Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2

Rodrigo Martins Soares1, Adriana Cortez1, Marcos Bryan Heinemann2, Sidnei Myioshi Sakamoto3, Vanderlei Geraldo Martins4, Maurício Bacci, Jr4, Flora Maria de Campos Fernandes5 and Leonardo José Richtzenhain1

1 Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Rua Professor Dr Orlando Marques de Paiva, 87 CEP 05508-900 Cidade Universitária, São Paulo-SP, Brazil
2 Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, Brasília-DF, Brazil
3 Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Biomédicas, Universidade Federal de São Paulo, São Paulo-SP, Brazil
4 Centro de Estudos de Insetos Sociais, Departamento de Bioquímica, Instituto de Biociências, Universidade Estadual Paulista campus Rio Claro-SP, Brazil
5 Laboratório de Ictiogenética, Instituto de Biociências, Universidade de São Paulo, São Paulo-SP, Brazil

Correspondence
Rodrigo Soares
rosoares{at}usp.br


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Porcine parvovirus (PPV) is considered an important cause of reproductive failure in swine. Embryonic death and resorption, mummified foetuses and stillbirth, as a result of prolonged farrowing intervals, are typical clinical signs of PPV-induced reproductive failure (Mengeling et al., 2000).

The icosahedral non-enveloped capsid of PPV is formed by multiple copies of VP1, VP2 and VP3 (Molitor et al., 1983). VP1 and VP2 are proteins translated in the same frame but from different start codons and VP3 is a product of VP2 proteolytic cleavage (Molitor et al., 1983; Ránz et al., 1989; Bergeron et al., 1993).

The PPV genome, a single-stranded DNA (minus-strand) comprising about 5000 nt, contains two large open reading frames (ORFs), both located in the same frame of the complementary strand. The left ORF encodes the non-structural protein NS1 and the right ORF encodes the three capsid proteins (Ránz et al., 1989; Bergeron et al., 1993).

In spite of the high level of conservation in the parvovirus sequence, PPV strains can be distinguished by their different pathogenicity (Mengeling & Cutlip, 1975; Mengeling et al., 1984; Kresse et al., 1985; Choi et al., 1987). Substitutions of only a few residues in the VP2 capsid protein (D378->G, H383->Q and S436->P) are responsible for distinct biological properties between the NADL-2 and Kresse strains of PPV (Bergeron et al., 1996).

Comparison between genomes of the two aforementioned PPV strains revealed that there are four silent nucleotide substitutions in the non-structural coding genes. In contrast, six of eight nucleotide substitutions located in the VP1/VP2 genes alter the coding sequence, with all but one of the non-synonymous substitutions causing mutations in the VP2 sequence (I215->T, D378->G, H383->Q, S436->P and R565->K). The exception was a mutation in the unique portion of VP1 (A92->R) (Bergeron et al., 1996).

Bergeron et al. (1996) also found that among the differences between NADL-2 and Kresse VP2 sequences, five were present consistently in the Kresse strain and field isolates (I215->T, D378->G, H383->Q, S436->P and R565->K). Moreover, a 127 nt repeat after the right ORF was detected in the NADL-2 strain and in other vaccine strains but not in the virulent isolates of PPV.

In a closely related virus, canine parvovirus (CPV), sequence variability in the capsid protein-encoding genes can be correlated directly with differences in epitopes (Parrish et al., 1991; Strassheim et al., 1994). Regions containing the main differences between CPV and feline parvovirus (FPV) and between CPV-2, -2a and -2b (the evolutionary products of CPV antigenic drift) are located on the threefold spike on the capsid protein (Parrish, 1991; Parrish et al., 1991; Martyn et al., 1990). A number of polymorphic sites and residues that are phenotypically important for several parvoviruses were mapped onto the three-dimensional (3D) structure of CPV and were antigenicity strongly associated to the highly variable externally exposed loops (Chapman & Rossmann, 1993).

