Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy

Transcription

1 Journal of General Virology (2001), 82, Printed in Great Britain... SHORT COMMUNICATION Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy Mara Battilani, 1 Alessandra Scagliarini, 1 Ernesto Tisato, 2 Carlo Turilli, 2 Irene Jacoboni, 3 Rita Casadio 4 and Santino Prosperi 1 1 Dipartimento di Sanita Pubblica Veterinaria e Patologia Animale, Universita degli Studi di Bologna, Via Tolara di Sopra 50, Ozzano Emilia (BO), Italy 2 Istituto Zooprofilattico Sperimentale delle Venezie, Via Romea 14/A, Legnaro (Padova), Italy 3,4 Unita di Biocomputing Centro Interdipartimentale per le Ricerche Biotecnologiche (CIRB) 3 and Dipartimento di Biologia 4, Universita degli Studi di Bologna, Via Irnerio 42, Bologna, Italy The VP2 genes of Italian canine parvovirus (CPV) type 2 strains isolated from dogs and wolves were sequenced and a three-dimensional model of the VP2 capsid protein was constructed. Two mutations were detected in the VP2 sequences of the Italian strains: one at residue 297 and one at residue 265. Variant 297 is the predominant CPV isolate in Europe, whereas variant 265 has never been detected before. The mutation at residue 265 causes a disruption in a G strand of the β-barrel in the VP2 protein. Data on strains isolated from wolves demonstrated that the same strain of CPV can circulate among domestic and wild canids; therefore, this result leads us to exclude the possibility that a separate parvovirus pool exists in wild populations. Canine parvovirus type 2 (CPV-2) is an important pathogen in domestic dogs and several wild carnivore species. It was first identified in USA in 1978 (Appel et al., 1979) and was found later to have spread worldwide in domestic and wild canid populations. 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). Author for correspondence: Mara Battilani. Fax mbattilani vet.unibo.it 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). Viruses were typed for antigenicity by using the haemagglutination inhibition (HI) test with specific monoclonal antibodies (MAbs), as described previously (Table 1 b) (Parrish et al., 1982; Parrish & Carmichael, 1983); the American reference strains CPV-d (type 2), CPV-15 (type 2a) and CPV- 39 (type 2b) were used as the comparison strains. The American reference strains and the MAbs were kindly supplied by Colin Parrish (Cornell University, Ithaca, NY, USA). During passage in cell culture, virus-induced CPE was detected for each sample. Virus growth was expressed as an SGM BFFF

2 M. Battilani and others increase in the HA titre of the infected supernatant and resulted in a titre greater than 1:4000. Viral DNA was extracted from the cryo-lysate of the third passage supernatant from infected FEA cells using the QIAamp DNA mini kit (QIAGEN), according to the manufacturer s instructions. The VP2 capsid protein gene was then amplified by PCR in four sections with four sets of primers, namely P1 and P2, N1 and N2, P3 and P4, and VPM and VPR. The sequences of the primers were selected from the conserved regions of the VP2 genes (Reed et al., 1988; Rhode, 1985) and are as follows: P1, 5 ATGA- GTGATGGAGCAGTTC 3 (nt ); P2, 5 TCAT- CTAAAGCCATGTTTC 3 (nt , complementary); N1 and N2 (Senda et al., 1995); P3, 5 CCATTTCTAAAT- TCTTTG 3 (nt ); P4, 5 AAGTCAGTATCAA- ATTCTT 3 (nt , complementary); VPM, 5 TGGAGGTAAAACAGGAATT 3 (nt ); VPR, 5 TTTCTAGGTGCTAGTTGAG 3 (Mochizuki et al., 1995). PCR was carried out using Pfu DNA polymerase (Stratagene), essentially as described by Mochizuki et al. (1995). 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 AF AF 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 Jukes Cantor 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 (S ali & Blundell, 1993). The template structure used was that of the full capsid protein (PDB code 4DPV) at a resolution of 2 9 A (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 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. Table 1. Source, antigenic characterization and mutations present in CPV isolates (a) Source of Italian CPV isolates used in this study Virus Year of isolation Origin Accession no. W Wolf AF W Wolf ND W Wolf ND W Wolf ND CPV Dog AF CPV Dog AF CPV Dog AF CPV Dog AF CPV Dog AF CPV Dog ND CPV Dog AF CPV Dog ND ND, Not deposited in GenBank. (b) Results of antigenic characterization of the CPV isolates using MAbs MAb* Virus A4E3 C1D1 B4A2 B4E1 Type CPV-d 2 CPV-15 2a CPV-39 2b W42 2b W44 2b W55 2b W88 2b CPV-584 2a CPV-616 2b CPV-618 2a CPV-632 2a CPV-637 2b CPV-660 2a CPV-677 2a CPV-687 2a *, HI titre 100;, HI titre 10. 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. 1 a). Nucleotide changes and the predicted amino acid sequence substitutions are shown in Table 1(c). To investigate the effect of the threonine to proline mutation (VP2 residue 265), a 3D model of the VP2 capsid protein was constructed to simulate the structure of the mutated sequence. In both CPV-616 and the wolf sequences, BFFG

