Virus Research 83 (2002) 149– 157 www.elsevier.com/locate/virusres Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy Mara Battilani a,*, Sara Ciulli a, Ernesto Tisato b, Santino Prosperi a a Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale, sez Malattie Infetti6e, Facoltà di Medicina Veterinaria Uni6ersità degli Studi di Bologna, Via Tolara di Sopra, 50, 40064 -Ozzano Emilia, Bologna, Italy b Istituto Zooprofilattico Sperimentale delle Venezie, Via Romea, 14 ¯A, 35020 -Legnaro, Pado6a, Italy Received 31 July 2001; received in revised form 26 November 2001; accepted 26 November 2001 Abstract Genetic and antigenic properties of 62 field isolates of canine parvovirus (CPV-2) collected from 1994 to 2001 in Italy were investigated. Antigenic characterisation was conducted using specific monoclonal antibodies (Mabs). The VP1¯VP2 gene was amplified by PCR and characterised with restriction endonucleases to detect the 297 and 265 variant. The VP2 gene of 16 isolates was sequenced and molecular genetic analysis was conducted. The antigenic type prevalent among our isolates is type 2a as well as the 297 variant, which is also prevalent in the rest of Europe. Only the 9.7% of the isolates have the T265P mutation. The VP2 sequences of CPV-2 isolates were very similar to recent Asian isolates. In the threefold spike of CPV-699 a coding change was detected in the 440 residue where threonine was substituted by alanine: the same mutation has been found in two Asian CPV-2 isolates from leopard cats [Virology 278 (2000) 13]. Phylogenetic analysis revealed that the Italian CPV-2 strains followed the same evolution as observed in other countries and they gave no indication of a separate lineage. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Canine parvovirus (CPV-2); Antigenic types; Italy; VP2 sequences; Phylogenetic analysis Canine parvovirus (CPV-2) is a significant pathogen for domestic dogs. It causes an acute haemorrhagic diarrhoea and sometimes fatal myocarditis in young dogs. CPV-2 emerged suddenly in 1978 (Appel et al., 1979) and then the original type 2 was replaced by new genetic and antigenic variants, type 2a (CPV2a) and type 2b (CPV-2b) (Parrish et al., 1991). * Corresponding author. Tel.: + 39-051-792-202; fax: + 39051-792-039. E-mail address: [email protected] (M. Battilani). The old and the new variants have different host range; new types, in fact, replicated efficiently in cats, even if the original CPV-2 did not replicate (Mochizuki and Hashimoto, 1986). The viral capsid protein VP2 determines host range and other biological differences in different parvovirus; only a few amino acid substitutions are responsible for critical genetic and antigenic properties (Parrish et al., 1991; Truyen et al., 1995a). These viruses show how very small changes can profoundly alter the fundamental biological properties of a virus. The high rates of 0168-1702/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 1 ) 0 0 4 3 1 - 2 150 M. Battilani et al. / Virus Research 83 (2002) 149–157 substitution during replication as in RNA viruses are not required for viruses to undergo rapid evolution (Truyen et al., 1995a,b). Interestingly, CPV-2 continues to show an ongoing evolution. Truyen et al. (2000) by analysing recent CPV isolates from Germany, Switzerland and Austria, observed the consistent appearance of an amino acid change at position 297 (S-A); now it is the predominant virus in Europe and occurs in both CPV-2a and CPV-2b viruses. In the VP2 sequences of Italian strains in dogs and wolves we detected a mutation in the 265 residue, where threonine is substituted by proline: this amino acid change causes a disruption in a G strand of the b-sheet in the protein, but the biological consequences of this variant are unknown (Battilani et al., 2001). In this study we examined 62 CPV-2 strains isolated between 1994 and 2001 from faecal samples of dogs showing clinical signs of haemorrhagic gastroenteritis; these samples were collected from different regions in Italy; the VP2 genes of some strains were sequenced to verify the evolution of Italian CPV-2. Faecal samples were processed and inoculated onto feline embryonic fibroblast (FEA) cells as described by Mochizuki and Hashimoto (1986). Viruses were propagated in culture cells for three blind passages; the third cell passage supernatant was used for antigenic typing by the haemogglutination inhibition test (HI) using typespecific