Using synthetic peptides and porcine or rabbit anti-PPV antisera, the antigenic structure of PPV was investigated. Several linear epitopes were revealed in the region corresponding to the major capsid protein, VP2 (Kamstrup et al., 1998). Most of these linear epitopes map to the exposed surface regions of the PPV structure (Simpson et al., 2002).

The largest structural differences among PPV, FPV and minute virus of mice (MVM) occur at residues on the outside surface of the capsid, with the largest changes associated with eight specific regions. The sequences of the exposed surface loops are less conserved than internal regions of the capsid (Simpson et al., 2002).

Here, we report genetic diversity among 29 sequences of field strains of PPV, revealed by partial sequencing of the VP2-encoding gene, and analyse the differences in terms of the structure of the putative amino acid sequence. Structural correlations were based on the structure of PPV capsids solved using X-ray crystallography (Simpson et al., 2002). Also, we studied the genealogical relationship of PPV strains with the aim of elucidating PPV lineages.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PPV samples.
Clinical samples of foetal tissues from naturally PPV-infected swine were used. Foetal tissues (lung, liver and kidney) from dams that had shown signs of reproductive disturbances were ground using a mortar and pestle and resuspended as a 20 % (w/v) suspension in PBS, pH 7·4. After three freeze–thaw cycles, homogenates were cleared by centrifuging at 2000 g for 20 min. Supernatants were tested by PCR using primers directed to the NS1 gene (Soares et al., 1999). The DNA from homogenates that proved positive by PCR was extracted following a method described elsewhere (Chomczynski, 1993). DNA extracts were stored at -20 °C until required. Clinical samples originated from different municipalities across the Brazilian territory (Fig. 1 and Table 1).



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Fig. 1. Origin of PPV samples from different municipalities of Brazil.

 

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Table 1. Identification, region of isolation, clinical presentation and GenBank accession numbers of PPV isolates

 
Amplification.
DNA extracts of PPV samples were amplified using a procedure that generated two overlapping nested PCR products encompassing the last 853 nt of the right ORF. The first nested PCR product was generated using the following primers: P1, 5'-AACTCACTCATGGCAAACAAACAGA-3'; P2, 5'-TGCCTCCAAAACTACTAACTGAACC-3'; P5, 5'-TGCTGTTAATGGTCCATATGTATTGA-3'; and P6, 5'-ACCATTTGGAAATACAGGTGCAGTA-3'. The second nested PCR product was generated using the following primers: P3, 5'-GGCACCACTAAACCTAGAAAATACA-3'; P4, 5'-CACTTTTACCTTCAGATCCAATAGG-3'; P7, 5'-CTGAGTTTTTATTTACAGAGTTATTT-3'; and P8, 5'-CAATGATAGTAGTACATGATTAACCAA-3'. The positions of primers in the PPV genome are 3637–3661 (P1), 3672–3696 (P2), 4048–4072 (P3), 4083–4107 (P4), 4140–4165 (P5), 4174–4198 (P6), 4550–4575 (P7) and 4580–4606 (P8); the numbering system is that adopted by Bergeron et al. (1996).

Amplification conditions for both nested PCRs were performed as follows: 5 µl of extracted DNA in a final volume of 50 µl, containing 0·2 mM of each dNTP, 50 pmol each primer (outer primers), 1·5 mM MgCl2, 1xPCR buffer (Gibco-BRL), 2 units Taq DNA polymerase (Gibco-BRL) and milliQ water QS. PCR amplification was performed in a MJ Research PTC-200 ThermalCycler under the following conditions: 1 cycle at 94 °C for 3 min, 35 cycles at 94 °C for 45 s, 55 °C for 60 s and 72 °C for 90 s, and 1 cycle at 72 °C for 10 min. Nested PCR was performed with 5 µl of the primary amplification template and the inner primers. The thermal cycles for the nested assay were the same, except for an amplification phase of 25 cycles. PCR products were electrophoresed in 2·0 % agarose gels in standard TBE and stained with 0·5 µg ethidium bromide ml-1 (Sambrook et al., 1989).

