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1 Department of Public Health and Animal Sciences, Faculty of Veterinary Medicine, Strada per Casamassima km 3, 70010 Valenzano (BA), Italy
2 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, via Manfredonia 20, 71100 Foggia, Italy
3 Istituto Zooprofilattico Sperimentale di Lombardia ed Emilia Romagna, via A. Bianchi 9, 25124 Brescia, Italy
Correspondence
N. Decaro
n.decaro{at}veterinaria.uniba.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU498680–EU498720.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Carnivore parvoviruses (family Parvoviridae, genus Parvovirus), albeit DNA viruses, are very prone to genetic evolution, showing substitution rates similar to those of RNA viruses, with values of about 10–4 substitutions per site per year. Their genetic evolution has been associated with the intrinsic variability related to the single-stranded DNA conformation, as well as to the positive selection pressure related to the host immunity (Shackelton et al., 2005). Feline panleukopenia virus (FPLV) was identified at the beginning of the 20th century (Verge & Christoforoni, 1928), maintaining a certain degree of genetic stability (Battilani et al., 2006a). However, molecular studies have not been carried out for several decades: therefore the antigenic properties of the strains currently circulating are not well known. Also, the genetic and immunological relatedness between FPLV field strains and the vaccinal strains has not been evaluated, even though all the vaccines are prepared using the old FPLV strains. Several studies have demonstrated that some feline panleukopenia outbreaks in cats are caused not by classical FPLV strains but by the antigenic variants of canine parvovirus type 2 (CPV-2) (Truyen, 2006). CPV-2, was first identified in the late 1970s, has likely arisen from FPLV after adaptation in an unknown wild-carnivore species (Truyen, 2006). Three antigenic variants of CPV-2 are currently known: namely CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original type 2 that is still used in most commercial vaccines (Parrish et al., 1985; Buonavoglia et al., 2001).
There are six or seven amino acid changes between FPLV and CPV-2 and an additional five or six amino acid changes between the variants CPV-2a/b/c and the original type 2. These few amino acid differences in the VP2 sequences account for important antigenic and biological changes. For instance, in comparison to the original type 2, the antigenic variants are more aggressive and have regained the ability to replicate in vivo in the feline host (Carmichael, 2005; Truyen, 2006).
In this study, genetic analysis of FPLV strains detected in the past 4 years was achieved by PCR amplification, sequence analysis and phylogeny carried out on the VP2 protein gene.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) collected between 2001 and 2007 from cats with clinical signs of feline panleukopenia and confirmed to be parvovirus positive by a TaqMan PCR assay able to detect CPV and FPLV (Decaro et al., 2005c) were analysed. Thirty-four samples were collected in Italy and an additional five samples were sent from veterinary clinics or laboratories in the UK. The FPLV strains contained in vaccines Felocell CVR (Pfizer) and Purevax RCP (Merial) were also analysed.
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). DNA extracts were subjected to a minor groove binder (MGB) probe assay for rapid discrimination between true FPLV strains and the antigenic variants of CPV-2 (Decaro et al., 2008). The assay was carried out in a 25 µl reaction containing 10 µl DNA, 12.5 µl IQ Supermix (Bio-Rad), 900 nM primers FPV/CPV-For and FPV/CPV-Rev and 200 nM probes FPV-Pb and CPV-Pb (Table 2). Absolute quantification of the parvovirus DNA loads was obtained by means of standard curves constructed by using 10-fold dilutions of known amounts of FPLV or CPV field samples (Decaro et al., 2008). The thermal protocol was done as follows: activation of iTaq DNA polymerase at 95 °C for 10 min, 45 cycles of denaturation at 95 °C for 30 s and primer annealing–extension at 60 °C for 1 min. All reactions were conducted in an i-Cycler iQ Real-Time Detection System (Bio-Rad) and the data were analysed with the appropriate software (version 3.0). Parvovirus strains were characterized as FPLV and CPV on the basis of the detected VIC and FAM signals, respectively.