monoclonal antibodies (MAbs) (Parrish et al., 1982; Parrish and Carmichael, 1983). The American reference strains CPV-d (type 2), CPV-15 (type 2a) and CPV-39 (type 2b) were used for control. MAbs and American reference strains were provided by Colin Parrish (Cornell University, Ithaca, NY, USA). For PCR amplification, the DNA of each viral strain was extracted from the third passage supernatant using a QIAamp DNA mini kit (QIAGEN, Germany) according to the manufacturer’s instructions. The VP1¯VP2 gene was amplified by using Pfu Turbo (Stratagene, USA) and primers VPF and VPR (Mochizuki et al., 1995); the amplicons were characterised by restriction endonucleases analysis using the AluI and Sau96I enzymes which are able to detect the non-synonymous substitutions in the nucleotides 3579 (A C) and 3675 nucleotides (TG) responsible for the coding changes in the 265 and 297 residues, respectively. For VP2 gene sequencing analysis, the region was amplified using a set of four primers as precedent described (Battilani et al., 2001). The nucleotide sequences obtained were compared with sequences available from the GenBank and the alignment of sequences was performed with the GeneDoc program (Nicholas et al., 1997). Phylogenetic and molecular evolutionary analysis were conducted using MEGA version 2.1 (Kumar et al., 2001): pair-wise genetic distance was calculated by using Jukes–Cantor Gamma distance. The phylogenetic relationship was analysed by using the minimum evolution (ME) method with Close-Neighbour-Interchange algorithm; we selected representative minimal trees according to the neighbour-joining method. The amino acid distance of the VP2 protein was calculated using amino Poisson-correction distance. The reliability of the phylogenetic tree obtained for the VP2 region was evaluated by running 500 replicates in the bootstrap test (Felsenstein, 1985). The ratio of synonymous (dS) and non-synonymous substitutions (dN) was estimated in order to investigate the evolutionary mechanism of the CPV isolates; they were estimated using the modified Nei and Gojobori method (Nei and Gojobori, 1986) which differs from the original formulation, because, it is necessary to provide the transition/trasversion ratio (R). The results of antigenic typing by MAbs showed a prevalence of type 2a confirming the results of other authors (Table 1) (Buonavoglia et al., 2000). The epidemiological situation of CPV-2 in Italy is similar to that in the rest of Europe (Truyen et al., 2000). The new antigenic types 2a and 2b completely replaced the old type 2; the extension of the host range of the new types to cats has probably had some influence in this substitution which has been observed world-wide (Steinel et al., 1998). M. Battilani et al. / Virus Research 83 (2002) 149–157 151 Table 1 Prevalence of antigenic types Year of isolation Number of specimens CPV-2 CPV-2a CPV-2b 1994 1995 1996 1997 1998 1999 2000 2001 6 17 15 4 3 6 8 3 0 0 0 0 0 0 0 0 6 12 15 4 3 6 8 3 0 5 0 0 0 0 0 0 The amplicons of the VP1/VP2 gene were analysed using restriction endonucleases to identify the 265 and 297 variants in the isolates. The AluI enzyme detected the 297 variant which is prevalent in the CPV isolates, in fact, 53 strains have the 297 mutation (85.4%); it is detected both in type 2a and in type 2b. Our results showed that the 297 variant is prevalent in Italy as it is in North Europe (Truyen, 1999). Sau96I can detect the 265 variant; six strains, both type 2a and type 2b showed this mutation (9.7%). The 265 residue is located in the jelly-roll b-barrel, a structure typical of the virus with icosahedral symmetry (Rossmann and Johnson, 1989). The capsid b-barrel motif is more conserved compared with the loops and it is usually less interested from mutation in the parvovirus species (Chapman and Rossmann, 1993). When analysing the 3D model of the 265 variant, it emerged that the substitution of threonine with proline causes the disruption of the G strand of the barrel and eliminates a hydrogen bond with the 142 residue. Furthermore, it was supposed that the mutation might also affect DNA–protein interaction (Battilani et al., 2001). It was observed that one mutation excludes the other, in fact, no strain exists with both mutations: we supposed that the two mutations could be the result of different evolutive lineages. Furthermore, only three strains do not have both changes (CPV-PD isolated in 1994, CPV-613 isolated in 1995 