Nucleotide sequencing and alignment.
Amplified products with the expected molecular masses were excised from the gel and purified using a commercial kit (Concert, Gibco-BRL). Sequencing reactions were performed using the dideoxynucleotide chain-termination method with the BigDye Terminator kit (Applied Biosystems) and sequences were determined with an automated sequencer (ABI model 377, Applied Biosystems), according to the manufacturer's instructions. Sequencing was performed in both directions using the primers P2 and P5 for the first nested PCR product and primers P4 and P7 for the second nested PCR product. All sequences were performed at least twice to avoid artefacts.

Nucleotide sequences were aligned by eye using CLUSTAL X (Thompson et al., 1997), SEQPUP (version 0.6f) (Gilbert, 1995) and sequence Navigator (version 1.01) (Applied Biosystems). The degree of similarity among sequences at both nucleotide and amino acid levels (the NADL-2 and Kresse strains of PPV were included in this analysis) was determined using BIOEDIT (Hall, 1999). BIOEDIT was also used to align and calculate the degree of similarity of VP2 sequences between PPV, CPV and MVM. The alignment of closely related viruses was done with the aim of plotting polymorphic sites in the PPV sequences.

For plotting the polymorphic sites of VP2 sequences on Figs 2 and 6, we have included the data from sequences IAF-3, IAF-22, IAF-76 (Bergeron et al., 1996) and 90HS (Sakurai et al., 1989).



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Fig. 2. A 3D model of the VP2 structure of PPV. Arrows represent {beta}-sheets (gold) and {alpha}-helixes (green). (A) Yellow segments represent the last 283 aa of VP2. (B–D) Different views of VP2 showing polymorphic sites in yellow. The 3D model of VP2 was taken from the molecular modelling database (accession number 17713) with the kind permission of Dr Michael G. Rossmann (Simpson et al., J Mol Biol 315, 1189–1198, 2002).

 


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Fig. 6. Alignment of the VP2 sequences of PPV, CPV and MVM. The fragment studied here corresponds to the interval between residues 297 and 579, following the numbering system of PPV (indicated A). Closed arrows ({blacktriangledown}) point to polymorphic sites detected among Brazilian sequences of PPV. Open arrows ({nabla}) point to polymorphic sites detected in this study and in the study of Bergeron et al. (1996). Crosses (+) mark polymorphic sites detected in the study of Bergeron et al. (1996). CPV residues 296–306 correspond to loop 3 and residues 409–444 correspond to loop 4. Identical sites are shaded in black boxes, conservative differences are shaded in grey. Less conserved regions are left open. The antigenic sites identified by Kamstrup et al. (1998) are: site 4 (residues 458–476), site 5 (residues 475–494), site 6 (residues 515–538), site 7 (residues 526–538), site 8 (residues 535–549), site 9 (residues 587–603). The GenBank accession numbers for PPV, CPV and MVM are U44978, M19296 and NP_041248, respectively.

 
Genealogical analyses and nucleotide diversity.
NADL-2, a vaccine strain (I18745), and the Kresse strains of PPV were included in these analyses. For the construction of genealogical trees, PAUP*, version 4.0, was employed (Swofford, 2000), using heuristic search and equal weighting in the maximum-parsimony analysis. Consistency indexes were calculated by MACCLADE, version 3.03 (Maddison & Maddison, 1992). Groups of sequences identified in the trees were tested by statistical analyses of nucleotide diversity and sequence variability using DNASP, version 3.53 (Rozas & Rozas, 1997). The following parameters were considered: n (sequences analysed), s (polymorphic sites), k [average number of nucleotide differences (Tajima, 1983, 1993)], and its variance (Vk), ss (synonymous substitutions), nss (non-synonymous substitutions), p1m2 (mutations polymorphic in population 1 and monomorphic in population 2), p2m1 (mutations polymorphic in population 2 and monomorphic in population 1), f (number of fixed differences), s' (number of shared mutations), k' (average number of nucleotide differences), k'' (average number of nucleotide differences between populations), Nm (Nei's pseudoparameter) and the estimator, GammaST (Nei, 1982).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Over the 853 sites of the 29 nucleotide sequences of Brazilian strains of PPV, no gaps were detected, and a total of 26 polymorphic sites was found scattered within sequences. At the amino acid level, we found 22 polymorphic sites. Including other published sequences of PPV (IAF-3, IAF-22, IAF-76, 90HS, Kresse and NADL-2), 39 polymorphic sites were detected at the nucleotide level and 29 polymorphic sites were detected at the amino acid level (Table 2). The polymorphic sites from protein data were plotted against the 3D structure of PPV VP2 (Fig. 2).


