Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale, Università degli Studi di Bologna, Via Tolara di Sopra 50, 40064-Ozzano Emilia (BO), Italy1
Istituto Zooprofilattico Sperimentale delle Venezie, Via Romea 14/A, 35020-Legnaro (Padova), Italy2
Unità di Biocomputing Centro Interdipartimentale per le Ricerche Biotecnologiche (CIRB)3 and Dipartimento di Biologia4, Università degli Studi di Bologna, Via Irnerio 42, 40126-Bologna, Italy
Author for correspondence: Mara Battilani. Fax +39 051 792039. e-mail mbattilani{at}vet.unibo.it
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Abstract |
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After its initial appearance, it was shown that antigenic drift continuously changes the antigenicity of CPV: the original CPV-2 strain has been completely replaced by the newer antigenic types CPV-2a and CPV-2b (Parrish et al., 1991 ), which have also extended their host range to include cats (Mochizuki et al., 1996
). The new types of CPV differ from the original type 2 strain in that there are some nucleotide changes (positions 3045, 3685, 3699, 4062 and 4449) in the gene encoding the VP2 coat protein (Parrish et al., 1991
; Truyen et al., 1995
). Sequences important for the determination of antigenic type and for the control of host range are located in the VP2 capsid protein (Parrish, 1991
; Chang et al., 1992
).
Several hypotheses have been proposed to explain the sudden emergence of CPV. The most probable of which suggests that CPV arose from wild carnivores that harboured the original CPV ancestor (Truyen, 1999 ).
The presence of CPV-2 in the wolf population in USA was confirmed through serological analyses (Goyal et al., 1986 ; Mech et al., 1986
) and virus isolation from faeces (Muneer et al., 1988
). Serological evidence of CPV-2 in wolf sera in Italy has been reported previously (Fico et al., 1996
); CPV has also been isolated from wolf faeces collected in the north-central Apennine mountains (Martinello et al., 1997
). As the CPV subtype circulating in the wolf population has not been identified, the aim of our research was to characterize several wolf strains by analysing the VP2 gene sequences and to compare these sequences with those from isolates originating from Italian dogs.
In this study, and as described previously, four Italian CPV strains isolated from samples of wolf faeces (Martinello et al., 1997 ) were analysed. Eight faecal samples from dogs showing clinical signs of haemorrhagic gastroenteritis were also examined. The specimens were first examined using the Canine Parvovirus Antigen Test kit (IDEEX). Viruses were propagated in feline embryonic fibroblast (FEA) cells as described by Mochizuchi & Hashimoto (1986)
. Cells were cultured for three blind passages and the supernatants were monitored for virus growth by using the haemagglutination (HA) test, as described by Carmichael et al. (1980)
. The viruses examined are listed in Table 1(a
).
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Nucleotide sequences of the PCR products were determined with an automated DNA sequencer (ABI PRISM 310, Perkin Elmer) and the sequences obtained were submitted to GenBank under the accession numbers AF306444AF306450. The sequences were then compared to those available in GenBank and aligned with the MegAlign program of the DNASTAR multiple program package (Lasergene) using the Clustal method (Higgins et al., 1992 ).
Phylogenetic analysis was performed using the MEGA program (Kumar et al., 1994 ): pairwise genetic distances were calculated by using the JukesCantor method and phylogenetic trees were constructed by using the neighbour-joining method. A bootstrap analysis with 500 replicates was carried out to assess the confidence level of each branch pattern: a bootstrap value of >70% was considered to be significant (Hills & Bull, 1993
).
A three-dimensional (3D) model of the VP2 protein was constructed using the Modeller program (ali & Blundell, 1993
). The template structure used was that of the full capsid protein (PDB code 4DPV) at a resolution of 2·9
(Xie & Chapman, 1996
).
All CPV strains isolated from wolves and two strains isolated from dogs (CPV-616 and -637) were antigenically and genetically identified as type 2b strains, while the other six strains isolated from dogs were found to be type 2a (Table 1b). These data were confirmed by sequence analyses.