3 Table 1 (cont.) (c) Variable nucleotides in the VP2 gene sequences analysed in this study Nucleotides differing from the CPV-584 isolate are indicated by a letter, whereas nucleotides identical to CPV-584 are indicated by dashes. Nucleotide position Complete genome Isolate VP2 gene BFFH CPV-584 T G G T T C A G G A A T G CPV-616 C A T C T G A CPV-618 C CPV-637 C C T T A G A CPV-660 C CPV-677 C A C CPV-687 C CPV-632 C T G C A W42 C A T C T G A W44 C A T C T G A W55 C A T C T G A W88 C A T C T G A Amino acid mutation T P (265)* S A (297)* N D (426)* * Deduced amino acid substitutions resulting from nucleotide changes are indicated, with the residue position of the VP2 protein in parentheses. Sequence analysis of Italian CPV isolates

4 M. Battilani and others (a) (b) Fig. 1. (a) Predicted amino acid sequences of VP2. Alignment of amino acids Residues 265 and 297 are indicated. (b) Phylogenetic tree constructed from the VP2 gene nucleotide sequences of CPV strains isolated in Italy and other parts of the world. this residue is a proline (compared with a threonine in the other dog sequences). In the non-mutated sequence, residue 265 is located in the interior part of the capsid in a β-sheet referred to as BIDG (Xie & Chapman, 1996); this structure, together with the CHEF-sheet forms the jelly-roll β-barrel in each of the monomers of the capsid. The 3D model of the mutated sequence is shown in Fig. 2(b) (the 3D structure of the nonmutated sequence is also shown for comparison). It is evident that the disruption of the G strand of the barrel (nomenclature refers to the PDB file) is the result of the threonine to proline substitution. Furthermore, the replacement of the threonine with the apolar proline eliminates the side-chain hydrogen bond that was found between the γ-oxygen atom of the side chain of threonine 265 and the ε2-oxygen of glutamate 142 (Xie & Chapman, 1996). In Fig. 2(a), the DNA fragment that is co-crystallized with the VP2 protein is also shown. By focusing on the threonine 265 residue, it can be observed that the DNA fragment is in close proximity to the residue, at a distance of less than 5 A. This observation suggests that the threonine to proline mutation may also affect DNA protein interaction, especially considering that the neighbouring phenylalanine 266 residue is hydrogen-bonded to the DNA (Chapman & Rossman, 1995). 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 BFFI

5 Sequence analysis of Italian CPV isolates Fig. 2. View of the β-sheet in the interior part of the monomer protein of the CPV structure (a) and that of the mutated CPV structure (b). Residue 265 is indicated in the blue space-filled region. The mutation disrupts the G strand of the β-sheet. The orange ribbon (a) is the section of DNA that is visible in the crystal structure. population: it is impossible to demonstrate which type is predominant in a small sample, as neither of them exhibits an evolutionaryadvantage (Truyenet 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 β- 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 BFFJ