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Table 2. Polymorphic sites within PPV VP2 sequences

Residues in bold correspond to the positions responsible for phenotypic differences between the NADL-2 and Kresse strains of PPV. Dashes represent amino acid identity. Amino acid positions are numbered according to Bergeron et al. (1996). The partial sequences of VP2-encoding genes of Brazilian strains of PPV are available in GenBank under accession numbers AY145472AY145500.

 
Nucleotide identity between each pair of PPV partial sequences varied from 97·6 (20 differences) to 100 %, and for amino acid sequences, from 93·9 (17 differences) to 100 %. We identified four groups of identical nucleotide and amino acid sequences; each group of identical sequences is shown in parentheses: (166–95 and 164–95), (100–95, 40–96, 49–96 and 29–97), (75–95 and 124–95) and (32–96, 20–97 and 39–96).

Using maximum-parsimony analyses of the 3'-terminal end of VP2-encoding sequences, we found 1035 trees with 61 steps and more than 9000 trees with 50 steps for the amino acid data. In each case, the strict consensus tree was computed from the shortest trees and represents the genealogy involving the alleles studied. Reconstructed trees presented a consistency index of 0·918 and 0·960, respectively. Consensus trees are shown in Figs 3 and 4.



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Fig. 3. Maximum-parsimony unrooted tree (consensus tree) based on the nucleotide sequence of the partial sequences of PPV VP2. There are four groups of identical nucleotide sequences. Each group of identical sequences is shown in parentheses: (166–95 and 164–95), (100–95, 40–96, 49–96 and 29–97), (75–95 and 124–95) and (32–96, 20–97 and 39–96). Only one arbitrary sequence from each group of identical sequences was included (marked with asterisks).

 


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Fig. 4. Maximum-parsimony unrooted tree (consensus tree) based on the amino acid sequence of the partial sequences of PPV VP2. There are four groups of identical amino acid sequences. Each group of identical sequences is indicated in parentheses: (166–95 and 164–95), (100–95, 40–96, 49–96 and 29–97), (75–95 and 124–95) and (32-96, 20-97 and 39-96). Only one arbitrary sequence from each group of identical sequences was included (marked with asterisks).

 
By including the homologous CPV sequence as the outgroup, we found a strict consensus rooted tree from 621 equally parsimonious trees. The consistency index of this consensus tree was 0·918 (Fig. 5). In all aforementioned analyses, we did not compute identical sequences. For example, from the group of identical sequences 100–95, 40–96, 49–96 and 29–97, we included only one arbitrary sequence, 49–96.



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Fig. 5. Dendrogram of the maximum-parsimony rooted tree based on the nucleotide sequence (consensus tree) of partial sequences of PPV VP2. CPV was used as the outgroup. There are four groups of identical nucleotide sequences. Each group of identical sequences is indicated in parentheses: (166–95 and 164–95), (100–95, 40–96, 49–96 and 29–97), (75–95 and 124–95) and (32–96, 20–97 and 39–96). Only one arbitrary sequence from each group of identical sequences was included (marked with asterisks).

 
The topology of the tree based on protein data shows two well-defined groups of sequences, named groups A and B (Fig. 4). Rooting the tree from Fig. 3, we obtained two other groups of sequences (sibling groups). One of them comprises sequences 32–96, 39–96, 83–95, 142–95, 20–97 and Kresse (named group Kresse), while the other group is formed by the rest of the sequences (Fig. 5).

