www.elsevier.com/locate/yviro

Virology 337 (20

Sequence analysis of the VP7 and VP4 genes identifies a novel

VP7 gene allele of porcine rotaviruses, sharing a common

evolutionary origin with human G2 rotaviruses

V. Martellaa,*, M. Ciarletb, R. Baselgac, S. Aristad, G. Eliaa, E. Lorussoa, K. Banyaie,

V. Terioa, A. Madioa, F.M. Ruggerif, E. Falconef, M. Cameroa, N. Decaroa, C. Buonavogliaa

aDipartimento di Sanita e Benessere Animale, Facolta di Medicina Veterinaria di Bari, S.p. per Casamassima km 3, 70010 Valenzano, Bari, ItalybBiologics-Clinical Research, Merck & Co., Inc., Blue Bell, PA, USA

cExopol S.L., Zaragoza, SpaindDepartment of Hygiene and Microbiology, University of Palermo, Palermo, Italy

eRegional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pecs, HungaryfDipartimento di Sanita alimentare e animale, Istituto Superiore di Sanita, Rome, Italy

Received 6 December 2004; returned to author for revision 1 February 2005; accepted 25 March 2005

Abstract

During an epidemiological survey encompassing several porcine herds in Saragoza, Spain, the VP7 and VP4 of a rotavirus-positive

sample, 34461-4, could not be predicted by using multiple sets of G- and P-type-specific primers. Sequence analysis of the VP7 gene

revealed a low amino acid (aa) identity with those of well-established G serotypes, ranging between 58.33% and 88.88%, with the highest

identity being to human G2 rotaviruses. Analysis of the VP4 gene revealed a P[23] VP4 specificity, as its VP8* aa sequence was 95.9%

identical to that of the P14[23],G5 porcine strain A34, while analysis of the VP6 indicated a genogroup I, that is predictive of subgroup I

specificity. Analysis of the 10th and 11th RNA segments revealed close identity to strains of porcine and human origin, respectively. The

relatively low overall aa sequence conservation (<89% aa) to G2 human rotaviruses, the lack of N-glycosylation sites that are usually highly

conserved in G2 rotaviruses, and the presence of several amino acid substitutions in the major antigenic hypervariable regions hampered an

unambiguous classification of the porcine strain 34461-4 as G2 serotype on the basis of sequence analysis alone. The identification of a

borderline, G2-like, VP7 gene allele in pigs, while reinforcing the hypotheses of a tight relationship in the evolution of human and animal

rotaviruses, provides additional evidence for the wide genetic/antigenic diversity of group A rotaviruses.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Rotavirus; VP7; VP4; G-type; P-type

Introduction one of the primary objectives of the World Health

Group A rotaviruses are the main etiologic agents of

acute viral gastroenteritis in humans and animals. In

humans, rotaviral diarrhea is responsible for 0.6–0.8 million

childhood deaths per year in non-developed countries, and

for about 20 deaths and 0.5 million hospitalizations per year

in the United States alone (Kapikian et al., 2001). Therefore,

the development of safe and effective rotavirus vaccines is

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

doi:10.1016/j.virol.2005.03.031

* Corresponding author. Fax: +39 080 4679843.

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

Organization.