and CPV-637 isolated in 1996). DNA sequence analysis of the VP2 gene of 16 strains confirmed the antigenic typing and the characterisation by restriction enzymes. In Table 2, the CPV isolates sequenced were listed. Comparison of VP2 gene sequences showed a complete identity between CPV-603 and CPV-616, CPV584 and CPV-589 as well as between CPV-618, CPV-660, CPV-687 and CPV-697. The other strains showed a sequence homology which varied from 99.9 to 99.5%. Nucleotide substitutions are summarised in Table 3. The coding changes were detected at nt. 35793675-4062-4104 resulting in amino acid substitutions in the 265 (T-P), 297 (S-A), 426 (N-D) and 440 (T-A) residues. The 426 residue distinguished between type 2a and 2b. Table 2 CPV isolates analysed by DNA sequencing Virus Year of isolation Origin GenBank accession no. CPV-584 CPV-589 CPV-598 CPV-603 CPV-616 CPV-618 CPV-632 CPV-637 CPV-660 CPV-677 CPV-684 CPV-685 CPV-687 CPV-689 CPV-697 CPV-699 1994 1995 1995 1995 1995 1995 1996 1996 1997 1998 1999 1999 1999 1999 2000 2000 Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, Dog, AF306446 ND ND ND AF306449 AF306447 AF306445 AF306450 ND AF306448 ND ND ND ND ND AF393506 Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy 30 A – – – – – – – – – – C – – – – Nt. VP2 gene CPV-584 CPV-589 CPV-598 CPV-603 CPV-616 CPV-618 CPV-632 CPV-637 CPV-660 CPV-677 CPV-684 CPV-685 CPV-687 CPV-689 CPV-697 CPV-699 A – – – – – – – – – G – – G – – 36 2822 T – C C C C C C C C C C C C C C 75 2861 G – A – – – – – – A – – – – – – 147 2933 T – – – – – – – – – C – – – – – 303 3089 G – – A A – – – – – – – – – – – 333 3119 T – – – – – – – – C – – – – – – 393 3179 T – – – – – – C – – – – – – – – 504 3290 C – T T T – T T – – T T – – – – 537 3323 T – C – – – – – – – – – – – – – 720 3506 A – G – – – – – – – – – – – – – 756 3542 A – – C C – – – – – – – – – – – aa265 T P 793 3579 G – – T T – – T – – – – – – – – aa297 SA 889 3675 G – – – – – – A – – – – – A – – 1083 3869 A – G G G – – G – – – – – – – – aa426 ND 1276 4062 A – – – – – – – – – – – – T – – 1310 4096 A – – – – – – – – – – – – – – G aa440 TA 1318 4104 A – – – – – G – – – – – – – – – 1602 4388 T – – – – – C – – – – – – – – – 1662 4448 G – A A A – A A – – A A – – – – 1710 4496 T – – – – – – – – – – G – – – – 1713 4499 The nucleotides that are identical to CPV-584 are indicated by dashes, while the nucleotides that differed from CPV-584 are indicated by letters. Nucleotide changes that result in amino-acid substitutions are indicated with residue position of VP2 protein and deduced amino acid changes. 2816 Nt. complete genome Table 3 Variable nucleotides in the sequences of the VP2 genes analysed in this study 152 M. Battilani et al. / Virus Research 83 (2002) 149–157 M. Battilani et al. / Virus Research 83 (2002) 149–157 The mutation in the 440 residue of the VP2 capsid protein has been found in the CPV-699 isolate. When comparing our Italian isolates with other CPV strains isolated in various parts of the world, the same mutation in two strains isolated from wild feline in Asia were found (GenBank reference number AB054221-AB054222). In fact, in the VP2 protein tree (Fig. 1b), the CPV-699 formed a distinct cluster with these Asian isolates. The 440 residue is located in the GH loop; this large loop is composed of 267– 498 residues and is located between the bG and bH strands. The GH loop intertwines with two other symmetry equivalents to form a protrusion around each threefold axis (Liljas, 1991). This region is exposed on the surface of the capsid and forms the 22 A, threefold spike; the greatest variability between parvoviruses was observed in this antigenic region (Chapman and Rossmann, 1993). For estimation of viral phylogenetic relationships, we constructed phylogenetic trees for the VP2 gene and the VP2 protein: phylogenetic tree analysis was performed using the ME method along with the sequences of the foreign isolates obtained from the GenBank and published by other groups. Representative minimal trees for the VP2 gene and the VP2 protein are shown in Fig. 1a and b. For the VP2 gene and the VP2 protein, more than 50 minimal trees were obtained using the Close-Neighbor-Interchange algorithm; the differences among those trees in each gene and protein did not seem to be significant. However, we selected the minimal trees showing the same topology as those obtained by neighbour joining method as representative trees method. The phylogenetic tree of the VP2 