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Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain
Virology 346 (2006) 301 – 311 www.elsevier.com/locate/yviro
Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain V. Martella a,*, M. Ciarlet b, K. Ba´nyai 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
c
Department of Animal Health and Well-being, University of Bari, Valenzano, Bari, Italy b Biologics – Clinical Research, Merck and Co., Inc., Blue Bell, PA 19422, USA Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pe´cs, Hungary d Department of Hygiene and Microbiology, University of Palermo, Palermo, Italy e 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 fulllength 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 icosahedral 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 * 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: [email protected] (V. Martella). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.11.001
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 nonstructural 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 proteasesensitive) and G (for glycoprotein) serotypes (Ciarlet and Estes,
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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.
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 rotavirus 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 (Ba´nyai 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 Go´mara 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 reassortment (Nakagomi and Nakagomi, 2002; Palombo, 2002). Thus, studying animal rotaviruses is paramount to acquire a more indepth understanding of rotavirus evolution and ecology, and rotavirus surveillance programs have been established in
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
Table 1 Distribution of rotavirus P genotypes across the various animal species Genotype Serotype Lineage Species
1 6
Man o Pig Monkey Cattle . Goat/Sheep . Horse Dog Cat Mouse Rabbit Birds
2 3 3 4 5 5A 5A 5B 1B 7
.
o
.
. o
. .
o o
. .
6 6 6 6 6 2A 2B 2C I II III IV V
. o . . .
o
o
7 9
. .
8 9 1A 3
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 4 8 4 11 10 12 13 14
. . . . o
o
o
. . .
.
. o o
. .
o
.
. .
o
.
. .
.
. .
. .
.
Full circle indicates that the P type is epidemiologically relevant for the species, or that it has been described only in that species to date. Open circle indicates sporadic description or low epidemiological relevance.
V. Martella et al. / Virology 346 (2006) 301 – 311
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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.
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V. Martella et al. / Virology 346 (2006) 301 – 311
Fig. 1 (continued ).
V. Martella et al. / Virology 346 (2006) 301 – 311
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 characterization to determine the molecular features of the noncultivable 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 Go´ mara 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 representative 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
305
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) S2 (human) 34461-4 (porcine) RRV (simian) Y0 (human) ST3 (human) OSU (porcine) A46 (porcine) JL94 H1 (equine) NCDV (bovine) Ch2 (avian) B37 (human) 116E (human) 61A (bovine) YM (porcine) L26 (human) L338 (equine) CH3 (equine) Hg18 (bovine)
1 2 2-like 3 3 4 5 5 5 5 6 7 8 9 10 11 12 13 14 15
1A[8] 1B[4] [23] 5B[3] 1A[8] 2A[6] 9[7] [13] [7] 9[7] 6[1] [17] ? 8[11] 7[5] 9[7] 1B[4] [18] 4[12] [21]
79.31 74.60 75.54 84.95 84.32 74.60 95.61 95.29 95.90 95.61 80.87 58.30 78.99 79.93 79.62 88.40 79.62 76.80 77.74 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).
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 MDR13, 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.
306 V. Martella et al. / Virology 346 (2006) 301 – 311 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 et al. / Virology 346 (2006) 301 – 311 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) SA11 (simian) RRV (simian) K9 (canine) GRV DS1 UK (bovine) Gottfried (porcine) M37 (human) OSU (porcine) KU (human) K8 (human) 69M (human) KK3 (bovine) F123 (equine) MDR-13 (porcine) A46 (porcine) Mc35 (human) Lp14 (ovine) EDIM (murine) 993/83 (bovine) L338 (equine) Mc345 (human) EHP (murine) Hg18 (bovine) 160/01 (lapine) A34 (porcine) TUCH (simian) Dhaka6 (human)
1 2 3 3 3 4 5 6 6 7 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21 22 23 24 25
6 5B 5B 5A n.d.