Rotaviruses belong to the Reoviridae family and are

characterized by a genome consisting of 11 segments of

double-stranded RNA (dsRNA) enclosed in a trilaminar

capsid (Estes, 2001). The two outer capsid proteins, VP7 and

VP4, are considered of high relevance for immune protection

and for vaccine development, as they both elicit serotype-

specific neutralizing antibodies (Estes, 2001). Based on the

two outer capsid proteins, a dual classification system by G

serotypes (VP7) and P serotypes (VP4) has been adopted

(Estes, 2001). So far, based on genetic characterization, 15

05) 111 – 123

V. Martella et al. / Virology 337 (2005) 111–123112

VP7 and 24 VP4 gene alleles (genotypes) have been

identified (Estes, 2001; Kapikian et al., 2001; Liprandi et

al., 2003; Martella et al., 2003a; McNeal et al., 2005; Rao et

al., 2000). Rotaviruses belonging to the same G serotype

usually share at last 90% aa sequence conservation (Green et

al., 1988, 1989; Kapikian and Chanock, 1996). Of the 15 G

genotypes established so far, only 14 have been defined

antigenically as G serotypes. Conversely, out of the 23

genotypes identified to date, only 14 P serotypes have been

defined antigenically using VP4-specific antisera or mono-

clonal antibodies (Mabs) (Hoshino et al., 2002; Kapikian et

al., 2001; Liprandi et al., 2003). With a few exceptions

(Ciarlet et al., 1997a), strains sharing more than 89% of aa

conservation are considered to belong to the same P

genotype. Designation of the VP4 specificity reflects the

difficulties to evaluate antigenically the protein, and the P

serotype is indicated with a number immediately after the

letter P, while the genotype is represented by a number in

square brackets (Estes, 2001; Hoshino et al., 2002; Kapikian

et al., 2001).

The inner capsid protein VP6 bears the subgroup (SG)

specificities that allow the classification of group A

rotaviruses into SGI, SGII, both SGI and II, or into neither

SG based on the reactivity with SG-specific monoclonal

antibodies (MAbs) (Estes, 2001). By polyacrylamide gel

electrophoresis (PAGE), the majority of SGI and SGII

animal and SGII human rotavirus strains display a long

electropherotype (e-type) of migration of the 11 dsRNA

gene segments (Kapikian et al., 2001), while almost all SGI

human rotavirus strains possess a ‘‘short’’ e-type.

The most common rotavirus G serotypes found in

humans are G1, G2, G3, G4, and G9. Direct interspecies

transmission and exchange of dsRNA segments via reassort-

ment may account for the detection in humans of animal-

like rotavirus strains, such as G5, G6, G8, and G10, with

either a sporadic or a non-sporadic pattern, highlighting the

potential zoonotic impact of animal rotaviruses for humans

(Table 1) (Adah et al., 2001; Banyai et al., 2003; Beards et

Table 1

Distribution of rotavirus G types across the various animal species

Species VP7 specificity (G type)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Human . . . . o o o . o o .Pig o * . . . o . o .Monkey .Cattle o o . o . . oSheep/Goat o . . o .Horse . o o o .Dog .Cat .Mouse .Rabbit .Avian .Full circle indicates that the G type is epidemiologically relevant for the

species. Open circle indicates sporadic description or low epidemiological

relevance. The asterisk indicates the G2-like strain described in the present

study.

al., 1992; Cunliffe et al., 1999; Das et al., 1993; Gerna et al.,

1992; Gouvea et al., 1994c; Griffin et al., 2002; Hoshino

and Kapikian, 2000; Iturriza Gomara et al., 2004; Leite et

al., 1996; Nakata et al., 1999; Palombo et al., 2000; Steele et

al., 1999, 2002). RNA–RNA hybridization has provided

powerful molecular evidence to show close interspecies

relationships between human and animal strains, or confirm

the existence of reassortant rotavirus strains that occurred in

nature (Nakagomi and Nakagomi, 1991, 2002).

Therefore, acquisition of information on the distribution

of rotavirus G and P types among the various animal species

is important to comprehend rotavirus ecology, and the

mechanisms by which rotaviruses evolve, cross the species

barriers, exchange their genes during reassortment, and

mutate via accumulation of single-point substitutions and/or

via genetic rearrangements.

Porcine and bovine rotaviruses are regarded as important

animal pathogens because of their economic impact in the

livestock as well as of their potential source of heterologous

rotavirus infections in humans. In pigs, the main G

serotypes are G3, G4, G5, and G11 (Estes, 2001), although

human-like types G1 and G9 (Ciarlet and Liprandi, 1994;

Racz et al., 2000; Santos et al., 1999) and bovine-like types

G6, G8, and G10 (Gouvea et al., 1994b; Martella et al.,

2001; Pongsuwanna et al., 1996; Racz et al., 2000;

Winiarczyk et al., 2002) have been occasionally reported

(Table 1). The most common porcine rotavirus P types are

P2B[6] and P9[7], Gottfried- and OSU-like, respectively.

