Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy

Virus Research 83 (2002) 149–157
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 Sanita` Pubblica Veterinaria e Patologia Animale,sez Malattie Infetti6e,
Facolta` di Medicina Veterinaria Uni6ersita` 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
www.elsevier.com/locate/virusres
Canine parvovirus (CPV-2) is a significant
pathogen for domestic dogs. It causes an acute
haemorrhagic diarrhoea and sometimes fatal my-
ocarditis 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 (CPV-
2a) and type 2b (CPV-2b) (Parrish et al., 1991).
The old and the new variants have different
host range; new types, in fact, replicated effi-
ciently 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
* Corresponding author. Tel.: [phone]; fax: +39-
051-792-039.
E-mail address
: [email protected] (M. Battilani).
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M.Battilani et al. / Virus Research 83 (2002) 149–157 150
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 on-
going 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 bio-
logical 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 sam-
ples of dogs showing clinical signs of haemor-
rhagic gastroenteritis; these samples were collected
from different regions in Italy; the VP2 genes of
some strains were sequenced to verify the evolu-
tion 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 super-
natant was used for antigenic typing by the
haemogglutination inhibition test (HI) using type-
specific 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 Univer-
sity, Ithaca, NY, USA).
For PCR amplification, the DNA of each viral
strain was extracted from the third passage super-
natant using a QIAamp DNA mini kit (QIA-
GEN, 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 Sau 96 Ienzymes which are able
to detect the non-synonymous substitutions in the
nucleotides 3579 (AC) and 3675 nucleotides
(TG) 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 com-
pared 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 se-
lected representative minimal trees according to
the neighbour-joining method.
The amino acid distance of the VP2 protein was
calculated using amino Poisson-correction dis-
tance.
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-synony-
mous 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 Go-
jobori, 1986) which differs from the original for-
mulation, 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
CPV-2 CPV-2aYear of isolation CPV-2bNumber of specimens
01994 66 0
1995 17 0 12 5
1996 15 0 15 0
044 01997
31998 0 3 0
61999 0 6 0
088 02000
2001 3 0 3 0
The amplicons of the VP1/VP2 gene were
analysed using restriction endonucleases to iden-
tify 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 de-
tected 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).
Sau
96 Ican 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 con-
served 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 muta-
tions: we supposed that the two mutations could
be the result of different evolutive lineages. Fur-
thermore, only three strains do not have both
changes (CPV-PD isolated in 1994, CPV-613 iso-
lated 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. Com-
parison of VP2 gene sequences showed a complete
identity between CPV-603 and CPV-616, CPV-
584 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. 3579-
3675-4062-4104 resulting in amino acid substitu-
tions 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
Origin GenBankYear ofVirus
accession no.isolation
1994 Dog, ItalyCPV-584 AF306446
1995CPV-589 Dog, Italy ND
CPV-598 NDDog, Italy1995
1995CPV-603 Dog, Italy ND
CPV-616 1995 Dog, Italy AF306449
Dog, ItalyCPV-618 AF3064471995
1996CPV-632 Dog, Italy AF306445
CPV-637 1996 Dog, Italy AF306450
CPV-660 1997 Dog, Italy ND
1998CPV-677 Dog, Italy AF306448
1999 NDCPV-684 Dog, Italy
NDDog, ItalyCPV-685 1999
1999CPV-687 Dog, Italy ND
1999CPV-689 Dog, Italy ND
CPV-697 2000 Dog, Italy ND
2000CPV-699 Dog, Italy AF393506

M.Battilani et al. / Virus Research 83 (2002) 149–157 152
Table 3 Variable nucleotides in the sequences of the VP2 genes analysed in this study
3119 3179 3290Nt. complete genome 33232816 3506 3542 3579 3675 3869 4062 4096 4104 4388 4448 4496 44992822 2861 2933 3089 333 393 504 537 720 756 793 889 1083 1276 1310 1318 1602 1662Nt. VP2 gene 171030 171336 75 147 303 GTTCTAA G GAG ACPV-584 AATGTTTAA
– –––––––– – –– –– ––––––CPV-589 –
–CPV-598 –––TCG–––G–– ––A–– CA– A––T––CT–G– –C– ––A––CPV-603 ––
–– A––T––CT–G–– ––A–– C–CPV-616 –– ––––––– – –– –– –––––C–CPV-618
–––T––– – ––– –CPV-632 –GCA––C–– ––CT–CPV-637 –– – TAG–– ––A–– C–– ––––––– – ––– –– – ––––CPV-660 –C–
–––C––––– – –– –– –––––CACPV-677 C– –––T––––––––––A–GC–CPV-684
–––T––– – ––– –CPV-685 – ––AG–C–C ––––––– – –– –– ––CPV-687 –– –– C–– ––––––– – A–– TC – –––––CPV-689 –G
–– ––––––– – –– –– –––––C–CPV-697
–––CPV-699 –– ––– – –– –G–––––C––
aa265 aa297 aa426 aa440
SANDTPTA
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.

M.Battilani et al. / Virus Research 83 (2002) 149–157 153
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 bGandbH strands. The GH
loop intertwines with two other symmetry equiva-
lents 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 par-
voviruses was observed in this antigenic region
(Chapman and Rossmann, 1993).
For estimation of viral phylogenetic relation-
ships, 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 differ-
ences among those trees in each gene and protein
did not seem to be significant. However, we se-
lected the minimal trees showing the same topol-
ogy 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 to-
gether with Asian isolates were classified as CPV-
2a: nine Italian CPV-2a isolates formed a cluster
including Asian isolates; three Italian CPV-2a
strains (CPV-684, CPV-685 and CPV-632) were
placed in different branches to form three inde-
pendent lineages; it probably depends on the si-
lent 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, CPV-
616 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 leop-
ard cats (Ikeda et al., 2000) formed an indepen-
dent lineage; an Asian isolate V208 (type 2b)
formed a cluster with CPV-598. CPV 699 formed
a cluster supported by 53% of the bootstrap repli-
cates with LCVP-V204 and LCVP-V139.
On the basis of phylogenetic analysis, the Ital-
ian strains showed great similarity to the recent
Asian isolates: all Asian strains examined were
isolated from domestic and leopard cats in Viet-
nam and Taiwan (Ikeda et al., 2000).