69.60 68.76 70.08 67.88 73.60 59.08 60.84 56.44 55.12 72.72 57.32 55.56 67.00 38.84 67.00 73.60 70.06 58.64 67.88 62.16 37.52 69.64 57.32 67.00 63.92 72.72 68.76 69.20 65.82
80.24 80.76 81.28 80.76 83.33 72.05 73.35 72.31 72.05 81.41 70.75 68.77 81.50 59.70 78.94 85.96 86.05 70.10 79.98 76.60 61.52 77.51 73.35 78.03 77.38 – – 81.28 –
7 2B 2A 9 1A 3 4 8 4 n.d. n.d. 11 n.d. 10 n.d. n.d. 12 13 n.d. n.d. 14 n.d. n.d.
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).
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). Therefore, 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 representative 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 PCRgenotyping and sequence analysis. Only one sample out of 35 untypeable samples was recognized by primer MV26, indicating that the new P genotype is rare, at least in the Italian porcine herds.
307
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; Ba´nyai 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 nextgeneration (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 diversification 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 understanding 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,
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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.
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 134/0415 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 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, 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
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) Con2* (872 – 891)a 170* (2338 – 2362) Inv1 (567 – 592) 176f (793 – 819) 184f (943 – 967) Ge17R* (321 – 344) Ge13R* (1476 – 1499) Ge15 (1904 – 1925) MV26* (564 – 588)
ggc tat aaa atg gct tcg ctc att tat aga ca att tcg gac cat tta taa cc ggt cac awc ctc tag mmr ytr ctt a agg tca ata cac aac aac caa cta tg tgg aaa gaa atg caa tat aac aga gat gtc ata gcg cat acc acg tgt tca g acg ttt ggt tcg act aaa ata atc tgt ctc tca aga tct tgt cta acg att ttg atg ata ttt cag ctg c ggt tgt tgt gta ttg acc tga c
Antisense primers (*) are reversed and complementary to the 5V– 3V nt sequence. Nucleotide position is determined on the sequence of strain 134/04-15. a Gentsch et al. (1992).
V. Martella et al. / Virology 346 (2006) 301 – 311
press, 2005b; Winiarczyk et al., 2002). However, the VP4 gene of PoRV strain 134/04-15 was not recognized by any of the Ptype-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 endspecific 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 Go´mara 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 Biosystems 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 genogroupspecific 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 assistance. We are extremely grateful to Professor Leland Eugene Carmichael for the continued encouragement throughout our studies.
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Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain V. Martella a,*, M. Ciarlet b, K. Ba´nyai 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
c
Department of Animal Health and Well-being, University of Bari, Valenzano, Bari, Italy b Biologics – Clinical Research, Merck and Co., Inc., Blue Bell, PA 19422, USA Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pe´cs, Hungary d Department of Hygiene and Microbiology, University of Palermo, Palermo, Italy e 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 fulllength 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 icosahedral 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 * 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: [email protected] (V. Martella). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.11.001
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 nonstructural 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 proteasesensitive) and G (for glycoprotein) serotypes (Ciarlet and Estes,
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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.
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 rotavirus 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 (Ba´nyai 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 Go´mara 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 reassortment (Nakagomi and Nakagomi, 2002; Palombo, 2002). Thus, studying animal rotaviruses is paramount to acquire a more indepth understanding of rotavirus evolution and ecology, and rotavirus surveillance programs have been established in
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
Table 1 Distribution of rotavirus P genotypes across the various animal species Genotype Serotype Lineage Species
1 6
Man o Pig Monkey Cattle . Goat/Sheep . Horse Dog Cat Mouse Rabbit Birds
2 3 3 4 5 5A 5A 5B 1B 7
.
o
.
. o
. .
o o
. .
6 6 6 6 6 2A 2B 2C I II III IV V
. o . . .
o
o
7 9
. .
8 9 1A 3
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 4 8 4 11 10 12 13 14
. . . . o
o
o
. . .
.
. o o
. .
o
.
. .
o
.
. .
.
. .
. .
.
Full circle indicates that the P type is epidemiologically relevant for the species, or that it has been described only in that species to date. Open circle indicates sporadic description or low epidemiological relevance.
V. Martella et al. / Virology 346 (2006) 301 – 311
303
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.
304
V. Martella et al. / Virology 346 (2006) 301 – 311
Fig. 1 (continued ).