Additional P genotypes, P[13] (MDR-13-like), P[19] (4F-

like), and P[23] (A34-like), have been identified but their

epidemiological significance is unknown (Burke et al.,

1994; Estes, 2001; Gouvea et al., 1994a; Huang et al., 1993;

Liprandi et al., 2003; Zaberezhny et al., 1994). In addition,

typical human P genotypes P[8] and P[6] (M37-like) (Racz

et al., 2000; Santos et al., 1999) and bovine P genotypes

P[1], P[5], and P[11] have been sporadically detected by

PCR genotyping in pigs (Gouvea et al., 1994b; Martella et

al., 2001; Pongsuwanna et al., 1996). On the whole,

additional epidemiological data are necessary to completely

understand the importance of the rarely detected rotavirus P

types in the porcine population.

In the present study, we report the molecular character-

ization of a non-typeable P[23] porcine rotavirus (PoRV),

which displayed an unusual VP7 gene, not classifiable into

any of the well-established G serotypes.

Results

Sequence analysis of the VP7

The basic structure of the VP7-encoding gene of the

PoRV strain 34461-4 was 1062 nucleotide long, with two

in-phase open reading frames (ORFs) beginning at nucleo-

tides 49 and 136, and a single TAG codon at nucleotides

1027, coding for a VP7 protein of 326 or 297 aa,

V. Martella et al. / Virology 337 (2005) 111–123 113

respectively. Nucleotide (nt) sequence identity to the

rotavirus VP7 genes available in the databases was

constantly <82% (data not shown). When comparing the

complete inferred aa sequence with those of rotavirus strains

representative of all 15 G serotypes (Table 2), the highest aa

identity was found to the human G2 prototype strains

HN126 (88.88%) and DS-1 (88.59%). Amino acid identity

to other human G2 rotaviruses ranged from 87.34% to

85.49%, while aa identity to non-G2 rotavirus strains ranged

from 58.33% to 75.92%. The VP7 protein of PoRV strain

34461-4 showed a potential N-linked glycosylation site at aa

69, which is unusually conserved among most rotavirus

strains (Ciarlet et al., 1995, 1997b; Nishikawa et al., 1989).

However, the other potential glycosylation sites at aa

positions 146 and 238, commonly found in G2 rotavirus

strains, were absent due to substitutions at aa positions 146

(NYT) and 238 (NYS) (Fig. 1). When comparing the VP7

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

208–223), and F (aa 235–242) (Ciarlet et al., 1997b; Dyall-

Smith et al., 1986; Kobayashi et al., 1991; Nishikawa et al.,

1989) to the reference serotype G2 strain DS-1, a high

number of aa substitutions were observed. The substitutions

observed included aa positions: region A, 87 (AYT), 96

(DYT), 99 (EYT); region B, 143 (RYK), 146 (NYT);

region C, 211 (DYE), 216 (IYT), 220 (SYQ); and region

Table 2

Amino acid comparison (% aa identity) of the VP7 of porcine strain 34461-

4 with those of well-established G serotypesa and with a selection of G2

rotavirusesa

Strain (origin) VP7 serotype VP4 genotype 34461-4 aa %

KU (human) 1 1A[8] 75.62

DS-1 (human) 2 1B[4] 88.59

S2 (human) 2 1B[4] 87.34

HN-126 (human) 2 ? 88.88

7PE/89 (human) 2 1B[4] 85.49

BS3585/99human) 2 2A[6] 85.49

RRV (simian) 3 5B[3] 75.6

Y0 (human) 3 1A[8] 75.01

ST3 (human) 4 2A[6] 73.15

OSU (porcine) 5 9[7] 75.62

NCDV (bovine) 6 6[1] 75.92

Ch2 (avian) 7 [17] 58.33

B37 (human) 8 ? 75.01

116E (human) 9 8[11] 75.82

61A (bovine) 10 7[5] 74.07

YM (porcine) 11 9[7] 75.3

L26 (human) 12 1B[4] 74.38

L338 (equine) 13 [18] 72.84

CH3 (equine) 14 4[12] 73.76

Hg18 (bovine) 15 [21] 73.14

Predicted G serotype on the basis of sequence identity.

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); NCDV (M12394); Ch2 (X56784); B37

(J04334); 116E (L14072); 61A (X53403); YM (M23194); L26

(M58290); L338 (D13549); CH3 (D25229), Hg18 (AF237666).

F, 238 (NYS), 241 (IYT) (Fig. 1). The changes 87-AYT

and 216-IYT were not unique to strain 34461-4 since they

were shared by other G2 strains. When considering only the

VP7 antigenic regions, aa sequence conservation to G2

rotaviruses was constantly <83.4%.