Comparison of the VP2 gene sequences showed 100% nucleotide identity between wolf isolates and CPV-616 as well as between CPV-618, -660 and -687. The other Italian strains differed by 0·5%. Sequence alignment analyses showed that there were different silent mutations and few coding changes in the VP2 gene. In particular, a coding or non-synonymous mutation was detected at nt 3579 in only the wolf strains and the dog strain CPV-616; this mutation results in residue 265 of the VP2 protein changing from a threonine to a proline residue. Another coding mutation at nt 3675 was observed in all of the type 2a strains, resulting in residue 297 of the VP2 protein changing from a serine to an alanine residue (Fig. 1a). Nucleotide changes and the predicted amino acid sequence substitutions are shown in Table 1(c
).
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Our results show that the new CPV antigenic types 2a and 2b have replaced the old type 2 in wolf populations, as is the case in dog populations.
In Italy, the prevalent antigenic type in canine populations is type 2a (Sagazio et al., 1998 ; Buonavoglia et al., 2000
). Our results seem to confirm these data, even though they show that both antigenic types 2a and 2b co-exist in the Italian canid population: it is impossible to demonstrate which type is predominant in a small sample, as neither of them exhibits an evolutionary advantage (Truyen et al., 2000
). The wolf isolates were all type 2b, but it is impossible to conclude that type 2b is predominant strain in the Italian wolf population because our data are limited to an exiguous number of samples and we do not have data regarding CPV-2 dog strains from the area where the wolf samples were collected. The complete sequence identity of the VP2 gene between the wolf strains and CPV-616 leads us to exclude the possibility that a separate CPV pool could exist in wolf populations.
The coding change at nt 3579 (VP2 residue 265, threonine to proline) is very interesting because it has not been detected previously in any other strain. This mutation cannot be referred to exclusively as antigenic type 2b because the same change was also found in several type 2a strains isolated in Italy (M. Battilani, unpublished data). The mutation at residue 265 was also unexpected, as this residue is located in the -barrel motif where residues are significantly more conserved compared with residues in the loops. This barrel region is not exposed at the virion surface and, therefore, is not subjected to the selective pressure of neutralizing antibodies of the host immune systems (Chapman & Rossmann, 1993
). However, our data showed that variant 265 is viable, as it is able to replicate in cell culture and gives an increasing HA titre with each passage. Furthermore, it is not a defective variant, as we were able to amplify the complete VP1/VP2 region at an expected PCR product of 2200 bp (data not shown). The 265 mutation was observed in both types 2a and 2b. As is the case for domestic and wild canids, this mutation is not selected for in the population, but may have arisen independently from various backgrounds.
Sequence analysis demonstrated a non-synonymous change at nt 3675, found only in type 2a: retrospective analysis revealed that this antigenic type first appeared in the USA in 1989 and in Germany around 1993, but it is now the predominant CPV antigenic type in Europe. Our results confirm that this variant is predominant among our isolates, especially in the type 2a isolates. Current studies are investigating if this change has any biological consequence (Truyen, 1999 ).
The VP2 gene sequence of CPV-632 showed two peculiar silent mutations at nts 4388 and 4448 which had not been reported previously in types 2a or 2b. Furthermore, two other mutations (nts 3323 and 4496) were found in only CPV-632, type 2b and wolf strains. In fact, phylogenetic analysis showed that CPV-632 does not belong to the Italian type 2a cluster but forms a separate virus lineage (Fig. 1b).
To analyse the phylogenetic relationships of the Italian isolates with other CPV strains isolated in various parts of the world, we constructed a neighbour-joining phylogenetic tree. A representative minimal tree for the VP2 gene is shown in Fig. 1(b). The phylogenetic tree shows three branches with high bootstrap values of >90%. One of the three groups consists of recent parvoviruses isolated from species other than the dog, the second group consists of type 2 and the third group consists of types 2a and 2b and includes our wolf and dog isolates. The CPV isolates were clearly subdivided between type 2 and types 2a and 2b, as described previously (Parrish et al., 1991
); no evidence of obvious grouping was observed with respect to the geographical origin of the isolate.
This report contributes to the study of the continuing evolution of CPV and is the first study to deal with the sequence analysis of CPV strains isolated in Italy. Some interesting results emerged, in particular, the mutation at residue 265 of the VP2 viral protein, which results in a change in the VP2 3D model. This mutation has not been detected before and further investigations will determine the biological consequences of this mutation.
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References |
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Received 15 November 2000;
accepted 4 March 2001.