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). The amplification was achieved by means of 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 or 55 °C for 30 s and polymerization at 72 °C for 1 min. After electrophoresis on a 1.5 % agarose gel and ethidium bromide staining, the PCR products were excised from the gel and purified by a commercial kit (QIAquick gel extraction kit; Qiagen).
Sequence analysis and phylogeny.
The purified products were sequenced in both directions by Genome Express and the obtained sequences were assembled and analysed using the BioEdit software package (Hall, 1999) and the NCBI's (htttp://www.ncbi.nlm.nih.gov) and EMBL's (http://www.ebi.ac.uk) analysis tools. The VP2 sequences obtained were compared with the following reference FPLV and CPV sequences retrieved from GenBank (accession numbers are reported in parentheses): FPLV CU-4 (M38246
[GenBank]
), 193/70 (X55115
[GenBank]
), FPV-483 (D88286
[GenBank]
), PLI-IV (D88287
[GenBank]
), V142 (AB054225
[GenBank]
), V208 (AB054226
[GenBank]
), V211 (AB054227
[GenBank]
), Gercules (AY665655
[GenBank]
), GT-2 (DQ003301
[GenBank]
), ZF-5 (DQ099430
[GenBank]
), JF-3 (DQ099431
[GenBank]
), Tiger/PT06 (EF418568
[GenBank]
), Lion/PT06 (EF418569
[GenBank]
), XJ-1 (EF988660
[GenBank]
), ARG01 (EU018145
[GenBank]
), ARG02 (EU018144
[GenBank]
), ARG03 (EU018143
[GenBank]
), ARG04 (EU018142
[GenBank]
); CPV-2 CPV-b (M38245
[GenBank]
), CPV-2a CPV-15 (M24003
[GenBank]
), CPV-2b CPV-39 (M74849
[GenBank]
), CPV-2c 56/00 (not available). To evaluate the selection pressure driving FPLV evolution, the ratio of synonymous substitutions per non-synonymous site (dS) and to non-synonymous substitutions per synonymous site (dN) was estimated using the Datamonkey web interface (http://www.datamonkey.org), a maximum-likelihood-based tool for the identification of sites prone to positive or negative selection.
The phylogenetic relationships were evaluated by using MEGA3 (Kumar et al., 2004); pairwise genetic distances were calculated by using the Kimura's two-parameter model and phylogenetic trees were constructed by the neighbour-joining and maximum-parsimony methods. A bootstrap analysis with 1000 replicates was done to assess the confidence level of the branch pattern. The maximum-parsimony method was also used to confirm the topology of the phylogeny.
Nucleotide sequence accession numbers.
The nucleotide sequences of the VP2 gene of the analysed FPLV strains have been deposited in GenBank under accession numbers listed in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Titres detected for vaccine strains Purevax and Felocell were 6.75x106 and 1.89x108 DNA copies vaccine suspension ml–1, respectively.
PCR amplification of the VP2 gene
The three overlapping fragments of the VP2 gene of the FPLV strains were successfully amplified from all the 42 samples, including 40 field strains and two FPLV vaccines.
Sequence analysis
The full-length sequences of the VP2 gene (1755 nt) of the strains analysed were obtained by assembling the nucleotide sequences of the different amplicons. Amino acid translation confirmed that the sequences encoded a VP2 protein of 584 aa.