The VP7 of the PoRV strain 34461-4, although

displaying the highest genetic relatedness to G2 HRVs,

contained several nt mismatches affecting the primer

binding sites recognized by the G2 type-specific primers,

aCT2 and 9T1-2 (Das et al., 1994; Gouvea et al., 1990),

thus preventing its genetic characterization as a G2 strain.

There were 11/25 nucleotide mismatches in the aCT2

primer-binding site (nt 411–425) and 7/20 nucleotide

mismatches in the 9T1-2 primer-binding site, many of

them at the 3V primer end that is critical for primer

extension (data not shown). Primer aCT2 was designed

over the hypervariable region C, and thus the variation

accumulating in the primer binding site reflects the aa

variations observed in region C of the VP7 of strain

34461-4.

Sequence analysis of the VP4

To determine the P genotype of the PoRV strain 34461-4,

we sequenced the VP8* trypsin-cleavage product of VP4,

which includes the region spanning aa 71 and 204 that

correlates with VP4 genotype specificity (Larralde and

Gorziglia, 1992; Larralde et al., 1991). The deduced aa

sequence of the VP8* of the PoRV strain 34461-4 was

compared with those of rotavirus strains representative of all

23 P genotypes (data not shown and Table 3). As with most

rotavirus strains, the potential trypsin-cleavage sites at

residues 231, 241, and 247, and the prolines at residues

68, 71, 225, and 226 were conserved in the VP8* of PoRV

strain 34461-4. The highest degree of nt (87.01%) and aa

(95.94%) identity was found to the PoRV strain A34,

P14[23],G5. With the remaining 22 P genotypes, the aa

identity of the VP8* of the PoRV strain 34461-4 ranged

from 77% (Hu/69M, P4[10]) to 39% (Bo/B223, P8[11]).

The high aa identity to the prototype PoRV strain A34

allowed the unequivocally classification of PoRV strain

34461-4 as genotype P[23] (Table 2).

Phylogenetic analysis of the VP7 and VP4

Phylogenetic analyses of the deduced aa sequence of

the VP7 of the strain 34461-4 provided a molecular basis

for its relatedness to G2 rotaviruses (Fig. 2A). In the

phylogenetic tree, the VP7 of the PoRV strain 34461-4

clustered close to those of all G2 human rotavirus strains

analyzed, suggesting a status of ancestry of strain 34461-

4 with respect to the G2 human strains. However, PoRV

strain 34461-4 is clearly divergent from the other G2

human strains. In the VP8* phylogenetic tree, PoRV

strain 34461-4 was grouped with the PoRV strain A34,

P14[23] (Fig. 2B).

Fig. 1. Deduced amino acid sequence of the VP7 protein of the porcine strain 34461-4 and a selection of G2 rotaviruses and of strains representative of well-

established G types. The VP7 antigenic regions A, B, C, and F are indicated. The glycosylation site NST (aa 69–71) is indicated by black triangles (4).

V. Martella et al. / Virology 337 (2005) 111–123114

Table 3

Amino acid comparison (% aa identity) of the VP8* of the porcine strain

34461-4 with well-established P genotypesa

Strain (origin) P Genotype P Serotype VP8* aa 1 to

247 34461-4

A5 (bovine) 1 6 76.12

SA11 (simian) 2 5B 75.22

RRV (simian) 3 5B 76.12

HCR3a (human) 3 5A 75.61

L26 (human) 4 1B 61.71

UK (bovine) 5 7 65.76

M37 (human) 6 2A 61.26

Gottfried (porcine) 6 2B 65.31

OSU (porcine) 7 9 73.42

KU (human) 8 1A 60.36

K8 (human) 9 3 55.85

69M (human) 10 4 77.02

B223 (bovine) 11 8 38.73

H-2 (equine) 12 4 72.97

MDR-13 (porcine) 13 n.d. 63.06

PA169 (human) 14 11 56.75

Lp14 (ovine) 15 n.d. 75.22

EW (murine) 16 10 63.96

993/83 (bovine) 17 n.d. 40.09

L338 (equine) 18 n.d. 69.81

Mc323 (human) 19 12 63.09

EHP (murine) 20 13 75.22

Hg18 (bovine) 21 n.d. 67.11

160/01 (lapine) 22 n.d. 61.71

A34 (porcine) 23 14 95.94

N.d.: not determined.a GenBank accession nos. of VP4 genes: A5 (D13395); SA11 (M23188);

