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Short Communication |
1 Institut für Mikrobiologie der Bundeswehr, Neuherbergstraße 11, 80937 München, Germany
2 Klinik für Schweine, Tierärztliche Fakultät, Ludwig-Maximilians-Universität München, Sonnenstraße 16, 85764 Oberschleißheim, Germany
3 Impfstoffwerke Dessau-Tornau GmbH, PF 400214, 06855 Rosslau, Germany
4 Institut für Tierhygiene und Öffentliches Veterinärwesen, Veterinärmedizinische Fakultät Universität Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany
Correspondence
U. Truyen
truyen{at}vmf.uni-leipzig.de
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AY684864–AY684872.
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). It is classified in the genus Parvovirus, the subfamily Parvovirinae and the family Parvoviridae, and contains a single-stranded (minus-strand) DNA genome of about 5 kb. The large ORF1 and the small ORF3, in the left half of the genome, code for the non-structural proteins NS1, NS2 and NS3. ORF2, in the right half of the genome, encodes the structural proteins VP1, VP2 and VP3. VP1 and VP2 are a result of alternative spliced RNAs giving VP1 a specific N terminus of 150 aa. VP3 is generated by proteolytic cleavage of VP2 (Ranz et al., 1989; Bergeron et al., 1993).
Although the three structural proteins form the icosahedral non-enveloped capsid, VP2 can assemble into virus-like particles (VLPs) by itself and the immunogenic activity of the VLPs is indistinguishable from that of inactivated whole-virus vaccines (Martinez et al., 1992). Nine linear B-cell epitopes have been defined within the major capsid protein VP2, but so far only peptides from the N terminus of VP2 are known to induce virus-neutralizing antibodies (Kamstrup et al., 1998).
The capsid proteins VP1/VP2 of the carnivore parvoviruses canine parvovirus (CPV) and Feline panleukopenia virus (FPV) determine the host range (Chang et al., 1992; Truyen et al., 1995; Shackelton et al., 2005). For PPV, however, not much is known about host-range determinants. Two amino acid differences (one in VP1/VP2 and one in NS1) extend the host range for PPV in cell culture from porcine to porcine and canine cells (Vasudevacharya et al., 1992). The pathogenicity of the highly virulent PPV Kresse isolate appears to be determined by only three substitutions in the VP1/VP2 protein (Bergeron et al., 1996). The three-dimensional structure of empty PPV capsids was determined by X-ray crystallography and was found to be similar to that of the related parvoviruses CPV and FPV. The three differences in amino acid sequence associated with the pathogenic phenotype of Kresse, as well as the known epitopes on VP2, are all located on the surface of the capsid (Simpson et al., 2002).
A recent study on the genetic variability of PPV isolates from Brazil based on partial VP2 sequences revealed a high variability in the 3'-terminal 853 nt of the capsid protein gene (Soares et al., 2003). Despite the high economic importance and wide distribution of PPV, very few DNA sequences of PPV isolates are available and have been analysed. The goal of this study was to evaluate the genetic variability of recent German isolates. Therefore, the complete VP1/VP2 gene (2187 nt) of seven recent field isolates from Germany, one isolate from the UK and one German vaccine strain were sequenced. These nine sequences were analysed along with the sequences of two American PPV isolates (NADL-2 and Kresse), 22 partial sequences from isolates from Brazil, two full-length sequences from China and one full-length sequence from Korea, all retrieved from GenBank.
Clinical samples of tissues (lung, liver and lymph nodes) from aborted fetuses and rectal swabs from sows with reproductive failure were tested for PPV by PCR. Genomic and viral DNA was purified from clinical samples and cell culture supernatant using the QIAamp DNA Mini kit (Qiagen), according to the supplier's instructions. The DNA was stored at –20 °C until further analysis. The primers used for detection of PPV by PCR were also used for amplification of the VP1/VP2 gene (D2593sense, 5'-TACTTCTTCAGAGCAAAGCG-3'; D3571antisense, 5'-CCTGTGGAGAATTCATCTCC-3'). PCR was performed using Taq DNA polymerase and 1·1x ReddyMix PCR master mix (Abgene) in a final volume of 50 µl. Amplification by PCR was performed under the following conditions: 1 cycle at 94 °C for 5 min; 35 cycles of 94 °C for 1 min, 55 °C for 2 min and 72 °C for 3 min; and 1 cycle at 72 °C for 5 min. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and custom sequenced by Seqlab. The obtained sequences were visually evaluated with CHROMAS (version 1.45) and assembled using GENEjOCKEYii and JELLYFISH (version 3.0). Multiple alignments were created with CLUSTAL_W (Thompson et al., 1994), GENEjOCKEYii and JELLYFISH (version 3.0).
Phylogenetic analyses were performed using PAUP version 3.1.1 (Swofford, 1993). Analyses were done using the maximum-parsimony method, particularly bootstrapping and the branch swapping algorithm as well as bootstrapping and the heuristic search approach. For the full-length sequences, the UPGMA method was used to confirm the topology of the phylogenies. For partial sequence analyses, which includes the Brazilian sequences, the PHYLIP package (Phylogeny Inference Package, version 3.6b, 2004; Felsenstein, 1989), namely the programs SEQBOOT, DNAPARS, PROTPARS and CONSENSE, was used. Trees were drawn using TREEvIEW (version 1.6.6, 2001). All PPV sequences analysed and their GenBank accession numbers are listed in Table 1.