Statistical analyses were used to test nucleotide diversity between and within the groups above. Sequence variability within group A was greater than variability within group B and Kresse, as inferred by analysis of the value of parameters k. For groups A, B, Kresse and A+B+Kresse, k values were 7·949, 4·333, 2·333 and 9·707, respectively.

Genetic variability within group A was not significantly lower than the sequence variability of all sequences (groups A+B+Kresse), as the variance of k for these groups was 2·405 and 1·704, respectively. The variance of k for groups B and Kresse was 0·875 and 1·111, respectively.

Considering the sequence variability between populations, the Nm and GammaST parameters for comparison between groups A and B were 0·8 and 0·386, respectively. The value of these parameters for comparison between group B and Kresse was, respectively, 0·36 and 0·583. The parameters k' and k'' for comparison between groups A and B were 9·707 and 12·859, respectively. For comparison between groups B and Kresse, k' and k'' equalled 8·750 and 15·625, respectively. Among the 24 nucleotide differences detected in sequences of group B and Kresse, eight were fixed differences.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Field strains of PPV cannot be propagated in conventional cell lines; hence, molecular studies of PPV DNA have been carried out directly on clinical material. In the present paper, we analysed the allele sequence of the VP2 gene from 29 PPV field strains from different states of Brazil and other published sequences (Table 1 and Fig. 1). The entire VP2-encoding sequence of PPV comprises 1740 nt (Bergeron et al., 1996; Ránz et al., 1989), over which we have amplified and sequenced the 853 residues within the 3'-terminal end. The putative amino acid sequence corresponding to this fraction is 283 residues long.

This segment was chosen for several reasons: (i) it encompasses the region containing four of six non-synonymous differences identified within the VP1/VP2-encoding sequences of strains NADL-2 and Kresse (Bergeron et al., 1996); (ii) it encodes the region containing four of five amino acid substitutions that are consistently present within VP2 of the Kresse strain and virulent field isolates (Bergeron et al., 1996); (iii) the degree of similarity among VP2 sequences of PPV, MVM and FPV varies from 54 to 61 % in the first half (amino-terminal portion) and from 43 to 51 % in the second half (carboxy-terminal extremity); (iv) it encodes five of eight regions of large conformational differences, as stated by Simpson et al. (2002); (v) it encodes a segment shared by all of the different capsid proteins of PPV (VP1, VP2 and VP3) (Molitor et al., 1983; Ránz et al., 1989; Bergeron et al., 1993); (vi) it encodes six of nine antigenic sites identified by Kamstrup et al. (1998) in the VP2 capsid protein of PPV.

Considering genetic analyses of other parvoviruses, phylogenetic analysis of partial sequences from the V9-related isolates combined with human erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-defined genotypes (Servant et al., 2002). On the other hand, phylogenetic analysis revealed that the Italian CPV strains followed the same evolution as that observed in other countries and gave no indication of a separate lineage (Battilani et al., 2002).

Parsimony analyses have proved to be highly informative for demographical and mutational studies concerning the genetic variability of the virus (Novitsky et al., 2002; Bok et al., 2002; Yanagihara et al., 2002; Heinemann et al., 2002; Fanning & Taubenberger, 1999). In this study, we could demonstrate using parsimony analyses the presence of specific sites that gave information for the reconstruction of the genealogy of PPV field strains.

In spite of the fact that group A is the most heterogeneous group and the topology of the maximum-parsimony unrooted tree is based on nucleotide sequences (Fig. 3), the unrooted amino acid tree identifies unequivocally at least two distinct groups of PPV prevalent in Brazil, one represented by a group of isolates named group B and the other represented by a group named group A (Fig. 4).

Rooting the maximum-parsimony unrooted tree based on nucleotide sequences using the homologous sequence of CPV as the outgroup identified a further subgroup, named subgroup Kresse (Fig. 5).

The assumption of the existence of two main groups (A and B) within the sequences was reinforced by data from inter-group analyses, which produced GammaST and Nm values estimated at 0·39 and 0·8, respectively. These results indicate that both groups should be considered distinct evolutionary units. The same considerations should be stated for the distinction between the Kresse subgroup and group B. In this case, those parameters indicate an even higher inter-group divergence (0·58 and 0·36 for GammaST and Nm, respectively).