RRV (M18736); HCR3a (L21666); L26 (M58292); UK (M22306); M37

(L20887); Gottfried (M33516); OSU (X13190); KU (M21014); K8

(D90260); 69M (M60600); B223 (D13394); H-2 (L04638); MDR-13

(L07886); PA169 (D14724); EW (U08429); 993/83 (D16352); L338

(D13399); Mc323 (D38052); EHP (U08424); Hg18 (AF237665); 160/01

(AF526376); A34 (AY174094).

V. Martella et al. / Virology 337 (2005) 111–123 115

Analysis of the VP6, NSP4, and NSP5/6 genes

Although it was not possible to visualize the dsRNA by

PAGE, by PCR amplification PCR amplicons of about 700

and 600 bp were obtained for the 10th and 11th genome

segments, respectively, that are consistent with a long

pattern of migration (long e-type).

Comparative analysis of the deduced amino acid

sequences of the fragment of VP6 (amino acids 241 to

367) known to correlate with SG specificity (Iturriza-

Gomara et al., 2002) allowed characterization of strain

34461-4 as genogroup I. The highest aa sequence identity

(95.5%) was found to the PoRV strain OSU (P[7],G5 and

SGI). The residues 305 and 315 were Ala and Ile,

respectively, a pattern that is consistent with SGI specificity.

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. 3A), with exception of the

porcine strains A411, that is SGII and that has Asn-305

instead of Ala.

A fragment (680 bp) of the NSP4 gene was obtained. By

comparison with the databases, the NSP4 sequence of strain

34461-4 was found to be 94.9 nt identical to PoRV strain

OSU and 96.1% aa identical to the PoRV strain A34, within

genogroup B (Wa-like), that includes both PoRVs and HRVs

(Fig. 3B).

The NSP5/6 sequence of strain 34461-4 was partially

determined (590 bp). The best match (97.9% nt and 97.4%

aa) in the databases was found to the G1 human strain

87H134. By phylogenetic analysis, strain 34461-4 was

grouped along with a clade of human and porcine rotavirus

strains (Fig. 3C).

Discussion

The acquisition of epidemiological data by molecular

characterization of cultivatable and non-cultivatable rotavi-

rus strains is quickly providing new insights into the

diversity of rotaviruses, and eventually into the mechanisms

driving rotavirus evolution. The antigenic variation seen in

rotaviruses appears to have evolved in a similar fashion to

that of the hemagglutinin and neuraminidase genes of

influenza A viruses: antigenic shift (resulting from gene

reassortment) and antigenic drift (resulting from the

successive accumulation of mutations) (Ciarlet et al.,

1997b). Several examples of genomic exchanges between

human and animal rotaviruses have been reported, suggest-

ing that the possibility of genetic reassortment during mixed

infections may easily allow for repeated and, sometimes,

stable introduction of rotavirus gene alleles of animal origin

into the allelic pool of human rotaviruses and vice versa. In

theory, new serotypes can emerge by the succession of

accumulation of mutations due to the host selective immune

pressure. Conversely, strains belonging to different G

serotypes may cluster separately but as mutations on VP7

accumulate, different interserotypic strains may start to

‘‘approach’’ another serotype (Ciarlet et al., 1997b). For

instance, up to 87.8–90.5% aa similarity may be described

between rotavirus strains belonging to the distinct G5 and

G11 serotypes, that are hypothesized to share a relatively

recent common ancestor (Timenetsky et al., 1997).