gene showed three branches with high confidence values (\ 70%). One of the three groups includes the FPV and the FPV-like virus isolated in species other than dog; a second group consists of old type 2. The third group includes all new type 2a, 2b isolated in various parts of the world. A cluster of the new types on the phylogenetic tree was divided into three subgroups. Twelve Italian isolates placed in a cluster together with Asian isolates were classified as CPV2a: nine Italian CPV-2a isolates formed a cluster 153 including Asian isolates; three Italian CPV-2a strains (CPV-684, CPV-685 and CPV-632) were placed in different branches to form three independent lineages; it probably depends on the silent changes in the VP2 gene of these isolates. In fact analysing the phylogenetic tree of the VP2 protein, CPV-684 CPV-685 and CPV-632 were included in the type 2a cluster. Four Italian isolates classified as type 2b formed a cluster denominated CPV-2b together with Asian, American and African isolates. Inside cluster 2b, the VP2 sequences of CPV-603, CPV616 and W42 (an Italian CPV-2b strain isolated from wolf) were differentiated from other 2b strains by 89% of the bootstrap replicates. This group was maintained in the VP2 protein tree: this may be due to the presence of the T265P mutation in these isolates, which had never been detected, in other foreign strains. CPV-598, an Italian CPV type 2b, was included in a subgroup together with Asian type 2b and type 2c isolates. Instead, in the VP2 protein tree, this subgroup is inserted in the 2a cluster; in fact, all these strains have the S297A mutation, which resembles the 2a strain. The third subgroup is composed of the original type 2a taken from American isolates (CPV-15 and CPV-31), a Japanese isolate (CPV-Y1) and the African isolate (CPV-Africa9). Unlike the phylogenetic tree of the VP2 gene, the internal branches in the cluster of the new types were not present in the phylogenetic tree of the VP2 protein, indicating that the branches of the phylogenetic tree of the VP2 gene are mainly composed of silent changes. In the VP2 protein tree, three CPV strains LCVP-V140 and LCVP-V203 isolated from leopard cats (Ikeda et al., 2000) formed an independent lineage; an Asian isolate V208 (type 2b) formed a cluster with CPV-598. CPV 699 formed a cluster supported by 53% of the bootstrap replicates with LCVP-V204 and LCVP-V139. On the basis of phylogenetic analysis, the Italian strains showed great similarity to the recent Asian isolates: all Asian strains examined were isolated from domestic and leopard cats in Vietnam and Taiwan (Ikeda et al., 2000). 154 M. Battilani et al. / Virus Research 83 (2002) 149–157 Fig. 1. Phylogenetic tree constructed from the VP2 gene nucleotide (a) and amino acids (b) sequences of the Italian CPV-2 strains generated in this study and other sequences obtained from the GenBank database. Principal bootstrap values are indicated. M. Battilani et al. / Virus Research 83 (2002) 149–157 Fig. 1. (Continued) 155 156 M. Battilani et al. / Virus Research 83 (2002) 149–157 The ratio of synonymous (dS) and non-synonymous substitutions (dN) was calculated by a comparison of 16 VP2 gene nucleotide sequences of Italian strains. Since, the modified Nei and Gojobori method requires the transition/trasversion ratio (R), we calculated it to be 3.243. The proportion of synonymous substitutions is higher than that of the non-synonymous substitutions when we consider the entire VP2 gene: the ratio of dN/dS is 0.37. On the contrary, the result changes if we consider the region ranging from the 295 to the 444 residue. We focused on this region, because, epitopes important for host-range and antigenic characteristics are concentrated in this part of the VP2 region and the major amino acid variations are located here (Horiuchi et al., 1998). In this region the dN is the same as dS (dN/dS: 1). Horiuchi et al. (1998) estimated the ratio of synonymous (dS) and non-synonymous substitutions (dN) in the VP2 gene of several Asian CPV strains and they found that in the region ranging from aa295 and aa444, dN is higher than the dS, with a ratio of dN/dS =2.42. This value is not in accordance with our results; in fact, in the Italian isolates, the ratio is dN/dS =1 as in the case of no selection. This different probably comes from the brief period during which our CPV-2 strains were isolated. 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