The sequences obtained were aligned with the FPLV strains detected in samples from Argentina (n=4), USA (n=1), Japan (n=5), China (n=4), Portugal (n=2), Russia (n=1) and Austria (n=1). Eighty-eight variable positions were detected in the VP2 gene, 50 of which were singletons. At position 1167, the polymorphism involved two different bases with the change T
C in all sequences, but strain 198/01 had the change TG. Considering all the nucleotide changes encountered in the VP2 sequences, synonymous substitutions predominated over non-synonymous substitutions. In fact, of the 88 nt changes, 57 were synonymous and 31 were non-synonymous. When only the phylogenetically informative changes were assigned for comparison, the proportion of non-synonymous substitutions increased, but still remained less than synonymous substitutions. To examine the evolutionary pressure determining genetic variation in the VP2 gene of FPLV, the dN/dS ratio was calculated and this value was estimated as 0.10. The single likelihood ancestor counting (SLAC) method did not detect any positively selected site, whereas nine sites subject to negative pressure selection were identified at codons 16, 233, 277, 291, 347, 389, 431, 524 and 534. By sequence analysis, the FPLV strains detected in samples from Italy and UK were found to be highly related to each other, with a nucleotide identity of 99.1–100 and 99.4–99.8 % among Italian and British strains, respectively, whereas the similarities between all the sequences analysed were 98.6–100 %. A 100 % nucleotide identity was found between the following Italian FPLVs: 189/03 and 143/04, 134/04-5 and 228/06, 189/03 and 143/04, 46/06-G6, 42/06-G14 and 22/06, 498/07 and 443/07, 134/04-2, 42/06-G7 and 42/06-G17. The Italian strains had the lowest degree of identity in comparison to the Argentinean isolates (98.8–99.0 %), whereas the strains most highly related were the Italian 134/04-2, 22/06, 42/06-G1, 42/06-G6, 42/06-G7, 42/06-G14, 42/06-G17 and the Portuguese Tiger/PT06 and Lion/PT96, the Italian 41/02, 189/03, 300/03, 143/04 and the Russian Gercules, the Italian 42/06-G3 and the Chinese JF-3, the Italian 189/03, 143/04 and the Chinese XJ-1. Also, the five British strains had a low genetic relatedness in comparison with the Argentinean isolates (99.0–99.2 % nucleotide identity), but the highest similarity (100 %) was found between FPLV 490/07 and the vaccine strain Felocell.
At the peptide level, several amino acid changes were found in the two regions forming the 22 Å (2.2 nm) long spike of the capsid surface on the threefold axis accumulating most residues that discriminate between FPLV and CPV (Horiuchi et al., 1998). Three changes were detected in region 1 comprising aa 80–106; in this region, all Italian and four of five British strains displayed the mutation I101T, which differentiates the CPV variants from the original type 2. Such a mutation is shared by all recent FPLVs, including that contained in the vaccine strain Felocell. An additional nine changes were identified in region 2 (aa 295–444), but no common pattern was found in this region, as each change was displayed by a single strain. Most residues discriminating between FPLV and CPV-2 were conserved, including 80-K, 93-K, 103-V, 323-D, 564-N and 568-A. However, four of five British strains and the two vaccinal strains displayed the change VI at residue 232. Such a mutation, which has been considered typical of CPV-2, is also shared by FPLVs samples from Argentina, Japan and China (Table 3). Noteworthy singleton mutations were observed at residues 297 and 440 in the Italian strains 134/04-1 and 42/06-G5, respectively. Another change, R377K, was detected in the British strain 50/07-1.
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Phylogeny
Phylogenetic analysis using the neighbour-joining method showed that FPLVs do not segregate on a clear temporal or geographical basis (Fig. 1). Two different clusters were formed by the VP2 nucleotide sequences analysed. The first cluster included only Italian strains and two Portuguese isolates from captive wild felids, but most viruses formed two groups into a larger cluster. Other Italian FPLVs segregated with Asian isolates into one of these groups, whereas the second group of the large cluster was formed by the remaining Italian and British strains along with vaccine viruses Purevax and Felocell, field strains from Argentina and old isolates (CU-4 and PLI-IV). The British strains 50/07-2 and 490/07 were tightly intermingled with vaccine and old FPLV strains, whereas another strain from the UK (50/07-1) was an outlier between the two main clusters. Two Italian FPLVs, 41/02 and 300/03, clustered together with the Russian isolate Gercules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Shackelton et al., 2005). The CPV variants can be shed in the faeces at much higher titres than the original CPV-2 (Carmichael, 1994; Decaro et al., 2005a, b), probably as a consequence of a further adaptation of the CPV variants to the canine host. There is also concern that the newer antigenic variants may cause a more severe disease than the original CPV-2 (Carmichael, 2005). These changes in the biological behaviour may be associated with the improved ability of CPV-2a and CPV-2b to bind to the transferrin receptor in comparison with the original type 2 (Hueffer & Parrish, 2003). In contrast, FPLV since its first identification in 1920 has not undergone significant changes in antigenic and biological properties. FPLV has evolved mainly by random genetic drift and maintained host-specificity. Analogous to RNA viruses, CPV evolution was partly driven by positive selection, leading to the emergence of new antigenic variants and expansion of the host range (Shackelton et al., 2005).