Herewith we describe the identification of an unusual,

non-typeable, rotavirus strain from piglets in Spain, resem-

bling G2 human rotaviruses, with P[23] VP4 genotype, long

e-type and SGI. Long e-type and SGI are commonly

associated in animal rotaviruses, while this association is

uncommon in human rotavirus strains (Kapikian et al.,

2001). Also, analysis of the 10th and 11th RNA segment

revealed close identity to strains of porcine and human

origin, respectively. Human G2 rotaviruses have been often

associated with more severe clinical forms of children

gastroenteritis than the other common serotypes circulating

in humans, and to infection in older children and adults,

because of their high genetic/antigenic diversity from the

other human rotavirus strains (Bern et al., 1992; Cascio et

al., 2001; Mikami et al., 2004; Yolken et al., 1978). Indeed,

unlike G1, G3, and G4 human rotavirus strains, G2 rotavirus

Fig. 2. Phylogenetic tree of the VP7 and VP8* of the porcine strain 34461-4. (A) VP7 tree displaying the relationships among a selection of strains

representative of well-established G types. The dendrogram is drawn to scale and rooted using the human strain Wa (P1A[8],G1). The arrow highlights the

ancestry status of the strain with 34461-4 with respect to G2 rotaviruses (B) VP8* tree displaying the relationships among strains representative of all the VP4

genotypes recognized to date. The dendrogram is drawn to scale and rooted using the avian-like bovine strain Bo/993/83 (P[17],G7).

V. Martella et al. / Virology 337 (2005) 111–123116

strains are usually linked to P1B[4] VP4 type, SGI VP6

specificity, and possess short electropherotypes (Kapikian et

al., 2001). Although G2 strains are commonly found in

humans, G2 rotaviruses have been never identified in

animals (Estes, 2001) (Table 1). Bellinzoni et al. (1990)

reported in the early 1990s the identification of a PoRV,

V. Martella et al. / Virology 337 (2005) 111–123 117

V. Martella et al. / Virology 337 (2005) 111–123118

V. Martella et al. / Virology 337 (2005) 111–123 119

strain C134, classified as G2 because of one-way reaction to

serotype G2 (and another to serotype G5) based on

neutralization tests, and reactivity with a serotype G2

VP7-specific MAb. However, further antigenic and molec-

ular analyses of strain PoRV C134 confirmed the G5

serotype specificity of PoRV C134 strain (Ciarlet et al.,

1995, 1996).

When considering the deduced aa sequence, strain

34461-4 displayed the highest aa identity to human G2

rotaviruses. However, the degree of aa sequence variation to

G2 strains (11.1–14.5%) did not allow to classify unam-

biguously strain 34461-4 as a serotype G2 rotavirus.

Detailed analysis of the VP7 antigenic regions A, B, C,

and F revealed several aa substitutions, and some of these

resulted in the elimination of two of the three potential

glycosylation sites that are usually conserved across the G2

strains. Glycosylation of the VP7 has been linked to

blocking or alteration of recognition by antibodies (Caust

et al., 1987). Lazdins et al. (1995) demonstrated that the

presence of an additional glycosylation site in the VP7 of a

G2 reassortant (RV-5 � SA11, P[2],G2), selected with a G2-

specific monoclonal antibody, was able to prevent binding

by a panel of G2-specific antibodies, though the additional

glycosylation did not block neutralization by hyperimmune

antiserum to the original RV-5 virus. Thus, the common

progenitor of G2 HRVs and strain 34461-4 may have lost

the two glycosylation sites in the VP7 during adaptation to

the porcine host, or G2 HRVs may have acquired those sites

to modify their antigenic profile during evolution and

adaptation to the human host. Anyway, branching of the

viruses in the VP7 phylogenetic tree supports more a status

of ancestry of strain 34461-4 with respect to G2 rotaviruses

than vice versa, as strain 34461-4 appears to be a precursor

and not a descendant of the G2 strains (Fig. 2A).

When inspecting the VP7 antigenic regions of strain

34461-4, several aa substitutions were either close to or in

those residues that have been mapped as critical for

maintenance of G2-specific epitopes, i.e., 146-NYT, 211-

DYE, 216-IYT, and 96-DYT. Differences at residues 147,

213, and 217 correlate with the loss of specific epitopes

recognized by G2 monotype-specific MAbs (Coulson et al.,

1996). Also, loss of reactivity to G2-specific MAbs due to a

unique change at aa 96 (DYN) has been observed in some

G2 strains (Iturriza-Gomara et al., 2001), but not in others

G2 strains with the same anti-G2 MAbs (Page and Steele,

2004). Accordingly, sequence analysis of the VP7 of the

34461-4 strain provides strong evidence for the genetic/

Fig. 3. Phylogenetic tree of the VP6, NSP4, and NSP5/6 genes of the porcine strai

379-bp cDNA fragments of the VP6 genes of animal and human rotavirus str

serologically determined SG are shown. For the other strains, the species of or

bootstrap values (>85%) are shown at the branching point of each cluster. (B) NSP

strains representative of the various NSP4 genetic groups. The branch of the mu

prototypes of the NSP4 genogroups A, B, and C (strains KUN, Wa, and AU-1,

relationships among a selection of animal and human strains. The tree is inferred

bovine; mu, murine; eq, equine; si, simian; ov, ovine; fe, feline; ca, canine; la, la

antigenic diversity of strain 34461-4 from other G2

rotaviruses.