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The German isolates PPV-15a, PPV-27a and PPV-Tornau 1/02 were directly amplified from tissues of infected fetuses, whereas isolates 21a, 106b, 143a, 225b, ‘challenge’ virus and the ‘IDT vaccine virus' were amplified from cell culture supernatants. Sequencing of the PCR amplificates showed that in all virus isolates the VP1 gene had a length of 2190 nt. The sequences obtained were aligned with NADL-2, Kresse, 22 published Brazilian, one South Korean and two Chinese isolates. For the Brazilian isolates, only 853 3'-terminal nucleotides were available. The alignment identified 81 variable positions for VP1 and 73 for VP2. Fifty-four of these positions were present in more than one isolate. In four positions, the polymorphism involved two different bases (Table 2). Fifty-two of the 81 changes were non-synonymous; 34 of those caused conservative amino acid changes. The ratio of non-synonymous to synonymous changes of 0·64 may indicate antigenic selection or selection by other factors. Compared with the Brazilian isolates, the German viruses had a high genetic diversity (up to 6·8 % at the amino acid level); this is even higher than that reported for the corresponding VP1/VP2 region of the B19 parvovirus (diversity of 0·09–4·22 %; Erdman et al., 1996). The similarities between all the PPV sequences studied in this report were 95·0–98·9 %; sequences of the German isolates had the lowest degree of similarity with that of NADL-2 (96·4 %) and were 96·8 % similar to the Brazilian isolates. However, it must be stated that, for the Brazilian isolates, only 853 nt were analysed and the similarity of that region is lower compared with the whole VP1/VP2 sequence.
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Y exchange in one isolate. Of the 52 variable nucleotide positions in the VP1 gene that are non-synonymous, 34 of which were observed in at least two isolates, 48 were found in the common VP1/VP2 coding sequence. Nineteen of 25 nt changes in the highly variable regions (peptide positions 511–627) were non-synonymous. At the peptide level, even more conserved domains were seen, i.e. positions 58–101, 103–145, 196–231, 244–276, 295–341, 343–377, 404–445, 474–510, 628–655 and 657–689. In the VP2 protein, a highly variable region with 19 aa substitutions in 117 aa (positions 511–627) is followed by a conserved section (positions 628–694), in which, of 67 positions, only one conservative amino acid substitution is seen in one isolate. Both domains are located on the surface of the capsid (Simpson et al., 2002). Five of the seven conserved regions are located on the surface and one is believed to mediate an internal accessibility to capsid surface residues. Interestingly, the alignment revealed another conserved domain of 90 aa in the VP1 unique region with only one conservative amino acid substitution. For this region, a phospholipase activity has been described for parvovirus B19 (Dorsch et al., 2002).
Three amino acid substitutions were reported to be responsible for the pathogenicity of Kresse (Bergeron et al., 1996). Two of these amino acids changes (H533Q and D528G) were also found in the German sequences. Interestingly, the D528G change was not found in the IDT vaccine virus. Although these changes were shown to be located on the surface of the capsid, it is unlikely that they are solely responsible for differing pathogenicity as Soares et al. (2003) found NADL-2-like sequences in isolates from clinical cases. However, the 127 nt repeat found in strain NADL-2 may be associated with virulence, because all but one of the field isolates examined so far by Soares et al. (2003), Bergeron et al. (1996) and in this study lacked this repeat.
Data about the genetic variability of PPV are very rare. Bergeron et al. (1996) sequenced 15 field isolates and described only five consistent non-synonymous nucleotide changes. Soares et al. (2003) published 26 polymorphic sites, 22 with non-synonymous changes in the 3'-terminal 853 nt of VP2. Comparing all published sequences of VP1/VP2 with our sequences, the diversity is extended. Aligning the 2140 nt of VP1 with 27 PPV sequences, a total of 81 variable positions was detected, 54 in more than one isolate.
Phylogenetic analysis of the full-length VP1 nucleotide sequence revealed one new cluster containing some of the German isolates (PPV-21a, PPV-15a and PPV-27a); the other German isolates formed a subcluster among the American (NADL-2 and Kresse), the English isolate challenge virus from 1986 and the three Asian isolates. Fig. 1(a) shows the consensus tree from a total of two most parsimonious trees calculated with the branch and bound option of the program PAUP 3.1.1. Phylogenetic trees based on the VP1 protein sequences showed basically the same topology. Fig. 1(b) shows the single most parsimonious tree resulting from the same analysis. The topology of the trees was constant regarding the German cluster irrespective of the algorithm (maximum parsimony and heuristic search option or branch and bound search, neighbour-joining or UPGMA) used for analysis (not shown). Among the substitutions that define the distinct cluster are the non-synonymous nucleotide changes leading to the change of aa 381 (VP1), 569 (VP1) and 586 (VP1), which correspond to aa 231, 419 and 436 when the VP2 numbering used by Soares et al. (2003) and Simpson et al. (2002) is followed. All three amino acids are exposed on the virus surface and aa 436 is one of the amino acids that differs between NADL-2 and Kresse (SP) and which defines the ‘allotropic determinant’ of these viruses. Amino acid 436 is located very closely to the threefold spike, a region that is known to be the main antigenic determinant (epitope B) in feline parvoviruses (Strassheim et al., 1994). When only the 3'-terminal 853 nt or the 284 C-terminal amino acids were analysed and the Brazilian sequences (Soares et al., 2003) were included, the German cluster was still seen, although isolate PPV-225b also grouped in that cluster (not shown). The amino acid responsible for that clustering was 586 (VP1) (Table 2
). The German subcluster was no longer defined.
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In conclusion, our results indicate that PPV is more diverse than previously thought and that the German isolates can be divided into two genetic clusters. Further studies on the genetic and antigenic variability of PPV isolated from different geographical regions are needed for understanding the pattern of evolution of PPV.
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Received 8 April 2005;
accepted 13 October 2005.
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