Although we may separate the samples into two groups, designed A and B, and further identify a subgroup within group A (subgroup Kresse), genotypic grouping could be associated with neither clinical presentation nor geographical origin of the samples.

However, it is worthwhile mentioning two paradoxical findings: firstly, two samples (83–95 and 148–95, which were both from the same herd; see Table 1 and Fig. 1), detected about 2 months apart in the same year, were also one of the most divergent pair of sequences (97·8 % identity). Samples 148–95, 75–95, 12–97 and 83–95 were all from the same herd but, according to the genotyping proposed here, sample 83–95 belongs to a different genotype. This result suggests that different genotypes may co-exist in a given region. In a similar work, Erdman et al. (1996), while evaluating the genetic diversity of human parvovirus B19, suggest that more than one genetic lineage appears to circulate in an outbreak.

Another noticeable finding was that two samples (58–00 and 71–00) originating from municipalities about 1000 km apart (Fig. 1) were one of the least divergent pairs of samples (99·7 %). Interchanges of infected sows or boars and contaminated genetic material between herds could explain closely related genotypes infecting animals of herds at distances of 1000 km. This finding highlights the need for intensive surveillance of PPV transmission between herds.

Interchange of residues at positions 378, 383 and 436 between the Kresse and the NADL-2 strains was sufficient to alter the cell tropism observed in vitro but was not sufficient to demonstrate the difference between their virulence (Bergeron et al., 1996). In our work, we have found field strains carrying exactly the same residues of NADL-2 at the aforementioned positions (Table 2); we may, therefore, speculate that these positions are not solely responsible for differences in virulence between virulent PPV isolates and the NADL-2 strain. Based on the results of the nested PCR using primers P3, P4, P7 and P8, which yielded a single band of the expected size for all PPV samples, we observed that all PPV samples in our study lacked the 127 nt repeat found in the NADL-2 strain.

All field samples analysed in this study were from foetal tissues farrowed by sows showing signals of reproductive failure and which were negative for other common causes of reproductive disturbance in swine, such as brucellosis, leptospirosis and Aujeszky's disease. Live attenuated virus vaccines against PPV infection are not available in Brazil, so all vaccines are made from inactivated virus. Therefore, detection of vaccine strains in the field samples may be ruled out and thus all field strains should be considered virulent.

As stated before, the VP proteins of PPV differ only in their amino-terminal initiation site. The capsid is formed by a mixture of VP1, VP2 and VP3, with VP1 and VP2 being translated from different start codons and VP3 being a product of VP2 proteolytic cleavage. Therefore, the amino acid sequence of the three components of the PPV capsid is identical in the carboxy terminus. Consequently, each amino acid substitution detected in the carboxyl termini of a product encoded by the right ORF of PPV would occur simultaneously in all three capsid proteins of the virus. Thus, all substitutions encountered in the segment studied here would be responsible for amino acid changes in all subunits of the virus particle.

We observed that the amino acid substitutions were not distributed evenly within the sequences of PPV samples. Instead, considering also the substitutions detected in some other vaccine and virulent strains sequenced elsewhere (Sakurai et al., 1989; Bergeron et al., 1996), we observed a highly conserved fraction flanked by two variable regions (Fig. 6). The segment between residues 468 and 550 has almost 100 % identity. The exception is the conserved substitution V485->A in the IAF-22 sequence (Table 2 and Fig. 6).

The pattern of amino acid substitutions within VP2 sequences of PPV agrees well with the pattern of similarity between homologous regions of VP2 sequences of PPV, CPV and MVM (Fig. 6). Polymorphic sites and conserved regions are mapped onto regions of higher and lower variability, respectively, in the alignment between the aforementioned viruses.