Usually, strains sharing >90% aa identity in the VP7 are

considered to belong to the same G serotype, while strains

belonging to different G serotypes share <85% aa con-

servation. Thus, the G serotype specificity of strains sharing

a VP7 aa identity >85% but <90% with those of known

serotypes is difficult to assign based on aa sequence analysis

alone because they may be considered as ‘‘borderline’’

rotavirus G genotypes. Rotavirus strains of a borderline

genotype could represent ancestor-like strains, remaining in

a sort of evolutionary stasis, as it has been observed for

avian influenza viruses (Webster, 2002), or strains that have

greatly diverged from a particular rotavirus G serotype due

to restricted ecological settings. Timenetsky et al. (1997)

identified a human strain, IAL28, with dual serotype

specificity (G5 and G11) with an aa conservation in the

range of 90–92% with serotypes G11 and G5, respectively.

Similarly, Racz et al. (2000) isolated an atypical porcine

strain, ICB2185, sharing about 90% aa conservation with

G4 rotaviruses but not recognized by G4-specific MAbs.

Therefore, the detection of strains with borderline genotype

would support the hypothesis that a continuum of declina-

tion of rotavirus gene alleles exist. Due to its genetic

relatedness to G2 rotaviruses, PoRV strain 34461-4 would

represent an example of borderline G2 VP7 genotype. The

detection and successful adaptation to cell culture of 34461-

4-like strains will help elucidate whether this novel VP7

genotype is antigenically distinct from G2 rotaviruses.

Hoshino et al. (1995) reported that the substitution by

selective genetic reassortment of genome segment 9 (coding

for VP7) of a virulent porcine strain (SB-1A) with the

cognate genome segment 9 of the human rotavirus G2 strain

DS-1 (reassortant 18-1, SB-1A � DS-1) attenuated the

virulence of the porcine strain SB-1A in gnotobiotic piglets.

Since a G2-like rotavirus strain (34461-4) infected and

induced disease in piglets, it is difficult to understand the

molecular basis of the virulence attenuation of the 18-1

reassortant (Hoshino et al., 1995). Accordingly, different

modulation and expression of the virulence phenotype can

be explained by the VP7 aa divergence observed between

the DS-1 and strain 34461-4, and/or by differences in the

genomic backgrounds of strain SB-1A and strain 34461-4.

The VP8* of the porcine strain 34461-4 shared the highest

nt and aa sequence identity, 87.01% and 95.94%, respec-

tively, with that of the porcine strain A34, P14[23],G4,

isolated from a diarrheic piglet in Venezuela in the mid-

n 34461-4 (shown in boldface type). (A) Phylogenetic tree constructed from

ains. For reference strains, the strain designation, species of origin, and

igin and, if available, the SG antigenic specificity is reported. Significant

4 tree displaying the relationships among a selection of animal and human

rine (genogroup D) and avian (genogroup E) strains are not to scale. The

respectively) are boldfaced and underlined. (C) NSP5 tree displaying the

from the nucleotide alignment. Abbreviations: hu, human; po, porcine; bo,

pine.

V. Martella et al. / Virology 337 (2005) 111–123120

1980s (Liprandi et al., 2003). During the epidemiological

survey conducted in Spain, in 2001–2003, additional P[23]

strains were identified (Martella et al., unpublished),

suggesting that the P14[23] VP4 specificity is present in

European pigs and that such VP4 type plays an important

epidemiological role in pigs, thus not representing a sporadic

finding.

In conclusion, we have identified a PoRV strain that

displays an unusual combination of VP7 and VP4 gene

alleles. A novel rotavirus VP7 gene allele was described in

pigs, providing additional evidence for the high genetic/

antigenic heterogeneity of group A rotaviruses and enlarg-

ing the knowledge on rotavirus diversity. The creation of a

baseline defining the distribution of G and P types in the

various animal species will be extremely helpful to under-

stand the global ecology of rotaviruses.

