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
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ABSTRACT |
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INTRODUCTION |
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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 (I215T, 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.
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METHODS |
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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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RESULTS |
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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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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.
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DISCUSSION |
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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 (8395 and 14895, 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 14895, 7595, 1297 and 8395 were all from the same herd but, according to the genotyping proposed here, sample 8395 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 (5800 and 7100) 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 504520).
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 19, were identified by PEPSCAN using anti-PPV antisera from pigs and rabbits. The segment of VP2 studied here contains sites 49. 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.
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ACKNOWLEDGEMENTS |
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Received 26 November 2002;
accepted 31 January 2003.