V. Martella et al. / Virology 346 (2006) 301 – 311
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 characterization to determine the molecular features of the noncultivable 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 Go´ mara 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 representative 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
305
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) S2 (human) 34461-4 (porcine) RRV (simian) Y0 (human) ST3 (human) OSU (porcine) A46 (porcine) JL94 H1 (equine) NCDV (bovine) Ch2 (avian) B37 (human) 116E (human) 61A (bovine) YM (porcine) L26 (human) L338 (equine) CH3 (equine) Hg18 (bovine)
1 2 2-like 3 3 4 5 5 5 5 6 7 8 9 10 11 12 13 14 15
1A[8] 1B[4] [23] 5B[3] 1A[8] 2A[6] 9[7] [13] [7] 9[7] 6[1] [17] ? 8[11] 7[5] 9[7] 1B[4] [18] 4[12] [21]
79.31 74.60 75.54 84.95 84.32 74.60 95.61 95.29 95.90 95.61 80.87 58.30 78.99 79.93 79.62 88.40 79.62 76.80 77.74 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).
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 MDR13, 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.
306 V. Martella et al. / Virology 346 (2006) 301 – 311 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 et al. / Virology 346 (2006) 301 – 311 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) SA11 (simian) RRV (simian) K9 (canine) GRV DS1 UK (bovine) Gottfried (porcine) M37 (human) OSU (porcine) KU (human) K8 (human) 69M (human) KK3 (bovine) F123 (equine) MDR-13 (porcine) A46 (porcine) Mc35 (human) Lp14 (ovine) EDIM (murine) 993/83 (bovine) L338 (equine) Mc345 (human) EHP (murine) Hg18 (bovine) 160/01 (lapine) A34 (porcine) TUCH (simian) Dhaka6 (human)
1 2 3 3 3 4 5 6 6 7 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21 22 23 24 25
6 5B 5B 5A n.d.
69.60 68.76 70.08 67.88 73.60 59.08 60.84 56.44 55.12 72.72 57.32 55.56 67.00 38.84 67.00 73.60 70.06 58.64 67.88 62.16 37.52 69.64 57.32 67.00 63.92 72.72 68.76 69.20 65.82
80.24 80.76 81.28 80.76 83.33 72.05 73.35 72.31 72.05 81.41 70.75 68.77 81.50 59.70 78.94 85.96 86.05 70.10 79.98 76.60 61.52 77.51 73.35 78.03 77.38 – – 81.28 –
7 2B 2A 9 1A 3 4 8 4 n.d. n.d. 11 n.d. 10 n.d. n.d. 12 13 n.d. n.d. 14 n.d. n.d.
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).
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). Therefore, 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 representative 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 PCRgenotyping and sequence analysis. Only one sample out of 35 untypeable samples was recognized by primer MV26, indicating that the new P genotype is rare, at least in the Italian porcine herds.
307
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; Ba´nyai 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 nextgeneration (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 diversification 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 understanding 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,
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V. Martella et al. / Virology 346 (2006) 301 – 311
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.
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 134/0415 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 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, 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
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) Con2* (872 – 891)a 170* (2338 – 2362) Inv1 (567 – 592) 176f (793 – 819) 184f (943 – 967) Ge17R* (321 – 344) Ge13R* (1476 – 1499) Ge15 (1904 – 1925) MV26* (564 – 588)
ggc tat aaa atg gct tcg ctc att tat aga ca att tcg gac cat tta taa cc ggt cac awc ctc tag mmr ytr ctt a agg tca ata cac aac aac caa cta tg tgg aaa gaa atg caa tat aac aga gat gtc ata gcg cat acc acg tgt tca g acg ttt ggt tcg act aaa ata atc tgt ctc tca aga tct tgt cta acg att ttg atg ata ttt cag ctg c ggt tgt tgt gta ttg acc tga c
Antisense primers (*) are reversed and complementary to the 5V– 3V nt sequence. Nucleotide position is determined on the sequence of strain 134/04-15. a Gentsch et al. (1992).
V. Martella et al. / Virology 346 (2006) 301 – 311
press, 2005b; Winiarczyk et al., 2002). However, the VP4 gene of PoRV strain 134/04-15 was not recognized by any of the Ptype-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 endspecific 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 Go´mara 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 Biosystems 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 genogroupspecific 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 assistance. We are extremely grateful to Professor Leland Eugene Carmichael for the continued encouragement throughout our studies.
309
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