; Hu, human; Go, goat; Ov, ovine; La, lapine.

Table 4

List of the primers used for sequence analysis of the VP4 gene of strain

134/04-15

Primer (nt position) Nucleotide sequence 5V to 3V

Con3m (1–32) ggc tat aaa atg gct tcg ctc att tat aga ca

Con2* (872–891)a att tcg gac cat tta taa cc

170* (2338–2362) ggt cac awc ctc tag mmr ytr ctt a

Inv1 (567–592) agg tca ata cac aac aac caa cta tg

176f (793–819) tgg aaa gaa atg caa tat aac aga gat

184f (943–967) gtc ata gcg cat acc acg tgt tca g

Ge17R* (321–344) acg ttt ggt tcg act aaa ata atc

Ge13R* (1476–1499) tgt ctc tca aga tct tgt cta acg

Ge15 (1904–1925) att ttg atg ata ttt cag ctg c

MV26* (564–588) ggt tgt tgt gta ttg acc tga c

Antisense primers (*) are reversed and complementary to the 5V–3V nt sequenceNucleotide position is determined on the sequence of strain 134/04-15.a Gentsch et al. (1992).

V. Martella et al. / Virology 346 (2006) 301–311308

Emilia Romagna, Umbria, Basilicata, Puglia) in Italy between

2003 and 2004. A total of 124 (16.1%) rotavirus-positive

samples were identified from 751 rectal swabs of piglets

affected with diarrhea. The samples were collected directly

from the rectum of animals affected with symptoms of

gastroenteritis. Rotavirus antigens in rectal swabs were

detected by electron microscopy or by a commercial

immuno-enzymatic assay specific for group A rotaviruses

(Rotascreen Dipstick, Microgen Bioproducts, Camberley, UK).

PoRV strain 134/04-15 was identified in the stool sample from

a diarrheic piglet of a farm in Reggio Emilia (Emilia

Romagna). Despite numerous attempts, PoRV strain 134/04-

15 could not be adapted to cell culture in African green monkey

kidney (MA-104) cells.

RNA extraction, RT-PCR genotyping, and sequence analysis of

the VP7 and VP4 genes

Viral dsRNAwas extracted by adsorption on cellulose CF11

as described previously (Wilde et al., 1990). Viral dsRNA was

denatured in dimethyl-sulphoxide (Sigma-Aldrich, Milano,

Italy) at 97 -C for 5 min. Reverse transcription (RT) of dsRNA

was carried out using MuLV-reverse transcriptase (Applied

Biosystems, Applera Italia, Monza), while PCR amplification

was carried out with AmpliTaq Gold DNA polymerase (Applied

Biosystems) following the manufacturer’s recommendations.

RT of the VP7 gene was carried out using primer Beg9

(Gouvea et al., 1990) and primer End9deg (Martella et al.,

2003b). Determination of the G genotype was performed using

different pools of primers specific for human and animals G

types (G1 to G6 and G8 to G11), as previously described (Das

et al., 1994; Gouvea et al., 1990, 1994b; Martella et al., 2004;

Winiarczyk et al., 2002). The VP7 specificity was subsequently

confirmed by sequence analysis of the VP7 gene.

The VP8* subunit of the VP4, the connecting peptide, and

the N terminus of the VP5* subunit of VP4 (about 880 bp)

were reverse transcribed and amplified with primer pair Con2–

Con3 (Gentsch et al., 1992). P genotyping was initially

attempted using different pools of primers specific for several

human and animal P genotypes (P1, P3 to P11, P14, and P22)

(Gentsch et al., 1992; Gouvea et al., 1994a; Martella et al., in

.

V. Martella et al. / Virology 346 (2006) 301–311 309

press, 2005b; Winiarczyk et al., 2002). However, the VP4 gene

of PoRV strain 134/04-15 was not recognized by any of the P-

type-specific primers used. Sequence analysis of the VP8* did

not allow unambiguous classification of PoRV strain 134/04-15

into any of the previously established VP4 genotypes, and

therefore, we determined the full-length gene sequence of VP4.

A consensus primer (based on alignments of the VP4 genes

from all the previously recognized P genotypes) was designed

for the 3V end of the VP4, and it was used along with the 5V end-specific primer designed from the actual sequence of PoRV

strain 134/04-15. The primers designed for this study and used

for sequence analysis are shown in Table 4.

Amplification of the VP6 and NSP4 genes

The VP6 genogroup, predictive of the VP6 subgroup

specificity, was determined by amplification of a 379-bp

fragment, spanning from amino acids 241 to 367 of the VP6,

with primer pair VP6F-VP6R (Iturriza Gomara et al., 2002).

The nearly full-length NSP4 gene was amplified with primer

pair 10Beg16-10End722 (Lee et al., 2000).

Sequence and phylogenetic analysis

After purification on Ultrafree DA Columns (Amicon

Millipore, Bedford, USA), the amplicons underwent sequence

analysis with ABI-PRISM 377 (Perkin-Elmer, Applied Biosys-

tems Division). Sequences were assembled and analyzed using

Bioedit software package (Department of Microbiology, North

Carolina State University, USA) (Hall, 1999) and NCBI’s and

EMBL’s analysis tools. GenBank accession numbers DQ061053

and DQ062572 were assigned to the VP4 and VP7 of the PoRV

strain 134/04-15, respectively. The sequence of the genogroup-

specific fragment of the VP6 and of the NSP4 gene of PoRV

strain 134/04-15 is freely available upon request. Phylogenetic

and molecular evolutionary analyses were conducted using

MEGA version 2.1 (Arizona State University, USA) (Kumar et

al., 2001). Phylogenetic trees were elaborated with both

parsimony and distance methods, supplying a statistical

support with bootstrapping over 100 replicates.

Acknowledgments

The work was supported by grants from the Ministero della

Sanita (Ministry of Health) (Progetto Finalizzato 2003,

‘‘Diversita genetica ed antigenica dei rotavirus, studio dei

meccanismi evolutivi ed implicazioni ai fini diagnostici e

vaccinali’’ and Progetto Finalizzato 2003, ‘‘Il ruolo del suino

quale serbatoio e vettore di zoonosi: valutazione del rischio e

proposte per nuove strategie’’) and from the Ministero

dell’Istruzione, dell’Universita e della Ricerca (Ministry of

Education, University and Research) (Fondi di Ateneo ex

60%). We thank Filomena Cariola, Caterina Nanna, Nicoletta

Tutino, and Donato Narcisi for their expert technical assis-

tance. We are extremely grateful to Professor Leland Eugene

Carmichael for the continued encouragement throughout our

studies.

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