Materials and methods

Origin of the virus and virological examinations

The PoRV strain 34461-4 was identified during an

epidemiological survey in the region of Saragoza, Spain,

in 2003. A total of 32 rotavirus-positive samples (32.6%)

were identified from 98 rectal swabs of piglets affected with

diarrhea. Rotavirus antigen was detected in the rectal swab

by an indirect immunoperoxidase (IPX) technique. Briefly,

mucosal cells were obtained by the washing rectal swab of

the animal in PBS–trypsin 0.1%. Cell suspensions were

washed with PBS and fixed onto multi-sphere slides. A

monoclonal antibody, MoAb851 (Chemicon International

Inc.), specific for the inner capsid protein VP6 of

rotaviruses, was used at 1:100 dilution to detect rotavirus

antigen, and an anti-mouse IgG (Fab-specific) conjugated to

peroxidase (Sigma-Aldrich, Milano, Italy) was used to

reveal the antibody–antigen complex. The reaction was

developed with AEC-DMF (Sigma-Aldrich, Milano, Italy),

and counter-stained with Mayer’s hematoxylin. Despite

numerous attempts, it was not possible to adapt the strain

to cultivation on African green monkey kidney (MA-104)

cells. Because of the low viral amounts in the sample, it was

not possible to visualize the dsRNA by electrophoresis in

polyacrylamide gel (PAGE) and silver staining.

RNA extraction, PCR genotyping of the VP7 and VP4 genes

Viral dsRNA was 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 Italy,

Monza), while PCR amplification was carried out with

AmpliTaq Gold\ DNA polymerase (Applied Biosystems

Italy, Monza).

As neither the VP7 nor the VP4 gene of strain 34461-4

was recognized by any of the G- and P-type-specific primers

used, the sequence of the VP7 and VP8* genes was

determined. The VP7 gene was reverse transcribed and

amplified using primer Beg9 (Gouvea et al., 1990) and

primer End9deg (Martella et al., 2003b). The VP8* subunit

of the VP4, the connecting peptide, and the N terminus of

the VP5* subunit of VP4 (876 bp) were reverse transcribed

and amplified with primer pair Con2-Con3 (Gentsch et al.,

1992). The G-typing was performed using different pools of

primers specific for human and animals G types (G1–G6,

G8–G11), as previously described (Das et al., 1994;

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

Winiarczyk et al., 2002). The VP4 characterization was

performed using different pools of primers specific for

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

(Gentsch et al., 1992; Gouvea et al., 1994a; Winiarczyk et

al., 2002).

Amplification of the VP6, NSP4, and NSP5/6 genes

The nearly full-length NSP4 gene was amplified with

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

NSP5/NSP6 full-length gene was amplified with a primer

pair described by Mohan and Atreya (2001). The VP6

genogroup, predictive of the VP6 subgroup specificity, was

determined by amplification of a 379-bp fragment, spanning

from amino acids 241–367 of the VP6, with primer pair

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

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 Biosystems Division). Sequences were assembled

and analyzed using Bioedited software package (Depart-

ment of Microbiology, North Carolina State University,

USA) (Hall, 1999) and NCBI’s and EMBL’s analysis tools.

GenBank accession number AY766085 and AY768809

were assigned to the VP7 and VP4 of the porcine strain

34461-4, respectively. The sequences of the VP6, NSP4,

and NSP5/6 genes of strain 34461-4 are freely available

upon request.

Phylogenetic and molecular evolutionary analyses were

conducted using MEGA version 2.1 (Arizona State Uni-

versity, USA) (Kumar et al., 2001). Phylogenetic trees based

on the VP7 gene products 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 CEGBA

(Centro di Eccellenza di Genomica in campo Biomedico

V. Martella et al. / Virology 337 (2005) 111–123 121

ed Agrario), from Ministero della Sanita (Progetto Final-

izzato 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’’). We thank Filomena Cariola, Caterina Nanna,

Nicoletta Tutino, and Donato Narcisi for their expert

technical assistance. We are extremely grateful to

Professor Leland Eugene Carmichael for continued

encouragement throughout our studies.

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