Plotting the polymorphism detected here in the 3D structure of PPV VP2 [see the X-ray crystallography of PPV capsid protein determined by Simpson et al. (2002)], we observed that the predominant regions of highest variability are also some of the most accessible. On the other hand, the conserved region is mostly formed by residues on the inner surface of the capsid, with the exception being just one segment formed by residues on the outer surface and which differs conformationally when compared to homologous fractions of FPV and MVM (residues 504–520).

Even though Kamstrup et al. (1998) have shown that neutralizing activities induced with oligopeptides derived from linear antigenic sites are low, the authors could demonstrate the antigenicity of nine regions (linear epitopes) scattered within VP2 sequences. These sites, originally numbered 1–9, were identified by PEPSCAN using anti-PPV antisera from pigs and rabbits. The segment of VP2 studied here contains sites 4–9. From all 22 amino acid substitutions, we have found 15 substitutions located within sites 4, 6, 7, 8 and 9 (Fig. 2).

There is a close correspondence of PPV amino acid changes and residues that characterize the differences between the three genotypes of CPV (CPV-2, -2a and -2b). Moreover, plotting the substitutions encountered here onto the alignment between PPV and CPV sequences has shown that many PPV substitutions mapped to regions within, or in the vicinity of, loops 3 and 4. Those regions are of recognized antigenicity and/or contain phenotypically important residues for CPV and other parvoviruses (Chapman & Rossmann, 1993).

The 100 % identity of some conserved segments of VP2 suggests that those fractions may be associated with some essential function, such as a receptor-binding site, or may take part in some vital process during virus replication. The function of the parvovirus capsid protein and its relation to virus replication has been demonstrated already (Gardiner & Tattersall, 1988).

Within a protein, different structural or functional domains are likely to be subject to differential constraints and to evolve at different rates. The basic structural requirements for some proteins to play a given role are functional constraints that impair the domain responsible for that protein to evolve at faster rates than other domains which do not take part in the functional activity of the molecule. Conversely, the faster rate of protein domain evolution, e.g. non-synonymous substitutions evolve faster than synonymous, is likely to be a consequence of advantageous selection for a given protein (Li, 1997).

The strong correlation between polymorphic residues and its accessibility and the high proportion between non-synonymous and synonymous substitutions encountered in the target sequences are conditions likely associated with attempts for antigenic changes in response to the selective pressure imposed by the host's immune system.

Public health issues concerning animal organ and tissue transplantation into humans have provoked intense debates because of the risk posed by animal pathogens crossing the species barrier (Murphy, 1996; Takeuchi, 2000; Chapman et al., 1999; Platt, 2000). Opportunities for species jumping in xenotransplantated patients are higher because of the immunosuppressed status of the recipient. As stated elsewhere (Baranowski et al., 2001), opportunistic contacts of variant viruses with potential new hosts have been considered a risk for disease emergence. Moreover, there is a putative precedent for parvoviruses jumping the species barrier; it has been postulated that CPV was derived as a variant of FPV or some other closely related virus (Parrish et al., 1988; Truyen, 1999). In fact, various properties that distinguish CPV and FPV were mapped in the capsid protein gene.

In conclusion, despite the high overall conservation of VP2-encoding sequences, the genealogical analyses presented here provide enough evidence to show that distinct genotypes exist between PPV strains. Moreover, results from protein data may indicate the existence of non-negligible changes in the antigenic make-up of PPV. This genetic drift is likely to generate new variant strains that may have important consequences in the epidemiology of PPV infection.


   ACKNOWLEDGEMENTS
 
We thank Dr Fábio Gregori and Dr Paulo Eduardo Brandão (Laboratório de Virologia, Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo, Brazil) for helpful discussions and their kind attention in the informatics' tasks. This work was supported by FAPESP (grant no. 98/12378-6).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baranowski, E., Ruiz-Jarabo, C. M. & Domingo, E. (2001). Evolution of cell recognition by viruses. Science 292, 1102–1105.[Abstract/Free Full Text]

Battilani, M., Ciulli, S., Tisato, E. & Prosperi, S. (2002). Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy. Virus Res 83, 149–157.[CrossRef][Medline]

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Received 26 November 2002; accepted 31 January 2003.