6 M. Battilani and others 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. We thank Uwe Truyen for his help regarding the sequence data, Colin R. Parrish for providing the CPV prototypes and MAbs, Michael G. Rossmann and Alan Simpson for their suggestions regarding CPV structure. This work was supported by grants from the University Ministry of Scientific Research and Technology. References Appel, M. J. G., Scott, F. W. & Carmichael, L. E. (1979). Isolation and immunization studies of a canine parvo-like virus from dogs with haemorrhagic enteritis. Veterinary Record 105, Buonavoglia, D., Cavalli, A., Pratelli, A., Martella, V., Greco, G., Tempesta, M. & Buonavoglia, C. (2000). Antigenic analysis of canine parvovirus strains isolated in Italy. Microbiologica 23, Carmichael, L. M., Joubert, J. C. & Pollock, R. V. (1980). Hemagglutination by canine parvovirus: serologic studies and diagnostic applications. American Journal of Veterinary Research 40, Chang, S.-F., Sgro, J.-Y. & Parrish, C. R. (1992). Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. Journal of Virology 66, Chapman, M. S. & Rossmann, M. G. (1993). Structure, sequence, and function correlations among parvoviruses. Virology 194, Chapman, M. S. & Rossmann, M. G. (1995). Single-stranded DNA protein interactions in canine parvovirus. Structure 3, Fico, R., Marsilio, F. & Tiscar, G. (1996). Indagine sulla presenza di anticorpi contro il virus della parvovirosi canina, del cimurro, dell epatite infettiva del cane, il coronavirus del cane e l Ehrlichia canis in sieri di lupo (Canis lupus) dell Italia Centrale. Supplemento alle Ricerche di Biologia della Selvaggina 24, Goyal, S. M., Mech, L. D., Rademacher, R. A., Khan, M. A. & Seal, U. S. (1986). Antibodies against canine parvovirus in wolves of Minnesota: a serologic study from 1975 through Journal of Wildlife Diseases 30, Higgins, D. G., Bleasby, A. J. & Fuchs, R. (1992). CLUSTAL V: improved software for multiple sequence alignment. Computer Applications in the Biosciences 8, Hills, D. M. & Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42, Kumar, S., Tamura, K. & Nei, M. (1994). MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers. Computer Applications in Biosciences 10, Martinello, F., Galuppo, F., Ostanello, F., Guberti, V. & Prosperi, S. (1997). Detection of canine parvovirus in wolves from Italy. Journal of Wildlife Diseases 33, Mech, L. D., Goyal, S. M., Bota, C. N. & Seal, U. S. (1986). Canine parvovirus infection in wolves (Canis Lupus) from Minnesota. Journal of Wildlife Diseases 22, Mochizuki, M. & Hashimoto, T. (1986). Growth of feline panleukopenia virus and canine parvovirus in vitro. Japanese Journal of Veterinary Science 48, Mochizuki, M., San Gabriel, M. C., Nakatani, H. & Yoshida, M. (1995). Comparison of polymerase chain reaction and haemagglutination assays for the detection of canine parvoviruses in faecal specimens. Research in Veterinary Science 55, Mochizuki, M., Horiuchi, M., Hiragi, H., San Gabriel, M. C., Yasuda, N. & Uno, T. (1996). Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia. Journal of Clinical Microbiology 34, Muneer, M. A., Farah, I. O., Pomeroy, K. E., Goyal, S. M. & Mech, L. D. (1988). Detection of parvoviruses in wolf faeces by electron microscopy. Journal of Wildlife Diseases 24, Parrish, C. R. (1991). Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones. Virology 183, Parrish, C. R. & Carmichael, L. E. (1983). Antigenic structure and variation of canine parvovirus type-2, feline panleukopenia virus, and mink enteritis virus. Virology 129, Parrish, C. R., Carmichael, L. E. & Antczak, D. F. (1982). Antigenic relationships between canine parvovirus type 2, feline panleukopenia virus and mink enteritis virus using conventional antisera and monoclonal antibodies. Archives of Virology 72, Parrish, C. R., Aquadro, C. F., Strassheim, M. L., Evermann, J. F., Sgro, J.-Y. & Mohammed, H. O. (1991). Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. Journal of Virology 65, Reed, A. P., Jones, E. V. & Miller, T. J. (1988). Nucleotide sequence and genome organization of canine parvovirus. Journal of Virology 69, Rhode, S. L. (1985). Nucleotide sequence of the coat protein gene of canine parvovirus. Journal of Virology 54, Sagazio, P., Tempesta, M., Buonavoglia, D., Cirone, F. & Buonavoglia, C. (1998). Antigenic characterization of canine parvovirus strains isolated in Italy. Journal of Virological Methods 73, S ali, A. & Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology 234, Senda, M., Parrish, C. R., Harasawa, R., Gamoh, K., Muramatsu, M., Hirayama, N. & Itoh, O. (1995). Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines. Journal of Clinical Microbiology 33, Truyen, U. (1999). Emergence and recent evolution of canine parvovirus. Veterinary Microbiology 69, Truyen, U., Gruenberg, A., Chang, S.-F., Obermaier, B., Veijalainen, P. & Parrish, C. R. (1995). Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo. Journal of Virology 69, Truyen, U., Steinel, A., Bruckner, L., Lutz, H. & Mostl, K. (2000). Distribution of antigen types of canine parvovirus in Switzerland, Austria and Germany. Schweizer Archiv fu r Tierheilkunde 142, (in German). Xie, Q. & Chapman, M. S. (1996). Canine parvovirus capsid structure, analyzed at 2 9 A resolution. Journal of Molecular Biology 264, Received 15 November 2000; Accepted 4 March 2001 BFGA