In the present study, 39 parvovirus strains from cats were analysed, all being characterized as true FPLVs. Sequence and evolutionary analyses of the 34 Italian and five British FPLVs confirmed that FPLV is in evolutionary stasis and its variation is driven by random genetic drift. Although several mutations were detected in the FPLV VP2 sequences examined, accumulation of point mutations was not consistent with a clear temporal or geographical distribution. Non-synonymous substitutions were detected in most strains analysed, but no genetic marker has been identified in the recent strains in comparison with the old isolates, with the exception of the change I101T, which is also present in the CPV-2 variants, but not in the old CPV-2. Important mutations were the S297F and T440S changes detected in the Italian strains 134/04-1 and 42/06-G5, respectively. While change at residue 297 is due to a second-base mutation, a first-base change is responsible for substitution T440S (Table 3). A different substitution (SA) at position 297 (due to the first-base change T889G) has been recently described for the antigenic variants of CPV-2 currently circulating throughout the world (Truyen, 1999, 2006; Battilani et al., 2001; Nakamura et al., 2004; Wang et al., 2005; Chinchkar et al., 2006; Martella et al., 2004, 2005, 2006; Meers et al., 2007). Residue 297 is located in the antigenic region close to epitope B and substitutions at this position may be responsible for changes in antigenicity of CPV-2 variants (Truyen, 2006). Analogously, a change at position 440 (TA) has been described for Italian, Indian, Korean and American CPV-2 isolates (Battilani et al., 2002; Chinchkar et al., 2006; Kang et al., 2008; Kapil et al., 2007) and for a clone from a mixed CPV-2 population detected in a domestic cat from Italy (Battilani et al., 2006b). As for the FPLV strains, that change is a consequence of a first-base mutation (A1318G). Both residues 297 and 440 are present in the GH loop region of the VP2 protein, where the greatest variability among parvoviruses has been observed (Battilani et al., 2002). Strain 40/07-1 showed the change R377K that was due to a first-base substitution. This change has been associated with the inability to bind erythrocytes in a non-haemaglutinating CPV mutant, as a consequence of the shortness of the 377-K side chain or different pH with respect to R (Barbis et al., 1992).
The vaccine strains Purevax and Felocell were more closely related to old FPLV strains, with the exception of British strains 490/07 and 50/07-2, which displayed a 99.9–100 and 99.7–99.8 % nucleotide identity to vaccine viruses, respectively. The genetic identity between the ‘field’ strain 490/07 and the vaccine virus Felocell suggests a possible isolation of the vaccine strain from a cat recently vaccinated. In fact, it is well known that modified-live parvoviruses contained in the vaccines are able to replicate in the intestinal mucosa of vaccinated animals and to be shed with the dog or cat faeces (Decaro et al., 2007; Patterson et al., 2007). In our case, the anamnesis of the cat shedding a Felocell-like FPLV strain was not known, thus preventing the unambiguous identification of strain 490/07 as vaccine virus.
Recently, several FPLV outbreaks in cats regularly vaccinated have been reported (C. Buonavoglia, personal observation). Whether the lower genetic relatedness of recent strains may affect the real efficacy of the FPLV vaccines currently used should be assessed by means of cross-neutralization studies using vaccine and field strains, as recently carried out for the CPV variants (Cavalli et al., 2008) and for the divergent porcine parvovirus isolates (Zeeuw et al., 2007).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 22 February 2008;
accepted 4 May 2008.
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